Puneet 

Kumar
Pran Kishore Deb  Editors

Frontiers in
Pharmacology
of Neurotransmitters
Frontiers in Pharmacology
of Neurotransmitters
Puneet Kumar • Pran Kishore Deb
Editors

Frontiers in Pharmacology
of Neurotransmitters
Editors
Puneet Kumar Pran Kishore Deb
Department of Pharmacology Department of Pharmaceutical Sciences
Central University of Punjab Faculty of Pharmacy
Bathinda, Punjab, India Philadelphia University
Amman, Jordan
Department of Pharmaceutical
Sciences and Technology
Maharaja Ranjit Singh
Punjab Technical University
Bathinda, Punjab, India

ISBN 978-981-15-3555-0 ISBN 978-981-15-3556-7 (eBook)

https://doi.org/10.1007/978-981-15-3556-7

# Springer Nature Singapore Pte Ltd. 2020

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the
material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
“This book is dedicated to our mentors,
students, and researchers.”
Preface

It is our pleasure and privilege to introduce the book Frontiers in Pharmacology of

Neurotransmitters covering a wide range of information about neurotransmitters and
their receptors involved in the pathophysiology of various neurological disorders. In
the last decade, substantial progress has been made in understanding the pathophys-
iological basis of various neurological disorders; however, the discovery and devel-
opment of drugs for the treatment of such disorders is still an uphill task to the world
of neuroscientists. Moreover, the prevalence of neurological disorders is increasing
day by day, thereby imposing a great challenge to the researchers to fulfill the unmet
demand and develop new therapeutic strategies. This is possible only with the
complete understanding of the pathophysiological role neurotransmitters play in
the development of such diseases.
This book provides an updated insight into the pathophysiology and pharmacol-
ogy of neurotransmitters in order to upgrade the knowledge and understanding of
researchers about the involvement of these neurotransmitters in the progression of
the disease as well as provide innovative ideas to develop new therapeutic
interventions targeting various neurological diseases. A total of 20 chapters have
contextualized in this book, covering the history, basic science, classification, and
pathophysiological mechanisms involved at molecular and ionic levels along with
the pharmacology of neurotransmitters. Various research findings in preclinical and
clinical settings, highlighting the current status and potentials of various novel
molecules targeting these neurotransmitters as promising therapeutic agents, are
also discussed. The authors have provided a wide range of literature reviews to
make this book an excellent source of information including various diagrammatic
pathways and tables in order to facilitate the reader not only to get deeper informa-
tion in minimum time but also to contribute towards better research in this field.
Readers will get to know the various types of pathological changes that occur in a
neurological disorder and which types of neurotransmitter is actually involved to
elicit specific signs and symptoms of a particular disease, and how it affects the
normal life of a person.
This book will be helpful to the undergraduate and postgraduate students and
researchers from academia and pharmaceutical industries to gain insight into the
different neurological pathways involving the neurotransmitters and their receptors
in the initiation and progression of various diseases. This book will attract and

vii
viii Preface

motivate the young scientists and researchers from the multidisciplinary interna-
tional research community to design innovative drug discovery strategies for the
development of novel therapeutic interventions to cure various neurological
disorders and improve the human health in future.

Bathinda, Punjab Puneet Kumar

Amman, Jordan Pran Kishore Deb
Acknowledgments

We thank all the authors for their sincere efforts and valuable contributions in this
book. Dr. Puneet Kumar would like to convey his sincere thanks to Prof. MPS Ishar
(Vice Chancellor, MRSPTU, Bathinda), Prof. S.K. Kulkarni (Emeritus Professor,
Panjab University), and Prof. Anil Kumar (UIPS, Panjab University) for their
valuable guidance and encouragement. Dr. Pran Kishore Deb would also like to
express his gratitude towards his mentor Prof. Raghuprasad M. (Sri Vishnu College
of Pharmacy, AP, India) for his valuable guidance, encouragement, and
contributions; and Dr. Wafa Hourani, Research Assistant Ms. Sara Nidal Abed
(Faculty of Pharmacy, Philadelphia University) for their valuable contributions
during the proofreading of various chapters of the book.

ix
Contents

1 Neurotransmitters and Their Receptors—State of the Art . . . . . . . 1

Puneet Kumar, Sara Nidal Abed, Yazan A. Bataineh,
and Mutaz Sheikh Salem
2 Drug-Receptor Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Balakumar Chandrasekaran, Haneen Al-Joubi, Sara Samarneh,
Ghadir Kassab, Pran Kishore Deb, Puneet Kumar, Bilal A. Al-Jaidi,
Yazan Al-Thaher, and Yazan A. Bataineh
3 Pharmacology of Acetylcholine and Cholinergic Receptors . . . . . . . 69
Sarah Falah Kokaz, Pran Kishore Deb, Sara Nidal Abed,
Amal Al-Aboudi, Nirupam Das, Fatimah Amin Younes,
Ruba Anwar Salou, Yazan A. Bataineh, Katharigatta N. Venugopala,
and Raghu Prasad Mailavaram
4 Pharmacology of Adrenaline, Noradrenaline, and Their
Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Bapi Gorain, Sulagna Dutta, Utpal Nandy, Pallav Sengupta,
and Hira Choudhury
5 Pharmacology of Dopamine and Its Receptors . . . . . . . . . . . . . . . . 143
Sunpreet Kaur, Shamsher Singh, Gagandeep Jaiswal, Sandeep Kumar,
Wafa Hourani, Bapi Gorain, and Puneet Kumar
6 Pharmacology of Serotonin and Its Receptors . . . . . . . . . . . . . . . . 183
Satyendra Deka, Ratnali Bania, Pobitra Borah, Sanjib Das,
and Pran Kishore Deb
7 Pharmacology of Histamine, Its Receptors and Antagonists
in the Modulation of Physiological Functions . . . . . . . . . . . . . . . . . 213
Bapi Gorain, Pallav Sengupta, Sulagna Dutta, Manisha Pandey,
and Hira Choudhury
8 Pharmacology of GABA and Its Receptors . . . . . . . . . . . . . . . . . . . 241
Sunpreet Kaur, Shamsher Singh, Anchal Arora, Parladh Ram,
Sachin Kumar, Puneet Kumar, and Sara Nidal Abed

xi
xii Contents

9 Pharmacology of Melatonin and Its Receptors . . . . . . . . . . . . . . . . 293

Shamsher Singh, Arti Rana, Sunpreet Kaur, Jasdeep Singh,
Vikrant Rahi, Hira Choudhury, and Puneet Kumar
10 Pharmacology of Adenosine Receptors . . . . . . . . . . . . . . . . . . . . . . 325
Pran Kishore Deb, Sarah Falah Kokaz, Sara Nidal Abed,
Balakumar Chandrasekaran, Wafa Hourani,
Abdulmuttaleb Yousef Jaber, Raghu Prasad Mailavaram,
Puneet Kumar, and Katharigatta N. Venugopala
11 Pharmacology of Angiotensin and Its Receptors . . . . . . . . . . . . . . . 361
Satyajeet Biswal, Rajat Ghosh, and Pratap Chandra Acharya
12 Pharmacology of Endogenous Opioids, Opiates and Their
Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Mohammed Noorladeen Al–Qattan, Nirupam Das,
and Rati Kailash Prasad Tripathi
13 Pharmacology of Endocannabinoids and Their Receptors . . . . . . . 415
Gaurav Gupta, Wafa Hourani, Pran Kishore Deb, Satyendra Deka,
Pobitra Borah, Juhi Tiwari, Sacchidanand Pathak, and Puneet Kumar
14 Hormones and Steroids as Neurotransmitters . . . . . . . . . . . . . . . . . 447
Sarapynbiang Marwein, Satyajeet Biswal,
and Pratap Chandra Acharya
15 Pharmacology of Neuropeptides: Substance P, Vasoactive
Intestinal Peptides, Neuropeptide Y, Calcitonin Peptides
and Their Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
Nabil A. Nimer, Najlaa S. Ismael, Ruwaida W. Abdo,
Sura Y. Taha Alkhammas, and Qutaiba A. Alkhames Aga
16 Neuropeptides and Neurotransmission . . . . . . . . . . . . . . . . . . . . . . 553
Anindita Mondal Gantait, Yazan A. Bataineh, Hiba Salim Surchi,
Arunava Gantait, G. Tulja Rani, Paramita Paul, Sarah Falah Kokaz,
Bilal A. Al-Jaidi, Puneet Kumar, Saumen Karan,
and Tanushree Singha
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon
Monoxide) and Their Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Rohitas Deshmukh, Ranjit K. Harwansh, Nabamita Bandyopadhyay,
Shantanu Bandopadhyay, and Puneet Kumar
18 Sodium Channels: As an Eye of the Storm in Various Clinical
Pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619
Vinod Tiwari, Ankit Uniyal, Akhilesh, Anagha Gadepalli,
Vineeta Tiwari, and Somesh Agrawal
Contents xiii

19 Pharmacology of Potassium Channels . . . . . . . . . . . . . . . . . . . . . . 635

Satyendra Deka, Pobitra Borah, Ratnali Bania, Sanjib Das,
and Pran Kishore Deb
20 Pharmacology of Calcium Channel . . . . . . . . . . . . . . . . . . . . . . . . . 683
Santanu Mallik and Pratap Chandra Acharya
Editors and Contributors

About the Editors

Puneet Kumar Bansal is an Associate Professor and

Head at the Department of Pharmacology, Central Uni-
versity of Punjab, Bathinda, Punjab, India. Previously,
Dr. Bansal worked as Assocaite Professor at Department
of Pharmaceutical Sceinces and Technology, Maharaja
Ranjit Singh Punjab Technical University, Bathinda,
Punjab, India. Dr. Bansal completed his Ph.D. from
the University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh. His research interests
encompass the pharmacological screening of herbal
and semisynthetic and synthetic drugs in animal models
of various diseases such as movement disorders. He has
published more than 115 peer-reviewed research and
review papers/book chapters and five books to his
credit. He has more than 14 years of teaching and
research experience. Dr. Bansal completed three major
research projects for government-funded agencies. He
received the International Brain Research Organization
(IBRO) fellowship for attending IBRO neuroscience
schools and presented his research work at various
national and international conferences. Dr. Bansal is
member of various highly recognized professional
national and international societies like MNAMSc.,
NAMSc., APTI, IPS, IAN, IPGA, SNCI, LASA,
IBRO, and MDS.

xv
xvi Editors and Contributors

Pran Kishore Deb is currently working as an Associ-

ate Professor at the Faculty of Pharmacy, Philadelphia
University, Jordan. He received his Ph.D. in pharmaceu-
tical sciences from the University Institute of Pharma-
ceutical Sciences (UIPS), Panjab University,
Chandigarh, India. His key research area of interest
includes e-Learning innovations, computer-aided drug
design (CADD), and development of new chemical
entities (NCEs) targeting adenosine receptors, cycloox-
ygenase enzymes, and TB. He has got one patent on TB
and published more than 90 scientific articles including
research, review, and book chapters in various peer-
reviewed international journals and books. He has
9 years of teaching and research experience in the field
of medicinal chemistry. He is a recipient of several
awards including CSIR-SRF (Ph.D., India), e-Learning
GURU Award, and Best Faculty Achievement Award
under e-Learning innovation. He is a recipient of several
National and International Research Grants from vari-
ous funding agencies including the Government of
India, Malaysia, and Jordan.

Contributors

Abdulmuttaleb Yousef Jaber Faculty of Pharmacy, Philadelphia University,

Amman, Jordan
Akhilesh Kotiyal Department of Pharmaceutical Engineering & Technology,
Indian Institute of Technology (B.H.U.), Varanasi, UP, India
Amal Al-Aboudi Department of Chemistry, Faculty of Science, The University of
Jordan, Amman, Jordan
Amandeep Kaur Department of Pharmaceutical Sciences, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Anagha Gadepalli Department of Pharmaceutical Engineering & Technology,
Indian Institute of Technology (B.H.U.), Varanasi, UP, India
Anchal Arora Department of Pharmaceutical Sciences, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Anindita Mondal Gantait Malla Reddy Pharmacy College, Hyderabad,
Telangana, India
Ankit Uniyal Department of Pharmaceutical Engineering & Technology, Indian
Institute of Technology (B.H.U.), Varanasi, UP, India
Editors and Contributors xvii

Arti Rana ISF College of Pharmacy, Moga, Punjab, India

Arunava Gantait Dr. Reddy’s Laboratory, IPDO, Hyderabad, Telangana, India
Balakumar Chandrasekaran Faculty of Pharmacy, Philadelphia University,
Amman, Jordan
Bapi Gorain School of Pharmacy, Faculty of Health and Medical Science, Taylor’s
University, Subang Jaya, Selangor, Malaysia
Bilal A. Al-Jaidi Faculty of Pharmacy, Philadelphia University—Jordan, Amman,
Jordan
Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy,
Yarmouk University, Irbid, Jordan
Fatimah Amin Younes Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
G. Tulja Rani Malla Reddy Pharmacy College, Hyderabad, Telangana, India
Gagandeep Jaiswal Department of Pharmaceutical Sciences, Maharaja Ranjit
Singh Punjab Technical University, Bathinda, Punjab, India
Gaurav Gupta School of Pharmacy, Suresh Gyan Vihar University, Jaipur, India
Ghadir Kassab Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Haneen Al-Joubi Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Hiba Salim Surchi Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Hira Choudhury School of Pharmacy, International Medical University, Bukit
Jalil, Kuala Lumpur, Malaysia
Jasdeep Singh Department of Pharmaceutical Sciences, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Juhi Tiwari Alwar Pharmacy College, Alwar, India
Katharigatta N. Venugopala Department of Pharmaceutical Sciences, College of
Clinical Pharmacy, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia
Department of Biotechnology and Food Technology, Durban University of Tech-
nology, Durban, South Africa
Manisha Pandey School of Pharmacy, International Medical University, Kuala
Lumpur, Malaysia
Mohammad Aldhoun Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
Mohammed Noorladeen Al-Qattan Department of Pharmacy, Al-Noor Univer-
sity College, Mosul, Iraq
xviii Editors and Contributors

Mutaz Sheikh Salem Faculty of Pharmacy, Philadelphia University, Amman,

Jordan
Nabamita Bandyopadhyay Molecular Biology Division, National Institute of
Malarial Research (NIMR), Delhi, India
Nabil A. Nimer Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Najlaa S. Ismael Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Nirupam Das Department of Pharmaceutical Science, Assam University, Silchar,
Assam, India
Pallav Sengupta Department of Physiology, Faculty of Medicine, Bioscience and
Nursing, MAHSA University, Kuala Lumpur, Malaysia
Paramita Paul Department of Pharmaceutical Technology, University of North
Bengal, Raja Rammohunpur, Dist-Darjeeling, West Bengal, India
Parladh Ram Department of Pharmaceutical Sciences, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Pobitra Borah Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam,
India
Pran Kishore Deb Department of Pharmaceutical Sciences, Faculty of Pharmacy,
Philadelphia University, Amman, Jordan
Pratap Chandra Acharya Department of Pharmacy, Tripura University
(A Central University), Suryamaninagar, Tripura (W), India
Priyanka Chandolia Department of Pharmaceutical Sciences, Maharaja Ranjit
Singh Punjab Technical University, Bathinda, Punjab, India
Puneet Kumar Department of Pharmacology, Central University of Punjab,
Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Qutaiba A. Alkhames Aga Faculty of Pharmacy, Philadelphia University,
Amman, Jordan
Raghu Prasad Mailavaram Department of Pharmaceutical Chemistry, Shri
Vishnu College of Pharmacy, Vishnupur (Affiliated to Andhra University),
Bhimavaram, W.G. Dist., AP, India
Rajat Ghosh Department of Pharmacy, Tripura University (A Central University),
Suryamaninagar, Tripura (W), India
Ranjit K. Harwansh Institute of Pharmaceutical Research, GLA University,
Mathura, Uttar Pradesh, India
Editors and Contributors xix

Rati Kailash Prasad Tripathi Department of Pharmaceutical Sciences, Sushruta

School of Medical and Paramedical Sciences, Assam University, Silchar, Assam,
India
Ratnali Bania Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam,
India
Rohitas Deshmukh Institute of Pharmaceutical Research, GLA University,
Mathura, Uttar Pradesh, India
Ruba Anwar Salou Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
Ruwaida W. Abdo Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Sacchidanand Pathak School of Pharmacy, Suresh Gyan Vihar University, Jaipur,
India
Sachin Kumar Department of Pharmaceutical Sciences, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Sandeep Kumar Department of Pharmaceutical Sciences, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Sanjib Das Department of Pharmaceutical Sciences, Assam University, Silchar,
Assam, India
Santanu Mallik Department of Pharmacy, Tripura University (A Central Univer-
sity), Suryamaninagar, Tripura (W), India
Bharat Pharmaceutical Technology, Amtali, Agartala, Tripura (W), India
Sara Nidal Abed Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Sara Samarneh Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Sarah Falah Kokaz Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
Sarapynbiang Marwein Department of Pharmacy, Tripura University (A Central
University), Suryamaninagar, Tripura (W), India
Satyajeet Biswal Department of Pharmacy, Tripura University (A Central Univer-
sity), Suryamaninagar, Tripura (W), India
Satyendra Deka Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam,
India
Saumen Karan Department of Pharmaceutical Technology, Jadavpur University,
Kolkata, West Bengal, India
Shamsher Singh ISF College of Pharmacy, Moga, Punjab, India
xx Editors and Contributors

Shantanu Bandopadhyay Faculty of Pharmacy, Naraina Vidyapeeth Group of

Institutions, Kanpur, Uttar Pradesh, India
Somesh Agrawal Department of Pharmaceutical Engineering & Technology,
Indian Institute of Technology (B.H.U.), Varanasi, UP, India
Sulagna Dutta Department of Oral Biology and Biomedical Sciences, Faculty of
Dentistry, MAHSA University, Kuala Lumpur, Malaysia
Sumit Jamwal Department of Pharmaceutical Sciences and Technology, Maharaja
Ranjit Singh Punjab Technical University, Bathinda, India
Sunpreet Kaur ISF College of Pharmacy, Moga, Punjab, India
Sura Y. Taha Alkhammas Faculty of Pharmacy, Philadelphia University,
Amman, Jordan
Tanushree Singha Department of Pharmaceutical Technology, Jadavpur Univer-
sity, Kolkata, West Bengal, India
Utpal Nandy PK-PD, Toxicology and Formulation Division, CSIR-Indian Institute
of Integrative Medicine, Jammu, India
Vikrant Rahi Department of Pharmaceutical Sciences, Maharaja Ranjit Singh
Punjab Technical University, Bathinda, Punjab, India
Vineeta Tiwari Department of Pharmaceutical Engineering & Technology, Indian
Institute of Technology (B.H.U.), Varanasi, UP, India
Vinod Tiwari Department of Pharmaceutical Engineering & Technology, Indian
Institute of Technology (B.H.U.), Varanasi, UP, India
Wafa Hourani Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Yazan A. Bataineh Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Yazan Al-Thaher Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Neurotransmitters and Their Receptors—
State of the Art 1
Puneet Kumar, Sara Nidal Abed, Yazan A. Bataineh,
and Mutaz Sheikh Salem

Abstract

Neurotransmitters are endogenous chemical messengers that are responsible for

neuronal communication throughout the body. These compounds serve to facili-
tate various functions controlled by the central nervous system via a process
known as chemical synaptic transmission. The discovery of various types of
neurotransmitters has taken place over the past years where the neurotransmitters
have been classified based on their chemical, functional, and molecular properties
along with their location in the body. This chapter highlights all the important
neurotransmitters including GABA and glycine, glutamate, melatonin, histamine,
serotonin, acetylcholine along with other neurotransmitters, taking into account
their synthesis, release, mechanism of action, and metabolism. This chapter also
briefly discusses the physiological roles of these neurotransmitters and their
contribution in different pathological conditions, in addition to the therapeutic
effects of their agonists/antagonists.

Keywords
Neurotransmission · Neurotransmitter · Neuroreceptors · Agonists · Antagonists

P. Kumar (*)
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India
S. N. Abed · Y. A. Bataineh · M. S. Salem
Faculty of Pharmacy, Philadelphia University, Amman, Jordan

# Springer Nature Singapore Pte Ltd. 2020 1

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_1
2 P. Kumar et al.

Abbreviations

5-HIAA 5-Hydroxyindoleacetic acid

5-HT 5-Hydroxytryptamine
Ach Acetylcholine
AchE Acetylcholinesterase
AD Alzheimer’s disease
BuchE Butyrylcholinesterase
cAMP Cyclic adenosine monophosphate
CBD Cannabidiol
CNS Central nervous system
DAO Diamine oxidase
ECs Endogenous cannabinoids
EOPs Endogenous opioid peptides
GABA γ-Aminobutyric acid
GPCRs G-protein-coupled receptors
HDC L-histidine decarboxylase
HNMT Histamine N-methyltransferase
KO Knockout
MAO- A Monoamine oxidase A
NO Nitric oxide
NOP Nociceptin opioid receptor
NOS Nitric oxide synthase
PKG Protein kinase G
PNS Peripheral nervous system
TCA Tricarboxylic acid
THC Tetrahydrocannabinol
VGAT Vesicular GABA transporter

1.1 Introduction

Neurotransmitters are endogenous chemical messengers that are responsible for

neuronal communication throughout the body. These compounds serve to provide
various functions facilitated by the brain via a process known as chemical synaptic
transmission (Rizo 2018). Neurotransmitters play essential roles in many processes
of early human development, which include differentiation, neurotransmission, the
growth of neurons, as well as the neural circuitry development (Herlenius and
Lagercrantz 2001, 2004). A compound is referred to as a neurotransmitter when it
meets four criteria. First, being synthesized in the neurons. Second, being present in
the presynaptic terminal and released in sufficient amounts enough for exerting a
certain action on the postsynaptic neuron or targeting receptors in effector organs.
Third, the exogenous administration has to mimic the endogenously produced
neurotransmitter’s action, and last, the presence of intrinsic mechanisms responsible
for its removal from the site of action (Kavalali 2015).
1 Neurotransmitters and Their Receptors—State of the Art 3

Neurotransmitters

Ester Amino acid Amines Peptides Other

Acetylcholine Glycine Endorphins ATP

Catecholamines Indolamines
GABA Encephalin NO

Glutamate Substance P CO
Epinephrine Serotonin
Aspartate
Norepinephrine Histamine
Dopamine Taurine

Fig. 1.1 Chemical classification of neurotransmitters

Fig. 1.2 Functional classification of neurotransmitters

The discovery of various types of neurotransmitters has taken place over the past
years where the neurotransmitters have been classified based on their chemical
(Fig. 1.1), functional (Fig. 1.2), and molecular properties along with their location
in the body (Fig. 1.3) (Zhou and Danbolt 2014). Neurotransmitters are classified into
(a) biogenic amines that include serotonin, dopamine, epinephrine (adrenaline), and
norepinephrine (noradrenaline), (b) neuropeptides that include substance P, as well
as (c) amino acids that include glutamate and γ-aminobutyric acid (GABA) (Rangel-
gomez and Meeter 2016). Neurotransmitters show their action via two classes of
receptors possessing distinctive modalities of synaptic transmission. The first class is
known as ionotropic receptors, which comprise the ligand-gated ion channels that
are responsible for eliciting fast synaptic transmission. The second class is
metabotropic receptors, which consist of GPCRs that bind to neurotransmitters
causing slow synaptic transmission by intracellular signaling pathways in addition
to inducing gene expression (Komatsu 2015). The aforementioned neurotransmitters
4 P. Kumar et al.

Fig. 1.3 Classification of neurotransmitters based on location

Fig. 1.4 Classification of neurotransmitters based on disease association

along with other important ones are briefly discussed in this chapter, taking into
account the roles they serve in the physiological/pathological conditions, including
their mechanisms of action and their pharmacological properties (Fig. 1.4).
1 Neurotransmitters and Their Receptors—State of the Art 5

1.2 GABA

The γ-aminobutyric acid (GABA) is a non-protein amino acid and serves as a key
inhibitory neurotransmitter in the adult brain. GABA is widespread in early embry-
onic life and has been shown to have an excitatory synaptic transmission activity in
the immature brain (Luján et al. 2005). The neurotransmission served by GABA is
known to be present in nearly all organisms, starting from bacteria to human beings
(Owens and Kriegstein 2002). GABA is synthesized by the GABAergic neurons
from L-glutamate, and the catalysis of this reaction is done by the glutamic acid
decarboxylase (GAD) enzyme (Olsen 2002; Bouche et al. 2003). Once GABA is
synthesized, it is then stored in GABAergic synaptic vesicles which is facilitated by
a vesicular GABA transporter (VGAT) and upon depolarization, it undergoes
calcium ion-dependent release. The release of GABA from the presynaptic terminals
will be then followed by the action on GABA receptors (GABAA and GABAB),
(Markwardt and Overstreet-wadiche 2008; Li et al. 2012). GABAA receptors are ion
channels, whereas GABAB receptors are G-protein-coupled receptors (GPCR).
Stimulating GABAA receptor was shown to increase the permeability to chloride
ions which therefore resulted in inhibition or hyperpolarization of neurons by an
increase in the potential of the postsynaptic membrane. Stimulating GABAB recep-
tor leads to modulating cyclic adenosine monophosphate (cAMP) production as well
as increasing the conductance of potassium and hence resulting in either hyperpo-
larization or inhibition of the voltage-gated calcium channels (Olsen 2002; Bouche
et al. 2003).
GABAergic neurotransmission has been shown to play a vital role in
proliferating, migrating, and integrating other neuronal progenitors that are of key
importance for modulating the brain patterns, and deliberates various trophic
functions along with its contribution in the synaptic plasticity (Schmidt and Mirnics
2015). Disruptions in the GABAergic neurotransmission mostly result in sequential
events in the brain development, which was revealed in numerous in vitro and in vivo
studies by using various receptor modulators and knockout (KO) mice models
(Fig. 1.5) (Owens and Kriegstein 2002).

1.3 Glutamate

Glutamate is an excitatory amino acid neurotransmitter that is highly distributed in

the brain tissue (Danbolt et al. 2016). Glutamate is biosynthesized from glucose-
determined tricarboxylic acid (TCA) derivatives in the mitochondria (Stanley et al.
2017). In the neurotransmission of glutamate, four enzymes are involved, where
glutamate may be acting either as a substrate or a final product. Glutamate dehydro-
genase, aminotransferase, glutamine synthetase as well as glutaminase are the
enzymes involved in the neurotransmission of glutamate (Walker and van der
Donk 2016). When the ionotropic glutamate receptors (NMDA, AMPA, and
Kainate) get activated, they open membrane channels that allow ions to pass
through. AMPA and Kainate receptors increase the permeability of sodium and
6 P. Kumar et al.

GABA Binding site

(δ, ε or θ)
(σ)

Benzodiazepine Binding site

GABA Binding site

A) The GABAA receptor consists of two α, two β, and one γ subunit arranged in αβαβγ way. The γ subunit may be
replaced with either δ, ε, or θ.

Glutamine
Pre synaptic terminal
Glutaminase

Mitochondrion

Glutamate
CAD 65/67
GABA
Glutamine
VMAT transporter Astrocyte
Post synaptic receptor and
ionic channel GABA Glutamine
Exocytosis Glutamine
synthetase

GABA Glutamate
Synaptic
cleft
Post synaptic receptor GABA-transporter
and ionic neuron
GABA

GABA-T
Extra synaptic
Post synaptic neuron
Phasic inhibition cleft

Tonic inhibition
(e.g. neuronal excitability)

Fig. 1.5 Downstream regulating pathway of GABA

potassium, whereas NMDA receptor increases membrane permeability for calcium

as shown in Fig. 1.6.
Glutamate has been shown to serve a major role in the learning as well as memory
of the normal brain that include cognition, behavioral patterns, and sensation
(Stanley et al. 2017). Glutamate mainly serves a role in the CNS development
including synapse’s induction and elimination; migration, differentiation as well as
apoptosis process of the cell. Moreover, glutamate also has a key role in the
peripheral tissues and endocrine cells. Despite the important roles of glutamate as
a neurotransmitter, it is also known for its lethality to neurons causing a phenomenon
known as “excitotoxicity” which causes excessive firing of the neurons in the brain
1 Neurotransmitters and Their Receptors—State of the Art 7

Fig. 1.6 Downstream regulating pathways of glutamate

and triggers development of the brain disorders such as hyperkinetic progressive

neurodegenerative disorder (Huntington disease), progressive loss of memory, cog-
nitive decline (Alzheimer’s disease), progressive muscle weakness (amyotrophic
lateral sclerosis), stroke, and epilepsy (Murrough et al. 2017).

1.4 Dopamine

Dopamine is considered as the major neurotransmitter in the brain from the cate-
cholamine group. Dopamine is responsible for various functions in the brain includ-
ing controlling the voluntary actions, reward, consciousness, circadian rhythm as
well as cognition (Hasbi et al. 2011). The dopaminergic neurons are present in the
midbrain and the associated regions that include the basal ganglia, ventral tegmental
area, and the retrorubral field (Haber 2016). The mediation of dopamine physiologi-
cal actions is done by its interaction with its receptors over dopaminergic synapses.
There are mainly five types of dopamine receptors that are categorized under two
subclasses, D1 and D2 dopamine receptors (Beaulieu et al. 2015). Dopamine
receptors are typically stable, however sharp and sometimes prolonged. The increase
or decrease in dopamine levels can downregulate or upregulate the number of
dopamine receptors as shown in Fig. 1.7.
8 P. Kumar et al.

Fig. 1.7 Regulation of dopamine

The distribution of D1 receptors takes place in nigrostriatal, mesolimbic, and

mesocortical areas, like the caudate-putamen (striatum), substantia nigra, olfactory
bulb, amygdala and at small level in the hippocampus, cerebellum, thalamic, and
hypothalamic areas. However, D2 receptors are expressed in the striatum, the
substantia nigra, ventral tegmental area, hypothalamus, cortical areas, septum,
amygdala, hippocampus, nucleus accumbens, and the olfactory tubercle regions
(Rangel-Barajas et al. 2015). These dopamine receptors serve diverse functions.
D2 auto-receptors that are localized in the presynaptic region are responsible for
inhibiting the release of dopamine, hence decreasing locomotor activity, whereas D2
receptors in the postsynaptic region are responsible for enhancing the release of
dopamine and hence stimulating the locomotion. Therefore, dopamine agonists
show biphasic response by the activation of both the presynaptic and postsynaptic
dopamine receptors (Sulzer et al. 2016). Likewise, D3 receptors exhibit a biphasic
response, however at a moderate level. D3 receptors are responsible for regulating
reward and reinforcement like functions of the human behavior. On the other hand,
D4 and D5 receptors are less expressed at motor control regions; therefore, they only
provide a low contribution to movement control (Beaulieu et al. 2015). Furthermore,
D1 and D2 receptors are also responsible for regulating learning, working memory
as well as executive functions. However, D3, D4 as well as D5 were shown to
provide less influence over cognition, because of the lower expression they have in
the hippocampus (Nyberg et al. 2016). Nowadays, clinical psychotic drugs show
their action by blocking D2 actions to treat diseases like schizophrenia and bipolar
disorder, due to the contribution of dopamine in the psychotic behavior. Dopamine
also serves other functions outside CNS including D2-mediated prolactin secretion
from the pituitary gland, D1-mediated renin secretion from kidney, and adrenal
1 Neurotransmitters and Their Receptors—State of the Art 9

gland-mediated D1-directed aldosterone secretion (Lv et al. 2018). Some specific

agonists and antagonists of dopamine are available for D1 and D2 class of receptors.
Selective agonists for D1 include A77636, SKF38393, SKF81297, and
dihydrexidine, while D2 selective compounds include quinpirole and N-0437. On
the other side, antagonists of D1 class involve SCH23390, SCH39166, and
SKF35566, while D2 antagonism is caused by domperidone, nemonapride,
raclopride, and sulpiride (Smee and Overstreet 1976).

1.5 Melatonin

Melatonin or 5-methoxy-N-acetyltryptamine was discovered and isolated from

bovine pineal in 1958 by Aaron Lerner (Lerner et al. 1958). Melatonin is considered
as the main hormone secreted by the pineal gland. However, it has been reported that
melatonin is also obtained from extrapineal sources including the retina, bone
marrow cells, platelets, skin, lymphocytes, harderian gland, cerebellum, as well as
the gastrointestinal tract of vertebrate species (Lerner et al. 1958; Dubbels et al.
1995; Hattori et al. 1995; Manchester et al. 2000; Reiter et al. 2014; Shi et al. 2016;
Tan et al. 2016). Melatonin was identified in various animals and plants (Lerner et al.
1958). The initial function of melatonin was thought to be the detoxification of the
free radicals resulting from processes like photosynthesis and metabolism (Tan
1993; Manchester et al. 2015; Galano et al. 2018). Later, melatonin became a
pleiotropic molecule that is responsible for resisting oxidation-related stress; how-
ever, it was also shown to influence the biological rhythms, suppression of inflam-
mation, as well as other actions (Tan et al. 2010; Lochner et al. 2018; Onaolapo and
Onaolapo 2018; Tamtaji et al. 2018). The molecular structure of melatonin has
determined its high efficiency in the detoxification of free radicals due to its ability
to donate an electron or a hydrogen atom, or in other means, depending on the radical
type (Reiter et al. 2014; Tan et al. 2015; Galano et al. 2018). The melatonin’s
superior antioxidant capacity of limiting the oxidative stress is attributed to the
cascade reaction that occurs as a result of generating derivatives that are likewise
free radical scavengers (Tan et al. 2013, 2015; Jou and Peng 2018). Furthermore,
melatonin was also shown to play a role in some age-related processes such as
retardation, anti-inflammatory activity, neurodegenerative changes resistance,
preventing apoptosis in normal cells, as well as the preservation of mitochondrial
and chloroplast physiology (Poeggeler 2005; Jou et al. 2007; Manchester et al. 2015;
Majidinia et al. 2018; Nabavi et al. 2019).
Melatonin is formed specifically from the amino acid tryptophan (Hardeland
2015). Tryptophan is consumed in the diet along with being synthesized via the
shikimic acid pathway that starts with D-erythrose-4-phosphate, phosphoenolpyr-
uvate, or carbon dioxide in certain species (Bochkov et al. 2012). Tryptophan is
initially converted to serotonin in a reaction that involves both a decarboxylation and
a hydroxylation reaction. Darkness and light are the two factors that are responsible
for melatonin release as well as its synthesis as shown in Fig. 1.8.
10 P. Kumar et al.

Fig. 1.8 Mechanism of

melatonin release

Melatonin demonstrates its activity via different molecular pathways; however,

the activation of two types of membrane-specific receptors, high-affinity ML1 sites
and low-affinity ML2 sites, was shown to be the best characterized pathways
(Morgan et al. 1994; Dubocovich 1995). Melatonin receptors have been reported
in both the central and the peripheral tissues, which include the heart and arteries,
adrenal gland, kidney, lung, liver, gallbladder, small intestine, adipocytes, ovaries,
uterus, breast, prostate, and skin (Ekmekcioglu 2006). Melatonin is responsible for
regulating circadian rhythms like the sleep-wake rhythm, neuroendocrine rhythms,
or body temperature cycles via its action on both MT1 and MT2 receptors (Axelrod
1974; Dahlitz et al. 1991; Morgan et al. 1994; Zisapel 2001; Von Gall et al. 2002; Jin
et al. 2003; Ekmekcioglu 2006; Karasek and Winczyk 2006; Liu et al. 2016).
Ingesting melatonin prompts fatigue, sleepiness, and a diminution of sleep latency
(Zhdanova et al. 1997). Besides, melatonin also plays a role in the regulation of
blood pressure and autonomic cardiovascular activity, as well as regulating the
immune system. Melatonin has been also reported for its various therapeutic
potentials in several disorders including certain tumors, cardiovascular or psychiatric
disorders (Tordjman et al. 2017).

1.6 Adrenaline and Noradrenaline

Adrenaline (epinephrine) is a hormone that is produced by the adrenal glands as well

as by a small number of neurons in the medulla oblongata of the CNS. Adrenaline
serves as a neurotransmitter that regulates visceral functions, e.g., respiration.
Adrenaline also plays a significant role in the fight-or-flight response by increasing
the blood flow to muscles, the heart output, pupil dilation, as well as the levels of
blood sugar as it binds to alpha and beta receptors (Goldstein 2010). Noradrenaline
(norepinephrine) shows its action as a neurotransmitter in both the CNS and PNS
1 Neurotransmitters and Their Receptors—State of the Art 11

Fig. 1.9 Conversion of

phenylalanine to adrenaline

(Wassall et al. 2009). Under stressful conditions, adrenaline is released into the
blood and therefore transmits signals to the organs of the body and creates a specific
response (Daubner et al. 2011). Adrenaline is synthesized from dopamine by the
action of phenylethanolamine N-methyltransferase as shown in Fig. 1.9.
The effects of both noradrenaline and adrenaline were shown to be mediated by
seven transmembrane G-protein-coupled receptors “adrenergic receptors” or
“adrenoceptors” (AR) which can be categorized into two major groups: α (α1, α2)
and β (β1, β2, β3) receptors (Strosberg 1993). The α1-ARs are activated by adrenergic
agonists stimulating Gq protein and phospholipase C (PLC). This activation
promotes the phosphatidylinositol bisphosphate (PIP2) hydrolysis, thereby produc-
ing inositol trisphosphate (IP3) and diacylglycerol (DAG) (Cotecchia 2010). The α2-
AR is referred to as the inhibitory receptors which, upon activation, stimulates the Gi
protein, thus inhibiting adenylate cyclase (AC) and reducing cAMP (Civantos
Calzada and Aleixandre De Artiñano 2001). The β1-AR is responsible for mediating
the cardiovascular responses to the circulating adrenaline as well as noradrenaline
that is released from the sympathetic nerve terminals. The β2-AR is present in the
airway smooth muscle cells including the bronchial muscles. The β3-AR is primarily
present in the adipocytes and can bind to Gi as well as Gs proteins. When these
receptors are stimulated, protein kinase A (PKA) will be activated, L-type Ca++
channels will be phosphorylated, and Ca++ entry will be in the state of relaxation
(Lefkowitz 2000; Ma and Huang 2002; Kohout and Lefkowitz 2003). AR ligands
12 P. Kumar et al.

Table 1.1 Subtypes of α and β receptors

Post receptor
Receptor Location mechanism
α1 Contraction: most vascular smooth muscle, pupillary radical IP3/DAG
muscle, pilomotor smooth muscle, prostate, sphincters (urinary cascade (Gq)
bladder and anal)
α2 Presynaptic CNS adrenoreceptors: decreases sympathetic flow, cAMP
GIT (heterotrophic receptors—relaxation), pancreas inhibition (Gi)
β1 Heart, juxtaglomerular cells cAMP
activation (Gs)
β2 Relaxation: smooth muscle of GIT, urinary bladder, uterus and cAMP
bronchial, ciliary epithelium, vascular smooth muscle activation (Gs)
(skeletal), liver
β3 Fat cells cAMP
activation (Gs)

have been tested and applied as drug therapeutics for the treatment of different
cardiovascular diseases including hypertension, angina pectoris, and congestive
heart failure. AR ligands have been also shown to be useful in the treatment of
diseases like asthma, depression, benign prostatic hypertrophy, glaucoma, shock,
premature labor, opioid withdrawal, and adjunct medications in general anesthesia
(Wassall et al. 2009). Agonists of β2-AR are used as first-line therapeutic agents for
the treatment of asthma and chronic obstructive pulmonary disease (COPD) as
shown in Table 1.1.

1.7 Acetylcholine

Acetylcholine (Ach) is a natural substance and the first neurotransmitter which was
identified by Otto Loewi. Ach alters many functions in the brain like neuronal
excitability, influences transmission, and coordinates the firing of neurons. Ach
also plays an important role in processing memory and learning behavior (Thorne
2010). In Alzheimer’s disease (AD), there is a decrease in the concentration as well
as the function of acetylcholine. Loss of Ach contributes to memory and attention
deficit. It is the major neurotransmitter in the parasympathetic nervous system
(PNS). It acts as a neurotransmitter at various sites, i.e., autonomic ganglia, neuro-
muscular junction, CNS, blood vessels, and postsynaptic receptors in the PNS (Oddo
and Laferla 2006). Acetylcholine is synthesized in cholinergic nerve terminal from
choline. Choline is actively taken up by the axonal membrane by a Na+: choline
transporter. Ach is hydrolyzed by acetylcholinesterase (AchE) (true cholinesterase)
and butyrylcholinesterase (BuchE) (pseudocholinesterase) into choline and acetate.
Acetylcholine acts on cholinergic receptors (muscarinic and nicotinic receptors).
Muscarinic receptors are of five types—M1, M2, M3, M4, M5; and nicotinic
receptors are of two types—Nm and Nn. All muscarinic receptors are G-protein
coupled receptors. The odd-numbered muscarinic receptors act through IP3/DAG
1 Neurotransmitters and Their Receptors—State of the Art 13

pathway, whereas the even-numbered muscarinic receptors act by inhibiting cAMP

production (Sarter and Parikh 2005). Nicotinic receptors are ion channel-gated
receptors. The M1 receptor is present on gastric ganglia and CNS. It stimulates the
secretion of HCl in stomach. The M2 receptor is present in the heart and has an
inhibitory effect on it. The M3 receptor is present in the eye, GIT, secretion glands,
and bladder and stimulates the secretion of glands and results in increased salivation
and lacrimation. The Nm receptor is present at the neuromuscular junction, and the
Nn receptor is present in autonomic ganglia, adrenal medulla, and the brain. M2
receptors act as autoreceptors on cholinergic terminals. The cholinergic neuron is
mainly present in basal forebrain and known as CNS cholinergic clusters. In the
substantia innominate of the basal forebrain, nucleus basalis of Meynert with neuron
projection is located throughout the cortex and amygdala. Degradation of these
neurons is the main cause of Alzheimer’s disease (AD). Muscarinic-mediated
inhibition is caused by the increase in K+ influx and inhibition of glutamergic
excitatory neuron which is present in the postsynaptic neuron and decreases Ca++
influx (Ishii and Kurachi 2006). From the aforementioned information, we conclude
that Ach is a major neurotransmitter with a modulatory function which acts by
inhibiting or stimulating the receptors, depending on which type of receptor is
present. It is distributed all over the brain and plays an important role in many
physiological functions such as learning, attention, sleep, wakefulness, and sensory
information as well as regulating sleep cycle (Boonstra et al. 2007). AD is treated by
giving cholinesterase inhibitors like donepezil, rivastigmine, and physostigmine,
which act by inhibiting NMDA receptors as shown in Fig. 1.10.

1.8 Histamine

Histamine (2-[3H-imidazol-4-yl]ethanamine) is a significant mediator that plays a

role in vasodilation, increasing vascular permeability and contributing to the ana-
phylactic reactions (O’Mahony et al. 2013). It has been shown that histamine also
plays other physiological functions like cell differentiation, proliferation, hemato-
poiesis, as well as cell regeneration. Histamine is synthesized by a decarboxylation
reaction of the amino acid histidine with the help of L-histidine decarboxylase (HDC)
enzyme, which is naturally expressed in neurons, parietal cells, gastric mucosal cells,
mast cells, as well as basophils. Histamine is degraded by the enzymes diamine
oxidase (DAO) and histamine N-methyltransferase (HNMT), which catalyze hista-
mine deamination as shown in Fig. 1.11 (Biegański 1983; Yoshikawa et al. 2013).
HNMT is expressed in the CNS and has a critical regulatory role, and its deficiency
was reported to lead to aggressive behavior and abnormal sleep-wake cycles in mice
(Naganuma et al. 2017).
Histamine was also shown to play a crucial role in the pathogenesis of several
allergic diseases, including atopic dermatitis, allergic rhinitis, as well as allergic
asthma via differential regulation of T helper lymphocytes (Jutel and Akdis 2007).
The characterization of histamine receptors (Table 1.2) (H1R–H4R) depends on their
function, structure, distribution, as well as their affinity to histamine (Leurs et al.
14 P. Kumar et al.

Fig. 1.10 Mechanism of synthesis, storage, and release of acetylcholine

Histidine
L-histidine
decarboxy
Histamine
Diamine oxidase
N-methyl transferase

N-methyl histamine Imidazole acetic acid

MAO-B Ribose Phosphoribosyl

transferase

Imidazole acid
N-methyl imidazole
acetic acid riboside

Fig. 1.11 Synthesis and metabolism of histamine

2009; Singh and Jadhav 2013). The H1 receptor is responsible for mediating cellular
migration, nociception, vasodilatation, and bronchoconstriction (Bakker et al. 2001).
On the other hand, the H2 receptor is responsible for modifying the secretion of
gastric acid, production of airway mucus, and vascular permeability (Seifert et al.
2013). The H3 receptor was reported to have a role in neuro-inflammatory diseases
1 Neurotransmitters and Their Receptors—State of the Art 15

Table 1.2 Characteristics of histamine receptors

H1 H2 H3 H4
G-protein Gq/11 Gs Gi/o Gi/o
coupling ("Ca2+ "cAMP) ("cAMP) (#cAMP) (#cAMP, "Ca2+)
(secondary
messenger)
Distribution Smooth muscle, Gastric parietal CNS: Cells of
endothelial cells, cells, cardiac presynaptic, hematopoietic
CNS muscle, mast myenteric origin
cells, CNS plexus
Representative 2-CH3-histamine Amthamine (R)α-CH3- 4-CH3-
agonist histamine Histamine
Representative Chlorpheniramine Ranitidine Tiprolisant JNJ7777120
antagonist

(Singh and Jadhav 2013), whereas the H4 receptor has been shown to have a role in
allergy and inflammation (Tiligada 2012; Thurmond 2015).
H1-antihistamines like azatadine, cetirizine, as well as mizolastine are useful for
treating mast cell activated diseases (Church and Church 2013). Other agents like
cimetidine, ranitidine, famotidine, and nizatidine are known as H2R selective
antihistamines that cause a reduction of gastric acid secretion (Shim and Kim
2017). H3R antihistamines include thioperamide, clobenpropit, BF2649,
PF-03654746, JNJ-17216498, and MK 0249. It has been evidenced that histamine
binding to H4 receptors cause exacerbation of allergy and inflammation. It has been
also demonstrated that mast cells have H4 receptors which, upon stimulation,
enhance degranulation and cytokine production. Consequently, antihistamines that
target both the H1 and H4 receptors might be effective treatments for mast cell-
mediated allergic reactions (Mishra et al. 2011).

1.9 Serotonin

Serotonin is a chemical that is found in almost all types of human tissues as well as in
plant and aerobic organisms like bacteria. Serotonin is a ubiquitous monoamine that
acts as both a neurotransmitter and hormone (Mohammad-Zadeh et al. 2008), which
is also known as 5-hydroxytryptamine (5-HT) (Shad 2017). The cell bodies of
serotonergic neuron are localized in the brainstem midline with broad axonal ridge
which extends to all the regions of the CNS (Peterlin and Rapoport 2007). 5-HT is
involved in various diseases like schizophrenia, anxiety, depression, hypertension,
migraine, carcinoid diarrhea, vomiting, irritable bowel syndrome (IBS), pulmonary
hypertension, eating disorders, and others (Pauwels 2003; De Ponti 2004).
5-HT is secreted by the nuclei in the median raphe of the CNS, and then
transferred to the spinal cord and other parts of the brain including the hypothalamus.
The synthesis of 5-HT is initiated through an active uptake of tryptophan in neurons
and enterochromaffin cell by a specific amino acid transporter (Upadhyay 2003;
16 P. Kumar et al.

Pytliak et al. 2011). Initially, in the presence of tryptophan hydroxylase, tryptophan

is converted into 5-hydroxytryptophan, which is finally converted into
5-hydroxytryptamine (serotonin) with the help of aromatic L-amino acid decarbox-
ylase (Berger et al. 2009). 5-HT is mainly degraded through oxidative deamination,
catalyzed by monoamine oxidase A (MAO- A), and followed by oxidation to
5-hydroxyindoleacetic acid (5-HIAA) (Ni and Watts 2006). In the brain, 5-HT is
secured from degradation by the storage in synaptic vesicles of the neurons (Hamel
and Current 2007).
5-HT and its receptors were shown to contribute to brain functioning. Therefore,
the dysregulation of serotonin system will in most cases end up with psychiatric and
neurological disorders (Berger et al. 2009). Serotonin has been also shown to play a
significant role in peripheral tissues, in addition to its contribution to the immune
system (Wu et al. 2019).

1.10 Adenosine

Adenosine is an endogenous nucleoside molecule which is ubiquitously present

throughout the body, mediating various pathophysiological functions (Borea et al.
2018). It is an integral component of ATP; therefore its synthesis is highly dependant
on the metabolic conditions of the cells. Extracellularly, adenosine concentration
ranges from 20 to 300 nM under physiological conditions, whereas metabolic
stressful conditions like hypoxia and inflammation might increase the adenosine
levels to micromolar concentrations (up to 30 μM) (Borea et al. 2016). Intracellu-
larly, adenosine is mainly produced from adenosine monophosphate (AMP) and S-
adenosyl-homocysteine (SAH) with the help of hydrolyzing enzymes endo-5-
0
-nucleotidase and/or SAH hydrolase, respectively. Adenosine is also generated
extracellularly due to the dephosphorylation of ATP, ADP, and AMP under meta-
bolic demand by ectonucleosidase triphosphate diphosphohydrolase (CD39) and
ecto-50 -nucleotidase (CD73), respectively (Chen et al. 2013; Zimmermann 2000).
Once generated, adenosine transportation across the cell membrane takes place
either by concentrative nucleoside transporters (CNTs) or by equilibrative nucleo-
side transporters (ENTs). However, there are three isoforms of CNTs which are
cation linked (Na+) energy dependent and four isoforms of energy independent
ENTs assisting the adenosine transportation across the cell membrane (Deussen
2000, Deussen et al. 1993, 1999).
In physiological conditions, adenosine kinase (AK) is the principal enzyme
involved in the biotransformation of adenosine inside the cell by phosphorylation
to AMP. On the other hand, adenosine deaminase (ADA) can cause deamination of
adenosine to inosine both intracellularly and extracellularly. Extracellular clearance
of adenosine is also facilitated by influx through ENTs. It should be noted that under
pathological conditions, ADA plays a major role in the intracellular clearance of
adenosine (Gracia et al. 2013; Pacheco et al. 2005).
Biological functions of adenosine are mediated by interactions with four GPCRs:
A1, A2A, A2B, and A3 adenosine receptors (ARs) (Fredholm et al. 2001; Fredholm
1 Neurotransmitters and Their Receptors—State of the Art 17

Fig. 1.12 Synthesis, biotransformation, and signal transduction mechanism of adenosine and A1,
A2A, A2B, and A3 receptors (reprinted with permission from Pran Kishore Deb 2019a)

et al. 2011; Borea et al. 2018). The primary signal transduction mechanism of ARs
involves the modulation of adenylyl cyclase (AC). Activation of A1 and A3 ARs
causes inhibition of AC activity leading to the reduction of cAMP and inhibition of
protein kinase A (PKA), whereas A2A and A2B ARs activation stimulates the AC
activity and consequently increases cAMP and PKA levels (Fredholm 2014; Merighi
et al. 2018). Figure 1.12 represents the synthesis, biotransformation, and signal
transduction mechanism of adenosine and its four receptor subtypes (Deb 2019a).
Ubiquitous distributions of ARs in the form of homomers, heteromers, or
oligomers have made them interesting drug targets for the therapeutic interventions
of various pathological conditions (Borea et al. 2018; Fredholm 2014; Merighi et al.
2018). In the last two decades a large number of agonists, antagonists, and allosteric
modulators of ARs have been discovered, some of which are under various phases of
clinical trial investigations, including those already in the market as shown in
Fig. 1.13 (Deb 2019a, b, c; Deb et al. 2019a, 2019b, 2018, 2011; Chandrasekaran
et al. 2018; Chandrasekaran et al. 2019; Shaik et al. 2019; Mailavaram et al. 2019;
Al-Attraqchi et al. 2019; Borah et al. 2019; Baraldi et al. 2008). In particular, recent
FDA approval of istradefylline as an A2A AR antagonist for the treatment of
Parkinson’s disease further boosted the research on ARs (Hoffman 2019; Voelker
2019).
18 P. Kumar et al.

NH2 O
O O
N N H O
N N N N
N N
O N N O N N
O
HO

HO HO
Name: Adenosine Name: Theophylline Name: Doxofylline
1. MoA: A1 AR agonist MoA: A1 AR antagonist MoA: A1 AR antagonist
Application: Paroxysmal supraventricular Application: Asthma Application: Asthma
tachycardia (PSVT)
2. MoA: A2A AR agonist
Application: Myocardial perfusion imaging
NH2
OH O
N N
N N N
O NH N
N N
O N N O
N O N O
N HO
N O
O N
HO
HO

Name: Bamifylline Name: Regadenoson Name: Istradefylline

MoA: A1 AR antagonist MoA: A2A AR agonist MoA: A2A AR antagonist
Application: Asthma Application: Myocardial perfusion imaging Application: Parkinson's disease

Fig. 1.13 Therapeutic applications of clinically approved drugs targeting ARs (reprinted with
permission from Pran Kishore Deb 2019a)

1.11 Angiotensin

The renin-angiotensin-aldosterone system (RAAS) is a critical regulator of blood

volume and systemic vascular resistance. Organ systems that are involved in RAAS
are kidneys, lungs, vasculature system, and brain (Liu et al. 2019). A powerful effect
of the RAAS is the obstruction in the pathophysiological condition like hypertension
and CHF. Kidney diseases, such as renal artery stenosis, secretion of a kidney tumor,
or other damage to the kidneys can lead to excessive levels of renin and angiotensin I
as well as high blood pressure (Hall et al. 2019). The renin-angiotensin system is
considered as a cardiovascular control system as it controls the extracellular fluid
volume as well as the blood pressure. Angiotensin I is considered as the active
product of peptide hormones from cascade renin enzymes. This hormone maintains
blood pressure and volume through various stages, including vasoconstriction,
stimulation of aldosterone secretion by the adrenal glands, increased absorption of
renal sodium activation of the sympathetic nervous system, and contractility of the
heart. Renin-angiotensin I plays an important role in the heart and kidney, with a
decrease in cardiac output in heart failure and an increase in the reactive
angiotensin-I and sodium retention in the kidneys due to blockage of blood circula-
tion as shown in Fig. 1.14.
1 Neurotransmitters and Their Receptors—State of the Art 19

Fig. 1.14 Renin-angiotensin

Liver
system

Angiotensinogen

Cathepsin D Renin

Angiotensin I
Chymase ACE

Angiotensin II
AT 1
Aminopeptides A
AT 2
Angiotensin III

Angiotensin II is the main stimulant for the production of aldosterone in the

adrenal glomeruli. It is most sensitive to the kidneys and also affects its function. It is
believed that some effects of ACE inhibitors and angiotensin receptor antagonists,
such as an increase in renal plasma velocity and a decrease in glomerular pressure,
are associated with antagonism of arterial vasoconstriction induced by selective
angiotensin (Bauer and Reams 1986). In the systemic circulation, a decrease in
resistant arterioles leads to an increase in blood pressure which causes vein contrac-
tion to worsen heart failure. The effect of angiotensin II on afterload (arterial
vasoconstriction) is more important in terms of CHF sensitivity than the effect on
preload (reduced nutrition). Angiotensin II can also act as a factor in the growth of
blood vessels, causing arterial hypertrophy of smooth muscles and increasing
peripheral resistance; this effect may be associated with vascular damage (Sasamura
et al. 1992). Angiotensin II can also contribute to the expansion of the extracellular
matrix by stimulating the production of TCF-β. Angiotensin II has a direct effect on
the renal tubules, thereby increasing proximal sodium reabsorption and subsequent
sodium retention (Cogan 1990; Kagami et al. 1994). It also acts directly on the
proximal renal tubules and acts against the normal functioning, causing contraction
of glomerular cells, reducing glomerular surfaces, and reducing glomerular filtration
rate. It also promotes the growth of myocytes and fibroblasts, a process that has a
direct effect on the heart and is believed to play an important role in the development
of left ventricular hypertrophy (Baker et al. 1992). There are two types of angioten-
sin II receptors, i.e., AT1 and AT2 receptors, which have different mechanisms of
signaling pathways and tissue distribution. The AT1 receptor binds with phospholi-
pase A2 and phospholipase C and negatively to adenylate cyclase as shown in
Table 1.3. These enzymes exert their action on specific substrates for generating
second messengers that will cause a transduction of the surface signal into a series of
biochemical events within the cell whereas AT2 inhibits guanylate cyclase
(Sasamura et al. 1992).
20 P. Kumar et al.

Table 1.3 Characteristics of AT1 and AT2 receptors

AT1 receptor AT2 receptor
Distribution Widely distributed in adult tissues, e.g., Widely distributed in fetal tissues,
blood vessels, kidney, adrenal gland, expression in the adult brain, adrenal
heart, liver, brain glands, ovary endothelium, uterus,
myocardium
Function Vasoconstriction, cardiac contractility, Possible role in growth and
aldosterone release, glomerular filtration, development (antiproliferation,
renal blood flow, cardiac and vascular inhibition of neointima, cell
hypertrophy, central osmoregulation differentiation)
Structure Seven transmembrane receptor, Seven transmembrane receptor,
G-protein coupling G-protein coupling
Ligands Losartan, Valsartan, Irbesartan, PD 123177, CGP 42112A, PD
Candesartan, Tasosartan 123319
Isoforms AT1a, AT1b

Fig. 1.15 Synthesis of nitric

Arginine nNOS/NOS1 Nitric
oxide
oxide

NADPH iNOS/NOS2 NADP

Oxygen eNOS/NOS3 Citrulline

1.12 Nitric Oxide

Nitric oxide (NO) production results from the oxidation of the terminal guanidine
nitrogen of the amino acid arginine. The catalysis of this reaction occurs by the
action of NADPH-dependent enzyme, nitric oxide synthase (NOS). When NO is
synthesized, it gets diffused outside the cell. It is produced by a group of enzymes
called nitric oxide synthases. These enzymes convert arginine into citrulline, pro-
ducing NO in the process as shown in Fig. 1.15 (Virarkar et al. 2013).
The concentration of NO differs under physiological conditions (Tieu et al.
2003). As a neurotransmitter, NO was shown to play a role in the activation of the
cGMP-dependent protein kinase G (PKG) pathway which is responsible for
phosphorylating synaptophysin, which is of significant importance for the fusion
of presynaptic vesicles, therefore leading to the potentiation and facilitating of
neurotransmission (Wang et al. 2005). NO was also shown to play a role in the
inhibition of GABAergic synaptic transmission by cGMP-dependent pathways in
addition to ion channels and exchangers (Yamamoto et al. 2015). NO plays a role in
supporting vascular homeostasis in endothelium-dependent vasodilatation; however,
its over- or underproduction was shown to be related to pathological conditions
(Džoljić et al. 2015). NO is therefore considered to be a critical mediator under
certain pathological conditions. For example, in brain ischemia-reperfusion injury,
1 Neurotransmitters and Their Receptors—State of the Art 21

the formation of NO is first increased serving a protective function by the induction

of collateral perfusion resulting from its powerful stimulatory effect on vasodilata-
tion as well as angiogenesis (Su et al. 2014). Nitric oxide, as mentioned above, is
considered as a key molecule for regulating physiological brain homeostasis (Nathan
1997).

1.13 Opioids

In general, any compound or substance that is related to opium is known as opioid.

Endogenous opioid peptides (EOPs) are naturally available endogenous substances
or ligands that mainly bind to the opioid receptors (Borg and Kreek 2003; Yaksh and
Wallace 2011). Opioid receptors are the receptors that are associated with opioid
analgesic activity. Mainly three classes of opioid receptors are found, namely, MOP
(μ), KOP (kappa), and DOP (delta). Additionally, subtypes of these receptors were
also identified that include epsilon, iota, lambda, and zeta. Nociceptin opioid recep-
tor (NOP) was also identified due to the structural similarities with opioid receptors.
Opioids do not have a high affinity to the NOP receptor but nociceptin, which is
structurally related to dynorphin, appears to be a ligand for NOP receptor. The
μ receptors are subdivided into two types, namely, μ1 and μ2. Endogenous ligands
for μ receptor are endomorphin1, endomorphin2, and enkephalin. The antagonists
for μ receptor include CTOP and beta-funaltrexamine. Upon stimulation of μ
receptor, relevant responses like analgesia (supraspinal and spinal), respiratory
depression, euphoria, miosis, sedation, physical dependence, and reduction in gas-
trointestinal motility were reported (Gentilucci and Tolomelli 2004). Kappa
receptors have higher affinity to ketocyclazocine and dynorphin A. Kappa receptors
are also subdivided into three types, kappa1, kappa2, and kappa3. Dynorphin A is
the endogenous ligand for kappa receptor and norbinaltorphimine, a bivalent deriv-
ative of naltrexone and specific antagonist for kappa receptors. Upon stimulation of
kappa receptors, relevant responses like analgesia (spinal), miosis, sedation,
respiratory depression, hallucination, and others were reported. Delta receptors
have a higher affinity for enkephalin and leuenkaphalin. Delta receptors are also
subdivided into two types, delta1 and delta2. Enkephalins are the endogenous
ligands for the delta receptors and naltrindole is the selective antagonist for delta
opioid receptors. Upon stimulation of delta opioid receptors, relevant responses like
analgesia (supraspinal and spinal), respiratory depression, feeding, inhibition of
dopamine release, and increase in the release of growth hormones have been
observed.
Mostly, opioid receptors are present on the peripheral sensory nerve, and they are
upregulated during the inflammation of the cell. EOPs are derived from the
immunocytes and occupied by the receptors on the sensory nerves that cause
analgesia by inhibiting the nerve excitability or inhibiting the release of
pro-inflammatory neuropeptides (Rasakham 2008). The opioid analgesic agonists
include morphine, codeine, dextropropoxyphene, fentanyl, heroin, methadone,
tramadol, and others. Morphine is known as a prototype opioid agonist that is widely
22 P. Kumar et al.

used for pain control. Well-known opioid antagonists are nalorphine, naloxone, and
naltrexone. Opioids like morphine and others produce their activity by the release of
the EOPs through direct action on opioid receptors (Koneru et al. 2009).

1.14 Cannabinoids

Cannabinoids are naturally occurring compounds found in the Cannabis sativa

plant, of which Δ9-tetrahydrocannabinol (THC) is the principal compound.
Cannabidiol (CBD) is another important component, which makes up to 40% of
the plant resin extract (Appendino and Chianese 2011). Cannabis has been used for
a variety of therapeutic applications such as pain, stimulation of appetite, nausea,
fever, infection, and gynecological disorders. Cannabinoids are a group of chemicals
which activate the cannabinoid receptors (CBRs) in the body such as endogenous
cannabinoids (ECs) which are present in human and animals, herbal cannabinoids
which are present in the cannabis plant, and synthetic cannabinoids which are
synthesized in the laboratory. The cannabinoid receptors are a class of cell mem-
brane receptors under the GPCRs group. The activation of CBRs occurs by ligands,
endocannabinoids, plant cannabinoids, and synthetic cannabinoids. There are cur-
rently two known cannabinoid receptors, namely, CB1R and CB2R. Both
the receptors are 7-transmembrane GPCRs, which inhibit the accumulation of
cAMP within the cells (Grotenhermen 2003). Like many other GPCRs, the CB1R
is primarily localized in the cell membrane. CB1Rs are abundantly present in
the brain and the periphery (i.e., liver, blood vessels, GIT, and peripheral nerve
endings).
CBD serves as an antagonist at the central CB1R causing inhibition of several CB1-
mediated THC effects. CBD causes a considerable reduction in the receptor activation
of a potent classical CB1R agonist. CBD causes inhibition of both the uptake and
hydrolysis of the endocannabinoid anandamide, hence causing an increase in its
concentration (Mackie 2006). The first identified EC was anandamide and the second
was 2-AG (2-arachidonoyl glycerol). Additional endocannabinoids include
virodhamine, noladin ether, and NADA (Alexander et al. 2018). Endocannabinoids
are retrograde synaptic messengers that are released from postsynaptic neurons and
travel backward across synapses which activate the CB1 receptors on presynaptic
neurons and suppress the transmitter release (Ahluwalia et al. 2000).
The physiological role and pharmacological effects of cannabinoids emphasize
that the activation of the cannabinoid system through THC, phytocannabinoids,
synthetic cannabinoids, and endocannabinoids causes numerous actions that include
effects on the central nervous system (anti-nociceptive, neuroprotection, alleviation
of painful spasms and spasticity, antiemetic, regulation of food intake, and energy
expenditure), reproductive system , immune system, vasodilation, anti-proliferative,
bronchodilation, as well as effect on intraocular pressure. Endocannabinoids are
agonists for CB1 receptors. CB2 receptor agonists have potentially useful effects in
many models of inflammatory and neuropathic pain and possibly involving the
release of endogenous opioids and can inhibit the growth of CB2 receptor expressing
1 Neurotransmitters and Their Receptors—State of the Art 23

glioma in vivo (Howlett 2002). The neuroprotective effects include short-term

adaptation to neuronal stress, limiting excitotoxicity, long-term adaptation, enhanc-
ing neurogenesis, and decreasing excitotoxicity (Jinwala and Gupta 2012).

1.15 Conclusion

Neurotransmitters are endogenous chemical messengers that are responsible for

neuronal communication throughout the body. Neurotransmitters have been classi-
fied based on their chemical, functional, and molecular properties as well as their
location in the body. Each neurotransmitter plays different roles to maintain
the proper functioning of the body. However, efforts are still under progress to
gain a complete understanding of the pathophysiological basis of various neurologi-
cal disorders in order to discover and develop new drugs for the treatment of various
diseases including inflammation, mental diseases, Alzheimer’s disease, Parkinson's
disease, cancers, and other disorders.

References
Ahluwalia J et al (2000) Cannabinoid 1 receptors are expressed in nociceptive primary sensory
neurons. Neuroscience 100(4):685–688
Al-Attraqchi O, Attimarad M, Venugopala K, Nair A, Al-Attraqchi N (2019) Adenosine A2A
receptor as a potential drug target-current status and future perspectives. Curr Pharm Des
25:2716–2740
Appendino G, Chianese G (2011) Cannabinoids: occurrence and medicinal chemistry. Curr Med
Chem 18(7):1085–1099
Axelrod J (1974) The pineal gland: a neurochemical transducer. Science 184(4144):1341–1348
Baker KM, Booz GW, Dostal DE (1992) Cardiac actions of angiotensin II: role of an intracardiac
renin-angiotensin system. Annu Rev Physiol 54(1):227–241
Bakker RA et al (2001) Histamine H(1)-receptor activation of nuclear factor-kappa B: roles for G
beta gamma- and G alpha(q/11)-subunits in constitutive and agonist-mediated signaling. Mol
Pharmacol 60(5):1133–1142
Baraldi PG, Tabrizi MA, Gessi S, Borea PA (2008) Adenosine receptor antagonists: translating
medicinal chemistry and pharmacology into clinical utility. Chem Rev 108:238–263
Bauer JH, Reams GP (1986) Renal effects of angiotensin converting enzyme inhibitors in hyper-
tension. Am J Med 81(4):19–27
Beaulieu J, Espinoza S, Gainetdinov RR (2015) Dopamine receptors–IUPHAR review 13. Br J
Pharmacol. Wiley Online Library 172(1):1–23
Berger M, Gray JA, Roth BL (2009) The expanded biology of serotonin. Annu Rev Med
60:355–366. https://doi.org/10.1146/annurev.med.60.042307.110802
Biegański T (1983) Biochemical, physiological and pathophysiological aspects of intestinal
diamine oxidase. Acta Physiol Pol 34(1):139–154
Bochkov DV et al (2012) Shikimic acid: review of its analytical, isolation, and purification
techniques from plant and microbial sources. J Chem Biol 5:5–17. https://doi.org/10.1007/
s12154-011-0064-8
Boonstra TW et al (2007) Effects of sleep deprivation on neural functioning: an integrative review.
Cell Mol Life Sci 64(7–8):934–946. https://doi.org/10.1007/s00018-007-6457-8
Borah P, Deka S, Mailavaram RP, Deb PK (2019) P1 receptor agonists/antagonists in clinical trials-
potential drug candidates of the future. Curr Pharm Des 25:2792–2807
24 P. Kumar et al.

Borea PA, Gessi S, Merighi S, Varani K (2016) Adenosine as a multi-signalling guardian angel in
human diseases: when, where and how does it exert its protective effects? Trends Pharmacol Sci
37:419–434
Borea PA, Gessi S, Merighi S, Vincenzi F, Varani K (2018) Pharmacology of adenosine receptors:
the state of the art. Physiol Rev 98:1591–1625
Borg L, Kreek MJ (2003) The pharmacology of opioids. In: Graham A et al (eds) Principles of
addiction medicine. Chevy Chase, MD, American Society of Addiction Medicine, pp 141–155
Bouche N, Lacombe B, Fromm H (2003) GABA signaling: a conserved and ubiquitous mechanism.
Trends Cell Biol 13(12):607–610
Chandrasekaran B, Deb PK, Kachler S, Akkinepalli RR, Mailavaram R, Klotz K-N (2018)
Synthesis and adenosine receptors binding studies of new fluorinated analogues of pyrido
[2,3-d] pyrimidines and quinazolines. Med Chem Res 27:756–767
Chandrasekaran B, Samarneh S, Jaber AMY, Kassab G, Agrawal N (2019) Therapeutic potentials
of A2B adenosine receptor ligands: current status and perspectives. Curr Pharm Des
25:2741–2771
Chen J-F, Eltzschig HK, Fredholm BB (2013) Adenosine receptors as drug targets—what are the
challenges? Nat Rev Drug Discov 12:265
Church MK, Church DS (2013) Pharmacology of antihistamines. Indian J Dermatol 58(3):219–224
Civantos Calzada B, Aleixandre De Artiñano A (2001) Alpha-adrenoceptor subtypes. Pharmacol
Res. https://doi.org/10.1006/phrs.2001.0857
Cogan MG (1990) Angiotensin II: a powerful controller of sodium transport in the early proximal
tubule. Hypertension 15(5):451–458
Cotecchia S (2010) The α1-adrenergic receptors: diversity of signaling networks and regulation. J
Recept Sig Transduct Res 30(6):410–419. Taylor & Francis
Dahlitz M et al (1991) Delayed sleep phase syndrome response to melatonin. Lancet 337
(8750):1121–1124
Danbolt NC, Furness DN, Zhou Y (2016) Neuronal vs glial glutamate uptake: resolving the
conundrum. Neurochem Int 98:29–45. Elsevier Ltd
Daubner SC, Le T, Wang S (2011) Tyrosine hydroxylase and regulation of dopamine synthesis.
Arch Biochem Biophys 508(1):1–12. NIH Public Access
De Ponti F (2004) Pharmacology of serotonin: what a clinician should know. Gut 53:1520–1535
Deb PK (2019a) Therapeutic potentials of adenosine receptors: the state of the art. Curr Pharm Des
25:2789–2791
Deb PK (2019b) Progress in the development of agonists, antagonists and allosteric modulators of
adenosine receptors. Curr Pharm Des 25:2695–2696
Deb PK (2019c) Recent updates in the computer aided drug design strategies for the discovery of
agonists and antagonists of adenosine receptors. Curr Pharm Des 25:747–749
Deb PK, Balakumar C, Rao AR, Roy PP, Roy K (2011) QSAR of adenosine receptor antagonists:
exploring physicochemical requirements for binding of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]
pyrimidine derivatives with human adenosine A3 receptor subtype. Bioorg Med Chem Lett
21:818–823
Deb PK, Mailavaram R, Chandrasekaran B, Kaki VR, Kaur R, Kachler S, Karl-Norbert K,
Akkinepally RR (2018) Synthesis, adenosine receptor binding and molecular modeling studies
of novel thieno[2,3-d]pyrimidine derivatives. Chem Biol Drug Des 91:962–969
Deb PK, Chandrasekaran B, Mailavaram R, Tekade RK, Muttaleb A, Jaber Y (2019a) Molecular
modeling approaches for the discovery of adenosine A2B receptor antagonists : current status
and future perspectives. Drug Discov Today 24:1854–1864
Deb PK, Deka S, Borah P, Abed SN, Klotz K (2019b) Medicinal chemistry and therapeutic
potential of agonists, antagonists and allosteric modulators of A1 adenosine receptor: current
status and perspectives. Curr Pharm Des 25:2697–2715
Deussen A (2000) Metabolic flux rates of adenosine in the heart. Naunyn Schmiedeberg's Arch
Pharmacol 362:351–363
1 Neurotransmitters and Their Receptors—State of the Art 25

Deussen A, Bading B, Kelm M, Schrader J (1993) Formation and salvage of adenosine by

macrovascular endothelial cells. Am J Phys Heart Circ Phys 264:H692–H700
Deussen A, Stappert M, Schäfer S, Kelm M (1999) Quantification of extracellular and intracellular
adenosine production: understanding the transmembranous concentration gradient. Circulation
99:2041–2047
Dubbels R et al (1995) Melatonin in edible plants identified by radioimmunoassay and by high
performance liquid chromatography-mass spectrometry. J Pineal Res 18:28–31
Dubocovich ML (1995) Melatonin receptors: are there multiple subtypes ? Trends Pharmacol Sci
16(2):50–56
Džoljić E, Grabatinić I, Kostić V (2015) Why is nitric oxide important for our brain? Funct Neurol
30(3):159–163
Ekmekcioglu C (2006) Melatonin receptors in humans: biological role and clinical relevance.
Biomed Pharmacother 60:97–108. https://doi.org/10.1016/j.biopha.2006.01.002
Fredholm BB (2014) Adenosine—a physiological or pathophysiological agent? J Mol Med
92:201–206
Fredholm BB, IJzerman AP, Jacobson KA, Klotz K-N, Linden J (2001) International Union of
Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev
53:527–552
Fredholm BB, IJzerman AP, Jacobson KA, Linden J, Müller CE (2011) International Union of
Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine
receptors—an update. Pharmacol Rev 63:1–34
Galano A, Tan D-X, Reiter RJ (2018) Melatonin: a versatile protector against oxidative DNA
damage. Molecules 23:E530. https://doi.org/10.3390/molecules23030530
Gentilucci L, Tolomelli A (2004) Recent advances in the investigation of the bioactive conforma-
tion of peptides active at the μ-opioid receptor. Conformational analysis of endomorphins. Curr
Top Med Chem 4(1):105–121
Goldstein DS (2010) ‘Adrenaline and Noradrenaline’, in Encyclopedia of Life Sciences. John Wiley
& Sons, Ltd, Chichester, pp 1–9. https://doi.org/10.1002/9780470015902.a0001401.pub2
Gracia E, Farré D, Cortés A, Ferrer-Costa C, Orozco M, Mallol J, Lluís C, Canela EI, McCormick
PJ, Franco R (2013) The catalytic site structural gate of adenosine deaminase allosterically
modulates ligand binding to adenosine receptors. FASEB J 27:1048–1061
Grotenhermen F (2003) Pharmacokinetics and Pharmacodynamics of Cannabinoids. Clin
Pharmacokinet 42(4):327–360
Hourani W, Alexander SP. (2018) Cannabinoid ligands, receptors and enzymes: Pharmacological
tools and therapeutic potential. Brain and Neuroscience Advances, 2, 2398212818783908.
Hall JE et al (2019) Obesity, kidney dysfunction and hypertension: mechanistic links. Nature
Reviews Nephrology 15:367–385. Springer US
Hamel E, Current H (2007) Serotonin and migraine: biology and clinical implications. Cephalalgia
27(11):1293–1300
Hardeland R (2015) Melatonin in plants and other phototrophs: advances and gaps concerning the
diversity of functions. J Exp Bot 66(3):627–646
Hasbi A, O’Dowd BF, George SR (2011) Dopamine D1-D2 receptor heteromer signaling pathway
in the brain: emerging physiological relevance. Mol Brain 4(1):26. BioMed Central
Hattori A et al (1995) Identification of melatonin in plants and its effects on plasma melatonin levels
and binding to melatonin receptors in vertebrates. Biochem Mol Biol Int 35(3):627–634
Herlenius E, Lagercrantz H (2001) Neurotransmitters and neuromodulators during early human
development. Early Hum Dev 65:21–37
Herlenius E, Lagercrantz H (2004) Development of neurotransmitter systems during critical
periods. Exp Neurol 190:8–21. https://doi.org/10.1016/j.expneurol.2004.03.027
Hoffman M (2019) Istradefylline approved for Parkinson add-on therapy
Howlett AC (2002) The cannabinoid receptors. Prostaglandins Other Lipid Mediat 68–69:619–631
Ishii M, Kurachi Y (2006) Muscarinic acetylcholine receptors. Curr Pharm Des 12(28):3573–3581
26 P. Kumar et al.

Jin X et al (2003) Targeted disruption of the mouse Mel1b melatonin receptor. Mol Cell Biol 23
(3):1054–1060. https://doi.org/10.1128/MCB.23.3.1054
Jinwala FN, Gupta M (2012) Synthetic cannabis and respiratory depression. J Child Adolesc
Psychopharmacol 22(6):459–462. https://doi.org/10.1089/cap.2011.0122
Jou M-J, Peng T-I (2018) Protective stabilization of mitochondrial permeability transition and
mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin’s cascade metabolites
C3-OHM and AFMK in RBA1 astrocytes. J Pineal Res:e12538. https://doi.org/10.1111/jpi.
12538
Jou M-J et al (2007) Melatonin protects against common deletion of mitochondrial
DNA-augmented mitochondrial oxidative stress and apoptosis. J Pineal Res 43:389–403.
https://doi.org/10.1111/j.1600-079X.2007.00490.x
Jutel M, Akdis CA (2007) Histamine as an immune modulator in chronic inflammatory responses.
Clin Exp Allergy 37:308–310
Kagami S et al (1994) Angiotensin II stimulates extracellular matrix protein synthesis through
induction of transforming growth factor-beta expression in rat glomerular mesangial cells. J Clin
Invest 93(6):2431–2437
Karasek M, Winczyk K (2006) Melatonin in humans. J Physiol Pharmacol 57:19–39
Kavalali ET (2015) The mechanisms and functions of spontaneous neurotransmitter release. Nat
Rev Neurosci 16(1):5–16. Nature Publishing Group
Kohout TA, Lefkowitz RJ (2003) Regulation of G protein-coupled receptor kinases and arrestins
during receptor desensitization. Mol Pharmacol 63(1):9–18
Komatsu H (2015) Novel therapeutic GPCRs for psychiatric disorders. Int J Mol Sci:14109–14121.
https://doi.org/10.3390/ijms160614109
Koneru A, Satyanarayana S, Rizwan S (2009) Endogenous opioids: their physiological role and
receptors. Global J Pharmacol 3(3):149–153
Lefkowitz RJ (2000) The superfamily of heptahelical receptors. Nat Cell Biol 2(7):E133–E136
Lerner AB et al (1958) Isolation of melatonin, the pineal gland factor that lightens melanocytes. J
Am Chem Soc 58:2587
Leurs R et al (2009) Molecular and biochemical pharmacology of the histamine H4 receptor. Br J
Pharmacol 157:14–23
Li Y et al (2012) A novel role of intestine epithelial GABAergic signaling in regulating intestinal
fluid secretion. Am J Physiol Gastrointest Liver Physiol 303(4):453–460. https://doi.org/10.
1152/ajpgi.00497.2011
Liu J et al (2016) MT1 and MT2 melatonin receptors: a therapeutic perspective. Annu Rev
Pharmacol Toxicol 56:361–383
Liu J et al (2019) (Pro) renin receptor regulates lung development via the Wnt/B-catenin signaling
pathway. Am J Physiol Lung Cell Mol Physiol 317(2):202–211
Lochner A, Marais E, Huisamen B (2018) Melatonin and cardioprotection against ischaemia/
reperfusion injury: what’s new? A review. J Pineal Res 65(1):e12490
Luján R, Shigemoto R, López-Bendito G (2005) Review glutamate And GABA receptor signalling
in the developing brain. Neuroscience 130(3):567–580
Lv C et al (2018) Dopamine D2-like receptors (DRD2 and DRD4) in chickens: Tissue distribution,
functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expres-
sion. Gene 651:33–43. Elsevier
Ma YC, Huang XY (2002) Novel signaling pathway through the beta-adrenergic receptor. Trends
Cardiovasc Med 12(1):46–49
Mackie K (2006) Cannabinoid receptors as therapeutic targets. Annu Rev Pharmacol Toxicol
46:101–122. https://doi.org/10.1146/annurev.pharmtox.46.120604.141254
Mailavaram R, Al-Attraqchi O, Kar S, Ghosh S (2019) Current status in the design and develop-
ment of agonists and antagonists of adenosine A3 receptor as potential therapeutic agents. Curr
Pharm Des 25:2772–2787
Majidinia M et al (2018) The role of melatonin, a multitasking molecule, in retarding the processes
of ageing. Ageing Res Rev 47:198–213. https://doi.org/10.1016/j.arr.2018.07.010
1 Neurotransmitters and Their Receptors—State of the Art 27

Manchester LC et al (2000) High levels of melatonin in the seeds of edible plants Possible function
in germ tissue protection. Life Sci 67:3023–3029
Manchester LC et al (2015) Melatonin: an ancient molecule that makes oxygen metabolically
tolerable. J Pineal Res 59:403–419. https://doi.org/10.1111/jpi.12267
Markwardt S, Overstreet-wadiche L (2008) GABAergic signalling to adult-generated neurons. J
Physiol 586(16):3745–3749. https://doi.org/10.1113/jphysiol.2008.155713
Merighi S, Gessi S, Borea PA (2018) Adenosine receptors: structure, distribution, and signal
transduction. In: Borea P, Varani K, Gessi S, Merighi S, Vincenzi F (eds) The adenosine
receptors, The receptors, vol 34, pp 33–57
Mishra GP et al (2011) Recent patents and emerging therapeutics in the treatment of allergic
conjunctivitis. Recent Patents Inflamm Allergy Drug Discov 5:26–36
Mohammad-Zadeh LF, Moses L, Gwaltney-Brant SM (2008) Serotonin: a review. J Vet Pharmacol
Ther 31:187–199
Morgan PJ et al (1994) Melatonin receptors: localization, molecular pharmacology and physiologi-
cal significance. Neurochem Int 24(2):101–146
Murrough JW, Abdallah CG, Mathew SJ (2017) Targeting glutamate signalling in depression:
progress and prospects. Nat Rev Drug Discov 16(7):472. Nature Publishing Group
Nabavi SM et al (2019) Anti-inflammatory effects of melatonin: a mechanistic review. Crit Rev
Food Sci Nutr 59(sup1):4–16. https://doi.org/10.1080/10408398.2018.1487927
Naganuma F et al (2017) Histamine N-methyltransferase regulates aggression and the sleep-wake
cycle. Sci Rep 7(1):15899. https://doi.org/10.1038/s41598-017-16019-8
Nathan C (1997) Inducible nitric oxide synthase: what difference does it make? J Clin Invest
100:2417–2423
Ni W, Watts SW (2006) 5-hydroxytryptamine in the cardiovascular system: focus on the serotonin
transporter (SERT). Clin Exp Pharmacol Physiol 33:575–583
Nyberg L et al (2016) Dopamine D2 receptor availability is linked to hippocampal–caudate
functional connectivity and episodic memory. Proc Natl Acad Sci 113(28):7918–7923. National
Acad Sciences
O’Mahony L, Akdis M, Akdis CA (2013) Regulation of the immune response and inflammation by
histamine and histamine receptors. J Allergy Clin Immunol 128(6):1153–1162
Oddo S, Laferla FM (2006) The role of nicotinic acetylcholine receptors in Alzheimer’s disease. J
Physiol Paris 99(2–3):172–179
Olsen R (2002) GABA’. In: Charney D et al (eds) Neuropsychopharmacology the fifth generation
of progress. Cambridge University Press, Cambridge, pp 159–168
Onaolapo AY, Onaolapo OJ (2018) Circadian dysrhythmia-linked diabetes mellitus: examining
melatonin’s roles in prophylaxis and management. World J Diabetes 9(7):99–114
Owens DF, Kriegstein AR (2002) Is there more to GABA than synaptic inhibition? Nat Rev
Neurosci 3:715–727
Pacheco R, Lejeune M, Climent N, Oliva H, Gatell JM, Gallart T, Mallol J (2005) CD26, adenosine
deaminase, and adenosine receptors mediate costimulatory signals in the immunological syn-
apse. PNAS 102:9583–9588
Pauwels PJ (2003) 5-HT receptors and their ligands. Neuropharmacology 1083:38–50
Peterlin BL, Rapoport AM (2007) Clinical pharmacology of the serotonin receptor agonist,
zolmitriptan. Expert Opin Drug Metab Toxicol 3(6):899–912
Poeggeler B (2005) Melatonin, aging, and age-related diseases. Endocrine 27(2):201–212
Pytliak M et al (2011) Serotonin receptors – from molecular biology to clinical applications. Physiol
Res 60:15–25
Rangel-Barajas C, Coronel I, Florán B (2015) Dopamine receptors and neurodegeneration. Aging
Dis 6(5):349. JKL International LLC
Rangel-gomez M, Meeter M (2016) Neurotransmitters and novelty: a systematic review. J
Psychopharmacol 30(1):1–13. https://doi.org/10.1177/0269881115612238
Rasakham K (2008) Kappa Opioid Receptor regulation of ERK1/2 MAP kinase signaling cascade:
molecular mechanisms modulating cocaine reward. Northeastern University, Boston, MA
28 P. Kumar et al.

Reiter RJ et al (2014) Melatonin: exceeding expectations. Physiology 29:325–333. https://doi.org/

10.1152/physiol.00011.2014
Rizo J (2018) Mechanism of neurotransmitter release coming into focus. Protein Sci 27:1364–1391.
https://doi.org/10.1002/pro.3445
Sarter M, Parikh V (2005) Choline transporters, cholinergic transmission and cognition. Nat Rev
Neurosci 6(1):48–56
Sasamura H et al (1992) Cloning, characterization, and expression of two angiotensin receptor
(AT-1) isoforms from the mouse genome. Biochem Biophys Res Commun 185(1):253–259
Schmidt MJ, Mirnics K (2015) Neurodevelopment, GABA system dysfunction, and schizophrenia.
Neuropsychopharmacol Rev 40:190–206. https://doi.org/10.1038/npp.2014.95. Nature Publish-
ing Group
Seifert R et al (2013) Molecular and cellular analysis of human histamine receptor subtypes. Trends
Pharmacol Sci 34(1):33–58. Elsevier Ltd
Shad KF (2017) Introductory chapter: Serotonin - the most ancient neurotransmitter, hormone and
trophic factor. In: Serotonin - a chemical messenger between all types of living cells. BoD –
Books on Demand, Norderstedt, pp 3–6
Shaik K, Deb PK, Mailavaram RP, Chandrasekaran B, Kachler S, Klotz KN, Jaber AMY (2019)
7-Amino-2-aryl/hetero-aryl-5-oxo-5,8-dihydro[1,2,4]triazolo[1,5-a] pyridine-6-carbonitriles:
synthesis and adenosine receptor binding studies. Chem Biol Drug Des 94:1568–1573
Shi H et al (2016) Fundamental issues of melatonin-mediated stress signaling in plants. Front Plant
Sci 7(1124):1–6. https://doi.org/10.3389/fpls.2016.01124
Shim YK, Kim N (2017) The effect of H2 receptor antagonist in acid inhibition and its clinical
efficacy. Korean J Gastroenterol 70(1):4–12
Singh M, Jadhav HR (2013) Histamine H3 receptor function and ligands: recent developments.
Mini-Rev Med Chem 13:47–57
Smee ML, Overstreet DH (1976) Alterations in the effects of dopamine agonists and antagonists on
general activity in rats following chronic morphine treatment. Psychopharmacology 49
(2):125–130. Springer
Stanley JA et al (2017) Functional dynamics of hippocampal glutamate during associative learning
assessed with in vivo 1H functional magnetic resonance spectroscopy. Neuroimage., Elsevier
153:189–197
Strosberg AD (1993) Structure, function, and regulation of adrenergic receptors. Protein Sci 2
(8):1198–1209. https://doi.org/10.1002/pro.5560020802
Su K et al (2014) The essential role of transient receptor potential vanilloid 1 in simvastatin-induced
activation of endothelial nitric oxide synthase and angiogenesis. Acta Physiol (Oxf)
212:191–204. https://doi.org/10.1111/apha.12378
Sulzer D, Cragg SJ, Rice ME (2016) Striatal dopamine neurotransmission: regulation of release and
uptake. Basal Ganglia 6(3):123–148. Elsevier
Tamtaji OR et al (2018) Melatonin, a toll - like receptor inhibitor: current status and future
perspectives. J Cell Physiol. https://doi.org/10.1002/jcp.27698
Tan D-X (1993) Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr J 1:57–60
Tan D et al (2010) The changing biological roles of melatonin during evolution: from an antioxidant
to signals of darkness, sexual selection and fitness. Biol Rev 85:607–623. https://doi.org/10.
1111/j.1469-185X.2009.00118.x
Tan D et al (2013) Mitochondria and chloroplasts as the original sites of melatonin synthesis: a
hypothesis related to melatonin’s primary function and evolution in eukaryotes. J Pineal Res
54:127–138. https://doi.org/10.1111/jpi.12026
Tan D et al (2015) Melatonin as a potent and inducible endogenous antioxidant: synthesis and
metabolism. Molecules 20:18886–18906. https://doi.org/10.3390/molecules201018886
Tan D-X et al (2016) On the significance of an alternate pathway of melatonin synthesis via
5-methoxytryptamine: comparisons across species. J Pineal Res 61:27–40. https://doi.org/10.
1111/jpi.12336
1 Neurotransmitters and Their Receptors—State of the Art 29

Thorne BM (2010) Neurotransmitters’. In: Weiner IB, Craighead WE (eds) The corsini encyclope-
dia of psychology. Wiley, Hoboken, NJ, pp 1–2
Thurmond RL (2015) The histamine H4 receptor: from orphan to the clinic. Front Pharmacol 6
(65):1–11
Tieu K, Ischiropoulos H, Przedborski S (2003) Nitric oxide and reactive oxygen species in
Parkinson’s disease. IUBMB Life 55(6):329–335. https://doi.org/10.1080/
1521654032000114320
Tiligada E (2012) Editorial: Is histamine the missing link in chronic inflammation. J Leukoc Biol
92:4–6
Tordjman S et al (2017) Melatonin: pharmacology, functions and therapeutic benefits. Curr
Neuropharmacol 15(3):434–443
Upadhyay SN (2003) Serotonin receptors, agonists and antagonists. IJNM 18(1 & 2):1–11
Virarkar M et al (2013) L-arginine and nitric oxide in CNS function and neurodegenerative
diseases. Crit Rev Food Sci Nutr 53(11):1157–1167. https://doi.org/10.1080/10408398.2011.
573885
Voelker R (2019) Add-on drug approved for “Off” episodes of Parkinson disease. JAMA
322:1246–1246
Von Gall C, Stehle JH, Weaver DR (2002) Mammalian melatonin receptors: molecular biology and
signal transduction. Cell Tissue Res 309:151–162. https://doi.org/10.1007/s00441-002-0581-4
Walker MC, van der Donk WA (2016) The many roles of glutamate in metabolism. J Ind Microbiol
Biotechnol 43(2–3):419–430. Springer
Wang H et al (2005) Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting
potentiation and aggregation of synaptic proteins. Neuron 45(3):389–403. https://doi.org/10.
1016/j.neuron.2005.01.011
Wassall RD, Teramoto N, Cunnane TC (2009) Noradrenaline. In: Encyclopedia of neuroscience.
Academic Press, Cambridge, pp 1221–1230
Wu H et al (2019) Beyond a neurotransmitter: The role of serotonin in inflammation and immunity.
Pharmacol Res 140:100–114. https://doi.org/10.1016/j.phrs.2018.06.015
Yaksh TL, Wallace MS (2011) Opioids, analgesia, and pain management. In: Goodman and
Gilman’s the pharmacological basis of therapeutics. McGraw-Hill Medical, New York, NY,
pp 481–526
Yamamoto K et al (2015) Presynaptic cell type-dependent regulation of GABAergic synaptic
transmission by nitric oxide in rat insular cortex. Neuroscience 284:65–77. IBRO
Yoshikawa T et al (2013) molecular mechanism of histamine clearance by primary human
astrocytes. Glia 61:905–916. https://doi.org/10.1002/glia.22484
Zhdanova IV, Lynch HJ, Wurtman RJ (1997) Melatonin: a sleep-promoting hormone. Sleep 20
(10):899–907
Zhou Y, Danbolt NC (2014) Glutamate as a neurotransmitter in the healthy brain. J Neural Transm
121(8):799–817. https://doi.org/10.1007/s00702-014-1180-8
Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn
Schmiedeberg's Arch Pharmacol 362:299–309
Zisapel N (2001) Circadian rhythm sleep disorders pathophysiology and potential approaches to
management. CNS Drugs 15(4):311–328
Drug-Receptor Interactions
2
Balakumar Chandrasekaran, Haneen Al-Joubi, Sara Samarneh,
Ghadir Kassab, Pran Kishore Deb, Puneet Kumar, Bilal A. Al-Jaidi,
Yazan Al-Thaher, and Yazan A. Bataineh

Abstract

Conventional treatment of any disease can be achieved by the administration of

drugs of natural and synthetic origin. The drug exhibits its pharmacological action
by altering cellular signaling or the biochemical events associated with the
respective target proteins such as receptors or enzymes. Functional groups/
pharmacophores of the drug interact with functional groups present in the
receptor’s binding site, complementarily thereby producing effective binding
interactions. Key interactions that occur between the drug and the receptor will
decide the potency and intrinsic activity of the drug. Major interactions observed
in the drug-receptor complexes are mostly of reversible type which consist of
electrostatic interactions, ion-dipole and dipole-dipole interactions, hydrogen
bonding, charge-transfer interactions, hydrophobic, and Van der Waals
interactions. In this chapter, we have discussed the types of receptors, theories
and types of drug-receptor interactions, the role of functional groups, and stereo-

B. Chandrasekaran (*) · H. Al-Joubi · S. Samarneh · G. Kassab · Y. Al-Thaher · Y. A. Bataineh

Faculty of Pharmacy, Philadelphia University, Amman, Jordan
e-mail: [email protected]
P. K. Deb
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
P. Kumar
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India
B. A. Al-Jaidi
Faculty of Pharmacy, Philadelphia University, Amman, Jordan
Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Yarmouk
University, Irbid, Jordan

# Springer Nature Singapore Pte Ltd. 2020 31

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_2
32 B. Chandrasekaran et al.

chemical aspects involved between the drug and the receptor. Further, we have
shed light on the development of adenosine receptor antagonists through in silico
interactions as a case study.

Keywords

Drug-receptor complex · H-bonding · VdW interaction · Antagonist · Agonist

Abbreviations

3D Three dimensional
Ach Acetylcholine
ARs Adenosine receptors
ATP Adenosine triphosphate
CRC Concentration-response curve
DBD DNA-binding domain
DRC Dose-response curve
GPCRs G-protein-coupled receptors
H-bond Hydrogen bonding
LBD Ligand-binding domain
PPARγ Peroxisome proliferator-activated receptor gamma
RTK Receptor tyrosine kinase
TK Tyrosine kinase
vdW Van der Waals

2.1 Introduction

Upon drug administration, it gets absorbed, transported to the site of action through
general circulation, and elicits a pharmacological response. The target site for the
drug action is ultimately a bio-macromolecule, known as a receptor. Receptors are
mostly membrane-bound proteins, consist of various amino acids, and receive
signals from outside the cell through a ligand molecule. When such small molecules
or ligands bind to the receptor and interact, subsequently they elicit some form of
cellular/tissue responses (Uings and Farrow 2000). There are three different ways
wherein the receptor responds to the chemical messenger/ligand such as relay of
signal, amplification, and integration. Most of the receptors are integral proteins
connected to the protein-lipid bilayer of the cell membrane. The two functionally
important components of receptors include the recognition component (ability of
recognizing specific molecules) and the amplification component (an ability of the
intermolecular complex formation between the ligand and the receptor) to initiate a
pharmacological response (Pierce et al. 2002). Based on the complementarity
between the ligand and binding site of a receptor, each receptor is connected to a
specific biochemical pathway by specific binding towards a particular ligand. Upon
ligand binding to its binding site present in the receptor, it may activate or inhibit
various receptor-associated biochemical events. Further, this single receptor will
2 Drug-Receptor Interactions 33

Unused binding
DRUG region DRUG Extra
interaction

Drug extension

Receptor Receptor

Binding region Binding group

Fig. 2.1 The binding of a drug to the binding site of the receptor based on shape complementarity

send chemical responses as signals to the surrounding receptors or nearby proteins,

thereby allowing them to amplify according to the first response (Pramanik 2015).
Figure. 2.1 presents points of binding of the drug to the receptor based on shape
complementarity.

2.2 Types of Receptors

The structure of receptors varies which depends entirely on the diversity of the
binding site and functions of the receptors. Principally, four types of receptors are
identified, namely ligand-gated ion channel receptors, guanine nucleotide binding
regulatory protein (G-protein)-coupled receptors (GPCRs), tyrosine kinase-linked
receptors, and nuclear receptors.

2.2.1 Type-I: Ligand-Gated Ion Channel Receptors or Ionotropic

Receptors

Various neurotransmitters such as acetylcholine, nicotine, and GABA will bind to

type-I receptors. As a result of this binding, activation of the movement of ions takes
place across the membrane. The general structure of these receptors is normally
heteromeric, and these receptors include an extracellular binding domain for ligands
and a transmembrane domain bearing α-helices. They contain three important
domains such as pores (for transport of ions), gates (open or close based on stimuli),
and sensors to receive the stimuli. There are specific channels for ions such as Na+,
K+, Ca2+, and Cl
. Three integral functions of these receptors include opening,
closing, and activation according to the conformational changes. Figure 2.2 indicates
the signaling process of ligand-gated ion channel receptors wherein the ligand binds
to an extracellular domain of the receptor; thereby the channel opens for the influx of
Ca2+ ions into the cell. Membrane permeation is another way in which ligands can
enter through these receptors (Sansom et al. 1998). Cation-permeable “nicotinic”
acetylcholine (Ach) receptors, ionotropic glutamate-gated receptors, ATP-gated
P2X receptors, and the anion-permeable γ-aminobutyric acid-gated GABAA recep-
tor are few examples of type-I receptors.
34 B. Chandrasekaran et al.

Fig. 2.2 The signaling process shown by ligand-gated ion channel receptors

2.2.2 Type II: G Protein-Coupled Receptors (GPCRs; Metabotropic

Receptors)

GPCRs are the largest family of receptors for some transmitters (metabotropic
glutamate and dopamine), light-sensitive compounds, various hormones, lipids,
glycoproteins, small molecules, peptides, and larger proteins. Upon binding of
these ligands to the GPCRs, they facilitate the interaction with members of the
G-protein receptor family. GPCRs are mostly identified in eukaryotes, yeasts, and
animals (Saengsawang and Rasenick 2015). The structure of GPCRs includes seven
transmembrane α-helices, and loops for their integration. As the name indicates,
these receptors are coupled to diversified intracellular-effector systems incorporating
both extracellular (a binding site for large ligand) and intracellular (separate binding
site) involving the activation of signal transduction pathways resulting in a cellular
response, accordingly (Rosenbaum et al. 2009). Most of the modern medicines
(about 40%) act through modulating the signaling processes associated with
GPCRs (Chandrasekaran et al. 2019). Since they occur as transmembrane proteins,
they are difficult to isolate, purify, and crystallize. To date, bovine rhodopsin,
β-adrenoceptors, and A2A adenosine receptors were crystallized in their pure forms
(Deb et al. 2019a). A typical orientation of a type II receptor involved in the signal
transduction process is shown in Fig. 2.3.

2.2.3 Type III: Kinase-Linked and Related Receptors

Tyrosine kinase (TK) and enzyme-linked receptors have an extracellular domain

bearing a binding site for ligands (normally glycosylated) and an intracellular
domain associated with the enzymatic activity, connected by a transmembrane
2 Drug-Receptor Interactions 35

Fig. 2.3 The orientation of Ligand

type II GPCRs

NH3+

GPCR

Cell membrane
COO-

α-helix. The TK receptor is located on the surface of the cell exhibiting higher
affinity for many of the ligands such as growth factors, hormones, and cytokines
(Cadena and Gill 1992). They are the key regulators for a number of cellular
biochemical processes including their critical role in the progression of cancer.
There are almost twenty different classes of TK-linked receptors and the insulin
receptor is one such example. The main function of TK-linked receptors is the
phosphorylation of tyrosine amino acid residue of target proteins by transferring a
phosphate group from high energy donor molecule (ATP) (Huang and Reichardt
2003). The receptor tyrosine kinase (RTK) pathway is regulated through a number of
positive and negative feedback loops. Since RTKs are involved in many cellular
functions (cell proliferation, differentiation, survival, metabolism, migration, and
cell cycle regulation), they must be regulated to avert cellular abnormalities like
fibrosis and cancer (Gschwind et al. 2004). Figure 2.4 illustrates a typical signaling
process mediated by kinase-linked receptors.

2.2.4 Type IV: Nuclear Receptors

They are normally present in the cytoplasm when there is no ligand binding to the
receptor. Once the ligand binds to such receptors, they migrate to the nucleus of the
cell, accordingly. They are composed of different binding domains such as
N-terminal regulatory domain, DNA-binding domain (DBD) containing two zinc
fingers, hinge region, ligand-binding domain (LBD), and C-terminal domain. In
particular, the N-terminal domain interacts with other cellular transcription factors in
a ligand-independent manner (Robinson-Rechavi and Laudet 2003). Depending on
such interactions, the binding/activity of the receptor can be affected, suitably.
Nuclear receptors are accountable for the recognition of steroidal and thyroid
hormones, fatty acids, bile acids, prostaglandins, and certain vitamins. As they
regulate the gene expression by binding directly to DNA, they are classified as
transcription factors and all these processes depend on the availability of a ligand.
Upon ligand binding to a nuclear receptor, conformational changes occur in the
receptor which activates the receptor; thereby upregulation or downregulation of
gene expression has been effected. Hence, they control the general embryonic
development, homeostasis, and metabolism (Aranda and Pascual 2001). Lipophilic
 36

Receptor tyrosine
kinase
cytokine

RAS SOS Grb2

RAF-1 Cytokine
p
p p receptor
MAPKK p p p
p Non receptor
p p p
MAPK p tyrosine
p
kinase
p p
MAP kinase STAT
MAPK signal
pathway
JAK/STAT signal
p p p pathway
STAT

Fig. 2.4 Signal transduction process mediated by kinase-linked receptors

B. Chandrasekaran et al.
2 Drug-Receptor Interactions 37

Fig. 2.5 Signal transduction process mediated by nuclear receptor

components like xenobiotics, endogenous hormones, endocrine disruptors, and

vitamin D are the examples of common ligands binding to the nuclear receptors.
Figure 2.5 represents a typical signaling process mediated by a nuclear receptor.

2.3 Drug-Receptor Complex

Drug targets are molecules such as proteins (enzymes, receptors, and transport
proteins), lipids, carbohydrates, or nucleic acids (as RNA or DNA) wherein the
drug is going to reach and bind. An important step for a drug action is the binding
and subsequent interaction with the target. While drug is traveling throughout the
body, it reaches its target and identifies the correct binding site in a receptor protein
and interacts to elicit a biological response. The binding site in a receptor also known
as a pocket or canyon is at the surface of the macromolecular target. It is very
important to gain an insight into the forces involved in the receptor-drug binding in
order to determine the mode of drug action (Hase et al. 2009). A receptor has unique
amino acids with specific side chains, so the functional groups in drugs have the
complementary shape characteristics to fit into the binding site or pocket. There are
points for the attachment between a drug and the receptor selectively to provoke the
drug action, and this binding always exists in an equilibrium state. The drug should
have strong interaction with the receptor for association and to allow signal trans-
duction; at the same time, it should also show weak interaction for dissociation and
to allow the drug to depart, after completion of its action. Hence, a fine balance
between strong and weak interactions is required for the better activity of the drugs
(Bs Suvarna 2011).
38 B. Chandrasekaran et al.

The three-dimensional (3D) structural determination of the receptors using X-ray

crystallography facilitated the identification of the binding site for the small organic
molecules binding process. Prior to the structural determination, the receptor should
be isolated, cloned, sequenced, purified, and crystallized using suitable methods. It is
a very challenging task, as the receptors have higher molecular weight, traverse the
cell membrane, are complex, and exist in a tiny amount or lower concentration
(Topiol and Sabio 2009). Furthermore, drug-target interactions are classified into
two types, namely, irreversible interaction (a permanent interaction where covalent
bonds are involved) and reversible interaction (a transient interaction where different
types of bonding involved except covalent bond). If weaker and noncovalent
interactions exist between the drug-receptor complex, it leads to the reversible
pharmacological response (Marc 2008). However, the drug must detach from the
binding site to terminate the pharmacological action to stay away from the toxicity
and adverse side effects. In such responses, the drug becomes inactive as soon as the
concentration of the drug decreases in the extracellular fluid. These reversible
interactions are often very useful in drugs acting on the CNS (stimulants and
depressants). Sometimes, irreversible interactions are anticipated between the drug
and receptor to elicit the drug action for a prolonged period of time (e.g., anticancer
drugs) (Baillie 2016). When a drug interacts with the binding site of a receptor, the
bonding will usually be intermolecular between the drug and receptor to elicit the
desired pharmacological actions. Drugs bind to the receptor and form a complex
(drug-receptor), and the low energy state of this complex is the reason for the driving
force that exists in such complex (Eq. 2.1).
K on
drug þ receptor Ð drug‐receptor complex ð2:1Þ
K off

where, K on is the rate constant for the drug-receptor complex formation and depends
on the concentrations of both the drug and the receptor and K off is the rate constant
for dissociation or the breakdown of the complex and entirely depends on the
concentration of the drug-receptor complex. The pharmacological response of a
drug is associated with its affinity towards the receptor. Moreover, the stability of
the drug-receptor complex can be determined based on the dissociation of the
complex as represented in Eq. 2.2.

½drug½receptor
Kd ¼ ð2:2Þ
½durg‐receptor complex

where, Kd is dissociation constant for the drug-receptor complex at equilibrium and

the lower Kd value indicates the higher concentration and stability of the complex
which in turn explains the higher affinity of the drug to the receptor.
The formation of a drug-receptor complex is an entropically unfavorable process
which ultimately generates lowering of conformational degrees of freedom in both
ligand (drug) and protein (receptor), at the same time decreasing rotational and
translational degrees of freedom, correspondingly. The interaction between a drug
2 Drug-Receptor Interactions 39

and the receptor is usually consists of solvation, attractive-repulsive forces, confor-

mational changes, and long-range interactions (Tallarida 2007). Drugs can binds to
the binding site of the receptor and exhibit different types of interactions or bonds
such as covalent bonding, ionic or electrostatic interactions, ion-dipole and dipole-
dipole interactions, hydrogen bonding, charge-transfer interactions, hydrophobic
interactions, cation-π interactions, halogen bonding, and van der Waals (vdW)
interactions. Many theories were proposed for the binding between the ligand and
a receptor.

2.4 Theories of Drug-Receptor Interactions

After studying the different types of interactions between the drugs and their
receptors, scientists have proposed and developed a set of hypothesis over the
years, known as theories of drug-receptor interactions. These theories can explain
the ability of the drug to interact with the receptor to induce or stimulate a suitable
biological response (Kenakin 2008).

2.4.1 Occupancy Theory

This theory was proposed by Gaddum and Clark in 1926 (Clark 1926). According to
this theory, the drug binds to its specific receptor (complementary structures like
lock and key) and gives a cellular response. Based on the study, scientists concluded
that the intensity of cellular response is directly proportional to the number of
receptors occupied which means that a maximum response occurs when all receptors
are occupied. Drugs exhibit all or no response on each receptor, so it will be either
fully activated or not at all activated. Moreover, there are no partial activation
because once the drug-receptor complex dissociates, the response will halt, accord-
ingly. This concept is illustrated in Fig. 2.6.
As this theory does not cover the concept of partial agonist, Ariens and
Stephenson (Stephenson 1956) have modified the occupancy theory to incorporate
the concept of partial agonist. They have used the main concept of the drug-receptor
interactions that generally involves two stages, namely, affinity and efficacy. Affin-
ity is a feature that determines the capacity of a drug to bind to the receptor for
activating and producing a desired response. The degree of affinity between the drug
and receptor depends on molecular complementarity. If drugs have different affinity

Drug + Receptor Drug-Receptor complex Response

(Dissociation)

No response

Fig. 2.6 The association and dissociation of the complex

40 B. Chandrasekaran et al.

A B

100% 100%

Drug 1
Drug 2

% Response
% Response

Drug 3

Drug 1

Drug 2
Drug 3

0% 0%

Dose of the Drug Dose of the Drug

Fig. 2.7 (A) Drugs have similar affinities but different efficacies; (B) drugs have similar efficacies
but different affinities

to specific receptors, then it is difficult to figure out which one is going to bind to the
receptor. The answer is when a drug with high binding capacity comes around the
receptor, it will inevitably bind to the receptor, even if it is associated with another
drug. In this situation, the former drug will remove another drug which has lower
affinity from the active site of the receptor. The nature of the binding or the
interaction between the receptor and the drug molecule relies mainly on the degree
of affinity (Shuker et al. 1996). On the other hand, the maximum response can be
achieved by the dose of the drug, termed as efficacy and intrinsic activity (Nelson
et al. 2016). Figure 2.7 elucidates the exact difference between the affinity and the
efficacy.
Figure 2.7A illustrates the percentage of biological response relative to the dose
of three different drugs, and all of them have similar affinities to the same receptor.
At the same time, they have quite different efficacies ranging from 100% and ended
with approximately 70% of the maximum. By taking into consideration that the drug
which reached 100% of the maximum response is known as a full agonist while
others are partial agonists. On the other hand, Fig. 2.7B presents the dose-response
curve for three drugs, all of which exhibit an equal efficacy but the affinities of each
one vary. It is also interesting to know that those three drugs are full agonists.
Moreover, both agonist and antagonist will bind to receptors but each one will
produce a different response. Agonist will activate the receptor, while antagonist
will halt the receptor action. Also, a drug may be an agonist when it is bound to one
of the receptors and exerts a positive response, while the same drug could be
antagonist for another receptor. The reason why it is possible for two drugs that
can fit a specific receptor exert different effects is not clarified by this modified
occupancy theory (Maehle et al. 2002). The concept of efficacy has been displayed
in Fig. 2.8.
2 Drug-Receptor Interactions 41

Efficacy

Negative Zero Positive

Full inverse Partial inverse Full agonist Partial agonist

agonist agonist Antagonist

Fig. 2.8 The concept of drug efficacy

Agonist Partial Antagonist

agonist

Fig. 2.9 The concept of ligands (agonist, partial agonist, and antagonist)

2.4.2 Rate Theory

The rate theory was proposed by the scientist Paton in 1961 (Paton 1961). This
theory suggested that the pharmacological activity of any drug depends on the rate of
association and dissociation of the drug with the receptor and will differ based on
agonist, partial agonist, or antagonist. Further, the intensity of the pharmacological
response depends on the number of the drug-receptor interactions per unit time, and
this is directly proportional to the rate of association and dissociation between the
drug and the receptor and the total number of molecules involved in such
interactions. Like in the occupancy theory, the rate theory was also unable to justify
why various types of compounds display their own features. The concept of agonist,
partial agonist, and antagonist has been presented in Fig. 2.9.
42 B. Chandrasekaran et al.

2.4.3 Induced-Fit Theory

Induced-fit theory is another expression of the “lock-key hypothesis,” proposed by

Koshland in 1958 (Koshland 1958). The concept of this theory is the fitting of the
substrate at the active site of the receptor or an enzyme after causing conformational
changes in that receptor or an enzyme to make it convenient in forming the essential
interactions or the bonds with the drug in the right sequence to obtain the desired
response. Also, the drug may undergo a process to change its shape and fit into the
active site. Once the substrate reaches the surface of the enzyme, many conforma-
tional changes could take place to make the catalytic groups closer to the substrate
and react to yield products which are released from the surface of the enzyme and the
enzyme being free for a new turn, subsequently. On the other hand, if the
non-substrate molecules reach the active site, it will change the shape in an undesir-
able way and the catalytic groups will be misaligned; even if the molecule is in an
exact match with the receptor, the response will not be achieved, adequately. The
elasticity of the proteins (receptors) is very crucial because it facilitates its return to
its normal shape after the drug has dissociated from the active site. For example, the
noncompetitive inhibitor molecules produce a response from substrate-protein bind-
ing without preventing the binding of the substrate to the active site of the enzyme;
these inhibitors bind to an allosteric site and make deformations of the active site and
lead to preventing the occurrence of the desired response. According to this theory,
the agonist will bind to the receptor and affect different conformational changes to
produce a response; due to these modifications the molecule binds less tightly to the
receptor, thereby easily dissociating the product. The partial agonist will produce a
partial conformational change once it binds to the corresponding receptor (Koshland
and Neet 1968). While in the case of antagonist, it binds without causing any of the
conformational changes. If there is no conformational change in the receptor, then
the resultant drug-receptor complex will be stable, and the antagonistic action will be
produced. From the induced-fit theory, other theories have also been developed such
as macromolecular perturbation theory, activation-aggregation theory, and multi-
state theory.

2.4.4 Macromolecular Perturbation Theory

Based on the consideration of the flexibility of the receptor, two general types of
macromolecular perturbation were proposed by the scientist Belleau (Belleau 1964).
Either one or both of them could result into the receptor functions once the interac-
tion between the drug and the receptor has occurred. According to the macromolec-
ular perturbation theory, the intensity of pharmacological response is directly
proportional to the rate of formation of perturbations. One type of perturbation is a
specific conformational perturbation which contributes to generate a biological
response when specific molecules bind to the receptor’s binding site (agonist). The
other type is nonspecific conformational perturbations. In this case, no response will
be produced because the receptor binds with other types of molecule (antagonist).
2 Drug-Receptor Interactions 43

However, if the molecule has both molecular perturbations, the result will be a
mixture of two complexes (partial agonist). This theory illustrates the physicochem-
ical properties of the molecules binding to the receptor, but does not cover the
significance of the concept of inverse agonism.

2.4.5 Activation-Aggregation Theory

Activation-aggregation theory and macromolecular perturbation theory are based on

the concept of induced-fit theory as mentioned previously. Activation-aggregation
theory was proposed by Monod et al. (1965) and Karlin (1967) (Monod et al. 1965;
Karlin 1967). According to this theory, if receptors are free from ligands (agonists,
partial agonists, and antagonists), they will be in an equilibrium state. It is a state
between the activated (elicits a biological response) and inactivated forms of the
receptor. As per this theory, agonists will move towards the receptor that exists in an
activated form, antagonists migrate to a receptor in an inactivated form, while partial
agonist will bind to both of these conformations. So, it can be concluded from this
model that the binding sites of receptors in an active form differ from those in an
inactive form, and these structural differences justify why the agonist is responsible
for giving the desired biological response. This theory also illustrates that partial
agonists can have both the agonist and antagonist characteristics. For example, when
partial agonists exist, they will interact with free receptors and this will enhance the
response and it could reach to the maximum. Further, they compete with other
molecules (like neurotransmitters) at the respective binding sites and displace
them. As a result, the amount of receptor forms will be affected in the following
way, the inactivated form will increase, and the response will decrease, substantially.
Nevertheless, the concept of inverse agonist is not explained by this theory.

2.4.6 The Two-State Model of Receptor Activation Theory

This theory has been developed because the activation-aggregation theory men-
tioned previously did not sufficiently explain the activation of a receptor. Hence, the
two-state model of receptor activation theory was developed which depends on the
competitive and noncompetitive kinetics. In addition, it is based on the performance
and results of experiments applied by direct binding to the receptor’s binding-site. In
this model, scientists relied on the previously proposed models but explained the
process of activation in a comprehensive way (Bridges and Lindsley 2008). Simply,
the theory assumes the presence of the receptor in two conformational shapes. The
first one makes the receptor in the active state (R) and then can give a downstream
effect. While the second concept keeps the receptor in an inactive (R) state; therefore
no pharmacological effects can be exerted. The receptor will be in the equilibrium
state if the ligand is not available in the binding site. This is a state between R
44 B. Chandrasekaran et al.

and R, which is known as the basal activity of the receptor. Depending on the
constant need for the formation of a drug-receptor complex (Kd and Kd), the drug
will either bind to one conformational state or to both of them (Colquhoun 1998).
Ligands such as full agonist, partial agonist, full inverse agonist, and antagonist will
bind with a specific state and give a completely different effect. In the following
subsections, the behavior of different ligands is discussed briefly.

2.4.6.1 The Behavior of Agonist in the Two-State Model

Full agonists bind selectively to the active state of the receptor and the resulting
binding is responsible for the liberation of free energy, and this will trap a portion of
the receptor in the active site. The remaining portions of the unbound receptor will
proceed further to reach the equilibrium between active and inactive state (Leff
1995). Moreover, the binding with the active conformation will lead to the full
shifting of the receptor state from inactive equilibrium to active and yield a maxi-
mum response, effectively. Figure 2.10 presents the agonist binding to the active
state of the receptor model.

2.4.6.2 The Behavior of Partial Agonist in the Two-State Model

Partial agonists are ligands which have the features of both agonists and antagonists.
They play an important role in regulating the activity of the receptor. Partial agonists
also play a significant role of competitive antagonists when both full agonistic and
partial agonistic properties co-exist together. In this case, it will minimize the activity
of the receptor because it competes with full agonists at the binding site of the
receptor. On the other hand, it can increase the activity of a receptor and produce a
submaximal response when there is a lack in the sufficient amounts of endogenous
ligands. The partial agonist prefers to bind with the active state receptors but not as
much to the extent observed for the full agonist. As the partial agonist has lower
efficacy than the full agonist, the maximum response will not be attained.

2.4.6.3 The Behavior of Full Inverse Agonist in the Two-State Model

Full inverse agonists are ligands that bind to the receptor as an agonist but give a
completely different effect; this binding stimulates the receptor to contribute the
opposite response. Most of the time, it prefers to bind to the inactivated state of the

Agonist

Giving maximum
effect

Active receptor

Fig. 2.10 The agonist binding selectively to the receptor in the active state
2 Drug-Receptor Interactions 45

receptor and shift the equilibrium to inactive state, thus decreasing the basal activity
of the receptor and a negative efficacy was observed (<0% efficacy).

2.4.6.4 The Behavior of Antagonist in the Two-State Model

Antagonists are two types, namely, competitive and noncompetitive antagonists.
Specifically, competitive antagonists compete with the same binding site wherein the
natural ligand or the agonist normally binds. If a competitive antagonist exists alone,
then it will interact with the receptor reversibly; however, agonists can replace the
antagonist from the binding site and will completely abolish the pharmacological
effect of antagonists. Conversely, noncompetitive antagonists interact either alloste-
rically or irreversibly to the receptor and prevent the effect of the agonist irrespective
of the agonist concentration (Lew and Ziogas 2004). In general, noncompetitive
antagonists have the same affinity towards both the states of receptor, and they do
not have any impact on the equilibrium or the basal activity of the receptor. As a
result, they will never associate with positive or negative efficacy. Moreover, the
noncompetitive antagonist molecule can replace either the agonist or inverse agonist
from the receptor binding site Figure 2.11 presents the antagonist binding to the
receptor model.

2.4.6.5 Three-State Receptor Model of Agonist Action

This theory has been developed by Leff and coworkers and proposed initially that
the model involves three states, two of them are in the active state and the third is in
the inactive state. Later stages the model has been extended to include more than two
active states which is known as multi-state model of receptor activation. This model
gives us a clear idea about why the agonist and inverse agonist have different
behaviors in both affinities and efficacies, while they are at the same receptor binding
site According to this theory (Leff et al. 1997), the differentiation that occurs
between different agonists in their efficiencies is due to their different affinities for
various active states.

Antagonist

In the absence of other

ligands, it will block the
response of the receptor

Inactive receptor

Fig. 2.11 The antagonist binding to the receptor model

46 B. Chandrasekaran et al.

2.5 Types of Drug-Receptor Interactions

The driving force for drug-receptor interaction is the low energy state of the drug-
receptor complex. The biological activity is related to the drug affinity for the
receptor, i.e., the stability of the complex. Dissociation constant of the drug-receptor
complex gives an idea about how potent is the drug. The binding interactions occur
through points of attachment; for a chemical compound they are the functional
groups. Functional groups use their electronic and shape characters in the binding
process. If we talk about reversible binding, binding of the drug to receptor should be
in equilibrium state. Drug-target interactions can be grouped into two types: Perma-
nent (irreversible)—covalent bonding and reversible interactions. All plausible
interactions existing between a receptor and the ligand are discussed in the following
subsections.

2.5.1 Covalent Interactions

A covalent bond is produced between two species by mutual sharing of electrons and
is the strongest bond, irreversible, and exhibits a stability of
40 to
110 kcal/mol.
This type of covalent bond is mostly observed in drug-enzyme or drug-DNA
complexes rather than the drug-receptor complex. It is also beneficial in avoiding
toxic effects of drugs through an irreversible inhibition of the receptor. This covalent
bond usually occurs between a nucleophile (molecule having negative charge, rich in
electrons) such as hydroxyl or thiol group in the receptor amino acids and an
electrophile (molecule having positive charge, deficient in electrons) such as epoxide
or allyl group in drug structure (Kumalo et al. 2015). Two types of covalent bonds,
namely polar and nonpolar, are possible during interactions. Water molecule is an
example of a polar covalent bond, whereas peptide bond is an example of
noncovalent bond. The antibiotic penicillin is an irreversible inhibitor of the enzyme
glycopeptide-transpeptidase, the enzyme which catalyzes an essential step in bacte-
rial cell wall synthesis. Penicillin covalently blocks the active-site amino acid serine
present inside the glycopeptide-transpeptidase through the formation of a
covalent bond.

2.5.2 Ionic or Electrostatic Interactions

Ionic bond is also known as electrostatic interaction which is weaker than the
covalent bond and can provide an ionic interaction energy of
5 kcal/mol. This
bond appears between two opposite (negative and positive) charges of amino acids
of a receptor and ionized species of the ligand (Klebe 2013). Under the normal
physiological pH, some of the basic amino acids like arginine, lysine, and histidine
bearing amino group in their side chain get protonated, thereby providing a cationic
environment. On the other hand, acidic amino acids such as aspartic acid and
glutamic acid having carboxylic group get deprotonated and become anionic in
2 Drug-Receptor Interactions 47

Fig. 2.12 The ionic Ionic interaction

interaction between
carboxylate anion
(pivagabine) and protonated
amino group (receptor)

nature. Thus, both positive (cationic) and negative (anionic) charges available in the
receptors take part in an ionic bonding with ionized groups of drugs. Ionic bond is a
most prevalent bond in drug-receptor interaction which depends entirely on the
extent of ionization and the distance between two opposite charges. An example
of ionic interaction is presented in Fig. 2.12.

2.5.3 Ion-dipole and Dipole-Dipole Interaction

Due to the electronegativity of hetero-atoms (O, N, S, and halogens over the carbon
atom), an electric dipole is formed subsequently generating the polarization in
bonds. In the polarized bond, one of the pole will be partially positive and the
other confers partially negative charge, respectively. These partially positive or
negative charges can form an electrostatic bond with either partially charged atoms
or ionized elements. As a result of higher electronegativity of one atom, an asym-
metric distribution of the electrons was observed. Hence, ion-dipole interaction
involves an ion (side chain amino acids of receptors) and a dipole (drug) or vice
versa, but dipole-dipole interaction occurs between two dipoles of the drug and
receptor, respectively. This bond is polar and electrostatic; also the dipole-dipole
interaction is weaker than ion-dipole interaction. This type of interaction involves
the energy of
1 to
7 kcal/mol (Du et al. 2016). An example for the drug is
zaleplon (Fig. 2.13) which is indicated for the treatment of insomnia.

2.5.4 Hydrogen Bonding Interactions

It is a type of dipole-dipole interaction in which hydrogen atom is a linker between

two electronegative atoms, of which one electronegative atom donates the available
hydrogen, while the other electronegative atom (bearing a pair of non-bonded
electrons) accepts it (Varma et al. 2010). Consider atoms such as N-H and O are
electronegative atoms, in which the “N” removes the electron density from “H,”
rendering “H” a partial positive charge, allowing it to attract towards other partially
negative atom “O.” Thus, N-H acts as a proton donor and “O” as a proton acceptor. If
two atoms had equivalent electronegativity and degree of ionization, then the proton
can be shared equally between them, thereby generating a low-barrier hydrogen
bond. In general, the distance of hydrogen bond between a carbonyl oxygen (C¼O)
48 B. Chandrasekaran et al.

Fig. 2.13 The dipole-dipole Dipole-dipole interaction

(top) and ion-dipole (bottom) N
interactions of zaleplon
N HO

N N

Ion-dipole interaction
N
O
O O

Fig. 2.14 Hydrogen bond H

distance between heteroatoms 2.75 Å
N
O H

2.90 Å O
O H

and a hydroxyl group proton (H-O) is 2.75 Å, whereas a carbonyl oxygen (C¼O)
and proton of N-H is 2.90 Å (Fig. 2.14).
A hydrogen bond is very important and unique to hydrogen atom, exclusively as
it is the only atom that can confer a positive charge at physiological pH (Balakumar
et al. 2010). Hydrogen (H)-bonding includes two types of interactions, that is,
intermolecular and intramolecular H-bonds, respectively. However, compounds
that tend to make intramolecular H-bond will be less active and unable to interact
with the receptor. The possibility of hydrogen bonding involves the orientation of
hydrogen atom in donor and acceptor group and depends on the distance between
two atoms (1.5–2.2 Å). Hydrogen bond acceptor has electron-rich atom and slightly
negative (carboxylate ion) charge, whereas the donor has an electro-deficient hydro-
gen and slightly positive charge (alkyl ammonium ion or secondary and primary
amines) (Kuhn et al. 2010). Two types of hydrogen bonding interactions are
presented in Fig. 2.15.

2.5.5 Charge Transfer Interactions

Charge transfer interactions happen between electron donor group in one molecule
(alkene) and an electron acceptor in another group (aromatic ring). Some amino
acids in the receptor have electron donor groups like -OH group in aromatic amino
acid tyrosine and carboxylate group (-COO) of aspartate (Zhang et al. 2016).
Similarly, few amino acids bearing electron acceptor group (sulfur-containing
amino acid cysteine) and amino acid residues like histidine, tryptophan, and
2 Drug-Receptor Interactions 49

O
H
Intramolecular H-bonding
O

O Receptor
H O
H

Intermolecular H-bonding

Fig. 2.15 Intramolecular and intermolecular hydrogen bonding interactions

CN
Cl Cl

Cl Cl
HO
CN

Fig. 2.16 Chlorthalonil interacting with tyrosine residue of the receptor through charge transfer
process

asparagine have both electron donating and accepting capabilities. An example of

charge-transfer interaction is illustrated using the drug, chlorthalonil, in Fig. 2.16.

2.5.6 Hydrophobic Interactions

Hydrophobic interaction is a type of noncovalent interaction that occurs in an

aqueous solution. This type of interaction is due to the stabilization of the
receptor-drug complex originated from higher entropy and lower free energy
(Varma et al. 2010). When a nonpolar lipophilic group on a drug and nonpolar
group in a receptor surrounded by ordered water molecule, it will be disordered to
associate each other leading to the stabilization of the drug-receptor complex
(Fig. 2.17).
Further, it includes π-π interaction involving a face-to-face arrangement of
aromatic ring of drug and another aromatic ring of amino acids in the receptor,
both of which have π electron system (Fig. 2.18).
50 B. Chandrasekaran et al.

Hydrophobic interaction
Water/solvent

Nonpolar chain of
receptor Receptor
Nonpolar
nonpolar Drug nonpolar chain
chain in
Drug chain

Fig. 2.17 Hydrophobic interaction between drug and receptor

Π-Π interaction

Fig. 2.18 Aromatic system of lacosamide drug interacting with the aromatic ring of the receptor
through π-π interaction

VdW interaction H-bonding

Ionic bonding
o
-OOC

Lipid bilayer

Protein

Fig. 2.19 Van der Waals interaction between the drug and the receptor is shown for epinephrine
including H-bonding and ionic bonding

2.5.7 Van der Waals Interactions

Sometimes a temporary nonsymmetrical distribution of electron density occurs in a

molecule generating a temporary dipole which interacts with another dipole in the
receptor. These bonds are much weaker in comparison to other types of bonds,
bearing
0.5 kcal/mole energy, and is known as Van der Waals interactions (Barratt
et al. 2005). An example of Van der Waals interactions is illustrated in Fig. 2.19.
An example of multiple interactions exhibited by Dibucaine (local anesthetic
drug) is illustrated in Fig. 2.20.
2 Drug-Receptor Interactions 51

Hydrophobic

Charge Transfer N
Hydrogen bond
H Ionic, ion-dipole
N
O N
Hydrophobic Hydrophobic
Hydrophobic
O

Dipole-dipole, Hydrophobic
H-bond,
or Halogen bond

Dibucaine
Fig. 2.20 Multiple interactions shown by Dibucaine towards the receptor

2.6 Determination of Drug-Receptor Interactions

2.6.1 Agonists

Some endogenous molecules are responsible for the regulation of certain physiolog-
ical functions through binding with receptors and interact in the tissues which lead to
a specific physiological response. Like a natural messenger to the receptor, an
agonist is a chemical messenger that can activate the receptor as a result of binding
to it. Due to the structural similarity with the natural messenger, agonists can also
exhibit intermolecular interactions/bonds by employing the same induced fit similar
to natural messenger performs. However, the binding of agonist to the receptor
should be a reversible binding. The knowledge about the properties of the binding
site including the geometry and topography, the chemical structure of the normal
substrate that binds to the receptor, the correct binding group of agonists, position of
interaction with complementary binding region, and the shape and size of agonists is
required for the design of novel agonists so as to fit the binding site of the receptor
(Auerbach 2016).
There are two terms which describe the agonists as partial and full based on dose-
response curve (DRC) or concentration-response curve (CRC). This curve is
obtained initially by administering endogenous compounds like Ach to animal
muscle tissue and allowing the contraction of muscle as a measure of response.
Initially, the concentration of Ach is low; therefore, only a small number of
molecules interact with the receptor, and at the time they had linear relationship
between the concentration and the response because all of the receptors are occupied
at 100%. If a similar kind of response is obtained by the use of an exogenous
compound to this muscle tissue, then such compound is known as a full agonist.
However, 50% response indicates that the administered compound is a partial
52 B. Chandrasekaran et al.

Fig. 2.21 CRC of different 100% Full agonist

types of agonists

Full agonist
in presence of
partial agonist

Effect
50%
Partial agonist

0%

Agonist concentration

agonist (Lambert 2004). The CRC pattern of different types of agonists is shown in
Fig. 2.21.

2.6.2 Antagonists

Receptor antagonists are compounds that can inactivate the receptor after binding to
it. The antagonist must also have the complementary shape and binding group to
orient towards the binding site of a receptor. In general, the chemical structure of
antagonists exhibits slightly higher size than the endogenous compound and is
classified into competitive and noncompetitive antagonists. If the administration of
Ach does not initiate a response in the presence of another exogenous compound or
there is a need for a higher concentration of Ach, it indicates that the exogenous
compound could be a competitive antagonist. It directly demonstrates that the
agonist and the antagonist compete each other for the same binding site. The
competitive antagonist possesses structural similarity to agonists in terms of size,
shape, and functional groups to allow binding on the same agonist binding site on the
receptor. As mentioned earlier, the response will be tardy after adding an increased
amount of the agonist; consuming the binding is reversible. The predominant
binding to a receptor depends on the higher concentration of either agonist or an
antagonist (Buchwald 2017). If the unknown drug exhibits 50% of response and no
further enhanced response obtained even with the addition of excess amount of Ach,
it is a noncompetitive antagonist. Figure 2.22 represents the CRC of both competi-
tive and noncompetitive antagonists.
There are two strategies to design the noncompetitive antagonists such as alloste-
ric antagonists (Fig. 2.23) and antagonists using umbrella effect (Fig. 2.24). If the
designed compound binds to an allosteric binding site (another site of the normal
agonist binding site) that may be located beside the major binding site, then it leads
to geometric changes in the binding site, thereby preventing the agonist binding is
called as noncompetitive antagonist. It could be a drug or an endogenous compound,
2 Drug-Receptor Interactions 53

A B
100% Agonist alone
100% Agonist alone

% Response
% Response

Agonist + antagonist
50%
50%
Agonist + antagonist

0% Antagonist alone 0% Antagonist alone

Agonist or antagonist concentration Agonist or antagonist concentration

Fig. 2.22 (A) Competitive antagonist; (B) noncompetitive antagonist

Catalytic site Catalytic site

substrate substrate

Conformational
Protein change Protein
Allosteric
site

Regulatory
Regulatory molecule
molecule

Fig. 2.23 Noncompetitive antagonism by allosteric effect

Fig. 2.24 Noncompetitive antagonism by umbrella effect

and there will be little or no response to agonist, so maximum achievable response

will be reduced in this type. The percentage of antagonism in the noncompetitive
type does not depend on the amount or the concentration of the agonist or antagonist
(Schwartz and Holst 2007). Agonists will take much more time to give a response
when it is mixed with a competitive antagonist, but in case of a noncompetitive
antagonist, the response will be lowered due to the allosteric effect.
The second strategy in the design of an antagonist is by umbrella effect, in which
the noncompetitive antagonist will bind closely to the agonist binding site, but the
54 B. Chandrasekaran et al.

antagonist displays a part of its structure like a tail that will cover the opening of the
binding site, thus preventing the binding of an agonist (Karschin et al. 1988). In this
case, the percentage of antagonism depends on the amount of both the agonist and
the antagonist.

2.7 Contribution of the Functional Groups to Drug-Receptor

Interactions

The strength of the association between the receptor and a drug can be determined by
total free energy of interaction. Nevertheless, it does not clarify about the quality of
interaction or the effect of the addition of new functional group. Hence, it is very
important to estimate the contribution of an individual functional group to drug-
receptor interactions. To understand drug-protein interactions, functional group
additives (Eq. 2.3) or the additivity of free enthalpy components (Eq. 2.4) is majorly
employed (Andrews et al. 1984).

ΔG ¼ ΔGMe þ ΔGOH þ ΔGPh ðPhÞ þ . . . . . . ð2:3Þ

ΔG ¼ ΔGH
bridge þ ΔGsolvation þ ΔGconformation þ . . . . . . TΔS . . . . . . ð2:4Þ

The free energy of binding is defined in terms of the binding energies for the
individual functional groups that construct a drug molecule according to Eq. 2.5.

ΔG ¼ TΔSt,r þ nr Er ¼ E Nx Ex . . . . . . ð2:5Þ

where TΔSt,r is the loss of overall translational and rotational entropy related to the
drug binding, nr is the number of internal degrees of conformational freedom lost on
binding the drug molecule, and Er is the energy equivalent of the entropy loss
associated with the loss of each degree of conformational freedom on receptor
binding (Andrews et al. 1984).

2.7.1 Intrinsic Binding Energy

If the specific functional group of a drug aligned to the specified functional group of
the receptor without any strain, the Ex is known as intrinsic binding energy or
apparent binding energy. In general, Ex is the combination of the various enthalpic
and entropic interactions including enthalpy of interaction between the drug and
receptor binding site. The change in enthalpy is associated with the removal of water
of hydration (the functional group and its target binding site), subsequent bond
formation between the displaced water molecules, and the corresponding entropy
terms associated with the displacement and subsequent bonding of water molecules.
Thus, intrinsic binding potentials can be used reasonably in an additive manner in the
determination of the drug-receptor interaction (Jencks 1981).
2 Drug-Receptor Interactions 55

2.7.2 Anchor Principle

Based on Eq. 2.5, the binding energy Ex can be determined by comparing the
binding energies for pairs of compounds that differ only in terms of functional
group “X.” This concept was employed by the scientist “Page” who declared it as
“Anchor Principle” (Page 1977). It is based on the fact that the difference in binding
of a drug molecule by the presence or the absence of the particular functional group
is mainly due to the number of factors associated with that functional group. In other
words, the binding energy Ex with loss of any degrees of conformational freedom
arose due to the binding of group “X.” The magnitude of the binding energies
deduced by the anchor principle will vary according to the quality of the interaction.
If the functional groups are unable to align correctly, then small or even repulsive
interaction may occur.

2.7.3 Average Binding Energy

Andrews et al. studied the average contributions of individual functional groups to

the observed binding energies of about 200 different ligand-protein interactions in
aqueous solution (Andrews et al. 1984). In this, the average loss of rotational and
translational entropy TΔSt,r (Eq. 2.5) was determined as 58.5 kJ/mol at 310 K. The
outcome of their study indicated that the loss of entropy associated with each internal
rotation (ΔGr) on receptor binding is equivalent to a decrease in the free energy of
binding by an average of 3 kJ/mol. The averages calculated were smaller than the
respective intrinsic binding energies.

2.7.4 Contribution of Methyl Group and Nonpolar Groups

The scientist Page (1977) determined that the intrinsic binding energies for the CH2
group (methylene) fall in the range of 12–14 kJ/mol based on the data of the
selectivity of amino acid-tRNA synthetases. Under physiological conditions, the
value of Gibb’s free energy is approximated (in kJ/mol) by Eq. 2.6.

ΔG ¼
5:85 log K d . . . ð2:6Þ

For example, the calculated binding energies for isoleucine and its desmethyl
analog (Fig. 2.25) to isoleucyl-tRNA synthetase are 29.7 and 15.9 kJ/mol,

Fig. 2.25 2D structures of O O

isoleucine and desmethyl
analog OH OH
NH2 NH2

Isoleucine Desmethyl isoleucine

56 B. Chandrasekaran et al.

respectively, demonstrating that the methyl group contributes a total of 13.8 kJ/mol
to overall interactions. In case of long chain hydrocarbons, the positive contribution
is observed mainly because of dispersion forces and hydrophobic interactions
generating the loss of conformational entropy on binding. Hence, 3 kJ/mol binding
energy average derived for sp2 and sp3 carbons is same to the “average” decrease in
free energy of binding. While comparing with the unsaturated or cyclic analogs, this
effect will be higher in case of saturated hydrocarbons (Andrews et al. 1984).

2.7.5 Contribution of Hydroxyl Group or Hydrogen Bonding

Groups

The contribution of hydrogen bonds mediated by hydroxyl groups in transition-state

analogs was explained by Wolfenden et al. Based on the experiments conducted and
by the application of the anchor principle, it was observed that apparent binding
energies for single hydroxyl groups ranged from 20 to 42 kJ/mol (Wolfenden and
Kati 1991). By comparing the binding of 1,6-dihydropurine ribonucleoside and its
6-hydroxy derivative (Fig. 2.26) towards the protein adenosine deaminase, the
authors witnessed a binding energy value of 41 kJ/mol (Kati and Wolfenden
1989). Based on this particular observation, the authors perceived that the hydroxyl
group at sixth position has limited movement and had a proper alignment within the
active site, thereby generating a hydrogen bond. This was further supported by the
X-ray solved crystal structure of the inhibitory complex between adenosine deami-
nase and 6-hydroxy-1,6-dihydropurine ribonucleoside. In this crystal structure,
interactions between the 6-hydroxyl group with a zinc atom, protonated histidyl
residue, and aspartic acid residue at the active site were observed (Wilson et al.
1991).

2.7.6 Acidic and Basic Substituents

Some acidic and basic entities of charged groups influence the binding interaction
between the ligand and its receptor. In particular, the phosphate (cation) binds to

OH
H H
HN N HN N

N N N N
O O
HO OH HO OH
HO HO

1,6-Dihydropurine ribonucleoside 6-Hydroxy 1,6-dihydropurine ribonucleoside

Fig. 2.26 2D structures of 1,6-dihydropurine ribonucleoside and 6-hydroxy 1,6-dihydropurine

2 Drug-Receptor Interactions 57

Table 2.1 Contributions of functional groups to overall binding energies

Determination of interaction energy methods
Anchor Site-directed Average
Type of functional groups principle mutagenesis energy
Nonpolar (each carbon) 12–14 1–3 3–6
H-bonding (uncharged) 16 2–6 5–14
H-bonding (charged) 20–42 15–19 -
Carboxyl, amine groups 18–28 12–25 34–48
(charged)
TΔSt,r 12–60 - 58.5
ΔGr (internal rotation) 5–6 - 3

alkaline phosphatase (Levine et al. 1969) and showed a ΔG value of 33 kJ/mol. By

considering the loss of rotational and translational entropy related to this interaction,
Eq. 2.5 resulted in a lower estimate for binding (45 kJ/mol) for the phosphate ion. If
the same value of TΔSt,r is applied to the binding of oxalate ion (anion) to the
enzyme transcarboxylase, Eq. 2.5 yields an apparent binding energy of 24 kJ/mol per
carboxylate group after the minimal loss of conformational entropy (3 kJ/mol).
These values are in accordance with the average values estimated by Andrews
et al. (34–48 kJ/mol) (Andrews et al. 1984). The contributions of some functional
groups and/or bond types to overall binding energies are collected in Table 2.1.

2.8 Stereochemical Considerations in Drug-Receptor

Interactions

2.8.1 Diastereomerism and Binding Interaction

They are the type of stereoisomers, but not mirror images of each other.
Diastereomers are actually the complexes formed between two enantiomers, thereby
yielding different energies and chemical properties consequently resulting in differ-
ent dissociation constants for drug-receptor complexes of enantiomeric drugs. There
are a special case of diastereomers such as geometric isomers (E and Z) and epimers
(compounds having the same chemical formula but differing in their spatial
arrangements around the single carbon atom). They can be separated conveniently
using chromatography or recrystallization techniques than the enantiomers.
Diastereomers exhibit different energies and stabilities due to the fact that they
demonstrate different interactions with the same receptor after binding to the binding
site (Kier 1997). For example, the neuroleptic potency of the Z-isomer of the
chlorprothixene (an antipsychotic drug) is 12 times greater than that of the
corresponding E-isomer. Conversely, the E-isomer of the diethylstilbestrol
(an anticancer drug) had 14 times better estrogenic activity than the corresponding
Z-isomer (Fig. 2.27).
58 B. Chandrasekaran et al.

Fig. 2.27 Structures of

diastereomers of N N
chlorprothixene and
H H
diethylstilbestrol
Cl Cl

S S

(Z)-Chlorprothixene (E)-Chlorprothixene
highly active less active

HO
HO
OH

HO
(E)-Diethyl stilbesterol (Z)-Diethyl stilbesterol
highly active less active

2.8.2 Enantiomerism and Binding Interaction

According to the nomenclature of Ariëns (1987), the potent isomer is called as the
eutomer and the weaker one is known as distomer. The potency ratio of higher
affinity enantiomer to lower affinity is termed as eudismic ratio. The distomer can be
regarded as an impurity in the mixture, which may contribute to undesirable side
effects or toxicity. In some cases, the distomer will be responsible for the biological
activity and the eutomer attributable to the side effects. D-ketamine (Fig. 2.28), a
hypnotic and analgesic agent, is responsible for the pharmacological actions,
whereas the isomer L-ketamine is known for the undesired side effects. It is also
possible that both isomers are active biologically, but one of them causes toxicity
(e.g., the local anesthetic prilocaine). In some cases, it is required to have the two
isomers for better pharmacological activity. Both isomers of bupivacaine (Fig. 2.28)
act as local anesthetic, but only the L-isomer shows vasoconstrictive activity (Aps
and Reynolds 1978). On the other hand, the D-isomer is responsible (i.e., eutomer)
for both the diuretic activity and the side effect (uric acid retention).
Enantiomers may have different therapeutic actions, for example, Darvon, an
analgesic drug and its enantiomer, Novrad is an antitussive drug. Thus, these
enantiomers are marketed (Darvon and Novrad) separately under different trade
names. Another case for enantiomers is that they may display opposite effects. The
(R) enantiomer of 1-methyl-5-phenyl-5-propylbarbituric acid (Fig. 2.28) acts as a
narcotic, while the (S) enantiomer works as a convulsant. Hence, the receptor has an
ability to select and recognize the isomers through the chiral nature. Enantiomers
may have different biological activities depending on the fact that one isomer may fit
into the receptor binding site much better than its counterpart to demonstrate better
2 Drug-Receptor Interactions 59

Cl Cl

NH NH

O O O NH

(S)-Ketamine (R)-Ketamine N
more active-hypnotic more active-hypnotic

(RS)-Bupivacaine
local anesthetic

O O

N N

O N O O N O
H H

(R)-1-methyl-5-phenyl-5- (S)-1-methyl-5-phenyl-5-
propylbarbituric acid propylbarbituric acid
narcotic analgesic convulsant

Fig. 2.28 Structures of enantiomers of Ketamine and 1-methyl-5-phenyl-5-propyl-barbituric acid

with racemic mixture of Bupivacaine

pharmacological activity profile (Arthur 1927). If a receptor has two binding site
points (Fig. 2.29 A, B), then it is difficult to recognize the specific enantiomer
(epinephrine); on the other hand, if a receptor has three binding site points
(Fig. 2.29 C, D), it can recognize a particular enantiomer and distinguishes between
pairs of enantiomers (Arthur 1927).
R-(
)-isomer of epinephrine has three points of interaction due to the specific
conformation to maximize molecular complementarity. However, the S-(+)-isomer
showed two sites of interaction (the hydroxyl group cannot interact with the binding
site) and exhibited lower binding energy (Fig. 2.29A, B).
Similarly, Talapatra et al. studied the crystal structure of the Eg5-K858 complex
and its implications in structure-based design of thiadiazole-containing inhibitors as
anticancer agents (Talapatra et al. 2018). In their study, the inhibitor molecule K-858
(Fig. 2.30) exists in a racemic mixture, which was resolved using chiral HPLC.
Interestingly, S-enantiomer of K-858 showed higher inhibition of Eg5 protein, while
R-enantiomer was unable to inhibit the protein. Figure 2.30 describes the favorable
interaction for the S-enantiomer of K-858 displaying the correct orientation of
methyl group to solvent accessible region, while the phenyl ring is involved in
aromatic π-π interaction (Trp127) and hydrophobic interactions (Arg119 and
Pro137). Thus, the S-enantiomer demonstrated good inhibition of Eg5 protein and
acts as a potential anticancer agent. On the other hand, the phenyl group with a
significantly larger hydrophobic character than the methyl would be placed towards
the solvent region resulting in larger unfavorable interactions for the R-enantiomer
which is responsible for the lack of inhibition of Eg5 protein.
60 B. Chandrasekaran et al.

HO HO
H OH
HO NH2 HO NH2
OH

Ar Ar

R-(-)-Epinephrine S-(+)-Epinephrine
A B

HO HO
H OH
HO NH2 HO NH2
OH

Ar Ar H
H

R-(-)-Epinephrine S-(+)-Epinephrine
C D

Fig. 2.29 Binding of epinephrine enantiomers to two-site receptor (A, B a, b) and three-site
receptor (C, D c, d)

Solvent O Solvent
region region O
N N Unfavorable
O
steric crash N N
S N O
H S N
H
Aromatic Π-Π interaction (Trp127)
Larger unfavorable interaction
Hydrophobic interaction (Arg119 and Pro137)
No activity
Favorable interaction for good activity
S-isomer of K-858 R-isomer of K-858
active Inactive

Fig. 2.30 Binding interactions of enantiomers of K-858 with crucial amino acids in the binding
site of Eg5 protein
2 Drug-Receptor Interactions 61

2.8.3 Conformational Isomerism and Binding Interaction

Conformational isomers are the type of isomers formed due to the free rotation of
single bonds and cannot be separated. The concept of pharmacophore is best
described in terms of the configuration of a set of atoms and the bio-active confor-
mation as well (Balakumar et al. 2018). The crucial amino acid residues in the
binding site of the receptor can bind to only one specific conformer. The conformer
that binds to the receptor’s binding site should have adequate energy which can be
determined by sophisticated instrumentations such as X-ray crystallography and
NMR spectrophotometry or by computation through molecular mechanics
calculations. It is very imperative to identify the bioactive conformation which is
an active conformation of the drug that is involved in binding to the receptor for the
design of ideal drug candidates. If there is a lead compound exhibiting low potency,
then it is mainly due to the existence of a low amount of the active conformer in a
solution. For example, the antidiabetic drug rosiglitazone (Fig. 2.31) binding to
peroxisome proliferator-activated receptor gamma (PPARγ); the favorable orienta-
tion must be in a “U” shaped conformation for better activity (Gampe et al. 2000). In
this particular conformation, the thiazolidinone moiety was buried into the binding
site so that it can display H-bonding interactions with crucial amino acids (Ser289
and Tyr473). Thus, the bioactive conformer of rosiglitazone demonstrated good
inhibition of PPARγ.
Li and Biel demonstrated the correlation between the conformers and the
tranquilizing action of 4-(4-Hydroxypiperidino)-40 -fluorobutyrophenone
(Fig. 2.32A) with mild anti-emetic property (Li and Biel 1969). The compound
had 2 chair (Fig. 2.32B,E ) and 2 twisted-boat (Fig. 2.32C, D) conformations.
Initially, a relative compound, N-methyl-4-piperidinol (R ¼ Me), was considered,
and the difference in free energy between the axial and equatorial hydroxyl
conformers was determined to be 0.94  0.05 kcal/mol at 40  C (the equatorial
conformer is most favorable by a factor of 4.56 over axial conformer). The energies
for the twist-boat conformers are 6 kcal/mol higher due to hydrogen bonding; thus
C was considered as more stable than B. Based on this assumption, three

O O
HN N N
O
S

Rosiglitazone

Fig. 2.31 2D structure of rosiglitazone and 3D interaction plot of rosiglitazone with PPARγ
62 B. Chandrasekaran et al.

OH
O
N

F
A

4-(4-Hydroxypiperidino)-4'-fluorobutyrophenone

B C D E

F G H

Fig. 2.32 Chemical structure of 4-(4-Hydroxypiperidino)-40 -fluorobutyrophenone (A a) and its

conformers (B-H b–h)

conformationally rigid chair analogs (F-H f–h) were synthesized and evaluated for
their muscle relaxant activity. The structure G was found to be conformationally less
stable with the axial hydroxyl group resulting in a better molecular complementarity
with the receptor, thereby yielding good pharmacological activity.
This is a distinguished approach that can be used to determine the bioactive
conformation of a drug molecule in the drug-receptor complex. This involves the
synthesis of conformationally rigid analogs, followed by biological evaluation. The
highest potent analog can be used as a prototype. The major disadvantage in this
approach is that in order to form the analogs, additional atoms must be added to the
original compound and this may affect both chemical and physical properties. In
such cases, it is essential that the drug and the analog must be similar in size, shape,
and mass.

2.9 Case study in the Design of Adenosine Receptor

Antagonists Through Interactions

Adenosine receptors (ARs) are classified into four subtypes A1, A2A, A2B, and A3
ARs belonging to the superfamily of GPCRs (Kaur et al. 2011; Agrawal et al. 2019;
Deb 2019a). At present, about 40% of modern medicines function through the
mediation of various signaling processes associated with GPCRs (Deb et al.
2 Drug-Receptor Interactions 63

2019b; Deb 2019c; Mailavaram et al. 2019). In particular, two important AR

subtypes, namely A2B AR and A3 AR signaling pathways, are implicated mainly
in asthma and COPD (Pran Kishore et al. 2011). Hence, the design of antagonists
(Deb 2019b) for these types depends on the availability of crucial amino acids in the
binding site and the interacting functional groups of receptor amino acids with the
designed antagonists (Banda et al. 2013; Chandrasekaran et al. 2018). Earlier,
fluorinated fused quinazolines were designed as A2B AR antagonists and
investigated in silico by molecular docking with the developed homology model
of A2B AR (Chandrasekaran et al. 2017). Recently, a number of potential AR
antagonists were discovered (Shaik et al. 2019).
Due to the lack of availability of X-ray crystal structures of the A2B and A3 ARs,
homology models were constructed and subsequently employed for the docking
studies (Balakumar et al. 2012; Chandrasekaran et al. 2017). We describe here the
case study involving a homology model of A3 AR interacting with different ligands
through an in silico molecular docking and molecular dynamics simulations. For the
structure-based modeling study, Deb et al. developed a homology model of A3 AR
using X-ray crystal structure of human A2A AR (PDB ID: 4EIY) and conducted
docking (GLIDE XP, IFD-Schrodinger) of novel thieno[2,3-d]pyrimidine
derivatives (Deb et al. 2018). The results of the docking study revealed majorly
hydrogen bonding (H-bonding) and hydrophobic interactions. The most active
compound 8 was subjected to molecular dynamics simulation (Desmond software)
for 50 ns with hA2A and hA3 ARs to study the stability and binding interaction. The
crucial amino acid residues Phe168, Val 169, Asn236, and Leu232 exhibited
significant interaction with the ligand through π–π stacking and H-bonding to impart
good binding affinity towards hA3 AR binding site. A 2D interaction plot of the most
active compound 8 in the binding site of hA3 AR (Deb et al. 2018) is shown in
Fig. 2.33.

2.10 Conclusion

Certainly, for the drug to function, it must interact with the target protein such as
receptors. The interactions between a drug and its receptor are mediated through the
structural features, mainly by functional groups. It is very imperative that probing the
real receptors for the establishment of essential binding features can offer crucial
information about the mode of drug action. Of equal importance, the binding
orientation of the drug structure inside the binding site of a receptor can influence
the pharmacological activity. Theories of drug-receptor interactions can provide
more useful information to deduce the probability of proposing the mode of action
of any drug under biological environment. In particular, noncovalent interactions
mostly are weaker and they work in conjunction with other types of interactions.
This is mainly due to the loss in translational entropy (first interaction) followed by
lower entropy (second interaction). Finally, the effect of this cooperativity causes the
conversion of weak interactions to yield strong interactions.
The selectivity of the drug is primarily decided by the strength and existence of
good interactions between the functionalities in drug-receptor complexes. Many
64 B. Chandrasekaran et al.

Fig. 2.33 Ligand interaction pattern showing percentage of contacts with crucial amino acid
residues of A3 AR

times charged functional groups tend to bind more effectively than polar functional
groups, which bind more tightly than nonpolar groups. In case of electrostatic
interactions, ammonium ions are efficient followed by phosphate ion, and then
carboxylate anion. In order to exhibit good pharmacological activity of the ligands,
they must have higher binding energy than the calculated average binding energy.
Conversely, compounds demonstrating less binding energy can fit into receptors
poorly. Hence, the determination of drug-receptor interactions is very essential for
the safety and efficacy of a drug.

Acknowledgments The authors wish to thank Prof. Mutaz Sheikh Salem (President) and Prof.
Marwan Kamal (University Counsellor) of Philadelphia University, Jordan, for the support and
research funding to BC (467/34/100 PU).

Consent for Publication Not applicable.

Conflict of Interest The author declares no conflict of interest, financial or otherwise.

References
Agrawal N, Chandrasekaran B, Al-Aboudi A (2019) Recent advances in the in-silico structure-
based and ligand-based approaches for the design and discovery of agonists and antagonists of
A2A adenosine receptor. Curr Pharm Des 25:774–782
Andrews PR, Craik DJ, Martin JL (1984) Functional group contributions to drug-receptor
interactions. J Med Chem 27:1648–1657
2 Drug-Receptor Interactions 65

Aps C, Reynolds F (1978) An intradermal study of the local anaesthetic and vascular effects of the
isomers of bupivacaine. Br J Clin Pharmacol 6:63–68
Aranda A, Pascual A (2001) Nuclear hormone receptors and gene expression. Physiol Rev
81:1269–1304
Ariëns EJ (1987) Stereochemistry in the analysis of drug-action. Part II. Med Res Rev 7:367–387
Arthur RC (1927) Biological relations of optically isomeric substances. Nature 120:152
Auerbach A (2016) Dose-response analysis when there is a correlation between affinity and
efficacy. Mol Pharmacol 89:297–302
Baillie TA (2016) Targeted covalent inhibitors for drug design. Angew Chemie Int Ed
55:13408–13421
Balakumar C, Lamba P, Pran Kishore D et al (2010) Synthesis, anti-inflammatory evaluation and
docking studies of some new fluorinated fused quinazolines. Eur J Med Chem 45:4904–4913
Balakumar C, Narayana BL, Kishore DP et al (2012) Design, microwave-assisted synthesis and in
silico docking studies of new 4H-pyrimido[2,1-b]benzothiazole-2-arylamino-3-cyano-4-ones as
possible adenosine A2B receptor antagonists. Indian J Chem Sect B Org Med Chem Chem
51:1105–1113
Balakumar C, Ramesh M, Tham CL et al (2018) Ligand- and structure-based in silico studies to
identify kinesin spindle protein (KSP) inhibitors as potential anticancer agents. J Biomol Struct
Dyn 36:3687–3704
Banda V, Chandrasekaran B, Köse M et al (2013) Synthesis of novel pyrido[3,2-e]- [1,2,4]triazolo
[1,5-c]pyrimidine derivatives: potent and selective adenosine A3 receptor antagonists. Arch
Pharm (Weinheim) 346:699–707
Barratt E, Bingham RJ, Warner DJ et al (2005) Van der Waals interactions dominate
ligand
protein association in a protein binding site occluded from solvent water. J Am Chem
Soc 127:11827–11834
Belleau B (1964) A molecular theory of drug action based on induced conformational perturbations
of receptors. J Med Chem 7:776–784
Bridges TM, Lindsley CW (2008) G-Protein-coupled receptors: from classical modes of modula-
tion to allosteric mechanisms. ACS Chem Biol 3:530–541
Buchwald P (2017) A three-parameter two-state model of receptor function that incorporates
affinity, efficacy, and signal amplification. Pharmacol Res Perspect 5:e00311
Cadena DL, Gill GN (1992) Receptor tyrosine kinases. FASEB J 6:2332–2337
Chandrasekaran B, Deb PK, Rao R (2017) Structure-based design and pharmacological study of
fluorinated fused quinazolines as adenosine A2B receptor antagonists. JSM Chem 5:1–10
Chandrasekaran B, Deb PK, Kachler S et al (2018) Synthesis and adenosine receptors binding
studies of new fluorinated analogues of pyrido[2,3-d]pyrimidines and quinazolines. Med Chem
Res 27:756–767
Chandrasekaran B, Samarneh S, Jaber AMY, Kassab G, Agrawal N (2019) Therapeutic potentials
of A2B adenosine receptor ligands: current status and perspectives. Curr Pharm Des
25:2741–2771
Clark AJ (1926) The reaction between acetyl choline and muscle cells. J Physiol 61:530–546
Colquhoun D (1998) Binding, gating, affinity and efficacy: the interpretation of structure-activity
relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125:923–947
Deb PK (2019a) Editorial: Progress in the development of agonists, antagonists and allosteric
modulators of adenosine receptors. Curr Pharm Des 25:2695–2696
Deb PK (2019b) Recent updates in the computer aided drug design strategies for the discovery of
agonists and antagonists of adenosine receptors. Curr Pharm Des 25:747–749
Deb PK (2019c) Therapeutic potentials of adenosine receptors: the state of the art. Curr Pharm Des
25(26):2789–2791
Deb PK, Mailavaram R, Chandrasekaran B et al (2018) Synthesis, adenosine receptor binding and
molecular modelling studies of novel thieno[2,3-d]pyrimidine derivatives. Chem Biol Drug Des
91:962–969
66 B. Chandrasekaran et al.

Deb PK, Chandrasekaran B, Mailavaram R et al (2019a) Molecular modeling approaches for the
discovery of adenosine A2B receptor antagonists: current status and future perspectives. Drug
Discov Today 24:1854–1864
Deb PK, Deka S, Borah P, Abed SN, Klotz K-N (2019b) Medicinal chemistry and therapeutic
potential of agonists, antagonists and allosteric modulators of A1 adenosine receptor: current
status and perspectives. Curr Pharm Des 25:2697–2715
Du X, Li Y, Xia YL et al (2016) Insights into protein–ligand interactions: mechanisms, models, and
methods. Int J Mol Sci 17:144
Gampe RT, Montana VG, Lambert MH et al (2000) Asymmetry in the PPARγ/RXRα crystal
structure reveals the molecular basis of heterodimerization among nuclear receptors. Mol Cell
5:545–555
Gschwind A, Fischer OM, Ullrich A (2004) The discovery of receptor tyrosine kinases: targets for
cancer therapy. Nat Rev Cancer 4:361–370
Hase T, Tanaka H, Suzuki Y et al (2009) Structure of protein interaction networks and their
implications on drug design. PLoS Comput Biol 5:e1000550
Huang EJ, Reichardt LF (2003) Trk receptors: roles in neuronal signal transduction. Annu Rev
Biochem 72:609–642
Jencks WP (1981) On the attribution and additivity of binding energies. Proc Natl Acad Sci U S A
78:4046–4050
Karlin A (1967) On the application of “a plausible model” of allosteric proteins to the receptor for
acetylcholine. J Theor Biol 16:306–320
Karschin A, Aizenman E, Lipton SA (1988) The interaction of agonists and noncompetitive
antagonists at the excitatory amino acid receptors in rat retinal ganglion cells in vitro. J Neurosci
8:2895–2906
Kati WM, Wolfenden R (1989) Contribution of a single hydroxyl group to transition-state discrim-
ination by adenosine deaminase: evidence for an “entropy trap” mechanism. Biochemistry
28:7919–7927
Kaur R, Kishore DP, Narayana BL et al (2011) A facile microwave-assisted synthesis of
8,9-cycloalkathieno[3,2-e] [1,2,4]triazolo[1,5-c]pyrimidin-5(6H )-ones. J Chem Sci 123:69–73
Kenakin T (2008) Receptor theory. Curr Protoc Pharmacol 41:1.2.1–1.2.28
Kier LB (1997) In: Wolff ME (ed) Burgers’ medicinal chemistry and drug discovery, vol 4. Wiley,
New York, NY, pp 5355–5423
Klebe G (2013) Protein–ligand interactions as the basis for drug action. In: Klebe G (ed) Drug
design. Springer, Berlin, Heidelberg, pp 61–88
Koshland DE (1958) Application of a theory of enzyme specificity to protein synthesis. Proc Natl
Acad Sci U S A 44:98–104
Koshland DE, Neet KE (1968) The catalytic and regulatory properties of enzymes. Annu Rev
Biochem 37:359–411
Kuhn B, Mohr P, Stahl M (2010) Intramolecular hydrogen bonding in medicinal chemistry. J Med
Chem 53:2601–2611
Kumalo HM, Bhakat S, Soliman MES (2015) Theory and applications of covalent docking in drug
discovery: merits and pitfalls. Molecules 20:1984–2000
Lambert DG (2004) Drugs and receptors. BJA Educ 4:181–184
Leff P (1995) The two-state model of receptor activation. Trends Pharmacol Sci 16:89–97
Leff P, Scaramellini C, Law C, McKechnie K (1997) A three-state receptor model of agonist action.
Trends Pharmacol Sci 18:355–362
Levine D, Reid TW, Wilson IB (1969) Free energy of hydrolysis of the phosphoryl-enzyme
intermediate in alkaline phosphatase-catalyzed reactions. Biochemistry 8:2374–2380
Lew MJ, Ziogas J (2004) The two-state model of antagonist-AT1 receptor interaction: an hypothesis
defended but not tested. Biochem Pharmacol 67:397–399
Li JP, Biel JH (1969) Steric structure-activity relationship studies on a new butyrophenone deriva-
tive. J Med Chem 12:917–919
2 Drug-Receptor Interactions 67

Maehle A-H, Prüll C-R, Halliwell RF (2002) The emergence of the drug receptor theory. Nat Rev
Drug Discov 1:637–641
Mailavaram RP, Al-Attraqchi OHA, Ghosh SK (2019) Current status in the design and develop-
ment of agonists and antagonists of adenosine A3 receptor as potential therapeutic agents. Curr
Pharm Des 25:2772–2787
Marc J (2008) Pharmacogenetics of drug receptors. EJIFCC 19:48–53
Monod J, Wyman J, Changeux J-P (1965) On the nature of allosteric transitions: a plausible model.
J Mol Biol 12:88–118
Nelson MR, Johnson T, Warren L et al (2016) The genetics of drug efficacy: opportunities and
challenges. Nat Rev Genet 17:197–206
Page MI (1977) Entropy, binding energy, and enzymic catalysis. Angew Chem Int Ed Engl
16:449–459
Paton WDM (1961) A theory of drug action based on the rate of drug-receptor combination. Proc R
Soc London Ser B Biol Sci 154:21–69
Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nat Rev Mol Cell
Biol 3:639–650
Pramanik D (2015) Receptor. Principles of physiology. 3rd edition, Jaypee Brothers Medical
Publishers, India, pp 661-669.
Pran Kishore D, Balakumar C, Raghuram Rao A et al (2011) QSAR of adenosine receptor
antagonists: exploring physicochemical requirements for binding of pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine derivatives with human adenosine A3 receptor subtype. Bioorg Med
Chem Lett 21:818–823
Robinson-Rechavi M, Laudet V (2003) Bioinformatics of nuclear receptors. Methods Enzymol
364:95–118
Rosenbaum DM, Rasmussen SGF, Kobilka BK (2009) The structure and function of G-protein-
coupled receptors. Nature 459:356–363
Saengsawang W, Rasenick MM (2015) G protein-coupled receptors. In: Encyclopedia of cell
biology. Academic Press, Cambridge, pp 51–55
Sansom MSP, Adcock C, Smith GR (1998) Modelling and simulation of ion channels: applications
to the nicotinic acetylcholine receptor. J Struct Biol 121:246–262
Schwartz TW, Holst B (2007) Allosteric enhancers, allosteric agonists and ago-allosteric
modulators: where do they bind and how do they act? Trends Pharmacol Sci 28:366–373
Shaik K, Deb PK, Mailavaram RP et al (2019) 7-Amino-2-aryl/hetero-aryl-5-oxo-5,8-dihydro
[1,2,4]triazolo[1,5-a]pyridine-6-carbonitriles: synthesis and adenosine receptor binding studies.
Chem Biol Drug Des 94:1568–1573
Shuker SB, Hajduk PJ, Meadows RP, Fesik SW (1996) Discovering high-affinity ligands for
proteins: SAR by NMR. Science (80-) 274:1531–1534
Stephenson RP (1956) A modification of receptor theory. Br J Pharmacol Chemother 11:379–393
Suvarna BS (2011) Drug - receptor interactions. Kathmandu Univ Med J 9:9–13
Talapatra SK, Tham CL, Guglielmi P et al (2018) Crystal structure of the Eg5 - K858 complex and
implications for structure-based design of thiadiazole-containing inhibitors. Eur J Med Chem
156:641–651
Tallarida RJ (2007) Interactions between drugs and occupied receptors. Pharmacol Ther
113:197–209
Topiol S, Sabio M (2009) X-ray structure breakthroughs in the GPCR transmembrane region.
Biochem Pharmacol 78:11–20
Uings IJ, Farrow SN (2000) Cell receptors and cell signalling. Mol Pathol 53:295–299
Varma AK, Patil R, Das S et al (2010) Optimized hydrophobic interactions and hydrogen bonding
at the target-ligand interface leads the pathways of drug-designing. PLoS One 5:e12029
Wilson DK, Rudolph FB, Quiocho FA (1991) Atomic structure of adenosine deaminase complexed
with a transition-state analog: understanding catalysis and immunodeficiency mutations. Sci-
ence 252:1278–1284
68 B. Chandrasekaran et al.

Wolfenden R, Kati WM (1991) Testing the limits of protein-ligand binding discrimination with
transition-state analogue inhibitors. Acc Chem Res 24:209–215
Zhang T, Wei T, Han Y et al (2016) Protein-ligand interaction detection with a novel method of
transient induced molecular electronic spectroscopy (TIMES): experimental and theoretical
studies. ACS Cent Sci 2:834–842
Pharmacology of Acetylcholine
and Cholinergic Receptors 3
Sarah Falah Kokaz, Pran Kishore Deb, Sara Nidal Abed, Amal Al-
Aboudi, Nirupam Das, Fatimah Amin Younes, Ruba Anwar Salou,
Yazan A. Bataineh, Katharigatta N. Venugopala,
and Raghu Prasad Mailavaram

Abstract

Acetylcholine is a neurotransmitter that plays a significant role in a variety of

physiological functions. Cholinergic neurons synthesize, store, and release ace-
tylcholine and are also responsible for sympathetic and parasympathetic
responses of the autonomous nervous system. The wide range of functions that
the cholinergic system plays explains the diverse range of therapeutic potential
that targets this system. Over the decades, cholinergic and anticholinergic drugs
are utilized as treatment options for various conditions including ophthalmology,
neurogenic bladder, myasthenia gravis, dementia, postoperative urinary retention,
xerostomia, anticholinergic overdose, snakebites, Parkinson’s disease, and

S. F. Kokaz · S. N. Abed · F. A. Younes · R. A. Salou · Y. A. Bataineh

Faculty of Pharmacy, Philadelphia University, Amman, Jordan
P. K. Deb (*)
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
e-mail: [email protected]
A. Al-Aboudi
Department of Chemistry, Faculty of Science, The University of Jordan, Amman, Jordan
N. Das
Department of Pharmaceutical Science, Assam University, Silchar, Assam, India
K. N. Venugopala
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-
Ahsa, Kingdom of Saudi Arabia
Department of Biotechnology and Food Technology, Durban University of Technology, Durban,
South Africa
R. P. Mailavaram
Department of Pharmaceutical Chemistry, Shri Vishnu College of Pharmacy, Vishnupur (Affiliated
to Andhra University), Bhimavaram, W.G. Dist., AP, India

# Springer Nature Singapore Pte Ltd. 2020 69

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_3
70 S. F. Kokaz et al.

Alzheimer’s disease. Alongside, they are also investigated for various promising
therapeutics. This chapter provides an overview of the cholinergic system phar-
macology, functions in the body, cholinergic and anticholinergic compounds, and
their potential role in the medical field. Further, the chapter highlights the updates
on the cholinergic compounds currently used to treat various conditions as well as
compounds under investigation.

Keywords
Acetylcholine · Cholinergic system · Nicotinic receptors · Muscarinic receptors ·
Acetylcholinesterase inhibitors · Cholinergic ligands

Abbreviation

ACh Acetylcholine
AChE Acetylcholinesterase
AD Alzheimer’s disease
ADHD Attention deficit hyperactivity disorder
ANS Autonomic nervous system
ASDs Autism spectrum disorders
BQCA Benzyl quinolone carboxylic acid+
BTX Botulinum toxin
BuChE Butyrylcholinesterase
ChAT Choline acetyltransferase
ChE Cholinesterase
CNS Central nervous system
CoA Coenzyme A
COPD Chronic obstructive pulmonary disease
DMN Default mode network
DS Down syndrome
ECT Electroconvulsive therapy
EDHF Endothelium-derived hyperpolarizing factor
EDRF Endothelium-derived relaxing factors
EPSP Excitatory postsynaptic potential
FDA Food and Drug Administration
GPCR G-protein-coupled receptor
LAMA Long-acting muscarinic receptor antagonist
mAChR Muscarinic acetylcholine receptor
MCI Mild cognitive impairment
MTL Medial temporal lobe
nAChR Nicotinic acetylcholine receptor
NAL Neutral allosteric ligand
NAM Negative allosteric modulator
NMJ Neuromuscular junction
NO Nitric oxide
3 Pharmacology of Acetylcholine and Cholinergic Receptors 71

OPC Organophosphate compound

OSCC Oral squamous cell carcinoma
OSF Oral submucous fibrosis
PAM Positive allosteric modulator
PD Parkinson’s disease
PLA2 Phospholipase A2
PNS Peripheral nervous system
UAB Underactive bladder

3.1 Introduction

Autonomic nervous system (ANS) is a collection of ganglia (motor nerves) situated

in pelvis, abdomen, thorax, neck, and head, in addition to motor neurons axonal
connections. Acetylcholine (ACh) is the neurotransmitter found in sympathetic and
parasympathetic preganglionic autonomic neurons (Blessing and Gibbins 2016).
ACh also elicits activity on the postsynaptic dendrite and nerve cell body of nicotinic
receptor subclass that innervate the ganglia (Taylor 2012). The first neurotransmitter
to be identified was ACh. It has also been detected in primitive plants, fungi, algae,
protozoa, and bacteria, which indicates the wide distribution of cholinergic system in
living organisms before its identification as a part of the nervous system (Greig et al.
2013). Acetylcholine was first synthesized in 1867. However, its biological impor-
tance was only discovered 50 years later (Bylund 2016). Cholinergic neurons are
responsible for ACh synthesis, storage, and release. The ionotropic neuronal nico-
tinic acetylcholine receptors (nAChRs) and the muscarinic metabotropic receptors
are the two primary receptors that transduced the signal of ACh (Picciotto et al.
2012). Moreover, cholinergic neurons control peripheral sympathetic and parasym-
pathetic responses of the ANS. The “rest and digest” functionalities are mediated by
the release of ACh by the parasympathetic terminals in the ANS (Tiwari et al. 2013).
While in the central nervous system (CNS), ACh acts as a neuromodulator and
neurotransmitter when released from cholinergic neurons and interneurons in the
brain and spinal cord (Naser and Kuner 2018). In the ANS, ACh is released from
postganglionic parasympathetic and sympathetic nerves and preganglionic neurons.
In the somatic system, Ach is released at the neuromuscular junction. ACh is a
quaternary ammonium parasympathomimetic compound; it produces a transient
action due to its rapid destruction by cholinesterase enzyme, which limits the
therapeutic application of ACh. Another limiting factor is the fact that acetylcholine
possesses no specificity as it interacts with all nicotinic and muscarinic receptors
making it particularly not a useful therapeutic agent (Bylund 2016). Nevertheless,
ACh chloride is used topically after cataract surgeries to reduce a possible postoper-
ative increase in intraocular pressure (Drudi et al. 2017). Many compounds that
target the cholinergic system are currently used for the treatment of various
conditions, and these compound include muscarinic antagonists (e.g., darifenacin,
fesoterodine, oxybutynin, and tolterodine) for the treatment of urinary incontinence,
scopolamine for motion sickness, and aerosol ipratropium for COPD management
(Ehlert 2019a). Acetylcholinesterase inhibitors have also shown great therapeutic
importance in the treatment of diseases such as myasthenia gravis, Alzheimer’s
72 S. F. Kokaz et al.

disease, and glaucoma (Potter and Kerecsen 2017). Nicotinic receptors stimulation
has shown to improve cognitive functioning after several studies on animal models.
Besides, some preclinical and clinical studies suggested that nicotinic receptors also
play a role in depression, mood, and anxiety (Aboul-Fotouh 2015; Quik et al. 2015).
In this chapter, we present an overview of the cholinergic system pharmacology,
functions in the body, cholinergic and anticholinergic compounds, and their role in
the medical field. Finally, an update on the cholinergic compounds currently used to
treat various conditions as well as compounds under investigation are discussed.

3.1.1 Chemistry of Acetylcholine

Acetylcholine (2-acetoxy-N,N,N-trimethylethanaminium) is a small molecule with

the chemical formula CH3COOCH2CH2N+(CH3)3 and a molecular mass of
146.2074 g/mol. It has a simple chemical structure composed of an ester of choline
and acetic acid as shown in Fig. 3.1 (Tunç et al. 2016; Ueda et al. 2016).
ACh plays a crucial role in maintaining homeostasis in the brain by acting as a
neurotransmitter in both the peripheral nervous system (PNS) and CNS. Choline
acetyltransferase is the enzyme responsible for synthesizing acetylcholine from the
substrates choline and acetyl CoA (coenzyme A) (Fig. 3.2) (Akaike and Izumi
2018). Pyruvate that results from glucose breakdown serves as the key source of
acetyl CoA inside the neurons. The rate-limiting factor of ACh synthesis is the
choline uptake by neurons. Choline acetyltransferase is found as a particulate
membrane-bound enzyme in cholinergic neurons and as a soluble enzyme in the
cytoplasm. Acetylcholine storage takes place in small synaptic spherical vesicles

Acetylcholine (ACh)

Fig. 3.1 Chemical structure of ACh

Choline Choline Choline

Acetylcholinest
acetyltransferase erase (AChE)
(ChAT)

Acetylcholine (ACh)

Acetyl CoA HS-CoA Acetic acid

Fig. 3.2 Synthesis of acetylcholine by choline acetyltransferase (ChAT) using choline and acetyl
coenzyme A, releasing coenzyme A (HS-CoA). Metabolism of acetylcholine is catalyzed by
acetylcholinesterase (AChE) producing acetic acid and choline
3 Pharmacology of Acetylcholine and Cholinergic Receptors 73

where it is protected from being destroyed by the enzyme acetylcholinesterase

(Browning 2010; Colzato et al. 2017).

3.1.2 Acetylcholine Functions

Acetylcholine (ACh) is a neurotransmitter that functions in both the PNS and the
CNS. The ANS (sympathetic and parasympathetic) uses acetylcholine to generate a
nerve impulse. In PNS, ACh mainly acts on the muscular system by activating
muscle contraction after being released in the neuromuscular junction. In the central
nervous system, ACh has various effects on cognitive functions, alertness, learning,
and memory (Haerter and Eikermann 2016; Maurer and Williams 2017; Panus et al.
2009). When ACh gets released from somatic nerve endings into the neuromuscular
junction, it causes the nicotinic ligand-gated ion channels to open, leading to sodium
entrance into muscle cells. The sodium ions produce an excitatory postsynaptic
potential (EPSP) that generates an action potential which eventually stimulates
muscle contraction (Pappano 2018). Also, serum ACh has shown to produce vaso-
dilation by activating vascular endothelial muscarinic receptors, which causes the
release of endothelium-derived relaxing factors (EDRF) including prostanoids,
endothelium-derived hyperpolarizing factor (EDHF), and nitric oxide (Tangsucharit
et al. 2016). The chapter further presents the role of acetylcholine and cholinergic
system together with few specific ligands and their clinical utility.

3.2 Cholinergic Receptors

Cholinergic receptors were named “cholinergic” due to their activation by Ach, and
these receptors are mostly parasympathetic and transduce signal in the autonomic
and somatic nervous system (Wehrwein et al. 2016). Based on the stimulation by
muscarine or nicotine, cholinergic receptors are classified into muscarinic and
nicotinic receptors, respectively. Nicotinic receptors are ionotropic ligand-gated
channels, unlike muscarinic receptors, which are G-protein-coupled receptors
(GPCRs) (Kruse et al. 2014; Papke 2014). Both receptor types are present within
the CNS; however, nicotinic receptors are also found at the neuromuscular junction.
The difference in signal transduction upon activation of the two receptor types
results in distinctive physiological functions.

3.2.1 Muscarinic Receptors

Muscarinic receptors are mostly present at parasympathetic target organs, but it can
also be found at specific sympathetic target organs such as the eccrine sweat glands
that produce copious secretion for thermoregulation purpose, and in blood vessels of
the skeletal muscles. In the PNS, muscarinic receptors are mainly found on auto-
nomic effector cells, which are innervated by postganglionic parasympathetic nerves
74 S. F. Kokaz et al.

Table 3.1 Classification of muscarinic receptors, their distribution and role

Subtype Distribution Role
M1 Brain (cortex, hippocampus), Cognitive functioning and memory, salivary
salivary glands secretion
M2 Heart, brain, smooth muscle Regulation of heart rate and heart rate variability,
behavioral flexibility
M3 Smooth muscle, glands, eye Smooth muscle contraction, gland secretion, iris
contraction
M4 Brain (forebrain, striatum) Modulation of several important dopamine-
dependent behaviors
M5 Brain (substantia nigra), eye Regulation of striatal dopamine release

Table 3.2 Muscarinic receptors subtypes localization and role in the brain
Subtype Presence in brain Functional aspect
M1 All major areas of the forebrain Highly responsible for cholinergic
including cerebral cortex, hippocampus, functions. Synaptic plasticity, learning
thalamus, and corpus striatum. Cellular and memory (cognition), neuronal
localization at striatum nigrum neurons differentiation during development, and
and glutamatergic pyramidal neurons neuronal excitability
M2 Throughout the brain especially in Inhibitory effect on dopaminergic action.
hippocampus and neocortex. Abundant Antinociceptive effect reported
in non-cholinergic neurons in these areas
M3 Highly expressed in hypothalamus, Major role in food intake, body growth
lesser expression in hippocampus
M4 Major presence in corpus striatum An important role in psychosis, an
involvement in pathology of Parkinson’s
disease, inhibits D1 receptor of dopamine
signaling
M5 Pars compacta of substantia nigra, Rewarding effect of abusive drugs
ventral tegmental region

(Tiwari et al. 2013). Muscarinic receptors are classified into five subtypes; each
subtype distribution and role are listed in Table 3.1 (Glavind and Chancellor 2011).
These five muscarinic receptor subtypes are expressed throughout the human
brain and are involved in various functional processes, such as learning, memory,
attention, sleep-wake cycles, sensorimotor processing, and arousal (Lebois et al.
2018). Table 3.2 summarizes the details concerning muscarinic receptors distribu-
tion and function in the brain (Verma et al. 2018). Muscarinic receptors are GPCRs
in which M1, M3, and M5 are Gq/11 G-proteins which mediate excitatory
neuromodulatory acetylcholine actions while M2 and M4 receptors are Gi/o
G-proteins that produce inhibitory neuromodulatory acetylcholine actions (Brown
2019; Felder 1995; Lebois et al. 2018).
All five muscarinic receptor subtypes express throughout the mammalian brain.
However, different regions in the brain contain different receptor subtype
concentrations exemplified by M1 and M2 receptors. In the major forebrain areas,
3 Pharmacology of Acetylcholine and Cholinergic Receptors 75

the expression of these receptors is comparatively higher to that of other subtypes.

M1 receptor is the highest muscarinic receptor expressed in the cortex, striatum, and
hippocampus. M2 receptor is highly expressed in the occipital cortex and nucleus
basalis, while M4 receptor is more prominent within caudate putamen and the
striatum. The least muscarinic receptor expressed compared to the other subtypes
is M5 receptor (Carruthers et al. 2015; Scarr et al. 2016).

3.2.2 Nicotinic Receptors

Nicotinic acetylcholine receptors (nAChRs) are hetero- or homo-pentameric

structured ligand-gated ion channels. Nicotinic receptors possess an essential role
in various biological processes such as learning, memory, locomotion, anxiety, and
attention. Recent researches showed that nicotinic receptors play a role in regulating
inflammation by α7 nicotinic acetylcholine receptors activation in macrophages
(Egea et al. 2015). The nAChRs are mainly divided into two subclasses: neuronal
and muscular. In the neuromuscular junctions, the muscular nAChRs contribute to
the neuromuscular transmission, and the neuronal nAChRs are located in both the
PNS and the CNS. The nAChRs have a large number of homologous subunits that
can form many different combinations of pentamers which produce various
receptors with diverse functionalities (Kulbatskii et al. 2018).
Composition of nicotinic acetylcholine receptors (nAChRs) consists of five
subunits surrounding a water-filled pore. Neuronal nAChRs are classified based on
the presence of adjacent cysteine groups within the extracellular part of the α
subunits into two types: the alpha and beta. The alphas are α2, α7, α9, and α10,
while the betas are β2–β4 (Dani 2015; Fasoli and Gotti 2015). There have been
17 subunits of nAChRs identified (α1–10, β1–4, γ, δ, and ε). All those subunits were
found in mammals except for α8, which is found in avian species. In muscular
nicotinic receptors, the binding sites are at the interfaces of the δ or γ subunits and
the α subunit, while in neuronal nicotinic receptors, the binding sites are found at the
interfaces of the β subunit and α subunit or at two adjacent α subunits. Alpha
subunits are the only ones containing two cysteine residues near the binding site
of acetylcholine (Lukas et al. 1999; Melroy-Greif et al. 2016). The characteristics of
nAChRs are listed in Table 3.3 (Akaike and Izumi 2018; Dani 2015).
In the brain, the predominant subtypes expressed are α7 subunit containing
receptors which are either homo- or heteromeric. Different subtypes of nAChRs
have shown the ability to modulate synaptic transmission in various brain parts,
including thalamic nuclei, cortical interneurons, the visual cortex, and supraoptic
nuclei. nAChR subtypes are distributed differently in the CNS (Table 3.4) (Akaike
and Izumi 2018; Dineley et al. 2015).
76 S. F. Kokaz et al.

Table 3.3 Nicotinic acetylcholine receptors characteristics

Subtype Primary subunit composition Ca2+ permeability Major location
α1 (α1)2β1γδ, (α1)2β1δε Low Neuromuscular junction
α2 α2β2, α2β4 Low CNS
α3 α3β2, α3β4 Low CNS, autonomic ganglion
α4 (α4)3(β2)2, (α4)2(β2)3 Low CNS
α5 α3β2α5, α3β4α5, High CNS, autonomic ganglion
(α4)2(β2)2α5
α6 α6β2β3, α6α4β2β3 High CNS
α7 (α7)5 High CNS, non-neuronal cells
α8 (α8)5 High CNS
α9 (α9)5, α9α10 High Mechanosensory hair cells
α10 α9α10 High Mechanosensory hair cells

3.3 Pharmacology of Cholinergic Receptors

Cholinergic ligands provide a wide range of therapeutic applications, many are

currently on the market for various medical conditions including scopolamine,
neostigmine, tacrine, oxybutynin, donepezil, tolterodine, ipratropium, ambenonium,
and edrophonium (Bukala et al. 2019; Potter and Kerecsen 2017; Ramaswamy et al.
2018; Vozmediano-Chicharro et al. 2018). Other compounds are under investigation
for further therapeutic options in the future such as xanomeline, cevimeline,
tazomeline, benzyl quinolone carboxylic acid (BQCA), and bispyridinium oximes
(Antonijevic et al. 2016; Lorke and Petroianu 2018; Verma et al. 2018).

3.3.1 Muscarinic Receptor

Muscarinic acetylcholine receptors play a significant role in modulating physiologi-

cal processes related to mental health, respiration, salivation, excretion, and motion
perception. In recent years, investigations on the mAChR ligands have accelerated
the discovery of various novel chemical entities and some were approved for certain
conditions, including psychosis (Foster and Conn 2017), Alzheimer’s disease
(Bradley et al. 2017), asthma, motion sickness, and incontinence. An ideal therapeu-
tic agent for these diseases should not exert any adverse effects that can be caused by
nonspecific interaction with other mAChR subtypes. However, the orthosteric sites
of the mAChR subtypes have high amino acid sequence similarities, which explains
the current difficulties in designing drugs that target specific receptor subtype
(Korczynska et al. 2018). For example, darifenacin and tolterodine (Fig. 3.3) are
mAChR antagonists that treat incontinence by targeting M3 receptors, often cause
adverse effects such as dry mouth by interacting with glandular M1 and M3
receptors, and increase heart rate due to effects on M2 receptors and increase
drowsiness (Glavind and Chancellor 2011; Korczynska et al. 2018; Naicker et al.
 3

Table 3.4 Distribution of nicotinic receptors in CNS

α2 α3 α4 α5 α6 α7
Cortex Cortex Cortex Cortex
Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus
Striatum Striatum Striatum
Amygdala Amygdala Amygdala
Thalamus
Hypothalamus Hypothalamus Hypothalamus
Substantia nigra Substantia nigra Substantia nigra Substantia nigra Substantia nigra
Cerebellum Cerebellum Cerebellum
Pharmacology of Acetylcholine and Cholinergic Receptors

Spinal cord Spinal cord Spinal cord

77
78 S. F. Kokaz et al.

Darifenacin Tolterodine

Fig. 3.3 Chemical structures of darifenacin and tolterodine

2017). Such adverse effects of muscarinic receptor antagonists have reduced their
convenience as treatment options; nevertheless they are highly active compounds
(Korczynska et al. 2018) with scope for further structural optimization.
In CNS disorders, the majority of studies are focused on compounds targeting M1
and M4 mAChRs subtypes. On the other hand, M5 mAChRs expresses itself in
highly variable regions of therapeutic interest, and it is the least mAChR expressed
in the brain by 2% of the total mAChRs population. Despite the fact, M5 receptors
are the only subtype with an identifiable mRNA transcript in dopamine-containing
neurons. This factor makes it a viable target for drug addiction treatment by
supporting the hypothesis that mAChR subtype may be responsible for regulating
the transmission of midbrain dopamine and reward mechanisms (Berizzi et al. 2016;
Gunter et al. 2018).

3.3.1.1 Muscarinic Agonists

Arecoline (methyl-1,2,5,6-tetrahydro-1-methyl-nicotinate), an alkaloid, is consid-
ered the primary active constituent of A. catechu. In recent years, many studies
have been carried out to further investigate arecoline’s pharmacological and toxic
effects (Bhat et al. 2017). Several pharmacological activities produced by arecoline
have been reported such as anti-parasitic effects, in addition to effects on the
digestive, cardiovascular, nervous, and endocrine systems. The main toxic effects
of arecoline are genotoxicity, oral squamous cell carcinoma, and oral submucous
fibrosis. However, arecoline has an agonistic effect on muscarinic receptors which
upon further investigation have shown to reverse memory impairment and
scopolamine-induced memory loss in male rats model of Alzheimer’s disease
(Kuca et al. 2016; Liu et al. 2016).
In preclinical studies, muscarinic receptor agonists have exhibited atypical anti-
psychotic effects. Xanomeline is a mAChR agonist that can reverse certain
dopamine-mediated behaviors. Cevimeline, milameline, sabcomeline, and
xanomeline have all progressed into different clinical development stages for possi-
ble treatment of Alzheimer’s disease (AD) and schizophrenia. Phase II clinical trials
of xanomeline have demonstrated its effect and efficacy for various cognitive
symptoms domains such as hallucinations and behavioral disturbances associated
3 Pharmacology of Acetylcholine and Cholinergic Receptors 79

Arecoline Xanomeline Cevimeline

Milameline Sabcomeline Bethanechol

Fig. 3.4 Structures of mAChR agonists

with AD (Congreve et al. 2018; Sivaraman et al. 2019). Figure 3.4 depicts the
chemical structures of some mAChR agonists mentioned in this section.
Bethanechol is a cholinergic agonist that acts on M1 and M2 muscarinic
receptors. It is a methyl analogue of acetylcholine and used clinically for women
with underactive bladder (UAB). Stimulation of mAChRs at the neuromuscular
junction of smooth muscle with bethanechol can induce the contraction of the
detrusor muscle, which eventually improves bladder emptying. However, various
clinical evidence supporting bethanechol efficacy for UAB is limited and poor
(Sivaraman et al. 2019; Gaitonde et al. 2018). Bethanechol has also been
investigated in the treatment of tracheomalacia. However, further trials are required
to ensure the safety and efficacy of bethanechol as a treatment regimen (Bass et al.
2018).
BuTAC ([5R-(exo)]-6-[4-butylthio-1,2,5-thiadiazol-3-yl]-1-azabicyclo-[3.2.1]-
octane) is a muscarinic receptor agonist that produces full agonist activity on M2
receptor and partial agonist activity on both M1 and M4 receptors. Contrarily, it
exhibits full antagonist activity on M3 and M5 receptors. BuTAC has shown to
produce antipsychotic activity on schizophrenic animal models (Andersen et al.
2015; Watt et al. 2013). Another muscarinic agonist is cevimeline, which activates
both M1 and M3 receptors, and it was originally developed for the treatment of
Alzheimer’s-type senile dementia. Currently, this compound is used to treat
xerostomia, which is a condition that causes numerous disorders in oral functions,
including swallowing, taste, speech, and mastication. Other than pilocarpine,
cevimeline is the only agent available for the therapeutic intervention of xerostomia
(Kishimoto et al. 2016; Mitoha et al. 2017). Table 3.5 summarizes various
80 S. F. Kokaz et al.

Table 3.5 Muscarinic receptor agonists in preclinical and clinical phases

Muscarinic
Agonist receptor Status Therapeutic application Reference
AF102B, mAChR In vivo passed Alzheimer’s disease Ferreira-vieira
AF150, et al. (2016);
AF267B, Fisher (2012);
AF292 Sehgal et al.
(2018)
77-LH-28-1 mAChR In vivo passed Alzheimer’s disease and Langmead et al.
schizophrenia (2008a, b); Zhao
et al. (2019)
VU0357017 M1 In vivo passed Cognitive deficits Digby et al.
and mAChR (schizophrenia, (2010); Lebois
VU0364572 Alzheimer’s disease) et al. (2017);
Rogers and
Gray (2012)
Xanomeline M1/M4 Phase 2 trials Schizophrenia Bender et al.
mAChR (2017); Khatwal
et al. (2014)
Arecoline M1, M2, In vivo Wide pharmacological Langmead et al.
M3, M4 activities (nervous, (2008a, b); Liu
mAChR cardiovascular, et al. (2016)
endocrine, digestive
system, and anti-
parasitic effects)
CI-1017 M1 In vivo Cognitive deficits Brady et al.
mAChR (Alzheimer’s disease and (2008); Weiss
schizophrenia) et al. (2000)
Cevimeline M3 Passed in Primary Sjögren’s Garlapati et al.
mAChR in vitro and syndrome (dry mouth) (2019);
in vivo study Langmead et al.
but failed in (2008a, b)
clinical trial
Tazomeline, M1 Passed in Schizophrenia Langmead et al.
talsaclidine, mAChR in vitro and (2008a, b)
milameline, in vivo study
and arecoline but failed in
clinical trial
AF102B mAChR Clinical trial Alzheimer's disease Digby et al.
phase 2/3 (2010); Kumar
and Kumar
(2018)
SB202026 M1, M2, Discontinued Alzheimer's disease Digby et al.
M3 due to (2010)
mAChR cholinergic
adverse effects
Benzyl M1 In vivo passed Alzheimer’s disease Digby et al.
quinolone mAChR (2010);
carboxylic Hepnarova et al.
acid (BQCA) (2018)
(continued)
3 Pharmacology of Acetylcholine and Cholinergic Receptors 81

Table 3.5 (continued)

Muscarinic
Agonist receptor Status Therapeutic application Reference
MK-7622 M1 Discontinued Alzheimer’s disease Cummings et al.
mAChR after phase (2016)
1 trials
AC-260854 M1 In vivo study Schizophrenia and Berizzi et al.
mAChR Alzheimer’s disease (2016)
VU0152099, M4 In vivo study Alzheimer’s disease Lebois et al.
VU0152100 mAChR (2017)
TBPB M1 In vivo study Schizophrenia Conn et al.
mAChR (2009)

muscarinic receptor agonists in different developmental stages (Berizzi et al. 2016;

Cummings et al. 2016; Verma et al. 2018).

3.3.1.2 Muscarinic Antagonists

As discussed earlier, muscarinic receptors are present in autonomic ganglia, periph-
eral tissues, and various regions of the brain innervated by parasympathetic nerves.
Antagonism of muscarinic receptors can produce therapeutic effects in several
clinical conditions. For instance, during eye examination, muscarinic receptor
antagonist is applied topically to the eye inducing relaxation in the pupillary
constrictor muscles and circular ciliary, which results in pupil dilation, thereby
making it easier to view the retina and measure refractive errors of the lens
(de Linder Henriksen et al. 2019; Yi et al. 2015). Muscarinic antagonists have also
been considered for the treatment of COPD and asthma due to the constricting effect
in vagal tone in the pulmonary airways (Busse et al. 2016; Oba et al. 2016).
Muscarinic antagonists are sometimes used to treat diarrhea associated with inflam-
matory bowel conditions and dysenteries due to their ability to reduce gastrointesti-
nal tract mobility (Aleem and Janbaz 2018). Muscarinic antagonists (trospium,
oxybutynin, tolterodine, solifenacin, fesoterodine, and darifenacin) can also relieve
symptoms of frequency, urgency, and incontinence of an unstable bladder, which
reduces micturition frequency, and are currently used for the treatment of overactive
bladder and urge incontinence (Andersson 2019). Muscarinic antagonists inhibit the
vestibular apparatus of the inner ear, which reduces motion sickness. Scopolamine
has been used topically as a patch to be placed on the skin behind the ear for
the treatment of motion sickness (Zhang et al. 2016). Muscarinic antagonists can
also act as antidotes in cases such as muscarine poisoning from mushrooms,
insecticides poisoning, and war gases that contain cholinesterase (ChE) inhibitors.
Pharmacokinetic properties of the muscarinic antagonist and the route of adminis-
tration are the main factors affecting which muscarinic antagonist is of choice to
treat each health condition. Table 3.6 summarizes the currently used muscarinic
antagonists (Ehlert 2019a, b).
Long-acting muscarinic receptor antagonists (LAMA) such as tiotropium has
been used for years in the management of COPD. LAMAs antagonize
82 S. F. Kokaz et al.

Table 3.6 Muscarinic receptor antagonists that are currently used and their therapeutic
applications
Agent Use
Tertiary amines
Atropine Treatment of anticholinergic poisoning
Scopolamine Treatment of motion sickness
Homatropine Mydriatic and cycloplegic; for mild uveitis
Dicyclomine Alleviates GI spasms, pylorospasm, and biliary distention
Darifenacin Treatment of urinary incontinence
Fesoterodine Treatment of urinary incontinence
Oxybutynin Treatment of urinary incontinence
Tolterodine Treatment of urinary incontinence
Oxyphencyclimine Antisecretory agent for peptic ulcer
Cyclopentolate Mydriatic and cycloplegic
Tropicamide Mydriatic and cycloplegic
Benztropine Treatment of Parkinson’s and Huntington’s diseases
Trihexyphenidyl Treatment of Parkinson’s and Huntington’s diseases
Pirenzepine Antisecretory agent for peptic ulcer
Quaternary ammonium derivatives
Methylatropine Mydriatic, cycloplegic, and antispasmodic
Methylscopolamine Antisecretory agent for peptic ulcer, antispasmodic
Ipratropium Aerosol for COPD
Glycopyrrolate Antisecretory agent for peptic ulcer, antispasmodic
Tolterodine Treatment of urinary incontinence
Propantheline GI antispasmodic
Tiotropium Aerosol for COPD

parasympathetic bronchoconstriction in the airways reversing airflow obstruction.

Recently, several new agents have been developed and studied for COPD manage-
ment such as aclidinium bromide, umeclidinium bromide, and glycopyrronium
bromide. For COPD management, many LAMAs are under development, and
some are already available, including bencycloquidium, V0162, CHF 5407,
AZD8683, AZD9164, and TD-4208 (Mark A. Mastrodicasa et al. 2017). Tiotropium
was the only LAMA in clinical use for almost two decades, and it was the first
LAMA prepared as daily single-dose treatment. Tiotropium exerts its anticholinergic
effects by binding to M1 and M3 receptors (Alvarado-Gonzalez and Arce 2015).
Tiotropium has shown to decrease hospitalization, symptoms, and exacerbations and
improve health status in COPD patients. It also showed an improvement in the
effectiveness of pulmonary rehabilitation. As compared to salmeterol, tiotropium has
proven to show a higher increase in the time to first exacerbation occurrence, thus
decreasing exacerbation rate in general (Han and Lazarus 2016).
3 Pharmacology of Acetylcholine and Cholinergic Receptors 83

3.3.1.3 Allosteric Modulators

Many efforts were made to produce specific M1 and M4 receptor targeting agents to
maintain therapeutic efficacy and reduce adverse side effects (Foster et al. 2014;
Jones et al. 2012). The targeting of a specific subtype of muscarinic receptors is
difficult due to the highly conserved orthosteric binding site between the receptor
subtypes. Allosteric modulators have been developed to overcome this problem
since the allosteric binding sites are considered less conserved among the receptor
subtypes. Allosteric modulators show higher selectivity and can modulate receptor
activation either by acetylcholine or other agonists, or it can activate the receptor.
Allosteric activators can either be allosteric agonists or positive allosteric modulators
(PAMs). Allosteric agonists act on the allosteric site of the receptor, hence activating
the receptor without the presence of acetylcholine, while PAMs cannot activate the
receptor directly but only potentiate the activation by acetylcholine (Jakubik and
El-Fakahany 2016). One compound can also act as both an allosteric agonist and
PAM. However, since allosteric mechanisms are affected by cooperativity factors
and affinity, they present challenges and practical implications for drug discovery
and development (Conn et al. 2014; Yohn and Conn 2018).

Positive Allosteric Modulators

As mentioned before, PAMs possess higher selectivity than muscarinic agonists
because the allosteric sites of the muscarinic receptors have higher sequence diver-
gence as compared to orthosteric site. Generally, PAMs are considered as
compounds with an excellent safety profile because they do not cause the induction
of independent agonistic activity, which means they cannot exert their effect without
the presence of the endogenous agonist. Thiochrome is a natural compound that
results from the oxidation and metabolism of thiamine. Thiochrome acts as a PAM to
M4 muscarinic receptors (Takai and Enomoto 2018). Recently, there has been much
successful development of selective mAChR PAMs, especially M1 and M4 selective
compounds that have shown promising results including excellent brain penetration
and pharmacokinetic profiles. After the discovery of the M4 selective PAMs
(VU10010 and LY2033298), many efforts were directed towards developing M4
selective compounds. M4 mAChR possesses a potential role in dopaminergic
systems’ regulations, which are highly related to the positive symptoms of schizo-
phrenia, and these receptors are also significant in reducing negative symptoms and
cognitive impairment in AD patients (Chan et al. 2008; Foster and Conn 2017). M4
receptor PAMs are also considered as a potential therapeutic approach for drug
addiction, Huntington’s disease, and Parkinson’s disease (PD). The thieno[2,3-c]
pyridazine analogues received most of the attention due to the limited chemical
diversity of M4 PAMs discovered, and one of the promising analogue for further
preclinical trials is VU6005806/AZN-00016130 (Engers et al. 2019). Recently, the
discovery of selective M1 receptor PAMs showed their ability to enhance both
hippocampal and prefrontal cortex-dependent forms of cognitive functions in
non-human primates and rodents, which agrees with many studies suggesting that
activating M1 receptors produce cognitive-enhancing effects (Digby et al. 2012;
Gould et al. 2015; Lange et al. 2015; Vardigan et al. 2015). For example, MK-7622,
84 S. F. Kokaz et al.

a potent and selective M1 PAM, is under preclinical and clinical trials to reduce
cognitive dysfunction in AD patients (Uslaner et al. 2018).

Negative Allosteric Modulators

Allosteric ligands for individual muscarinic receptors subtypes are currently under
investigations. Allosteric ligands exhibit selective cooperativity between the ligand
and the receptor at the receptor after targeting allosteric site(s) (Berizzi et al. 2018).
Lately, ML375 (VU0483253) became the first negative allosteric modulator (NAM)
that displayed selectivity for M5 muscarinic receptor (Gentry et al. 2013). Studies
have shown that there is a functional interlink between M5 receptor and addiction/
reward pathways, and further substantiated by the attenuation of cocaine addiction
liabilities in a rodent model with cocaine addiction by ML375 (Gunter et al. 2018).
However, ML375’s elimination half-life is excessively long, with approximately
80 h in rats. An equipotent M5 receptor NAM (VU6008667) has been developed
later with a more appropriate shorter elimination half-life (2.3 h) in rats, excellent
selectivity, and high CNS penetration (Bender et al. 2019; Gentry et al. 2013;
McGowan et al. 2017).

3.3.2 Nicotinic Receptor

The nAChRs are a part of the “Cys-loop” receptor superfamily, which are multi-
subunit transmembrane receptors. nAChRs mediate excitatory neurotransmission in
autonomic ganglia, specific synapses in both the spinal cord and brain, and at the
vertebrate neuromuscular junction (Albuquerque et al. 2009; Alcaino et al. 2017).
These nAChRs generate complex calcium signals that influence neuronal processes
and signaling molecules. The combination of the nicotinic receptor subtype, Ca2+
signaling pathway, neuronal type, and developmental stage all together translate the
stimulation of the nicotinic receptors into a neuronal response and eventually into a
physiological outcome (Dajas-Bailador and Wonnacott 2004; Kabbani and Nichols
2018). As stated before, nicotinic receptors are ligand-gated ion channels, which are
pentamers assembled from five subunits either as homomeric or heteromeric
pentamers (Fig. 3.5) (Improgo et al. 2010; Millar and Gotti 2009; Wu et al. 2016).

Fig. 3.5 Schematic

representation of homomeric
and heteromeric nicotinic
α7 β4
receptors with the ACh
binding site location α7 α7 α3 α3

α7 α7 β4 β4

Homomeric nAChR Heteromeric nAChR

(e.g., α7) (e.g., α3β4)
3 Pharmacology of Acetylcholine and Cholinergic Receptors 85

During the inactivated state, the pentamers exist as a funnel shape structural motif
with a central core, and it remains closed. When activated by ACh, the channel
undergoes a conformational change in all subunits, and the core opens to allow Na+
and K+ flow according to electrochemical gradients leading to cellular response
(Svorc 2018). The nAChRs in the CNS are responsible for regulating various
processes including neuronal integration, neurotransmitter release, and cell
excitability and can also affect certain physiological functions including cognition,
pain, arousal, sleep, and mood (Rahman et al. 2015).
Nicotine, a prototypic tobacco alkaloid, had received interest as a potential
therapeutic compound 30 years ago, which was the time when tobacco smoking
was discovered to affect the performance of a particular cognitive task positively.
Further investigations substantiated that nicotine administration indeed affects
pro-cognitive performances (Terry and Callahan 2018; Heishman et al. 1994;
Sherwood 1993). Nowadays, growing pieces of evidence corroborate nicotine’s
effect in enhancing cognitive function and information processing in both experi-
mental animals and human nonsmokers (Levin et al. 2013). Additionally, other
effects of nicotine have been reported from in vivo and in vitro studies such as
sustained attention, working memory (Newhouse 2019) and recognition memory in
rats (Nikiforuk et al. 2015), attention, processing of visual information, and short
term memory in humans. In vivo and in vitro studies have also shown that nicotine
possesses neuroprotective activity in specific disease models which proposes that
nicotine can be used not only for symptomatic control but also to produce effects that
enhance cognition in neurodegenerative diseases such as AD and PD (Newhouse
2019). However, nicotine has limited therapeutic potentials for psychiatric and
neurologic conditions due to several factors including cardiovascular side effects,
abuse potentials, and its relatively short half-life. Nicotine transdermal patches used
for smoking cessation have shown no cardiovascular side effects while exhibiting
improved cognition in patients with mild cognitive impairment (MCI) (Newhouse
et al. 2012). Various types of nicotine formulations have been developed for
smoking cessation such as tablets, patches, nasal sprays, gums, inhalers, and
lozenges to reduce cravings and withdrawal symptoms (Shahab et al. 2013). Nico-
tinic acetylcholine receptor ligands other than nicotine have various therapeutic
potentials which are listed in Fig. 3.6 (Terry and Callahan 2018).
Nicotinic cholinergic receptors (nAChRs) are pentameric proteins that are
activated by acetylcholine at central and peripheral synapses. Neuronal nicotinic
receptors in the brain, autonomic ganglia, adrenal gland, and immune cells are
composed of either α subunits or both α and β subunits. One of the primary
phenomena that nicotinic receptors exhibit upon continued nicotinic agonist expo-
sure is desensitization, which occurs in both muscular and neuronal nicotinic
receptors.

3.3.2.1 Nicotinic Agonists

Nicotinic agonists are any compound that can mimic ACh action on nAChRs.
Nicotine (Fig. 3.7) is the most popular nicotinic agonist. After the discovery of the
positive effects of nicotine, the approach to investigate and design more nicotinic
86 S. F. Kokaz et al.

Nicotine
Dependence

Pain and Alcohol Use

Inflammation Disorder

Therapeutic
Parkinson’s Potential of
Depression
Disease nAChR
Ligands

Attention-
Deficit/Hype Alzheimer’s
ractivity Disease
Disorder
Schizophrenia

Fig. 3.6 Potential therapeutic applications of nicotinic acetylcholine receptors (nAChRs) ligands

Lobeline Nicotine

Cytisine Varenicline

Fig. 3.7 Chemical structures of nicotinic agonists

agonists started in the early 1990s. ABT-418 was one of the first nicotinic agonists
developed by Abbott Labs. Further investigations of ABT-418 have shown
cognitive-enhancing effects that are three to ten times more potent than nicotine.
Clinical trials on ADHD patients with ABT-418 have shown promising results in
3 Pharmacology of Acetylcholine and Cholinergic Receptors 87

reducing ADHD symptoms (Buccafusco 2004; Takechi et al. 2016). Lobeline

(Fig. 3.7) like nicotine is a naturally occurring alkaloid and has similar effects with
less potency as compared to nicotine. Both nicotine and lobeline are full agonists;
they lead to nicotinic receptor activation in sympathetic and parasympathetic
ganglia, on immune cells, in the brain, and at the adrenal medulla which leads to a
wide range of physiological effects. Lobeline has been investigated for drug abuse
treatment such as cocaine, alcohol abuse, opioid, and methamphetamine (Martin
et al. 2018; Zheng and Crooks 2015). Both the cytisine (natural plant alkaloid) and
varenicline (a synthetic compound) act as full agonists on (α3β4 and α7) nicotinic
receptor subtypes and as partial agonists on (α4β2) receptors. Both the compounds
were investigated for smoking cessation (Walker et al. 2018).
Nicotinic receptors are involved in various behavioral and cognitive functions,
which makes nicotinic ligands good candidates for the treatment of conditions with
cognitive dysfunction, such as schizophrenia. Postmortem studies of schizophrenic
patients have shown that both protein expression and binding levels of α7 nicotinic
acetylcholine receptor (α7 nAChR) were decreased in prefrontal cortex and hippo-
campus (cognition-related areas). A study was conducted on sub-chronic
schizophrenic rats, by using α7 nAChR partial agonist (A-582941) for treatment.
Results showed improvement in cognitive dysfunctions and social deficits on spatial
and visual memory (Unal and Aricioglu 2017; Verma et al. 2018).
ABT-126, an α7 nAChR agonist, showed high potency and affinity in mouse, rat,
and human in nonclinical studies. 25 mg once daily of ABT-126 in a phase 2a study
resulted in cognitive improvement in subjects with both mild and moderate
Alzheimer’s dementia (AD) (Gault et al. 2016; Valle’s et al. 2014).

3.3.2.2 Nicotinic Antagonists

Nicotinic antagonists block acetylcholine action at the autonomic ganglia, adrenal
medulla, and neuromuscular junction. Neuromuscular blockers act on muscle-type
nicotinic receptors, and ganglionic blockers act on neuronal ganglionic nicotinic
receptors. Neuromuscular blockers are categorized based on the mechanisms of
action and their use into two categories: depolarizing and non-depolarizing agents
(Ding 2017). Neuromuscular blockers produce skeletal muscle relaxation which
makes them useful in specific clinical situations such as electroconvulsive therapy
(ECT), scoping procedures, endotracheal intubation, orthopedic procedures, and as a
surgical adjunct (Keating 2016). Ganglionic blockers, on the other hand, can be used
to control hypotension during procedures and for the treatment of hypertensive
emergencies. Non-depolarizing neuromuscular blockers include
benzylisoquinolines (doxacurium, mivacurium, atracurium), aminosteroids
(rocuronium, pipecuronium, pancuronium), and natural alkaloids like
d-tubocurarine. All these compounds are considered relatively large and contain
multiple rings, making them bulky molecules that block acetylcholine from binding
to the receptor. Besides, these compounds are competitive antagonists that produce
flaccid paralysis and increasing acetylcholine concentration at synapsis by acetyl-
cholinesterase inhibitors reverses their effect (D’Souza and Johnson 2019; Smetana
et al. 2017).
88 S. F. Kokaz et al.

Similarly, depolarizing neuromuscular blockers such as succinylcholine that

binds and activates the receptor causes muscle relaxation that also leads to flaccid
paralysis. Although succinylcholine activates the receptor, it resists hydrolysis by
acetylcholinesterase, remaining bound to the receptor for a longer time than acetyl-
choline leading to blockage (Bertrand and Terry 2018; Hager and Burns 2018).
Trimethaphan and mecamylamine are ganglionic blockers with different structures
and mechanisms of action. While mecamylamine is a voltage-dependent noncom-
petitive antagonist that prevents activation by blocking the open cation channel,
trimethaphan is a competitive antagonist that competes with ACh for receptor
binding. Trimethaphan is used rarely for hypertensive emergencies during surgeries
to produce controlled hypotension (Petrakis et al. 2018; Sear 2019).

3.3.2.3 Nicotinic Allosteric Modulators

Acetylcholine binds at a site on the interface of two subunits from the extracellular
part of the nicotinic receptors. Many compounds were discovered that compete on
the same site and exert either an agonistic or antagonistic effect. The site where
acetylcholine binds is called the orthosteric binding site. However, researchers have
identified compounds that show a modulatory effect on nicotinic receptors by
binding to different sites other than the orthosteric-binding site, including the
transmembrane domain. These compounds are termed as allosteric modulators,
and the sites where they bind to are called allosteric sites (Abdel-Magid 2015;
Chatzidaki and Millar 2015).
Allosteric modulators do not all exert the same effect, as mentioned in Sect.
3.3.1.3. The allosteric modulators that initiate an agonist-mediated response are
termed as positive allosteric modulators (PAMs), while negative allosteric
modulators (NAMs) are those that inhibit the effects of agonist activation. PAMs
are different from allosteric agonists, as allosteric agonists can activate the receptor
directly when binding to the allosteric site. Conversely, neutral allosteric ligands
(NALs) do not cause changes in the receptor activity or its binding ability to the
ligands (Cecchini and Changeux 2015; Chatzidaki and Millar 2015; Gentry et al.
2015).
PAMs binding to the receptor causes potentiation of agonist-evoked responses,
which means they neither activate the receptor nor compete with acetylcholine
binding. PAMs acting on α7 nicotinic receptors show therapeutic potentials for the
treatment of inflammatory and neurological disorders. PAMs possess advantageous
characteristics that make them more promising as compared to exogenous agonists,
such as higher target selectivity, less tolerance due to desensitization, and their
ability to maintain the spatial and temporal characteristics of the endogenous activa-
tion processes (Corradi and Bouzat 2016; Uteshev 2014). Based on the effect on
macroscopic currents of receptors, PAMs are classified into two types, type I and
II. Type I PAMs such as NS-1738, genistein, 5-HI, and ivermectin do not signifi-
cantly affect current decay but enhance the agonist-induced peak currents. Type II
PAMs, on the other hand, show slow desensitization onset and reactivate receptors
that are desensitized (Bouzat and Sine 2018; Corradi and Bouzat 2016).
3 Pharmacology of Acetylcholine and Cholinergic Receptors 89

3.3.3 Cholinesterase Inhibitors

There are two types of cholinesterases in the body, acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE), which differ in substrate specificity and their distri-
bution within the body. While AChE terminates the action of ACh at synapses in the
nervous system, BuChE concentrates in non-neuronal sites such as liver and plasma.
BuChE is also responsible for metabolizing certain drugs (e.g., ester-type local
anesthetics, succinylcholine) (Colovic et al. 2013; Kishore et al. 2012; Mehrpouya
et al. 2017).
Most compounds used clinically to inhibit cholinesterase work on both the
enzymes without discrimination. Cholinesterase inhibitors are classified into two
main types based on the nature of the compound-enzyme binding, viz. reversible and
irreversible. Reversible compounds can either be covalent or non-covalent
inhibitors. Edrophonium is a non-covalent inhibitor that binds reversibly to the
anionic part of the acetylcholinesterase enzyme. Drug-enzyme bond characteristics
mainly determine the duration of these compounds. For instance, edrophonium binds
weakly to the enzyme and is rapidly cleared by the kidney, which results in a short
duration of action that is approximately 10 min long (Romano et al. 2017; dos
Coelho et al. 2018). Other examples of non-covalent ChE inhibitors are donepezil
and tacrine used for the treatment of AD. These two compounds possess a longer
duration of action due to their partition into lipids and their higher affinities (Birks
and Harvey 2018; Sameem et al. 2017). Carbamic acid ester derivatives such as
neostigmine and physostigmine are covalent inhibitors, sometimes termed as carba-
mate inhibitors. After their binding to the AChE enzyme, they get hydrolyzed, which
results in carbamylation of the serine residue in the active site of the enzyme (Arens
and Kearney 2019; Lauretti 2015).
On the other hand, irreversible ChE inhibitors act on the serine group in the active
site of the enzyme and phosphorylate it. Nerve gases such as tabun, sarin, and soman
are examples of irreversible ChE inhibitors. Other examples include insecticides
(malathion, parathion) and therapeutic agents such as isofluorophate and
echothiophate referred to as organophosphate or organophosphorus ChE inhibitors.
The reason why these compounds are considered irreversible is that the resultant
phosphorylated enzyme is exceptionally stable, which means that the dephosphory-
lation process can take hours to occur. The possibility of dephosphorylation of
phosphorylated enzyme by secondary and tertiary alkyl-substituted phosphates
(soman and isofluorophate) cannot be achieved to restore the enzyme activity until
further biosynthesis of new enzyme molecules (Sánchez-Santed et al. 2016; Thapa
et al. 2017).
Inhibition of ChE enzyme by anticholinesterase results in augmentation of
acetylcholine and the effects get relayed throughout the whole nervous system
leading to both therapeutic actions and associated adverse effects. Hence those
compounds lead to a nonselective indirect activation of both muscarinic and nico-
tinic receptors. Therefore, the receptors in the peripheral nervous system and the
brain get activated, including peripheral tissues innervated by parasympathetic
nerves, sympathetic and parasympathetic ganglia, and at the neuromuscular junction
90 S. F. Kokaz et al.

(Ehlert 2019b). Many acetylcholinesterase inhibitors are available on the market for
the treatment of various conditions (Table 3.7) (Potter and Kerecsen 2017).

3.3.4 Release Inhibitors

Release inhibitors’ primary mechanism of action is preventing ACh release from the
presynaptic end of the neuron. Botulinum toxin (BTX) is produced by Clostridium
botulinum bacteria that target cholinergic receptors at neuromuscular junctions in
skeletal muscles, inhibiting ACh release leading to neuromuscular blockage and
paralysis. The inhibition of ACh occurs after the toxin enters the nerve and reaches
the cytoplasm, then cleaves the (SNARE) proteins that are responsible for vesicle
fusion mediation. Prevention of vesicle fusion means that the ACh vesicles can no
longer bind into the intracellular cell membrane and release ACh into the synaptic
cleft (Dressler and Saberi 2005; Zhao et al. 2016). Some snake venoms produce an
irreversible blockade of neuromuscular transmission such as Crotoxin (CTX). CTX
is a heterodimeric phospholipase A2 (PLA2) neurotoxin produced by a Brazilian
rattlesnake (Crotalus durissus terrificus). This neurotoxin causes prevention of
acetylcholine release from presynaptic ends, which depends on intrinsic PLA2
activity (Cavalcante et al. 2017). Blockage of ACh release leads to interruption of
neuromuscular transmission and eventual muscle paralysis. Crotoxin exerts postsyn-
aptic level effects too by stabilizing desensitized acetylcholine receptors (Faure et al.
2017).

3.4 Therapeutic Applications

Cholinergic and anticholinergic drugs have a wide range of therapeutic application

including myasthenia gravis, dementia, ophthalmology, neurogenic bladder,
xerostomia, anticholinergic overdose, snakebites, and postoperative urinary reten-
tion including tensilon test. Further explanation will be presented later in this section.

3.4.1 Nicotinic Receptor Ligands

The most established therapeutic outcomes of nicotinic stimulation are cognitive

improvements. Nicotine was the first compound to bring attention to this since it can
improve performance cognitively and attention requiring vigilance tasks, which
suggests that nicotine possesses an optimizing ability to response and attention
mechanisms and enhances working memory (Gandelman et al. 2018; Grundey
et al. 2015). Figure 3.8 illustrates a potential model explaining how nicotinic
stimulation can enhance network function leading to improved cognitive perfor-
mance. Nicotine has been shown to enhance the efficacy of cognitive/function
control networks in frontal attention while decreasing the activity in default mode
network nodes in parietal regions, frontal lobe, and cingulate. These effects
 3

Table 3.7 Currently used acetylcholinesterase inhibitors (AChEIs) for various therapeutic applications
CNS
Drug Brand name Class/type of binding Duration effect Uses
Physostigmine Eserine Carbamate, reversible 0.5–2 h Yes Treatment of atropine poisoning, glaucoma
Neostigmine Prostigmin Carbamate, reversible 0.5–4 h No Reversal of NMJ blockade, myasthenia gravis
Pyridostigmine Mestinon, Carbamate, reversible 4–6 h No Reversal of NMJ blockade, myasthenia gravis, prevention of
Regonol organophosphate poisoning
Ambenonium Mytelase Carbamate, reversible 3–8 h No Reversal of NMJ blockade, myasthenia gravis
Tacrine Cognex Pyridine, reversible 4–6 h Yes Alzheimer’s disease (discontinued)
Donepezil Aricept Piperidine, non-covalent, >6 h Yes Yes Alzheimer’s disease
reversible
Galantamine Reminyl, Tertiary alkaloid, >6 h Yes Yes Alzheimer’s disease
Razadyne reversible
Rivastigmine Exelon Carbamate, pseudo- 10–12 h Yes Alzheimer’s disease
Pharmacology of Acetylcholine and Cholinergic Receptors

irreversible
Edrophonium Tensilon Electrostatic, rapidly 5–15 min No Diagnosis of myasthenia gravis, reversal of NMJ blockade
reversible
Echothiophate Phospholine Organophosphate, >24 h N/A Glaucoma
irreversible (topical)
91
92 S. F. Kokaz et al.

Nicotinic receptor
stimulation

Efficiency in
DMN activity in
attention and
frontal, cingulate
executive function
& parietal regions
networks

Attention
Distraction
Encoding of relevant info
Processing speed

Working memory Enhanced episodic

(frontal/parietal) memory
(MTL/Hippocampal)

Enhanced memory,
Planning and functioning

Fig. 3.8 Simplified model of nicotinic receptor stimulation effect in the enhancement of cognitive
functioning. Abbreviations: MTL: medial temporal lobe; DMN: default mode network

altogether may enhance processing speed, attention, and response inhibition, even-
tually leading to improved memory and attention to external stimuli and decreased
internally focused processing (Hahn 2019; Newhouse 2019).
Nicotine and nAChRs ligands have shown a different level of enhancement in
cognitive functions after many studies carried out on animal models, including
attention, memory, and learning. Further clinical trials have shown that these
cognitive effects are also present when varenicline has improved cognitive
impairment in schizophrenic patients and increased lapses in attention in smokers.
Recent clinical trials have shown that activating α7 nAChRs has procognitive effects
on patients with AD and schizophrenia. Likewise, a randomized placebo-controlled
double-blinded study with TC-1734, a β2-selective nicotinic agonist, had shown an
improved cognition when given to patients with age-associated memory impairment
(Hoskin et al. 2019; Quik et al. 2015). Some preclinical and clinical studies
supported that nicotinic receptors have a role in anxiety, mood, and depression. A
study revealed that the treatment of Tourette’s disorder with mecamylamine, a
nAChR antagonist, showed considerable improvement in depression symptoms
(Aboul-Fotouh 2015; Silver et al. 2001).
Nicotinic receptors are the principal receptors affecting the regulatory pathways
of nicotinic and cholinergic signaling, as suggested by recent studies. These signals
3 Pharmacology of Acetylcholine and Cholinergic Receptors 93

regulate both cell growth and migration, as well as regulation of angiogenesis of

endothelial cells during pathological and physiological conditions (Schaal and
Chellappan 2016). The nicotinic receptor that showed most of the oncogenic
responses in cancer is α7-nAChR. However, in breast cancer cells (estrogen
receptor-positive), the α9-nAChR was found to be upregulated. α9-nAChR coalition
with estrogen receptors stimulates both the initiation and progression of breast
cancer. Accordingly, these recent studies suggest that nicotinic receptors-mediated
oncogenic signaling has a significant role in cancer initiation and progression, which
supports that cytotoxic and mutagenic effects of tobacco smoke promote growth and
angiogenesis of tobacco-related cancers. Hence, nicotinic receptors are presenting
promising new targets for diagnosis, treatment, and prevention of cancers associated
with tobacco consumptions (Dang et al. 2016; Zhao 2016).
Currently, there has not been a medication developed that can target defects in
social communication and repetitive, restrictive patterns of behaviors in patients with
autism spectrum disorders (ASDs). Although individuals with Down syndrome
(DS) exhibit a progressive decline in adaptive functioning, that is also associated
with AD patients. Unfortunately, for DS patients and people with ASDs, there has
not been any effective medication strategy to stop or retard the worsening of adaptive
functions. However, α7-nAChRs present a potential therapeutic target in the treat-
ment of these disorders due to their involvement in the pathophysiology of these
disorders (Deutsch et al. 2014, 2015).

3.4.2 Muscarinic Receptor Ligands

Xanomeline, an M1/M4 receptor muscarinic agonist, exhibited positive effects on

psychotic and cognitive-like symptoms, including delusions and hallucinations in
patients with AD, which also makes it a therapeutic option to treat patients with
schizophrenia. Studies supported this notion when xanomeline had an antipsychotic
effect on rodent models (Yohn and Conn 2018). These studies and trials have
revealed the importance of muscarinic receptors in the modulation of neuronal
activity and release of neurotransmitter in various brain regions. Muscarinic
receptors additionally play a crucial role in shaping neuronal plasticity and affect a
wide range of functions from sensory and motor function to cognitive processes. In
recent years, with the development in gene targeting technology, knockout mice
offer valuable insight into the physiology and pathophysiology of the brain, thus
accelerating the efforts to develop new medications for analgesia and certain
conditions such as schizophrenia, addiction, PD, and AD (Thomsen et al. 2017).
Analgesic therapy by muscarinic agonists has been proposed in preclinical and
clinical models long ago. In a recent study, an M1 selective agonist has shown
analgesic activity in vivo. However, the pharmacokinetic/pharmacodynamic
measures have shown that to have the productive analgesic activity, a tenfold of
higher exposure is needed as compared to cognitive effects in a rodent model (Wood
et al. 2017).
94 S. F. Kokaz et al.

3.4.3 Cholinesterase Inhibitors

Acetylcholinesterase (AChE) inhibitors prevent acetylcholine breakdown. There are

already many AChE inhibitors currently used, such as galantamine, donepezil,
tacrine, and physostigmine in the treatment of AD (Fig. 3.9) (Verma et al. 2018).
These compounds have shown to positively affect psychosis, visual hallucinations,
and cognitive dysfunction in schizophrenia. Additionally, preclinical studies showed
that clinically used AChEIs could improve memory and learning in rodent models
for schizophrenia. However, clinical trials on schizophrenic patients treated with
AChEIs showed dose-limiting adverse effects due to their intrinsic ability to activate
the peripheral receptors (Thakurathi et al. 2013; Yohn and Conn 2018).
The two popular hypotheses, the cholinergic and the amyloid hypothesis, explain
the pathological mechanism for AD progression based on observed biochemical and
morphological changes in the brain of AD patients (Huang et al. 2014). The amyloid
hypothesis focuses on deposits accumulation in extracellular space formed from
β-amyloid peptides and tau proteins, which causes oxidative stress on neurons
causing inflammation leading to neurons degeneration (Jiang et al. 2014). On the
other hand, the cholinergic hypothesis is characterized by alterations in the biochem-
ical state of the brain due to ACh decrease in the cholinergic neurons present in the
cerebral cortex and the hippocampus. AD patients have a stunted Meynert nucleus
basalis, which is responsible for producing the enzyme ChAT that plays a central
role in acetylcholine synthesis and results in acetylcholine deficiency (Kuhn et al.
2015; Liu et al. 2015). The last treatment approved by the FDA for AD was
memantine, an NMDA (N-methyl-D-aspartate) glutamate receptor antagonist.
Memantine is currently indicted for moderate to severe phases of AD (Matsunaga
et al. 2015; de Souza et al. 2016). Postmortem studies have shown that patients with
PD have concomitant degeneration of nigrostriatal and cholinergic pathways. This
impairment in both neurotransmitter systems is related to specific motor and
non-motor features of the disease, such as cognitive and gait dysfunction (Aarsland
et al. 2017). Although the neurochemical trait in patients with PD is depletion in
dopamine, cognitive decline in PD is also associated with defects in the activity of
cortical acetylcholinesterase. Concomitant of dementia and classical gait

Alzheimer's
disease therapy

AChE NMDA
inhibitors antagonist

Tacrine
(withdrawn)
Galantamine Rivastigmine Donepezil Memantine

Fig. 3.9 Currently approved therapy regiment for Alzheimer’s disease

3 Pharmacology of Acetylcholine and Cholinergic Receptors 95

dysfunction in those patients increases the risk of falls and hospitalizations. 80% of
PD patients develop dementia after 20 years, which makes dementia the leading risk
factor for falls. Treatments that interfere with both dopaminergic and cholinergic
imbalance can improve long-term outcomes of PD patients (Hiller et al. 2015;
Pagano et al. 2015).
Organophosphorus compounds (OPCs) are primarily a group of pesticides that
inhibit acetylcholinesterase, and they are considered a severe health hazard. Current
therapy for OPCs exposure is oxime-type enzyme reactivators such as obidoxime
and pralidoxime, while atropine is administered for symptomatic treatment some-
times with unsatisfactory therapeutic outcomes. Better therapeutic results are
observed when AChE inhibitors (reversible) are administered before the exposure
to organophosphorus compounds. Recent studies showed that bispyridinium oximes
K027 and K203, AChE inhibitors under development, are highly effective (K027
more than K203) in protecting against OPCs when administered pre-exposure. This
strategy makes it a promising prophylactic agent and preventing possible occupa-
tional hazard (Antonijevic et al. 2016; Lorke and Petroianu 2018).

3.5 Conclusion

Cholinergic ligands possess great importance for various medical conditions. Over
the years, studies and research have provided substantial evidence of positive
outcomes from cholinergic ligands, such as nicotinic stimulators and muscarinic
agonist and their ability to improve cognitive performance. Currently, there are
potential cholinergic ligands approved for medical applications such as acetylcho-
linesterase (AChE) inhibitors including galantamine, donepezil, and rivastigmine for
the treatment of AD. Furthermore, several AChE inhibitors displayed promising and
appreciable therapeutic benefit for glaucoma, atropine poisoning, myasthenia gravis,
and reversal of NMJ (neuromuscular junction) blockade. Another example is mus-
carinic receptor antagonists (tertiary amines, quaternary ammonium derivatives)
used for conditions such as urinary incontinence, motion sickness, GI antispas-
modic, COPD, Parkinson’s disease, Huntington’s disease, and as an antisecretory
agent for peptic ulcer. The involvement and pathophysiology of cholinergic
receptors in certain medical conditions are subsequently investigated to provide
more potential therapeutic targets in the future. For example, α9-nAChR
upregulation in estrogen-positive breast cancer suggests that nicotinic receptors
can mediate oncogenic signaling, making them a potential new target in treating
and preventing certain cancers. Gene knockout technology is currently being used to
provide a further understanding of the cholinergic receptors, especially in the brain
to present new potential therapeutic agents for various conditions.
96 S. F. Kokaz et al.

References
Aarsland D, Creese B, Politis M, Chaudhuri KR, Ffytche DH, Weintraub D, Ballard C (2017)
Cognitive decline in Parkinson disease. Nat Rev Neurol 13:217–231
Abdel-Magid AF (2015) Allosteric modulators: an emerging concept in drug discovery. ACS Med
Chem Lett 6:104–107
Aboul-Fotouh S (2015) Behavioral effects of nicotinic antagonist mecamylamine in a rat model of
depression: prefrontal cortex level of BDNF protein and monoaminergic neurotransmitters.
Psychopharmacology 232:1095–1105
Akaike A, Izumi Y (2018) Overview. In: Akaike A, Shimohama S, Misu Y (eds) Nicotinic
acetylcholine receptor signaling in Neuroprotection. Springer, New York, NY, pp 1–16
Albuquerque EX, Pereira EFR, Manickavasagom Alkondon SWR (2009) Mammalian nicotinic
acetylcholine receptors: from structure to function. Physiol Rev 89:73–120
Alcaino C, Musgaard M, Minguez T, Mazzaferro S, Faundez M, Iturriaga-Vasquez P, Biggin PC,
Bermudez I (2017) Role of the Cys loop and transmembrane domain in the allosteric modulation
of α4β2 nicotinic acetylcholine receptors. J Biol Chem 292:551–562
Aleem A, Janbaz KH (2018) Involvement of dual mechanisms of anti-muscarinic and Ca++
antagonistic activities to validate the folkloric uses of Cyperus niveus Retz. in multiple gastro-
intestinal ailments. J Ethnopharmacol 213:138–148
Alvarado-Gonzalez A, Arce I (2015) Tiotropium bromide in chronic obstructive pulmonary disease
and bronchial asthma. J Clin Med Res 7:831–839
Andersen MB, Croy CH, Dencker D, Werge T, Bymaster FP, Felder CC, Fink-Jensen A (2015)
Antipsychotic-like effect of the muscarinic acetylcholine receptor agonist BuTAC in
non-human Primates. PLoS One 10:1–13
Andersson K-E (2019) Future considerations in overactive bladder pharmacotherapy. In: Cox L,
Rovner ES (eds) Contemporary pharmacotherapy of overactive bladder. Springer, New York,
NY, pp 219–229
Antonijevic E, Musilek K, Kuca K, Djukic-Cosic D, Vucinic S, Antonijevic B (2016) Therapeutic
and reactivating efficacy of oximes K027 and K203 against a direct acetylcholinesterase
inhibitor. Neurotoxicology 55:33–39
Arens AM, Kearney T (2019) Adverse effects of physostigmine. J Med Toxicol 15(3):184–191
Bass R, Santiago M, Smith L, Quinlan C, Panitch H, Giordano T, Piccione J (2018) Bethanechol in
Tracheomalacia: two case series and a review of the literature. Pediatr Allergy Immunol
Pulmonol 31:180–183
Bender AM, Jones CK, Lindsley CW (2017) Classics in chemical neuroscience : Xanomeline. ACS
Chem Neurosci 8:435–443
Bender AM, Garrison AT, Lindsley CW (2019) The muscarinic acetylcholine receptor M5:
therapeutic implications and allosteric modulation. ACS Chem Neurosci 10:1025–1034
Berizzi AE, Gentry PR, Rueda P, Den Hoedt S, Sexton PM, Langmead CJ, Christopoulos A (2016)
Molecular mechanisms of action of M5 muscarinic acetylcholine receptor allosteric modulators.
Mol Pharmacol 90:427–436
Berizzi AE, Perry CJ, Shackleford DM, Lindsley CW, Jones CK, Chen NA, Sexton PM,
Christopoulos A, Lawrence CJLAJ (2018) Muscarinic M5 receptors modulate ethanol seeking
in rats. Neuropsychopharmacology 43:1510–1517
Bertrand D Jr, Terry AV (2018) The wonderland of neuronal nicotinic acetylcholine receptors.
Biochem Pharmacol 151:214–225
Bhat SK, Ashwin D, Mythri S, Bhat S (2017) Arecanut (Areca catechu L) decreases Alzheimer’s
disease symptoms: compilation of research works. J Med Plants Stud 5:4–9
Birks JS, Harvey RJ (2018) Donepezil for dementia due to Alzheimer’s disease. Cochrane Database
Syst Rev 6:1465–1858
Blessing B, Gibbins I (2016) Autonomic nervous system. In: Prescott TJ, Ahissar E, Izhikevich E
(eds) Scholarpedia of touch. Springer, New York, NY, pp 467–477
3 Pharmacology of Acetylcholine and Cholinergic Receptors 97

Bouzat C, Sine SM (2018) Nicotinic acetylcholine receptors at the single-channel level. Br J

Pharmacol 175:1789–1804
Bradley SJ, Bourgognon J-M, Sanger HE, Verity N, Mogg AJ, White DJ, Butcher AJ, Moreno JA,
Molloy C, Timothy Macedo-Hatch JME, Wess J, Pawlak R, Read DJ, Sexton PM, Broad LM,
Steinert JR, Mallucci GR, Christopoulos A, Felder CC, Tobin AB (2017) M1 muscarinic
allosteric modulators slow prion neurodegeneration and restore memory loss. J Clin Invest
127:487–499
Brady AE, Jones CK, Bridges TM, Kennedy JP, Thompson AD, Heiman JU, Breininger ML,
Gentry PR, Yin H, Jadhav SB, Shirey JK, Conn PJ, Lindsley CW (2008) Centrally active
allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-
induced hyperlocomotor activity in rats. J Pharmacol Exp Ther 327:941–953
Brown DA (2019) Acetylcholine and cholinergic receptors. Brain Neurosci Adv 3:1–10
Browning RA (2010) Neurotransmitters and pharmacology. In: Ashley MJ (ed) Traumatic brain
injury: rehabilitation, treatment, and case management. CRC Press, Boca Raton, pp 98–166
Buccafusco JJ (2004) Neuronal nicotinic receptor subtypes: defining therapeutic targets. Mol Interv
4:285–295
Bukala BR, Browning M, Cowen PJ, Harmer CJ, Murphy SE (2019) Overnight transdermal
scopolamine patch administration has no clear effect on cognition and emotional processing
in healthy volunteers. J Psychopharmacol 33:255–257
Busse WW, Dahl R, Jenkins C, Cruz AA (2016) Long-acting muscarinic antagonists: a potential
add-on therapy in the treatment of asthma? Eur Respir Rev 25:54–64
Bylund D (2016) Acetylcholine. In: Reference module in biomedical sciences. Elsevier,
Amsterdam, pp 1–3
Carruthers SP, Gurvich CT, Rossell SL (2015) The muscarinic system, cognition and schizophre-
nia. Neurosci Biobehav Rev 55:393–402
Cavalcante WLG, Noronha-Matos JB, Timóteo MA, Fontes MRM, Gallacci M, Correia-de-Sá P
(2017) Neuromuscular paralysis by the basic phospholipase A2 subunit of crotoxin from
Crotalus durissus terrificus snake venom needs its acid chaperone to concurrently inhibit
acetylcholine release and produce muscle blockage. Toxicol Appl Pharmacol 334:8–17
Cecchini M, Changeux J-P (2015) The nicotinic acetylcholine receptor and its prokaryotic
homologues: structure, conformational transitions & allosteric modulation. Neuropharmacology
96:137–149
Chan WY, McKinzie DL, Bose S, Mitchell SN, Witkin JM, Thompson RC, Christopoulos A,
Lazareno S, Birdsall NJM, Bymaster FP, Felder CC (2008) Allosteric modulation of the
muscarinic M4 receptor as an approach to treating schizophrenia. PNAS 105:10978–10983
Chatzidaki A, Millar NS (2015) Allosteric modulation of nicotinic acetylcholine receptors.
Biochem Pharmacol 97:408–417
Colovic MB, Krstic DZ, Lazarevic-Pasti TD, Bondžić AM, Vasic VM (2013) Acetylcholinesterase
inhibitors: pharmacology and toxicology. Curr Neuropharmacol 11:315–335
Colzato LS, Schlauch K, Kullmann JK, Wild T (2017) Choline. In: Colzato LS (ed) Theory-driven
approaches to cognitive enhancement. Springer, New York, NY, pp 31–41
Congreve MS, Brown GA, Cansfield JE, Tehan BG (2018) Muscarinic M1 receptor agonists.
15/886,249
Conn PJ, Jones CK, Lindsley CW (2009) Subtype-selective allosteric modulators of muscarinic
receptors for the treatment of CNS disorders. Trends Pharmacol Sci 30:148–155
Conn PJ, Lindsley CW, Meiler J, Niswender CM (2014) Opportunities and challenges in the
discovery of allosteric modulators of GPCRs for treating CNS disorders. Nat Rev Drug Discov
13:692–708
Corradi J, Bouzat C (2016) Understanding the bases of function and modulation of α7 nicotinic
receptors: implications for drug discovery. Mol Pharmacol 90:288–299
Cummings J, Morstorf T, Lee G (2016) Alzheimer’s drug-development pipeline: 2016. Alzheimers
Dement (N Y) 2:222–232
98 S. F. Kokaz et al.

D’Souza RS, Johnson RL (2019) Nondepolarizing paralytics [WWW document]. StatPearls Publ,
Treasure Island, FL
Dajas-Bailador F, Wonnacott S (2004) Nicotinic acetylcholine receptors and the regulation of
neuronal signalling. Trends Pharmacol Sci 25:317–324
Dang N, Meng X, Song H (2016) Nicotinic acetylcholine receptors and cancer (review). Biomed
Rep 4:515–518
Dani JA (2015) Neuronal nicotinic acetylcholine receptor structure and function and response to
nicotine. Int Rev Neurobiol 124:3–19
de Linder Henriksen M, Lim C, Sharp JL (2019) Treatment of canine postoperative ocular
hypertension with combined latanoprost 0.005% and atropine 1% ophthalmic solutions. Vet
Ophthalmol 22(4):448–461
de Souza LG, Rennó MN, Figueroa-Villar JD (2016) Coumarins as cholinesterase inhibitors: a
review. Chem Biol Interact 254:11–23
Deutsch SI, Burket JA, Benson AD (2014) Targeting the α7 nicotinic acetylcholine receptor to
prevent progressive dementia and improve cognition in adults with Down’s syndrome. Prog
Neuro-Psychopharmacol Biol Psychiatry 54:131–139
Deutsch SI, Burket JA, Urbano MR, Benson AD (2015) The α7 nicotinic acetylcholine receptor: a
mediator of pathogenesis and therapeutic target in autism Spectrum disorders and down
syndrome. Biochem Pharmacol 97:363–377
Digby GJ, Shirey K, Conn PJ (2010) Allosteric activators of muscarinic receptors as novel
approaches for treatment of CNS disorders. Mol BioSyst 6:1345–1354
Digby GJ, Noetzel MJ, Bubser M, Utley TJ, Walker AG, Byun NE, Lebois EP, Xiang Z, Sheffler
DJ, Cho HP, Davis AA, Nemirovsky NE, Mennenga SE, Camp BW, Bimonte-Nelson HA,
Bode J, Italiano K, Morrison R, Daniels JS, Niswender CM, Olive MF, Lindsley CW, Jones CK,
Conn PJ (2012) Novel allosteric agonists of M1 muscarinic acetylcholine receptors induce brain
region-specific responses that correspond with behavioral effects in animal models. J Neurosci
32:8532–8544
Dineley KT, Pandya AA, Yakel JL (2015) Nicotinic ACh receptors as therapeutic targets in CNS
disorders. Trends Pharmacol Sci 36:96–108
Ding X-Q (2017) Drugs affecting nicotinic receptors. In: Dowd FJ, Johnson BS, Mariotti AJ (eds)
Pharmacology and therapeutics for dentistry. Elsevier, Amsterdam, pp 98–109
dos Coelho ST, Gomes TM, Serra PBA, Camara AL, Paes AMA (2018) Naturally occurring
Acetylcholinesterase inhibitors and their potential use for Alzheimer’s disease therapy. Front
Pharmacol 9:1–14
Dressler D, Saberi FA (2005) Botulinum toxin: mechanisms of action. Eur Neurol 53:3–9
Drudi FM, Lima C, Freitas L, Yogi M, Nascimento H, Junior RB (2017) Acetylcholine chloride 1%
usage for intraoperative cataract surgery miosis. Rev Bras Oftalmol 76:247–249
Egea J, Buendia I, Parada E, Navarro E, León R, Lopez MG (2015) Anti-inflammatory role of
microglial alpha7 nAChRs and its role in neuroprotection. Biochem Pharmacol 97:463–472
Ehlert F (2019a) Muscarinic antagonists and their clinical uses. In: Wecker L (ed) Brody’s human
pharmacology mechanism-based therapeutics. Elsevier, Amsterdam, pp 268–282
Ehlert F (2019b) Muscarinic agonists, Cholinesterase inhibitors, and their clinical uses. In: Wecker
L (ed) Brody’s human pharmacology mechanism-based therapeutics. Elsevier, Amsterdam, pp
241–267
Engers DW, Melancon BJ, Gregro AR, Bertron JL, Bollinger SR, Felts AS, Konkol LC, Wood MR,
Bollinger KA, Luscombe VB, Rodriguez AL, Jones CK, Bubser M, Yohn SE, Wood MW,
Brandon NJ, Dugan ME, Niswender CM, Conn PJ, Bridges TM, Lindsley CW (2019)
VU6005806/AZN-00016130, an advanced M4 positive allosteric modulator (PAM) profiled
as a potential preclinical development candidate. Bioorg Med Chem Lett 29:1714–1718
Fasoli F, Gotti C (2015) Structure of neuronal nicotinic receptors. In: Balfour DJK, Munafò MR
(eds) The neurobiology and genetics of nicotine and tobacco, Current topics in behavioral
neurosciences, vol 23. Springer, New York, NY, pp 1–17
3 Pharmacology of Acetylcholine and Cholinergic Receptors 99

Faure G, Porowinska D, Saul F (2017) Crotoxin from Crotalus durissus terrificus and crotoxin-
related proteins: structure and function relationship. In: Gopalakrishnakone P, Cruz LJ, Luo S
(eds) Toxins and drug discovery. Springer, New York, NY, pp 1–18
Felder C (1995) Muscarinic acetylcholine receptors: signal transduction through multiple effectors.
FASEB J 9:619–625
Ferreira-vieira TH, Guimaraes IM, Silva FR, Ribeiro FM (2016) Alzheimer’s disease: targeting the
cholinergic system. Curr Neuropharmacol 14:101–115
Fisher A (2012) Cholinergic modulation of amyloid precursor protein processing with emphasis on
M1 muscarinic receptor: perspectives and challenges in treatment of Alzheimer’s disease. J
Neurochem 120:22–33
Foster DJ, Conn PJ (2017) Allosteric modulation of GPCRs: new insights and potential utility for
treatment of schizophrenia and other CNS disorders. Neuron 94:431–446
Foster DJ, Choi DL, Conn PJ, Rook JM (2014) Activation of M1 and M4 muscarinic receptors as
potential treatments for Alzheimer’s disease and schizophrenia. Neuropsychiatr Dis Treat
10:183–191
Gaitonde S, Malik RD, Christie AL, Zimmern PE (2018) Bethanechol: is it still being prescribed for
bladder dysfunction in women? Int J Clin Pract 73:e13248. 1–6
Gandelman JA, Kang H, Antal A, Albert K, Boyd BD, Conley AC, Newhouse P, Taylor WD
(2018) Transdermal nicotine for the treatment of mood and cognitive symptoms in non-smokers
with late-life depression. J Clin Psychiatry 79:1–17
Garlapati K, Kammari A, Badam RK, Surekha BE, Boringi M, Soni P (2019) Meta-analysis on
pharmacological therapies in the management of xerostomia in patients with Sjogren’s syn-
drome. Immunopharmacol Immunotoxicol 41:1–7. https://doi.org/10.1080/08923973.2019.
1593448
Gault LM, Lenz RA, Ritchie CW, Meier A, Othman AA, Tang Q, Berry S, Pritchett Y, Robieson
WZ (2016) ABT-126 monotherapy in mild-to-moderate Alzheimer’s dementia: randomized
double- blind, placebo and active controlled adaptive trial and open-label extension. Alzheimers
Res Ther 8:1–13
Gentry PR, Kokubo M, Bridges TM, Kett NR, Harp JM, Cho HP, Smith E, Chase P, Hodder PS,
Niswender CM, Daniels JS, Conn PJ, Wood MR, Lindsley CW (2013) Discovery of the first
M5-selective and CNS penetrant negative allosteric modulator (NAM) of a muscarinic acetyl-
choline receptor: (S)-9b-(4-Chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo
[2,1-a]isoindol-5(9bH)-one (ML375). J Med Chem 56:9351–9355
Gentry PR, Sexton PM, Christopoulos A (2015) Novel allosteric modulators of G protein-coupled
receptors. J Biol Chem 290:19478–19488
Glavind K, Chancellor M (2011) Antimuscarinics for the treatment of overactive bladder: under-
standing the role of muscarinic subtype selectivity. Int Urogynecol J 22:907–917
Gould RW, Dencker D, Grannan M, Bubser M, Zhan X, Wess J, Xiang Z, Locuson C, Lindsley
CW, Conn PJ, Jones CK (2015) Role for the M1 muscarinic acetylcholine receptor in top-down
cognitive processing using a touchscreen visual discrimination task in mice. ACS Chem
Neurosci 6:1683–1695
Greig NH, Reale M, Tata AM (2013) New advances in pharmacological approaches to the
cholinergic system: an overview on muscarinic receptor ligands and cholinesterase inhibitors.
Recent Pat CNS Drug Discov 8:123–141
Grundey J, Amu R, Ambrus GG, Batsikadze G, Paulus W, Nitsche MA (2015) Double dissociation
of working memory and attentional processes in smokers and non-smokers with and without
nicotine. Psychopharmacology 232:2491–2501
Gunter BW, Gould RW, Bubser M, McGowan KM, Lindsley CW, Jones CK (2018) Selective
inhibition of M5 muscarinic acetylcholine receptors attenuates cocaine self-administration in
rats. Addict Biol 23:1106–1116
Haerter F, Eikermann M (2016) Reversing neuromuscular blockade: inhibitors of the acetylcholin-
esterase versus the encapsulating agents sugammadex and calabadion. Expert Opin
Pharmacother 17:819–833
100 S. F. Kokaz et al.

Hager HH, Burns B (2018) Depolarizing muscle relaxants [WWW document]. StatPearls Publ,
Treasure Island, FL
Hahn B (2019) The therapeutic potential of the cognitive-enhancing effects of nicotine and other
nicotinic acetylcholine receptor agonists. In: Preedy VR (ed) Neuroscience of nicotine. Aca-
demic Press, Cambridge, pp 305–311
Han MK, Lazarus SC (2016) COPD: clinical diagnosis and management. In: Broaddus VC, Mason
RJ, Ernst JD, King TE, Lazarus SC, Murray JF, Nadel JA, Slutsky AS, Gotway MB (eds)
Murray and Nadel’s textbook of respiratory medicine. Elsevier, Amsterdam, pp 767–785
Heishman SJ, Taylor RC, Henningfield JE (1994) Nicotine and smoking: a review of effects on
human performance. Exp Clin Psychopharmacol 2:345–395
Hepnarova V, Korabecny J, Matouskova L, Jost P, Muckova L, Hrabinova M, Vykoukalova N,
Kerhartova M, Kucera T, Dolezal R, Nepovimova E, Spilovska K, Mezeiova E, Pham NL,
Jun D, Staud F, Kaping D, Kuca K, Soukup O (2018) The concept of hybrid molecules of tacrine
and benzyl quinolone carboxylic acid (BQCA) as multifunctional agents for Alzheimer’s
disease. Eur J Med Chem 150:292–306
Hiller A, Nutt J, Mancini M, Horak F, Kareus S, Schoneburg BM, Chung KA (2015) Are
cholinesterase inhibitors effective in improving balance in Parkinson’s disease? J Neurol Disord
2:1–4
Hoskin JL, Al-Hasan Y, Sabbagh MN (2019) Nicotinic acetylcholine receptor agonists for the
treatment of Alzheimer’s dementia: an update. Nicotine Tob Res 21:370–376
Huang L, Miao H, Sun Y, Meng F, Li X (2014) Discovery of indanone derivatives as multi-target-
directed ligands against Alzheimer’s disease. Eur J Med Chem 87:429–439
Improgo MRD, Scofield MD, Tapper AR, Gardner PD (2010) The nicotinic acetylcholine receptor
CHRNA5/A3/B4 gene cluster: dual role in nicotine addiction and lung cancer. Prog Neurobiol
92:212–226
Jakubik J, El-Fakahany EE (2016) Allosteric modulation of muscarinic receptors. In: Myslivecek J,
Jakubik J (eds) Muscarinic receptor: from structure to animal models. Springer, New York, NY,
pp 95–130
Jiang N, Li S-Y, Xie S-S, Li Z-R, Wang KDG, Wang X-B, Kong L-Y (2014) Design, synthesis and
evaluation of multifunctional salphen derivatives for the treatment of Alzheimer’s disease. Eur J
Med Chem 87:540–551
Jones CK, Byun N, Bubser M (2012) Muscarinic and nicotinic acetylcholine receptor agonists and
allosteric modulators for the treatment of schizophrenia. Neuropsychopharmacol Rev 37:16–42
Kabbani N, Nichols RA (2018) Beyond the channel: metabotropic signaling by nicotinic receptors.
Trends Pharmacol Sci 39:354–366
Keating GM (2016) Sugammadex: a review of neuromuscular blockade reversal. Drugs
76:1041–1052
Khatwal RB, Dubala A, Kosaraju J, Chinni S (2014) Pharmacokinetics and tissue distribution of a
M1 muscarinic acetylcholine receptor positive allosteric potentiator , benzyl quinolone carbox-
ylic acid. Anal Methods 6:2672–2678
Kishimoto N, Stegaroiu R, Shibata S, Ito K, Inoue M, Ohuchi A (2016) Changes in the Oral
moisture and the amount of microorganisms in saliva and tongue coating after Oral ingestion
resumption: a pilot study. Open Dent J 10:79–88
Kishore P, Anuradha D, Piplani P, Rao R (2012) Molecular docking and receptor-specific
3D-QSAR studies of acetylcholinesterase inhibitors. Mol Divers 16:803–823
Korczynska M, Clark MJ, Valant C, Xu J, Von Moo E, Albold S, Weissa DR, Torosyan H,
Huang W, Kruse AC, Lyda BR, May LT, Baltos J-A, Sexton PM, Kobilka BK,
Christopoulos A, Shoichet BK, Sunahara RK (2018) Structure-based discovery of selective
positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor.
PNAS 115:2419–2428
Kruse AC, Kobilka BK, Gautam D, Sexton PM, Christopoulos A, Wess J (2014) Muscarinic
acetylcholine receptors: novel opportunities for drug development. Nat Rev Drug Discov
13:549–560
3 Pharmacology of Acetylcholine and Cholinergic Receptors 101

Kuca K, Soukup O, Maresova P, Korabecny J, Nepovimova E, Klimova B, Honegr J, Ramalho TC,

França TCC (2016) Current approaches against Alzheimer’s disease in clinical trials. J Braz
Chem Soc 27:641–649
Kuhn J, Hardenacke K, Lenartz D, Gründler TOJ, Ullsperger M, Bartsch C, Mai JK, Zilles K,
Bauer A, Matusch A, Schulz RJ, Moreik M, Bührle CP, Maintz D, Woopen C, Häussermann P,
Hellmich M, Klosterkötter J, Wiltfang J, Maarouf M, Freund HJ, Sturm V (2015) Deep brain
stimulation of the nucleus basalis of Meynert in Alzheimer’s dementia. Mol Psychiatry
20:353–360
Kulbatskii DS, Bychkov ML, Lyukmanova EN (2018) Human nicotinic acetylcholine receptors:
part I—structure, function, and role in neuromuscular transmission and CNS functioning. Russ J
Bioorgan Chem 44:595–607
Kumar A, Kumar A (2018) Alzheimer’s disease therapy: present and future molecules. In: Compu-
tational modeling of drugs against Alzheimer’s disease. Springer, New York, NY, pp 3–22
Lange HS, Cannon CE, Drott JT, Kuduk SD, Uslaner JM (2015) The M1 muscarinic positive
allosteric modulator PQCA improves performance on translatable tests of memory and attention
in rhesus monkeys. J Pharmacol Exp Ther 355:442–450
Langmead CJ, Austin NE, Branch CL, Brown JT, Buchanan KA, Davies CH, Forbes IT, Fry VAH,
Hagan JJ, Herdon HJ, Jones GA, Jeggo R, Kew JNC, Mazzali A, Melarange R, Patel N,
Pardoe J, Randall AD, Roberts C, Roopun A, Starr KR, Teriakidis A, Whittington M, Wu Z,
Watson J (2008a) Characterization of a CNS penetrant , selective M1 muscarinic receptor
agonist, 77-LH-28-1. Br J Pharmacol 154:1104–1115
Langmead CJ, Watson J, Reavill C (2008b) Muscarinic acetylcholine receptors as CNS drug
targets. Pharmacol Ther 117:232–243
Lauretti GR (2015) The evolution of spinal/epidural neostigmine in clinical application: thoughts
after two decades. Saudi J Anaesth 9:71–81
Lebois EP, Schroeder JP, Esparza TJ, Bridges TM, Lindsley W, Conn PJ, Brody DL, Daniels JS,
Levey AI (2017) Disease-modifying effects of M1 muscarinic acetylcholine receptor activation
in an Alzheimer’s disease mouse model. ACS Chem Neurosci 8:1177–1187
Lebois EP, Thorn C, Edgerton JR, Popiolek M, Xi S (2018) Muscarinic receptor subtype distribu-
tion in the central nervous system and relevance to aging and Alzheimer’s disease. Neurophar-
macology 136:362–373
Levin ED, Cauley M, Rezvani AH (2013) Improvement of attentional function with antagonism of
nicotinic receptors in female rats. Eur J Pharmacol 702:269–274
Liu AKL, Raymond Chuen-Chung Chang RKBP, Gentleman SM (2015) Nucleus basalis of
Meynert revisited: anatomy, history and differential involvement in Alzheimer’s and
Parkinson’s disease. Acta Neuropathol 129:527–540
Liu Y-J, Peng W, Hu M-B, Xu M, Wu C-J (2016) The pharmacology toxicology and potential
applications of arecoline: a review. Pharm Biol 54:2753–2760
Lorke DE, Petroianu GA (2018) Reversible cholinesterase inhibitors as pretreatment for exposure to
organophosphates. A review. J Appl Toxicol 39:101–116
Lukas RJ, Changeux J-P, Le Novère N, Albuquerque EX, Balfour DJ, Berg DK, Bertrand D,
Chiappinelli VA, Clarke PB, Collins AC, Dani JA, Grady SR, Kellar KJ, Lindstrom JM, Marks
MJ, Quik M, Taylor PW, Wonnacott S (1999) International Union of Pharmacology.
XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits.
Pharmacol Rev 51:397–401
Martin CA, Nuzzo PA, Ranseen JD, Kleven MS, Guenthner G, Williams Y, Walsh SL, Dwoskin LP
(2018) Lobeline effects on cognitive performance in adult ADHD. J Atten Disord 22:1–6
Mastrodicasa MA, Droege CA, Mulhall AM, Ernst NE, Panos RJ, Zafar MA (2017) Long acting
muscarinic antagonists for the treatment of chronic obstructive pulmonary disease: a review of
current and developing drugs. Expert Opin Investig Drugs 26:161–174
Matsunaga S, Kishi T, Iwata N (2015) Combination therapy with cholinesterase inhibitors and
memantine for Alzheimer’s disease: a systematic review and meta-analysis. Int J
Neuropsychopharmacol 18:1–11
102 S. F. Kokaz et al.

Maurer SV, Williams CL (2017) The cholinergic system modulates memory and hippocampal
plasticity via its interactions with non-neuronal cells. Front Immunol 8:1–14
McGowan KM, Nance KD, Cho HP, Bridges TM, Jones CK, Conn PJ, Jones CK, Lindsley CW
(2017) Continued optimization of the M5 NAM ML375: discovery of VU6008667, an M5
NAM with high CNS penetration and a desired short half-life in rat for addiction studies. Bioorg
Med Chem Lett 27:1356–1359
Mehrpouya M, Ataei S, Nili-Ahmadabadi A (2017) Potential drug interactions with cholinesterase
inhibitors in Alzheimer patients: a guideline for neurologists. J Appl Pharm Sci 7:223–226
Melroy-Greif WE, Stitzel JA, Ehringer MA (2016) Nicotinic acetylcholine receptors: upregulation,
age-related effects and associations with drug use. Genes Brain Behav 15:89–107
Millar NS, Gotti C (2009) Diversity of vertebrate nicotinic acetylcholine receptors. Neuropharma-
cology 56:237–246
Mitoha Y, Ueda H, Ichikawa H, Fujita M, Kobashi M, Matsuo R (2017) Effects of cevimeline on
excitability of parasympathetic preganglionic neurons in the superior salivatory nucleus of rats.
Auton Neurosci 206:1–7
Naicker P, Anoopkumar-Dukie S, Grant GD, Kavanagh JJ (2017) Anticholinergic activity in the
nervous system: consequences for visuomotor function. Physiol Behav 170:6–11
Naser PV, Kuner R (2018) Molecular, cellular and circuit basis of cholinergic modulation of pain.
Neuroscience 387:135–148
Newhouse PA (2019) Therapeutic applications of nicotinic stimulation: successes, failures, and
future prospects. Nicotine Tob Res 21:345–348
Newhouse P, Kellar K, Aisen P, White H, Wesnes K, Coderre E, Pfaff A, Wilkins H, Howard D,
Levin ED (2012) Nicotine treatment of mild cognitive impairment: a 6-month double-blind pilot
clinical trial. Neurology 78:91–101
Nikiforuk A, Kos T, Potasiewicz A, Popik P (2015) Positive allosteric modulation of alpha
7 nicotinic acetylcholine receptors enhances recognition memory and cognitive flexibility in
rats. Eur Neuropsychopharmacol 25:1300–1313
Oba Y, Sarva ST, Dias S (2016) Efficacy and safety of long-acting β-agonist/ long-acting musca-
rinic antagonist combinations in COPD: a network meta-analysis. Thorax 71:15–25
Pagano G, Rengo G, Pasqualetti G, Femminella GD, Monzani F, Ferrara N, Tagliati M (2015)
Cholinesterase inhibitors for Parkinson’s disease: a systematic review and meta-analysis. J
Neurol Neurosurg Psychiatry 86:767–773
Panus PC, Katzung B, Jobst EE, Tinsley SL, Masters SB, Trevor AJ (2009) Drugs affecting the
cholinergic system. In: Pharmacology for the physical therapist. McGraw Hill Professional,
New York, NY, pp 54–70
Papke RL (2014) Merging old and new perspectives on nicotinic acetylcholine receptors. Biochem
Pharmacol 89:1–11
Pappano AJ (2018) Cholinoceptor-activating & cholinesterase-inhibiting drugs. In: Katzung BG
(ed) Basic & clinical pharmacology. McGraw Hill Professional, New York, NY, pp 107–123
Petrakis IL, Ralevski E, Gueorguieva R, O’Malley SS, Arias A, Sevarino KA, Jane JS, O’Brien E,
Krystal JH (2018) Mecamylamine treatment for alcohol dependence: a randomized controlled
trial. Addiction 113:6–14
Picciotto MR, Higley MJ, Mineur YS (2012) Acetylcholine as a neuromodulator: cholinergic
signaling shapes nervous system function and behavior. Neuron 76:116–129
Potter PE, Kerecsen L (2017) Cholinesterase inhibitors. In: Rahman A, Choudhary MI (eds)
Frontiers in CNS drug discovery. Bentham Science, Sharjah, pp 201–239
Quik M, Bordia T, Zhang D, Perez XA (2015) Nicotine and nicotinic receptor drugs: potential for
Parkinson’s disease and drug-induced movement disorders. In: International review of neurobi-
ology. Elsevier, Amsterdam, pp 247–271
Rahman S, Engleman EA, Bell RL (2015) Nicotinic receptor modulation to treat alcohol and drug
dependence. Front Neurosci 8:426
3 Pharmacology of Acetylcholine and Cholinergic Receptors 103

Ramaswamy S, Chhabra SK, Gupta M, Dash DJ, Bansal V (2018) Salbutamol but not ipratropium
shifts autonomic balance towards sympathetic in chronic obstructive pulmonary disease. Curr
Respir Med Rev 14:166–171
Rogers BN, Gray DL (2012) Treating the cognitive deficits of schizophrenia. In: Rankovic Z,
Bingham M, Nestler EJ, Hargreaves R (eds) Drug discovery for psychiatric disorders. RSC
Publishing, Cambridge, pp 123–158
Romano M, Martin-Flores M, Sakai DM, Tseng CT, Campoy L, Gleed RD (2017) Effects of
neostigmine or edrophonium on force of contraction when administered at a train-of-four ratio
of 0.9 in anesthetized dogs. Vet Anaesth Analg 44:1313–1320
Sameem B, Saeedi M, Mahdavi M, Shafiee A (2017) A review on tacrine-based scaffolds as multi-
target drugs (MTDLs) for Alzheimer’s disease. Eur J Med Chem 128:332–345
Sánchez-Santed F, Colomina MT, Hernández EH (2016) Organophosphate pesticide exposure and
neurodegeneration. Cortex 74:417–426
Scarr E, Seo MS, Aumann TD, Chana G, Everall IP, Dean B (2016) The distribution of muscarinic
M1 receptors in the human hippocampus. J Chem Neuroanat 77:187–192
Schaal C, Chellappan S (2016) Nicotine-mediated regulation of nicotinic acetylcholine receptors in
non-small cell lung adenocarcinoma by E2F1 and STAT1 transcription factors. PLoS One
11:1–23
Sear JW (2019) Antihypertensive drugs and vasodilators. In: Hemmings HC, Egan TD (eds)
Pharmacology and physiology for anesthesia. Elsevier, Amsterdam, pp 535–555
Sehgal SA, Hammad MA, Tahir RA, Akram HN, Ahmad F (2018) Current therapeutic molecules
and targets in neurodegenerative diseases based on in silico drug design. Curr Neuropharmacol
16:649–663
Shahab L, Brose LS, West R (2013) Novel delivery Systems for Nicotine Replacement Therapy as
an aid to smoking cessation and for harm reduction: rationale, and evidence for advantages over
existing systems. CNS Drugs 27:1007–1019
Sherwood N (1993) Nicotine and psychomotor performance. Hum Psychopharmacol 8:155–184
Silver AA, Shytle RD, Sheehan KH, Sheehan DV, Ramos A, Sanberg PR (2001) Multicenter,
double-blind, placebo-controlled study of mecamylamine monotherapy for Tourette’s disorder.
J Am Acad Child Adolesc Psychiatry 40:1103–1110
Sivaraman D, Anbu N, Kabilan N, Kumar MP, Shanmugapriya P, Christian GJ (2019) Review on
current treatment strategy in ALZHEIMER’S disease and role of herbs in treating neurological
disorders. Int J Trans Res Ind Med 1:33–43
Smetana KS, Roe NA, Doepker BA, Jones GM (2017) Review of continuous infusion neuromus-
cular blocking agents in the adult intensive care unit. Crit Care Nurs Q 40:323–343
Svorc P (2018) Introductory chapter: autonomic nervous system - what we know about it. In: Svorc
P (ed) Autonomic nervous system. IntechOpen, London, pp 3–12
Takai K, Enomoto T (2018) Discovery and development of muscarinic acetylcholine M4 activators
as promising therapeutic agents for CNS diseases. Chem Pharm Bull 66:37–44
Takechi K, Suemaru K, Kiyoi T, Tanaka A, Araki H (2016) The α4β2 nicotinic acetylcholine
receptor modulates autism-like behavioral and motor abnormalities in pentylenetetrazol-kindled
mice. Eur J Pharmacol 775:57–66
Tangsucharit P, Takatori S, Zamami Y, Goda M, Poungrat Pakdeechote HK, Takayama F (2016)
Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine- induced
endothelium-independent vasodilatation in rat mesenteric arteries. J Pharmacol Sci 130:24–32
Taylor P (2012) Nicotinic receptors. In: Robertson D, Biaggioni I, Burnstock G, Low PA, Paton
JFR (eds) Primer on the autonomic nervous system. Academic Press, Cambridge, pp 79–82
Terry AV, Callahan PM (2018) Nicotinic acetylcholine receptor ligands, cognitive function, and
preclinical approaches to drug discovery. Nicotine Tob Res 21:383–394
Thakurathi N, Vincenzi B, Henderson DC (2013) Assessing the prospect of donepezil in improving
cognitive impairment in patients with schizophrenia. Expert Opin Investig Drugs 22:259–265
Thapa S, Lv M, Xu H (2017) Acetylcholinesterase: a primary target for drugs and insecticides. Mini
Rev Med Chem 17:1665–1676
104 S. F. Kokaz et al.

Thomsen M, Sørensen G, Dencker D (2017) Physiological roles of CNS muscarinic receptors

gained from knockout mice. Neuropharmacology 136:1–21
Tiwari P, Dwivedi S, Singh MP, Mishra R, Chandy A (2013) Basic and modern concepts on
cholinergic receptor: a review. Asian Pac J Trop Dis 3:413–420
Tunç AT, Koyuncu EA, Arslan F (2016) Development of an acetylcholinesterase choline oxidase
based biosensor for acetylcholine determination. Artif Cells Nanomed Biotechnol
44:1659–1664
Ueda Y, Zouzumi Y, Maruyama A, Nakano S, Sugimoto N, Miyoshi D (2016) Effects of
trimethylamine N-oxide and urea on DNA duplex and G-quadruplex. Sci Technol Adv Mater
17:753–759
Unal G, Aricioglu F (2017) A-582941, cholinergic alpha 7 nicotinic receptor agonist, improved
cognitive and negative symptoms of the sub-chronic MK-801 model of schizophrenia in rats.
Psychiatry Clin Psychopharmacol 28:4–13
Uslaner JM, Kuduk SD, Wittmann M, Lange HS, Fox SV, Min C, Pajkovic N, Harris D, Cilissen C,
Mahon C, Mostoller K, Warrington S, Beshore DC (2018) Preclinical to human translational
pharmacology of the novel M1 positive allosteric modulator MK-7622. J Pharmacol Exp Ther
365:556–566
Uteshev VV (2014) The therapeutic promise of positive allosteric modulation of nicotinic receptors.
Eur J Pharmacol 727:181–185
Valle’s AS, Borroni MV, Barrantes FJ (2014) Targeting brain a7 nicotinic acetylcholine receptors
in Alzheimer’s disease: rationale and current status. CNS Drugs 28:975–987
Vardigan JD, Cannon CE, Puri V, Dancho M, Koser A, Wittmann M, Kuduk SD, Renger JJ,
Uslaner JM (2015) Improved cognition without adverse effects: novel M1 muscarinic potentia-
tor compares favorably to donepezil and xanomeline in rhesus monkey. Psychopharmacology
232:1859–1866
Verma S, Kumar A, Tripathi T, Kumar A (2018) Muscarinic and nicotinic acetylcholine receptor
agonists: current scenario in Alzheimer’s disease therapy. J Pharm Pharmacol 70:985–993
Vozmediano-Chicharro R, Madurga B, Blasco P (2018) Efficacy of transdermal oxybutynin in the
treatment of overactive bladder syndrome: does it make sense using it in 2017? Adv Urol
2018:1–7
Walker N, Smith B, Barnes J, Verbiest M, Kurdziel T, Parag V, Pokhrel S, Bullen C (2018) Cytisine
versus varenicline for smoking cessation for Māori (the indigenous people of New Zealand) and
their extended family: protocol for a randomized non-inferiority trial. Addiction 114:344–352
Watt ML, Rorick-Kehn L, Shaw DB, Knitowski KM, Quets AT, Chesterfield AK, McKinzie DL,
Felder CC (2013) The muscarinic acetylcholine receptor agonist BuTAC mediates
antipsychotic-like effects via the M4 subtype. Neuropsychopharmacology 38:2717–2726
Wehrwein EA, Orer HS, Barman SM (2016) Overview of the anatomy, physiology, and pharma-
cology of the autonomic nervous system. Compr Physiol 6:1239–1278
Weiss C, Preston AR, Oh MM, Schwarz RD, Welty D, Disterhoft JF (2000) The M1 muscarinic
agonist CI-1017 facilitates trace eyeblink conditioning in aging rabbits and increases the
excitability of CA1 pyramidal neurons. J Neurosci 20:783–790
Wood MW, Martino G, Coupal M, Lindberg M, Schroeder P, Santhakumar S, Valiquette M,
Sandin J, Widzowski D, Laird J (2017) Broad analgesic activity of a novel, selective M1
agonist. Neuropharmacology 123:233–241
Wu J, Liu Q, Tang P, Mikkelsen JD, Shen J, Whiteaker P, Yakel JL (2016) Heteromeric α7β2
nicotinic acetylcholine receptors in the brain. Trends Pharmacol Sci 37:562–574
Yi S, Huang Y, Yu S-Z, Chen X-J, Yi H, Zeng X-L (2015) Therapeutic effect of atropine 1% in
children with low myopia. J Am Assoc Pediatr Ophthalmol Strabismus 19:426–429
Yohn SE, Conn PJ (2018) Positive allosteric modulation of M1 and M4 muscarinic receptors as
potential therapeutic treatments for schizophrenia. Neuropharmacology 136:438–448
Zhang L-L, Wang J-Q, Qi R-R, Pan L-L, Li M, Cai Y-L (2016) Motion sickness: current knowledge
and recent advance. CNS Neurosci Ther 22:15–25
Zhao Y (2016) The oncogenic functions of nicotinic acetylcholine receptors. J Oncol 2016:1–9
3 Pharmacology of Acetylcholine and Cholinergic Receptors 105

Zhao P, Sun H-X, Chu M, Hou Y-P (2016) Inhibitory effects of Botulinum toxin type a on pyloric
cholinergic muscle contractility of rat. Chin J Physiol 59:218–224
Zhao L, Chen M, Qian Y, Yang Q, Ge Y, Chen H, Qiu Y (2019) M1 muscarinic receptor activation
rescues β-amyloid-induced cognitive impairment through AMPA receptor GluA1 subunit.
Neuroscience 408:239–247. https://doi.org/10.1016/j.neuroscience.2019.04.007
Zheng G, Crooks PA (2015) Synthesis of lobeline, lobelane and their analogues. A review. Org
Prep Proced Int 47:317–337
Pharmacology of Adrenaline,
Noradrenaline, and Their Receptors 4
Bapi Gorain, Sulagna Dutta, Utpal Nandy, Pallav Sengupta,
and Hira Choudhury

Abstract

Adrenaline and noradrenaline are important catecholamines of the biological

system, responsible for the regulation of major functions of the body via their
action on the brain. This noradrenaline is the chief neurotransmitter of the
sympathetic nervous system, whereas adrenaline is an important metabolic hor-
mone, known to play a vital role in the cardiovascular system and a mediator of
the fight-or-flight response. These catecholamines act in the system through the
membrane-bound GPCRs, adrenergic receptors (ARs). Two major classes of
ARs, α-ARs and β-ARs, facilitate a number of functions at central and peripheral
sites. There are two subtypes of α-ARs (α1-AR and α2-AR), whereas three
different subtypes of β-ARs have been identified—β1-AR, β2-AR, and β3-AR.
Based on their role, different AR modulators have been introduced clinically for
their therapeutic application. In this chapter, we focus on the pharmacology of the
two catecholamines through their action on different ARs within the biosystem

B. Gorain (*)
School of Pharmacy, Faculty of Health and Medical Science, Taylor’s University, Subang Jaya,
Selangor, Malaysia
S. Dutta
Department of Oral Biology and Biomedical Sciences, Faculty of Dentistry, MAHSA University,
Kuala Lumpur, Malaysia
U. Nandy
PK-PD, Toxicology and Formulation Division, CSIR-Indian Institute of Integrative Medicine,
Jammu, India
P. Sengupta
Department of Physiology, Faculty of Medicine, Bioscience and Nursing, MAHSA University,
Kuala Lumpur, Malaysia
H. Choudhury
School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia

# Springer Nature Singapore Pte Ltd. 2020 107

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_4
108 B. Gorain et al.

and the modulators of ARs towards the treatment of potentially life-threatening

conditions.

Keywords

Adrenaline · Noradrenaline · Adrenergic receptors · Agonists · Antagonists ·

Biological function

Abbreviations

AR Adrenergic receptor or adrenoceptor

BP Blood pressure
BPH Benign prostatic hyperplasia
cAMP Cyclic adenosine monophosphate
cGMP Cyclic guanosine monophosphate
CHF Congestive heart failure
CNS Central nervous system
GIT Gastrointestinal tract
GPCRs G-protein-coupled receptors
HT Hydroxytryptamine
KO Knockout
L-DOPA L-dihydroxyphenylalanine
MDMA Methylenedioxymethamphetamine
mRNA Messenger ribonucleotide
NO Nitric oxide
PKA Protein kinase A
TM Transmembrane

4.1 Introduction

Adrenaline and noradrenaline are two important catecholamines, which are respon-
sible for foremost activities in the maintenance of the “inner world” of the brain
body. The existence of these substances in the adrenal gland, which turns red upon
oxidation, was first discovered by Vulpian (Gaddum and Holzbauer 1957); however,
the pressor effect of the adrenal extracts was first recognized by Oliver and Schafer
(Oliver and Schäfer 1895). Later, adrenaline was isolated and within a few years,
adrenaline was synthesized chemically (Szymonowicz 1896). Later, the component
was known by two different names due to progress in research in two different
countries where Takamine referred to it as “adrenalin” (Takamine 1902) and “epi-
nephrin” was preferred by Abel (1899). Later, adrenaline is considered as its official
name in Britain, whereas in the United States, epinephrine is preferred.
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 109

Simultaneously, Addison revealed the necessity of the adrenal gland for life, and
in continuation to that some of the researchers thought cortisol from adrenal medulla
is the necessary principle responsible for the pressor effect (Addison 1855; Daven-
port 1982). Following researches over decades, noradrenaline is recognized as the
chief neurotransmitter from the postganglionic sympathetic nerve fibers (Gaddum
and Holzbauer 1957). Noradrenaline is also recognized as norepinephrine (similar to
adrenaline), which is the chief sympathetic neurotransmitter. It is known to control
tonic and reflexive changes in cardiovascular tone. Alternatively, adrenaline is a key
factor for the metabolic responses or for global challenges to homeostasis, including
expression of emotional distress. This adrenomedullary hormone is also involved in
preserving homeostasis in emergencies (Goldstein 2010).

4.1.1 Noradrenaline

4.1.1.1 Synthesis
Tyrosine, the circulatory neutral aromatic amino acid, is obtained biologically from
the hepatic hydroxylation of phenylalanine or from diet. This amino acid, upon
uptake into the para-aortic enterochromaffin cells of adrenomedullary cells, sympa-
thetic neurons, and specific centers in the brain, begins in synthesis of
catecholamines (Fig. 4.1). Tyrosine is then converted to L-dihydroxyphenylalanine
(L-DOPA) by the catalytic effect of tyrosine hydroxylase, where ionized iron,
tetrahydrobiopterin, and molecular oxygen potentiate this rate-limiting step of cate-
cholamine synthesis (Wassall et al. 2009). Later this L-DOPA is rapidly converted to
dopamine in the cytoplasm of neuronal cells by the help of DOPA decarboxylase
(L-aromatic-amino-acid decarboxylase), found mainly in the brain, liver, gut, and
kidney, in the presence of the cofactor pyridoxal phosphate. Finally, noradrenaline is
synthesized from dopamine by the catalytic action of dopamine-beta-hydroxylase,
the enzyme mostly confined to the noradrenergic cell vesicles, in the presence of
copper and ascorbic acid cofactor (Wassall et al. 2009). During increased
adrenomedullary and sympathetic outflow, may be due to stressor effect, the rate
of synthesis and presence of tyrosine hydroxylase increase simultaneously (Daubner
et al. 2011).

Fig. 4.1 Synthesis and

release of noradrenaline from
the sympathetic nerve A
terminals and metabolic
pathway of noradrenaline by
uptaking into the nerve
terminal and to the vesicle or B
via enzymatic degradation to
form vanillyl mandelic acid
and 3-methoxy-4-hydroxy
phenylglycol C
110 B. Gorain et al.

According to the exocytic theory, depolarization of the terminal nerve cell

membrane results due to the action of acetylcholine, which increases permeation
of sodium into the cell. Increased permeation of sodium into the cell directly or
indirectly influences voltage-gated calcium channels to increase transmembrane
calcium influx (Awatramani et al. 2005). Thereafter, an increased level of cytoplas-
mic calcium induces a series of biochemical reactions to bring fusion of axoplasmic
and vesicular membranes. Thus, diffusion of the intra-vesicular contents occurs in
the extracellular environment (Müller and Schier 2011). There are a huge variety of
components that act on specific noradrenergic receptors at the nerve terminals to
stimulate release of noradrenaline during cellular activation. Alternately, up- or
downregulation of extracellular receptors or modulation of intracellular biochemical
reactions following receptor activation also affects the responses of the agonists
(Collins et al. 1991).

4.1.1.2 Metabolism
Noradrenaline is inactivated by taking up by the nerve cells, followed by storage of
the neurotransmitter or consequent intracellular metabolism. In this process, reup-
take by the noradrenaline transporters (uptake-1) on the nerve terminal cell mem-
brane plays the major pathway for terminating the action of noradrenaline
(Trendelenburg 1991). This uptake-1 process is carrier mediated and thus requires
energy to fulfill this action. Binding of adrenaline to the uptake-1 transporter does
not require catechol backbone; only one phenolic hydroxyl group containing drugs
are substrate to that site. Furthermore, the transport efficiency of the uptake-1
transporter decreases with increase in alkylation of the primary amino group of the
neurotransmitter. For example, reuptake efficiency of noradrenaline, adrenaline, and
isoprenaline (Fig. 4.2) is different, where reuptake of noradrenaline is highest
followed by adrenaline and isoprenaline (the extensively alkylated catechol)
(Burgen and Iversen 1965; Graefe and Bönisch 1988; Trendelenburg 1991).
As mentioned earlier, the uptake of noradrenaline via cell membrane mediated to
the axoplasm may undergo two fates, deamination to degrade by the action of
monoamine oxidases (Fig. 4.1) or may translocate into storage vesicles (Burgen
and Iversen 1965).
Thus, vesicular storage and degradation of the axoplasmic noradrenaline result in
an intra-neuronal “sink” with very low concentration of noradrenaline in cytoplasm.
The metabolism of catecholamines through oxidative deamination by the mono-
amine oxidases is common in neural and non-neural tissues. Rapid uptake of the
axoplasmic noradrenaline in the vesicles maintains the concentration of monoamine
oxidases onto the surface of mitochondria for continuous functioning of catechol-
aminergic systems (Goldstein 2010). Anything that prevents these monoamine
oxidases can increase the cytoplasmic concentration of catecholamines, stimulate
the outward transport of noradrenaline, and thereby promote cardiac smooth
muscles, vasoconstriction, leading to hypertension.
These monoamine oxidase enzymes are the chief intracellular enzyme that is
involved in metabolizing xenobiotic or biogenic amines throughout the biological
system (Westlund et al. 1988). Over a half of a century ago, Johnston brought two
 4

Synthesis and action Metabolism

Tyrosine
OH

3,4-dihydroxy phenylglycol
L-DOPA
CHO COMT

Dopamine Norepinephrine aldehyde

Aldehyde reductase
α2 OH
Noradrenaline COMT
3-methoxy-4hydroxyl phenylglycol

OH

MAO O
CHO
Noradrenaline 3,4-dihydroxy mandelic acid

Normetanephrine Normetanephrine aldehyde COMT

OH
Aldehyde dehydrogenase

β Vanillyl mandelic acid

α1
Pharmacology of Adrenaline, Noradrenaline, and Their Receptors

α2

Gq Gs
Gi

Fig. 4.2 Chemical structure of (a) noradrenaline, (b) adrenaline, (c) isoprenaline
111
112 B. Gorain et al.

different isoforms of monoamine oxidases in the human system, monoamine oxidase

A and monoamine oxidase B (Johnston 1968). It has been postulated that mono-
amine oxidase A is expressed mostly in sympathetic neurons, whereas extra-
neuronal cells are known to produce both forms of the enzyme (Westlund et al.
1988).
Alternatively, uptake-2 process is involved in metabolism of noradrenaline by
non-neuronal cells, where the carrier protein has low specificity and affinity for
catecholamines. Concurrently, the uptake-2 system involves catechol-O-
methyltransferase for degrading the uptaken catecholamines (Babin-Ebell and
Gliese 1995). Accordingly, the catalytic action of catechol-O-methyltransferase
leads the conversion of noradrenaline to normetanephrine, where S-adenosyl methi-
onine acts as the methyl group donor. Further process of the parent and product in
the presence of monoamine oxidase and aldehyde reductase or aldehyde dehydroge-
nase forms the two end products of noradrenaline metabolism, vanillylmandelic acid
and 3-methoxy-4-hydroxy phenylglycol (Fig. 4.1) (Babin-Ebell and Gliese 1995;
Wassall et al. 2009).

4.1.2 Adrenaline

Adrenaline is one of the important adrenomedullary hormones in humans, which

maintains the function of different body organs. Adrenomedullary secretion of this
adrenomedullary hormone covers prominently in neuroendocrine patterns attending
distress. Adrenaline is only sourced from adrenal medulla, and thus its role in the
maintenance of physiological functions is more explored for endogenous adrenaline
than for endogenous noradrenaline (Hillarp and Hokfelt 1955).

4.1.2.1 Synthesis
Adrenaline synthesis follows the similar synthetic pathway from tyrosine, as
described in the synthesis process of noradrenaline. Following the synthesis of
noradrenaline, it is converted to adrenaline by the catalytic action of
phenylethanolamine-N-methyltransferase, where adrenocortical steroidal hormones
(e.g., cortisol) act as trophic for this catalytic enzyme (Hillarp and Hokfelt 1955;
Wurtman and Axelrod 1966).
It has been postulated that the uptake-1 in sympathetic nerve fibers usually takes
up noradrenaline along with the circulatory adrenaline. Upon stimulation of the
sympathetic fibers, the nerve terminals release noradrenaline along with the up taken
adrenaline. This coreleased adrenaline binds to specific β-adrenoceptors on the nerve
terminal, subsequently increasing the release of noradrenaline from the terminal
(Iversen 1971). This hypothetical phenomenon brings up a model where the endog-
enous components are taken up into the nerve terminals, coreleased with the
neurotransmitter, and finally exaggerate or prolong the release pattern through
binding at facilitatory presynaptic receptors. However, confirmation of this hypo-
thetical assumption in isolated tissue preparation in in vitro models has failed
(Goldstein 2010).
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 113

4.1.2.2 Metabolism
The presence of catechol-O-methyltransferase in the medullary cells of adrenal
cortex leads to the formation of adrenaline from noradrenaline, simultaneously
resulting in inactivation of adrenaline following a similar pathway of noradrenaline.
As a result of metabolism, catechol-O-methyltransferase produces metanephrine,
thereby decreasing cytosolic concentration of adrenaline. Thus, the production of it
is continuous in the cytosol, whereas the release of adrenaline from the
adrenomedullary vesicles is episodic, upon receiving the impulse. Pheochromocy-
toma is a type of chromaffin cell tumors, where synthesis of catecholamines
increases heavily and thus, increases the metanephrines in the body. Therefore, the
detection of the level of metanephrines in plasma provides information on increased
levels of catecholamines, to aid effective treatment of the disease (Lenders et al.
2002; Spence et al. 2018).

4.2 Pharmacological Responses of Catecholamines Acting

Through Specific Receptors

Understanding the action of the catecholamine, particularly adrenaline and noradren-

aline, is crucial because of the diverse physiological responses of the catecholamines
acting through different categories of adrenoceptors or adrenergic receptors (ARs)
(alpha (α) and beta (β) adrenoceptors) (Strosberg 1993). All these adrenoceptors are
G-protein-coupled receptors (GPCRs), where these receptors are linked to
heterotrimeric G proteins. Each major type of receptors is preferentially linked to a
specific class of G protein, for example, Gq is observed in α1 adrenoceptors, Gi can
be found in α2 adrenoceptors, whereas β adrenoceptors are linked to Gs (Fig. 4.1).
Although these receptors are structurally related, individual receptors possess partic-
ular response to the physiological and biochemical pathways through the production
of particular second messengers. Thus, the activities following activation of these
adrenoceptors are solely dependent on the G protein-mediated generated second
messengers and on the action of different ion channels. Hereafter, individual
adrenoceptors are elaborated in detail.

4.2.1 Alpha (a) Adrenoceptors

Studies in the last few decades enabled developing concepts regarding different
receptors that mediate actions owing to the binding of endogenous catecholamines.
These were previously divided into only α- and β-adrenoceptors or ARs, while later
in the 1970s the α adrenoceptors are further divided into α1 and α2 ARs. The α1 ARs
are generally designated to mediate the responses in the effector organ. The α2 ARs
are generally localized in the presynaptic knob to regulate the release of
neurotransmitters, while reports also suggest their postsynaptic locations. Both α1
and α2 ARs are important in regulating the vascular tone, while none are known to
include homogenous groups. The initial division of the α1- and the α2-AR subtypes
114 B. Gorain et al.

was purely based upon the pharmacological properties of the same, but later detailed
structural analyses were performed by isolation and identification via cloning
methods. Today, α1-ARs have been classified into α1A-AR subtype (formerly
α1a/c, cloned α1c), α1B-AR subtype (cloned α1b), and α1D-AR subtype
(formerlyα1a/d, cloned α1d). It may convincingly be suggested that the α1A-AR
subtype is more prominently designated for vascular basal tone and arterial blood
pressure (BP) regulation, while α1B-AR subtype is better implicated for responses
towards exogenous agonists. The α1B-AR subtype expressions may also get altered
or modified owing to various pathophysiological modifications. The α2-AR
classifications include α2A/D-AR subtype, α2B-AR (cloned as α2b), and α2C-AR
(cloned as α2c). The α2A and α2B ARs are mainly used to mediate arterial
contraction, while α2C-ARs are mainly designated for venous vasoconstriction.
The functions of the α-ARs are subtype specific as well as common, depending
upon the required responses of the effector tissue. In most of the cases, the responses
evoked different α‐AR subtypes do overlap due to the lack of receptor specificity of
some ligands. Thus, it is essential to design drugs that will be specific in their mode
of actions in vivo, for therapeutic intervention using these receptors.

4.2.1.1 a1-Adrenoceptors

Structural Biology
α1-ARs mediate the essential role to regulate the physiological action of norepineph-
rine and epinephrine. The α1A, α1B, and α1D represent the three α1-AR subtypes.
They belong to the GPCR family and act via the Gq/11 signaling pathway. They
display individualistic patterns of tissue distributions and pharmacological
properties (Cotecchia 2010).
The pioneer hypothesis of Easson-Stedman had initiated bulk of research directed
towards drug designing for the ARs. Once ARs were cloned and the subtypes had
surfaced, the studies on drug design had oriented to reveal the detailed structure of
the receptors and new interactions were found. The α1 subtype and β-ARs were
found to greatly differ in their ligand-binding pocket which accounted for selective
drug design (Easson and Stedman 1933). Ahlquist, in later years, had used a series of
agonists to reveal the AR subtypes as α and β, and further into α1 and α2 receptors as
post- and pre-junctional ARs respectively (Starke et al. 1989; Ahlquist 1948). The
development of advanced pharmacological techniques, such as the radioligand
binding assay, leads to the classifications of α1-ARs into α1A and α1B-subtypes
(Morrow and Creese 1986). This was followed by the interventions of Art Hancock
who revealed the structure-function association of α1-ARs (Hancock et al. 1988) and
led to the synthesis of the first synthesized selective agonist α1A-AR, A-61603
(Knepper et al. 1995).
Initially four subtypes of α1-AR were revealed by cloning techniques. The α1b-
AR subtype was the first cloned α1-AR subtype, which displayed similar binding
properties like that of α1B-AR. Besides this, there were α1a from rats (Cotecchia
et al. 1988), bovine α1c- (Schwinn et al. 1990) and rat α1d-AR (Perez et al. 1991),
which had 99.8% homogeneity and considered a same subtype. At present, it is
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 115

conceived that the α1a/α1d clones refer to a novel α1D subtype. The latest classifi-
cation includes α1A (former α1a/c), α1B (former α1b), and α1D (former α1a/d)
(Perez et al. 1994).

Biological Distribution
The tissue distribution for α1-AR varies with their subtypes and depends on the
specific functions. Experiments in human cells, rats, and rabbits have showed that
the α1A/c-ARs are highly expressed in the heart, liver, salivary gland, lung, and vas
deferens (Table 4.1) (Schwinn et al. 1990; Perez et al. 1994). The mRNA expression
and receptor protein expressions also revealed the expression of this receptor
subtype in rat kidney, mediating vasoconstriction. In situ hybridization analysis of
α1-AR-mRNAs in rat brain displayed substantial levels of α1A-ARs expressions in
the olfactory system, hypothalamus, brainstem, and spinal cord, especially in the
structures that play roles in motor functions. The expression of α1A-ARs has also
been reported in glial cells, thus suggesting their non-neuronal mode of action in
mediating several brain functions (Day et al. 1997).
The α1B-AR has been reported to be highly expressed in rat liver and heart while
less in the hippocampus, aorta, and salivary gland. The expression of this receptor
subtype is high in the pineal gland, the lateral nucleus of the amygdala, most of the
thalamic nuclei, and in the raphe nuclei (Day et al. 1997).
The α1D-AR has been found to be abundant in the deferens and aorta and less
expressed or absent in the liver, spleen, kidney, and salivary gland. In neural tissues,
the distribution of this subtype is the most unique. It is highly expressed in the
olfactory system, hippocampus, cortical layers II–V, amygdala, the reticular
thalamic nucleus, the motor nuclei of the brainstem, and in the spinal cord (Day
et al. 1997).

Role in Biological System

The α1-AR subtypes carry out innumerable biological effects by binding their
ligands, epinephrine and norepinephrine. There is still a gap in the understanding
of the exact mechanism and specificity of the α1-AR subtypes. Besides their discreet
functions, many functions of the α1-ARs overlap with the α2-ARs.

Subtype-Specific Functions
Functions Mediated by a1A-ARs
The α1A-ARs have been suggested to play a vital role in mediating contractions in
several tissues such as in the vas deferens (Moriyama et al. 1997; Burt et al. 1998),
renal artery (Villalobos-Molina et al. 1997), rabbit ear artery (Fagura et al. 1997), tail
artery in rats (Lachnit et al. 1997), right atrium (Yu and Han 1994), internal anal
sphincter (Mills et al. 2008), and in the prostate (Marshall et al. 1995). α1A-ARs in
the rat vas deferens display two responses. One of these responses is phasic, may be
due to calcium ion release from ryanodine-sensitive compartments, and the other one
is tonic response which may be mediated via the pathway involving protein
kinase C, diacylglycerol, and calcium ion influx through L-type and may be also
the T-type channels (Burt et al. 1998). The rat submandibular gland may contain
 116

Table 4.1 Classifications, functions, and phenotypes of α1-adrenoreceptor subtype in mice with genetic modifications
AR Previous
group Present pharmacology Cloning Functional responses Genetic modification Phenotype
α1Ha α1A α1A α1c Control of blood pressure; Overexpression/ Increased contractile response, no
(α1A/ vasoconstriction; smooth muscle heart-specific hypertrophy
c) contraction promoter
α1B α1B α1b Regulatory; minor contractile role Gene deletion Decreased resting blood pressure,
decreased vasoconstriction
α1D α1D α1a, Control of blood pressure; Gene deletion Decreased resting blood pressure,
α1d, vasoconstriction; smooth muscle decreased vasoconstriction
α1a/d contraction
α1Lb α1Nc α1N –
α1L α1L – Vasoconstriction
a
High affinity for prazosin vs α1L-AR group
b
Members of this group have not yet been cloned
c
High affinity for HV 732 vs α1L receptors. Poorly classified with available probes
B. Gorain et al.
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 117

both α1A- and α1B-ARs, but in several experimental setup, it serves as a model for
the α1A-AR ligand-binding sites (Bruchas et al. 2008). It has been observed that
positive inotropic effects of phenylephrine in mouse are mediated via its binding to
the α1A-ARs (Ross et al. 2003). Moreover, noradrenaline-mediated contractions in
the prostate are also evident to occur via the α1A-AR (Gray et al. 2008). It is
suggested that overexpression of the α1A-AR elevates β-AR-supported cardiac
muscle contractility, thereby ameliorating the outcome from myocardiac infarction
(Woodcock 2007).

Functions of a1L-ARs: a1A-ARs

Under the classification of α1-ARs, based on the high and low affinity for prazosin,
the receptors have α1H, α1N, and α1L subtypes (Table 4.2). α1L-ARs have been
found to be expressed in longitudinal but not circular smooth muscle (Amobi et al.
2002) in the aorta, mesenteric, and carotid arteries (Muramatsu et al. 1990), rat
anococcygeus muscle (Civantos Calzada and Aleixandre de Artiñano 2001), and vas
deferens (Ohmura et al. 1992). Contractions of rat vas deferens in response to
exogenous agonists are suggested to be arbitrated by the α1A-ARs (Cleary et al.
2004). α1L-ARs are also found in the rabbit cutaneous resistance arteries (Smith
et al. 1997), rabbit iris (Nakamura et al. 1999), prostatic arteries in pigs (Recio et al.
2008), small mesenteric artery in rats (Graaf et al. 1996), guinea-pig aorta
(Muramatsu et al. 1990), rabbit bladder neck (Kava et al. 1998), pig internal anal
sphincter (Mills et al. 2008), in human, rat, and dog urethra, and in human prostate
(Muramatsu et al. 1990; Gray et al. 2008). α1-ARs are suggested to show the ligand

Table 4.2 Summary of tissue distribution of α1-adrenoreceptor subtype

mRNA abundance
Humans Rats
Tissue α1A/c α1B α1D α1A/c α1B α1D
Brain ID ND ND Yes Yes Yes
Cerebellum Yes ID ID ID ID ID
Cerebral cortex Yes ID ID Yes Yes Yes
Hippocampus ID ID ID Yes Yes Yes
Pituitary Yes ND ND ID ID ID
Heart Yes Yes Yes Yes Yes Yes
Aorta Yes Yes Yes Yes Yes Yes
Vena cava ID ID ID Yes ID ID
Lung Yes Yes Yes Yes Yes Yes
Kidney Yes Yes Yes Yes Yes Yes
Vas deferens ND ND ND Yes Yes Yes
Liver Yes Yes Yes Yes Yes Yes
Spleen Yes Yes Yes Yes Yes Yes
Salivary gland ND ND ND Yes Yes Yes
Prostate Yes Yes Yes ID ID ID
Epididymis ND ND ND Yes Yes Yes
ND not determined; ID insufficient data
118 B. Gorain et al.

binding properties of α1A-AR, as well as the functional characteristics of α1L-AR

(Daniels et al. 1999). Using mRNA-based techniques, it has been shown that α1L-
AR expressions are predominant in the tissues that mostly express α1A-ARs (Martí
et al. 2005). It is evident that human α1A-AR splice variants (Shibata et al. 1996)
have similar pharmacological features to that of the homo- and heterodimers of
human α1A variants (Ramsay et al. 2004). Thus, it can be inferred that α1L-ARs
represent a functional phenotype of α1A-AR, however, identification of these
functional phenotypes remain contentious.

Functions Mediated by a1B-ARs

Research regarding the functions of α1B-AR is scant owing to the unavailability of
selective antagonists. In several tissues, these receptors reportedly are involved in
mediating contractions, such as in mouse spleen (Eltze 1996), rat spleen (Noble et al.
1997), rat right atrium (Yu and Han 1994), rabbit cutaneous resistance arteries
(Smith et al. 1997), and in corpus cavernosum (Noble et al. 1997) and human
prostate (Teng et al. 1994). Knockout (KO) technology could put forth certain
functional aspects of α1B-ARs; for example, α1B-KO mice showed reduced or no
noradrenaline or phenylephrine potency in aorta, while combined α1B/α1D-KO led
to complete absence of noradrenaline and phenylephrine-mediated contractions in
aorta (Hosoda et al. 2004). Daly et al. (2002) used knockout technology to show that
α1B-ARs participate in mediating contractions in mouse arteries (such as aorta and
tail artery). α1B-AR overexpression has been associated with reduced
β-AR-mediated contractility in the heart (Woodcock 2007) and leads to cardiac
muscle hypertrophy and hypotension (Zuscik et al. 2001), increasing the risk of
heart failure. The α1B-AR overexpression also may diminish the positive inotropic
effects on phenylephrine in the heart owing to decrease in α1A-ARs. This suggests
rather a regulatory role of these receptors than contractile effects (Ross et al. 2003).

Functions Mediated by a1D-ARs

In several tissues, contractions of vascular smooth muscle are suggested to be
regulated at least partly by α1D-ARs. These tissues include human prostate, rat
aorta, mesenteric artery, pulmonary artery, iliac artery, renal artery, carotid artery,
rabbit ventricle and aorta. In certain occasions, α1D-ARs may also be the cause of
endothelium-dependent relaxation, as found by binding of phenylephrine to α1D-
AR in rat mesenteric vascular bed. The stimulation of α1D-AR may exert trophic
effects on endothelial cells. The ARs that are responsible for nerve stimulation-
mediated contractions are found to be predominantly α1D, while exogenous
noradrenaline-mediated contractions are predominantly functions of α1A-AR.
Experiments in sympathectomized rats have led to the conclusion that α1D-ARs
mostly mediate phasic contractions while α1A-ARs are attributed mainly for tonic
contractions (Cleary et al. 2004). These observations suggest that α1D-ARs are
mostly localized at the junctional region to stimulate nerve activities, but in case
of absence of the nerves, their locations extend along the smooth muscles.
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 119

Physiological Functions
Control of Blood Pressure
α1-ARs in the vascular system mainly mediate contractions and contribute greatly in
blood pressure regulation and in the baroreceptor reflex response. Piascik et al.
(1990) had put forth that in the conscious rat, α1A-AR subtype mediates tonic
maintenance of blood pressure, while α1B-AR subtype mediates responses towards
the exogenous agonists. In the unconscious or pithed rat, it has been reported that
both the blood pressure reflex and actions of exogenous noradrenaline involve both
α1A- and α1D-ARs, while blood pressure regulation via noradrenaline is mostly α1-
AR mediated (Vargas and Gorman 1995).
Temperature Control
Temperature regulation is another essential function of vascular α1-ARs since
vasoconstriction of superficial blood vessels is the primary mechanism for the core
body heat conservation. Methylenedioxymethamphetamine (MDMA) is an exten-
sively used recreational drug, which may lead to life-threatening hyperthermia. In
animal experimentations, MDMA reportedly affects thermoregulation, often leading
to hypothermia or hyperthermia in cold or hot ambient temperatures, respectively.
The α1-AR (α1A/D-ARs) antagonists could potentially covert the MDMA-mediated
monophasic hyperthermic response to a biphasic response characterized by hypo-
thermia followed by hyperthermia (Docherty 2010).
Depression
Depression, a multifactorial disorder, involves innumerable neural circuits and
neuronal processes. Noradrenaline plays significant roles in depression via the
ARs (Morilak et al. 2005). The ARs were found to undergo alterations followed
by the administration of antidepressant drugs (Blier 2003). Brain α1-adrenergic
neurotransmission impairment has been reported in certain depressive illnesses. It
is not yet clearly understood what is the exact mechanism by which individual α1-
AR subtypes mediate antidepressant effects. It had been suggested that α1-AR is
specifically associated with antidepressant action via a study using chronic
treatments with imipramine and electroconvulsive shock. The observations revealed
an increase in α1A-AR and not in the α1B-AR mRNA land receptor expressions in
the rat hippocampus and cerebral cortex (Stone and Quartermain 1999).
Nociception
The noradrenergic system is well known for its analgesic effects (Jinushi et al. 2018).
A majority of the studies that are focused on noradrenaline-mediated analgesic
effects have reported the antinociceptive role of α2-AR, while studies on the role
of α1-AR are mostly behavioral ones (Wei et al. 2016; Di Cesare et al. 2017). The
α1-AR binding modulations due to pain mostly take place in the areas of the central
nervous system (CNS) involved in pain processing. The receptor modulations,
including those of the α1B-AR subtype in acute pain phase, have been suggested
to be often lateralized and to vary according to the pain phases, whereas α1-ARs
involved in the late phases of pain were other than the α1B. Research on α1D-AR-
deficient mice [19] that focused on responses to different noxious stimuli put forth
the concept that α1D-ARs in the spinal cord participate in mediating responses of
thermal pronociception (Dogrul et al. 2006).
120 B. Gorain et al.

Genitourinary Functions
Three α1 AR subtypes have been found to be localized in the lower urinary tract:
α1A, α1B, and α1D. α1A receptors are predominant (about 70%) in the stroma of the
prostate gland, α1B in the prostate gland epithelium, and α1D mostly in the prostate
blood vessels as well as in the stroma. The α1ARs are also located in the urinary
bladder and urethral smooth muscle, as well as in the spinal cord and ganglia. The
extraprostatic sites of α-receptors lead to reduced organ selectivity and cause side
effects of treatments that focus on modulations of these receptors for lower urinary
tract symptoms. The α-blockers are used to treat benign prostatic hyperplasia (BPH)
owing to direct α-adrenergic antagonism of smooth muscle tonicity in prostatic
stroma. Current research has revealed longer-term effects of α-blockers on prostate
cellular differentiation and apoptosis (Cavallo 2018).

Receptor Modulators
Selective α1-adrenergic modulators retain a tiny low market share among all antihy-
pertensive drug medications within the United States (Griffith 2003). Prazosin,
introduced in 1976, was the primary marketed drug in this category. Afterwards,
two more α1-adrenergic blockers, doxazosin and antihypertensive drug, were avail-
able on the market, and their potential for once-daily dosing has provided extra
treatment flexibility (Xie et al. 2005). Within the last decade, sustained release
formulations for alpha-blocker and doxazosin are revealed. Tamsulosin and
alfuzosin are so-called uroselective agents with a better affinity for prostate α1-
ARs and are unremarkably employed in the management of patients with BPH
(White and Moon 2005).
α1-adrenergic antagonists, both selective and nonselective, are clinically avail-
able. Phenoxybenzamine, a nonselective, noncompetitive blocker, is currently
reserved for the preoperative treatment of pheochromocytoma-associated high
blood pressure. Nonselective α-blockade means that presynaptic α2-receptors,
which cut back the discharge of noradrenaline, are downregulated because of
inhibited feedback mechanism. Phentolamine could be a short-acting, competitive,
nonselective α-blocker parenterally administered and used for severe sorts of high
blood pressure prompted by extreme release of catecholamines. Prazosin, the pio-
neer selective α blocker, contains high affinity for the α1-AR associated with an
immediate-release formulation and has speedy onset of action. These features most
likely account for its comparatively higher rate of syncope and postural hypotension
compared with terazosin and doxazosin. Syncopal episodes may be decreased by
limiting the initial dose to 1 mg, administering the primary dose at night before sleep,
and gradually increasing the dosage. Doxazosin and terazosin have lower lipid
solubility than prazosin and thus bear lesser affinity for α1 receptors.
Different α1-adrenergic antagonists are pharmacologically discrete. Prazosin
features a comparatively short period of action and should be provided in a minimum
of double doses daily (Stanaszek et al. 1983). Doxazosin and terazosin with longer
half-lives might be given once daily. Doxazosin should be administered at bedtime,
which appears to be safe and effective in reducing morning hypertension (Pickering
et al. 1994; Fulton et al. 1995). α1-adrenergic antagonists ought to be used
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 121

cautiously in pregnant women and in children, since the effectualness and/or safety
of those compounds have not been estimated.
α1-adrenergic-specific antagonists hinder the noradrenaline-mediated vasocon-
striction via selective inhibition of postsynaptic α1 receptors activation by
catecholamines (Lund-Johansen and Omvik 1991). The presynaptic α2-ARs are
unblocked with these selective compounds, so inhibition of vasoconstriction by a
feedback mechanism of α2-AR stimulation is preserved.
The norepinephrine inhibition may be the cause of the absence of tachycardia,
higher cardiac output, and increased renin levels by the antagonists of both presyn-
aptic α2 ARs and the postsynaptic α1 ARs such as phentolamine (Baez et al. 1986).
As these drugs do not affect the renin-angiotensin-aldosterone system, they are
suited to be employed for hypertension regulation in patients with disorders related
to this axis (Webb et al. 1987). These drugs have ameliorative effects on
hemorheology as well including blood cell disorders, blood viscosity, and endothe-
lial function (Gomi et al. 1997).
The α1-AR antagonists may cause orthostatic hypotension in patients with either
volume-depletion disorders or susceptible to the loss of α1-ARs-induced vasocon-
striction. The actions of the α1-AR antagonists are at per with the sympathetic
activation, which is the reason for unchanged blood pressure after administration
of α1-AR antagonists in normotensive persons with normal activities of sympathetic
nervous system.

4.2.1.2 Alpha 2 (a2) Adrenergic Receptor

The α2-ARs belong to GPCR family and is linked to Gi heterotrimeric G protein. Its
vastly homologous subtypes include α2A, α2B, and α2C ARs. Few species excluding
humans possess an extra α2D AR subtype. α2-AR mediates the actions of its ligands,
such as the catecholamines (noradrenaline and adrenaline), in the peripheral and
central nervous systems (Civantos Calzada and Aleixandre De Artiñano 2001a, b).

Structural Biology
As discussed earlier, ARs were first classified as α- and β-receptors by Raymond
Ahlquist in 1948 on the basis of their pharmacological actions in various tissues
(Ahlquist 1948). With the advent of research in this realm, the adrenergic or
“adrenotropic” receptors were divided into three primary classes and into nine different
mammalian subtypes (Ahlquist 1948). The recent classification of receptors into three
main classes, α1-, α2-, and β-ARs, mainly relies on their amino acid sequences along
with pharmacological characteristics. Each of these classes of the ARs has again been
sequestered into three subtypes (Hieble et al. 1995). For α2-adrenoceptor, three subtypes
were previously referred to as α2-C10 (Kobilka et al. 1987), α2-C2 (Lomasney et al.
1990), and α2-C4 (Regan et al. 1988) according to their gene locations on human
chromosomes. These are now better recognized as AR subtypes α2A, α2B, and α2C.
α2-ARs are membrane glycoproteins whose most structural features bear resem-
blance and are common to other GPCRs. The common feature includes the presence
of seven transmembrane (TM) domains with extracellular amino terminus and
intracellular carboxyl terminus. The receptor binding sites must be accessible to
the specific ligands like adrenaline and noradrenaline. Thus, the binding sites of α2-
122 B. Gorain et al.

ARs are positioned within the core of the receptor proteins comprising the seven
α-helical TM domains (Laurila 2011).
The unique structural characteristics have rendered the α2-ARs-ligand
interactions to be highly specific. The ligand binding cavity in these receptors has
been reported to be structured by the residues in the third, fifth, sixth, and seventh
TM as well as the second extracellular loop. The relative lower affinity of dopamine
at α2-ARs than that of noradrenaline may be due to the absence of a specialized
β-hydroxyl moiety in dopamine molecule (Nyrönen et al. 2001). Besides these, the
other residues those supposedly have hydrophobic interactions with the N-methyl
group (positively charged) of catecholamine ligands are two phenylalanines posi-
tioned at 7.38 and 7.39 in the seventh TM. In the fifth TM of the human α2A-
adrenoceptor, two serine residues expose sites for hydrogen bonding to two
catecholic hydroxyl groups of the catecholamine ligands. These interactions may
be altered by conformational changes through receptor activation. They are also vital
for ligand orientations within the ligand-binding site of the α2-ARs. Some residues
mediate π-π stacking interactions with the aromatic ring present in the
phenylethylamine-type ligands (Nyrönen et al. 2001).
Unlike the agonists of α2-ARs, the concepts regarding receptor structural
characteristics of antagonists binding for these receptors are less clear. The α2-AR
antagonists bear way higher chemical diversity than that of agonists. Thus, the
antagonists are much complex and possess more divergent modes of binding.
There is no report that could specify the “antagonist binding site” for adrenoceptors
or for other GPCRs. However, in α2-ARs, a monoamine-binding GPCR, antagonists
have been predicted to bind almost at the same orthosteric agonist-binding sites
(Nyrönen et al. 2001).

Biological Distribution
The distributions of α2-AR subtypes in the mammalian tissues, especially in humans
and in rodents, have been detected using various techniques. Receptor radioligand
binding assays and autoradiography were employed to detect ligand binding
(Boyajian and Leslie 1987); in situ hybridization and other mRNA quantification
methods were used to determine receptor gene expression (Nicholas et al. 1993), and
antibody-based techniques such as immunohistochemistry and Western blotting
were used for receptor characterization (Tran et al. 2004). The three α2-AR subtypes
have exclusive tissue distribution patterns in the CNS, peripheral nervous system,
and in the peripheral tissues. The expressions of α2A-AR have been found exten-
sively in peripheral tissues and in the CNS, i.e., brainstem (especially the locus
coeruleus), midbrain, hypothalamus, hippocampus, spinal cord, cerebral cortex,
cerebellum, and septum. The expressions of α2B-adrenoceptor have been observed
mostly in the peripheral tissues and in lower levels in the CNS, mainly in the
thalamus, olfactory system, pyramidal layer of the hippocampus, and cerebellar
Purkinje layer. The α2C-ARs seem to be expressed mostly in the CNS, with different
expression patterns from those of α2A-ARs. They are most abundant in the cerebral
cortex, midbrain, amygdala, thalamus, olfactory system, dorsal root ganglia, hippo-
campus, basal ganglia substantia nigra, striatum, and ventral tegmentum (Brodde
et al. 2001) (Table 4.3).
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 123

Table 4.3 Tissue distributions of α2-adrenoceptor subtypes in the central nervous system and
peripheral tissues with their functions
Receptor Peripheral
subtype CNS tissues Physiological functions
α2A Amygdala Kidney Analgesia
Locus Vasculature Bradykinesia and hypotension
coeruleus
Lateral Urethra Hypothermia
septum
Brainstem Heart Inhibition of epileptic seizures
Cerebral Platelets Presynaptic inhibition of neurotransmitter
cortex release
Thalamus Spleen Anxiety like behavior
Hypothalamus Salivary Sedation and anesthesia
glands
Hippocampus Pancreas Regulation and blood glucose and insulin
Spinal cord Fat cells Decrease in intraocular pressure
Olfactory Inhibition of gastrointestinal motility
nucleus
Retina
α2B Thalamus Kidney Placental angiogenesis
Placenta Salt-induced hypertension
Liver Vascular smooth muscle contraction
Vasculature
α2C Striatum Kidney Presynaptic inhibition of catecholamine
release
Olfactory Adrenal Modulation of motor behavior
tubercle gland
Locus Vasculature Regulation of dopamine and serotonin
coeruleus balance in the brain
Hippocampus Pancreas Vascular smooth muscle contraction
Cerebral
cortex
Amygdala
Substantia
nigra

Role in Biological System

The α2-AR is a GPCR, which is classically localized on vascular prejunctional nerve
terminals, while several of its postsynaptic and extrasynaptic actions are also
evident. Its biological functions are mainly based on its ability to inhibit norepineph-
rine (noradrenaline) release via a negative feedback mechanism (Starke et al. 1989)
(Table 4.3). It is also localized on the vascular smooth muscle cells in specific blood
vessels for example on skin arterioles as well as on veins alongside other ARs. The α2-
AR comprises inhibitory G protein—Gi, whose α subunit dissociates from itself upon
activation and binds with adenylyl cyclase, thereby inhibiting the same. This causes
124 B. Gorain et al.

reduction in intracellular cAMP level and thereby protein kinase A (PKA) remains
inactivated. The PKA-mediated phosphorylation of downstream proteins such as
phosphorylase kinase, an enzyme for glycogen metabolism, is thus inhibited (Starke
et al. 1989).
The α2-ARs have slightly higher affinity in binding noradrenalin released from
the sympathetic postganglionic fibers than adrenaline from the adrenal medulla
(Boron and Boulpaep 2003). These receptors mediate several common functions
as the α1-AR, while having few specific physiological roles as well, including
sympathetic inhibition analgesia, sedation, hypotension, increased opioid and alco-
hol withdrawal symptoms, pupil control, body temperature regulation, seizure sus-
ceptibility modulation, and blood glucose homeostasis. The α2-AR agonists are
often used as veterinary anesthetics owing to its functions to cause analgesia,
sedation, and muscle relaxation via acting upon the CNS (Khan et al. 1999).

Presynaptic Regulation
In central adrenergic nerves as well as in sympathetic nerves, α2A- and α2C-ARs
regulate the neurotransmitter release by acting as inhibitory autoreceptors. α2B-
receptors on postsynaptic cells are activated by catecholamines released via the
sympathetic nerves. By doing so, they mediate various physiological functions
such as vasoconstriction. Functional differentiation of the presynaptic α2A- and
α2C-ARs has been proposed such that α2A-ARs have been found to inhibit the
release of norepinephrine from sympathetic nerves initially at high stimulation
frequencies, while the operational frequency for the α2C receptor can be very low
in order to regulate basal norepinephrine release (Philipp et al. 2002).

Blood Pressure Regulation

α2A-ARs mediate blood pressure regulation by impeding central sympathetic out-
flow and release of norepinephrine (Altman et al. 1999). On the contrary, the α2B-
ARs may counteract this function of α2A-Ars by inducing direct hypertension and
vasoconstriction. α2C-ARs play a role in α2-induced vasoconstriction on exposure
to extreme cold temperatures (Philipp et al. 2002).

Analgesia
All the three α2-AR subtypes play vital roles in the regulation of pain perception
(Bücheler et al. 2002). α2-ARs present in high levels in the spinal cord, specifically
in the superficial layers of the dorsal horns, are reported to regulate incoming
nociceptive impulses. α2A-ARs are essential to mediate the analgesic effect of α2-
AR agonists, the spinal α2C-ARs are reported to effectuate the moxonidine-
mediated analgesia, while the α2B-ARs are needed for spinal antinociception caused
by nitrous oxide (Philipp et al. 2002).

Sedation
α2-ARs agonists serve as potent sedatives and hypnotic agents in intensive care as
well as in the postoperative phase. The hypnotic effects mediated by α2-ARs are
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 125

suggested to be regulated by the locus ceruleus, since its neurons express high levels
of α2A-ARs (Philipp et al. 2002; Nguyen et al. 2017).

Behavior
α2-ARs mediate several behavioral functions owing to their high levels of
expressions in multiple sites in the CNS (Schramm et al. 2001; Frances Davies
et al. 2004). They have been shown to impede sensory information processing by the
CNS. They may also cause locomotor inhibition. α2A- and α2C-ARs have comple-
mentary actions in the integration of CNS function and behavior (Björklund et al.
2001; Philipp et al. 2002).

Other Physiological Functions

α2-ARs are potent mediators of body temperature regulation and control of seizure
threshold owing to their high antiepileptogenic effect (Janumpalli et al. 1998). α2A
and α2C-ARs have been suggested to bear hypothermic effects (Hunter et al. 1997).
They also mediate downregulation of intraocular pressure as suggested by the effects
of their agonists, apraclonidine and brimonidine (Liu et al. 1991; Kanagy 2005). The
α2-ARs have been reported to inhibit lipolysis in adipose tissue and thus are target
receptors to modulate conditions in obesity (Lafontan and Berlan 1980; Lafontan
et al. 1997; Morigny et al. 2016).

Receptor Modulators
α2-blockers represent the alpha blocker subset of drugs that are antagonists to the
α2-ARs (Rang et al. 1999). These drugs are mostly used in research arena, still not
widely applied in clinical interventions for humans. Their functions are based on
blockade of α2-ARs and thereby increase in the release of noradrenaline. These
α2-AR antagonists have been shown to significantly increase the release of dopami-
nergic and serotonergic besides the adrenergic neurotransmitters, trigger insulin
secretion, and reduce blood sugar levels (Rang et al. 1999).
Yohimbine is the most commonly used antagonist of α2-ARs (Rang et al. 1999).
It still finds immense applications in veterinary medicine, alongside its potent
alternative, atipamezole (Haapalinna et al. 2003; Lemke 2004). They operate to
reverse the effects of α2-AR agonists (such as medetomidine) that are used as
sedatives during surgery (Lemke 2004). Other α2-AR antagonists are efaroxan,
idazoxan, phentolamine, and rauwolscine (Chopin et al. 1999).
α2-AR antagonists find implications in the treatment of depression. The
tetracyclic antidepressants mirtazapine and mianserin are two of the antidepressants
belonging to this class of antagonists, but their antidepressant effects are also
supported by activation of other receptor sites as well. Sudden withdrawal from
use of the α2-AR antagonist drugs can prove detrimental because an immediate
global downregulation of release of the neurotransmitters may lead to various
neurological problems, depressions and sudden hyperglycemia, and reduction in
insulin sensitivity triggering diabetes. Moreover, downregulation of microcircula-
tion as well as adrenaline hypersensitivity in organs like the liver may also result if
there is a sudden withdrawal of a regular α2-AR treatment (Rang et al. 1999).
126 B. Gorain et al.

4.2.2 Beta (b) Adrenoceptors

Three different types of β adrenoceptors (β1, β2, and β3) are identified so far with a
homology of approximately 60% amino acid sequence. Ligands, such as adrenaline
and noradrenaline, bind to the ligand-binding pocket of the receptor site to control
various functional responses including contractility and heart rate, relaxation of smooth
muscle, and numerous metabolic events. As discussed earlier, all these receptor
subtypes are coupled to G-protein Gs and simultaneously activate adenylyl cyclase
(Table 4.4). However, research findings suggest variance in moderation of events and
signals, such as receptor downregulation or desensitization, following activation of
three different β-ARs (Lefkowitz 2000; Ma and Huang 2002; Kohout and Lefkowitz
2003). However, the magnitude of such regulation largely depends on the affinity of the
receptor to bind with the ligand, and thus, β2 ARs are found more susceptible. Overall,
the activation process of β-receptors leads to the accumulation of cyclic adenosine
monophosphate (cAMP) followed by activation of PKA and phosphorylation, thereby
alteration of function of cellular protein (Table 4.4).

4.2.2.1 b1-Adrenoceptor

Structural Biology
One of the major subtypes of the β-ARs is the β1 receptor (Bylund et al. 1994). The
receptors consist of seven membrane-spanning domains, three intra- and three extracel-
lular loops, one extracellular N-terminal domain, and one intracellular C-terminal tail.
Structural determination of β1 ARs was challenging because of its purification
difficulty and instability in detergent (Warne et al. 2008). However, β1 ARs from
frog and turkey red blood cells were estimated to have a molecular weight of 54,000
and 43,500, whereas β1 ARs obtained from mammalian lungs were shown to have a
molecular weight of 60,000–65,000 (Shorr et al. 1985). In fact, the first high
resolution picture of the ARs is obtained from β ARs, where the ligand binding
pockets were clear to fit a selective ligand (Suryanarayana et al. 1992).

Biological Distribution
β1-ARs are dominantly present in the cardiac tissue, coronary artery, adipose tissue,
and juxtaglomerular cells of kidney (Wallukat 2002). The presence of β1-AR has
also been found in the central nervous system, in brain (Gingrich and Caron 1993).

Table 4.4 Beta (β) adrenergic receptors and their effector system
Receptor subtype G protein Examples of some biochemical effectors
β1 Gs " adenylyl cyclase,
" L-type Ca2+ channels
β2 Gs " adenylyl cyclase,
β3 Gs " adenylyl cyclase,
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 127

Role in Biological System

β1-ARs act through GPCRs to stimulate adenylyl cyclase that hydrolyzed ATP to
cAMP. Then, the generated cAMP activates cAMP-dependent PKA, which
phosphorylates troponin that interacts with calcium ion to increase the rate and
force of contraction in cardiac muscle (Wallukat 2002).
The stimulation of β1-ARs leads to an increased heart rate and force of contraction
(positive inotropic action) as well as to an increased heart rate (positive chronotropic
action) and rhythm for enhanced conduction velocity (positive dromotropic action)
(Sakuma et al. 2001; Wallukat 2002). It also mediates to raise markedly the cardiac
output and consumption of oxygen in the heart. An increase in BP also occurs upon its
activation but significant enhancement in BP directed to reflex bradycardia mediated
by the stimulation of vagus nerve. β-AR triggers the release of rennin and ghrelin
(Zhao et al. 2010). It also stimulates the secretion of amylase from the salivary gland.
Isoprenaline/isoproterenol, adrenaline/epinephrine, and noradrenaline/norepinephrine
display agonism (sympathomimetics) towards β1-ARs where the order of potency is as
follows: Isoprenaline>adrenaline>noradrenaline (Rang et al. 1999).

Receptor Modulators
β1-AR agonists are used for cardiac stimulation in the treatment of cardiogenic shock
and severe congestive heart failure (CHF) (Wallukat 2002). Dobutamine,
denopamine, and xamoterol are the important members in this category. Though
dobutamine acts on both α and β-ARs, its specificity towards β1-AR is relatively
higher for which it has been considered as a selective β1-AR agonist (Parker et al.
2008). Denopamine is also a β1-AR agonist and is used in the treatment of angina
(Ishide 2002; Nakajima et al. 2006). It can also be used to treat CHF and pulmonary
edema (Nishio et al. 1998; Sakuma et al. 2001). In contrast, xamoterol is a partial
agonist to β-AR where it stimulates the heart at rest and blocks during exercise (Rang
et al. 1999). On the other hand, β-AR blockers are classified into β1-AR blocker
(cardioselective) and nonselective blocker, i.e., they block both β1 and β2-AR. In all
cases associated with β-AR blockers, ‘selectivity’ is a relative term and not absolute.
Therefore, selective β1-AR blocking may be associated with β2-AR-mediated
bronchoconstriction but less likely to produce these side effects (Baker et al. 2017).
Metoprolol, atenolol, acebutolol, bisoprolol, esmolol, betaxolol, celiprolol, etc. are the
more potent blockers of β1-AR than β2-AR and produce better cardioselective actions
depending upon the dose. These drugs are used to treat patients depending upon the
associated properties of their own viz. with intrinsic sympathomimetic action, without
sympathomimetic action, with additional α-AR blocking ability, membrane stabilizing
action etc. as well as patient’s viz. diabetes, asthmatics, etc. (Rang et al. 1999). On this
basis, there are numerous clinical uses of β-AR antagonists like hypertension, angina
pectoris, myocardial infractions, cardiac arrhythmias, glaucoma, etc. Metoprolol is the
classical molecule as a selective β1-AR blocker. It shows weak membrane stabilizing
activity but devoid of intrinsic sympathomimetic activity. Acebutolol is another
cardioselective drug with partial agonistic effects, i.e., it activates β1 and/or
β2 submaximally and membrane stabilizing actions. It is preferred for patients prone
to bradycardia. It has also intrinsic sympathomimetic activity. Diacetolol is also a
128 B. Gorain et al.

β-adrenergic blocker which is the primary metabolite of acebutolol and used as an anti-
arrhythmic agent (Basil and Jordan 1982). Atenolol and bisoprolol are selective β1-AR
blockers without any intrinsic sympathomimetic action and primarily used to treat high
blood pressure as well as heart-associated chest pain. Bisoprolol has a higher degree of
β1-AR selectivity compared to other selective β1-AR β blockers like atenolol, meto-
prolol, and betaxolol. However, nebivolol has better selectivity than bisoprolol.
Esmolol is an ultrashort acting β1-AR blocker and preferably used during cardiac
surgery to control heart rate and BP (Jaillon and Drici 1989; Deng et al. 2006).
Betaxolol is a selective β1 AR blocker, which is used for the treatment of hypertension
as well as glaucoma. Its therapy may be advantageous over timolol with respect to
fewer β2-AR blocking mediated side effects. The levo form of betaxolol is also
available on the market. Celiprolol is a selective β1-AR blocker with additional β2-
AR partial agonistic activity that is beneficial for asthmatic patients to avoid worsening
of disease conditions. It is also a weak α2 receptor antagonist. It has orphan drug status
for the treatment of vascular Ehlers–Danlos syndrome through promoting normal
collagen synthesis in the blood vessels and thereby shifting the pressure load away
from the vessels prone to rupture (Beridze and Frishman 2012). Flusoxolol is also a
selective β-AR blocker. Landiolol is a selective β1-AR antagonist that reduces heart
rate effectively with less negative effect on blood pressure or myocardial contractility
(Ikeshita et al. 2008; Wada et al. 2016). This is also an ultra short-acting drug like
esmolol but shows better cardioselectivity (Baker 2005; Okajima et al. 2015).

4.2.2.2 b2-Adrenoceptor

Structural Biology
β2 ARs are another key subtype of β-ARs which also belong to GPCRs superfamily
(Bylund et al. 1994). The representation of the binding pockets of β2 ARs from
humans is made in Fig. 4.3, where only one antagonistic binding site differs between
β1 ARs and β2 ARs constituting 15 amino acids. The presence of tyrosine in β2 AR
at the top of TM7 differs by the presence of phenylalanine in β1 AR (Suryanarayana
et al. 1992). The represented extracellular surface of β2 AR in Fig. 4.3b shows the
differences with β1 AR in yellow. The interior of the β2 AR has been represented in
Fig. 4.3c, where the binding pockets are split, with TM6-TM7 on the left and
TP-TM5 on the right (Kobilka 2011). The figure represents the identical nature
between the structures of β1 AR and β2 AR from the binding pocket to the
cytoplasm. Thus, it had been inferred that the structural diversity of the amino
acids are much higher into the binding pockets (Kobilka 2011).

Biological Distribution
β2-ARs are located mainly in the smooth muscles of the biological system, which
include vascular, airway, gastro-intestinal tract, uterus, bladder sphincter, seminal
tract, iris (radial muscle), and ciliary muscle (Bylund et al. 1994).
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 129

Fig. 4.3 Subtype-specific ligand binding in the β2AR. Amino acids that differ between β1AR and
β2AR are shown in yellow. The inverse agonist carazolol is shown with green carbons. (a) The
binding pocket of the human β2AR. Only one of the 15 amino acids that constitute the antagonist
binding pocket differs between β1AR and β2AR. Tyr 308 at the top of TM7 in the β2AR is Phe in
the β1AR. (b) The extracellular surface of the β2AR. (c) The interior surface of the β2AR that has
been split along the plane of the binding pocket (Kobilka 2011)
130 B. Gorain et al.

Role in Biological System

The pathway to exert the pharmacological response through this receptor is similar to β1
ARs except PKA-phosphorylated phospholamban that interacts with calcium ion for
faster relaxation of the smooth muscle. The stimulation of selective β2-ARs causes
relaxation of smooth muscle without affecting the function of the heart. The activation of
β2-ARs leads to glycogenolysis in both liver and skeletal muscle. An increase in release
of histamine occurs through stimulation of β2-ARs. The agonism to β2-ARs augments
the secretary action of ciliary muscle (Bylund et al. 1994). Isoprenaline/isoproterenol,
adrenaline/epinephrine, and noradrenaline/norepinephrine display agonism
(sympathomimetics) towards β2-ARs where the order of potency is as follows:
Isoprenaline>adrenaline>noradrenaline which is similar to β1-ARs-mediated agonism.

Receptor Modulators
Many selective β2-ARs agonists such as salbutamol, terbutaline, salmeterol, and
formoterol have been developed for their bronchodilator action for the relief of
reversible airway obstruction like asthma. These drugs have lower incidence of side
effects mediated by β1-ARs whereas long-term use of it led to muscle tremor through
change in β2-ARs-mediated rate and force of contraction. The activation of β2-ARs in
the α-cells of pancreatic islets causes increase in glucagon secretion. Isoxsuprine is a
selective β2 ARs agonist that causes direct relaxation of uterine and vascular smooth
muscle via β2-ARs. Isoxsuprine is used as a tocolytic for the treatment of premature
labor and as a vasodilator for the treatment of cerebral vascular insufficiency. Ritodrine
is a short-acting β2-ARs agonist and is also used to suppress threatened abortion.
Butaxamine/butoxamine is a selective antagonist to β2-ARs and has no clinical use. It
is used for experimentation where selective blocking of β2-ARs is required to estimate
the effect of other receptors. In contrast to cardioselective β-AR blocker, nonselective
β-blockers are widely used to treat hypertension, myocardial infraction, cardiac
arrhythmia, glaucoma, angina pectoris, CHF, atrial fibrillation etc. The blockade of
facilitatory effect of presynaptic β-ARs on noradrenaline release may also contribute to
the antihypertensive effect. Propanolol is the classical example as the nonselective
category of β-AR blocker. Prior to treating patients with β-blocker, associated adverse
effects of these drugs should be considered, viz. bronchoconstriction for patients with
airway disease like asthmatics, or obstructive pulmonary disease; bradycardia leading
to heart block for coronary disease patients; adequate cardiac output for cardiac failure
patients to impart a degree of sympathetic drive to the heart; hypoglycemia for diabetic
patients; and cold extremities due to block of beta receptor mediated vasodilation in
cutaneous vessels. Topical β-blockers are the first-choice drugs for open angle glau-
coma because these drugs lower intraocular tension by reducing aqueous formation
without affecting pupil size, tone of ciliary muscle, or outflow facility. Timolol is a
nonselective β-AR blocker. It has no local anesthetic or intrinsic sympathomimetic
activity. Cartiolol is a β blocker with intrinsic sympathomimetic activity. It can also act
as HT1A and 5-HT1B receptor antagonist. It has the ability to preserve ocular perfusion
and increase retinal blood flow. This may prevent optic nerve damage independent of
intraocular tension reduction. Hydroxycarteolol is the active metabolite of carteolol.
Levobunolol is an alternative to timolol for the management of ocular hypertension
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 131

and open-angle glaucoma. Metipranolol and mepindolol are also similar to timolol but
have different corneal anesthetic character. Befunolol is a β-AR blocker with intrinsic
sympathomimetic activity. This also acts as a β-ARs partial agonist. Pindolol is a
nonselective β-ARs blocker. It preferentially blocks 5-HT1A receptor as well as acts as
a weak partial agonist to this receptor. It also shows intrinsic sympathomimetic activity
and membrane-stabilizing effects. Tertatolol also displays similar action like
propanolol and pindolol to serotonin receptor (Langlois et al. 1993). Carvedilol is a
nonselective β-AR blocker as well as α1 blocker. Both S() and R(+) enantiomers are
responsible for later action whereas S() enantiomer is responsible for former action
(Ruffolo et al. 1990). Like carvedilol, labetalol also possesses α1 and β-ARs antago-
nistic activity. It has intrinsic sympathomimetic activity as well as membrane
stabilizing activity (Craig and Stitzel 2004; Mottram and Erickson 2009). Nebivolol
is generally categorized as a nonselective β-AR antagonist, but this unique drug shows
cardioselectivity at a dose of 5 mg and loses β1-ARs selectivity at a dose of above
10 mg (Nuttall et al. 2003). Moreover, nebivolol is devoid of cardioselectivity in
patients who are genetically poor metabolizer or who are coadministering this agent
with CYP2D6 inhibitors (Nuttall et al. 2003). Alprenolol is a nonselective β-AR
blocker with additional 5-HT1A and 5-HT1B receptor antagonist (Langlois et al.
1993). This is used for the treatment of angina pectoris (Hickie 1970). The activity
as well as application of oxprenolol is closely related to alprenolol (Langlois et al.
1993). Alprenoxime is a prodrug to alprenolol and also block β-ARs (Prokai et al.
1995). Adimolol is a nonselective α1, α2, and β-AR antagonist (Palluk et al. 1986).
Bevantolol is a β-AR blocker as well as a calcium channel blocker (Vaughan Williams
1987). Bopindolol is a β-adrenergic blocker which acts as a prodrug for its active
metabolite 4-(3-t-butylamino-2-hydroxypropoxy)-2-methylindole (Nagatomo et al.
2001). Bufuralol is a potent β-AR antagonist with partial agonist activity (Pringle
et al. 1986). Bupranolol is a nonselective β-AR blocker with strong membrane
stabilizing activity but devoid of intrinsic sympathomimetic activity. Sotalol is a
nonselective β-AR blocker with both class II and class III antiarrhythmic properties.
Like other nonselective blockers, it has also potential side effects but used generally to
treat abnormal heart rhythms associated with ventricular arrhythmias, atrial fibrillation,
or atrial flutter (Bertrix et al. 1986). Nipradilol is a β-AR antagonist and also acts as a
nitric oxide donor (Inoue et al. 2011). Medroxalol is a dual inhibitor for both α and β
ARs. Flestolol is a short-acting β-AR antagonist. Other than those mentioned above,
here are some β-AR blockers which have been developed for desired therapeutic
applications with lesser side effects like adaprolol, alfurolol, amosulalol, ancarolol,
arnolol, bornaprolol, brefonalol, bucumolol, bufetolol, bunitrolol, Butidrine,
carpindolol, cicloprolol, cinamolol, cloranolol, cyanopindolol, dalbraminol, ecastolol,
epanolol, ericolol, ersentilide, exaprolol, eugenodilol, falintolol, indenolol,
indopanolol, isoxaprolol, levomoprolol, moprolol, nadolol, nadoxolol, nifenalol,
pacrinolol, pafenolol, pamatolol, pargolol, primidolol, procinolol, ridazolol,
ronactolol, soquinolol, spirendolol, sulfinalol, talinolol, tazolol, tienoxolol, tilisolol,
tiprenolol, tolamolol, toliprolol, xibenolol, and xipranolol.
132 B. Gorain et al.

4.2.2.3 b3 Adrenoceptors
The existence of β3 ARs was first revealed in 1984, where the author reported the
existing β ARs (β1 and β2) are not specific to the novel β-adrenergic ligands (BRL
35135A, BRL 33725A, BRL 26830A). These ligands were shown to produce
remarkable anti-obesity actions on experimental animals with severe diabetes and
obesity (Arch et al. 1984). Later, this novel AR was cloned by Emorine and team
(Emorine et al. 1989).

Structural Biology
This β3 ARs, similar to β1 and β2, belong to serpentine seven transmembrane
GPCRs. Each segment of the transmembrane consists of 22–28 amino acids, with
three extracellular and three intracellular loops. Thus, altogether, these β3 receptors
contain 396 amino acids, where the C-terminal is intracellular and the N-terminal is
extracellular. This N-terminal ending is glycosylated, whereas the intracellular
C-terminal ending is phosphorylated by the action β-receptor kinases or PKA
(Coman et al. 2009). The interaction of specific ligands to this receptor and its
subsequent activity depend on the disulfide linkage between the two amino acids
(Cys110 and Cys361) in the second and third extracellular loops (Coman et al.
2009).
Additionally, four transmembrane domains are also reported crucial for the
interaction between receptors and ligands, such as transmembrane 3, 4, 5, and
6. Further, transmembrane 2 and 7 are associated with the activation of G protein
to promote the formation of a second messenger (Skeberdis 2004).

Biological Distribution and Role

The distribution of β3-ARs is observed in the myocardium, where the activation of
the atrial myocytes results in the phosphorylation of the calcium channels. Such
phenomenon gives rise to the transmembrane current of calcium (Kaumann 1996). It
has also been postulated that β3-ARs are also known to produce negative ionotropic
action on the ventricles. It is established that the stimulation of ARs in the ventricular
region activates endothelial NO synthase, thereby increasing nitric oxide production.
This increased nitric oxide in turn increases cyclic guanosine monophosphate
(cGMP) with a subsequent activation of phosphodiesterase 2 or inhibition of phos-
phodiesterase 3; thus there is a reduction in myocardium contractility (Gauthier et al.
1996).
The presence of β3-ARs was found in the isolated canine pulmonary rings, where
in vitro stimulation of this receptor has indicated the production of cAMP-dependent
vasodilation (Tagaya et al. 1999). Similarly, the presence of β3-ARs was also
evidenced in peripheral microvascular muscle of dogs and anesthetized rhesus
monkeys, where stimulation of the receptors resulted in vasodilator effect to
decrease the blood pressure of the respective animals (Berlan et al. 1994; Hom
et al. 2001).
The presence of β3-ARs has also been established in the respiratory system,
particularly in the nasal epithelium in rabbits to maintain salt and water movement
through epithelium (Danner et al. 2001). The presence of this AR in dog’s bronchi
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 133

muscle is reflected by the increase in cAMP production, leading to bronchodilation

(Tamaoki et al. 1993). The presence of β3-ARs in the respiratory system is species
dependent, as a specific agonist to this receptor showed relaxation of bronchial
muscle in dogs without producing any effect in sheep, guinea pig, and even in
humans (Martin and Advenier 1995).
The presence of β3-ARs has also been observed in different areas of the brain,
particularly lower in cerebellum, brain stem, and hypothalamus and higher in
striatum, cortex, and hippocampus (Summers et al. 1995). The administration of
β3-AR agonists directly to the CNS of experimental animals results in neuronal
activation, leading to increase in appetite (Castillo-Meléndez et al. 2000).
The presence of β3-AR in the human retinal endothelial cell lysate has been
established, and its role in the migration and proliferation of human retinal endothe-
lial cells and choroidal endothelial cells is investigated (Steinle et al. 2003, 2005).
The existence of β3-ARs has been reported in the gastrointestinal tract (GIT),
pancreas, and gall bladder. Agonists to this receptor have shown a decrease in
gastrointestinal motility, and thus a decrease in gastric emptying and intestinal transit
of the gastric contents. Stimulation of β3-ARs in the GIT of guinea pig showed
cAMP-dependent relaxation of stomach fundus and cAMP-independent relaxation
of the duodenum (Arch and Kaumann 1993; Horinouchi et al. 2002). It has also been
reported that the stimulation of β3-ARs produces a gastro protective action, may be
by increasing mucous production or by inhibition of gastric secretion (Vinay et al.
2002; Adami et al. 2003).
The role of β1 and β2 ARs is well assumed and established in the management of
obstructive airway diseases or coronary heart disorders, and the role of β3-ARs is
focused on overactive bladder syndrome (Chapple et al. 2008). Apart from the
described distribution of β3-ARs, it has also been observed in genital apparatus
(Berkowitz et al. 1995; Cirino et al. 2003) and skeletal muscles (Chamberlain et al.
1999).

Receptor Modulators
Although β3-AR modulators along with noradrenaline-serotonin uptake inhibitor
have been focused towards the treatment of obesity, there is no β3-AR-selective
agonist being approved so far for this purpose currently (Jesudason et al. 2011).
Alternatively, the activation of β3-ARs through brown adipose tissue recently focus
on moderate sleep, eating habit and metabolic responses (Szentirmai and Kapás
2017). Several β3-ARs agonists, such as vibegron and mirabegron, are presently
approved by regulatory agencies and introduced clinically to overcome problems
associated with overactive bladder syndrome, where solabegron and ritobegron are
still under clinical investigation towards approval (Warren et al. 2016; Schena and
Caplan 2019). Amibegron has also crossed the barrier of laboratory, but
discontinued during progression in clinical research for application as antidepressant
and antianxiolytic (Schena and Caplan 2019).
134 B. Gorain et al.

4.2.3 Conclusion

The role of catecholamines in the biological system is eminent and cannot be

compromised for a healthy life. Their action largely depends on engagement to the
particular receptor followed by conformational change and generation of the second
messenger. Understanding the activities of different receptors-based modulatory
action has led to the development of several modulators in the treatment of different
physiological ailments, and several are yet to be explored.

References
Abel J (1899) On epinephrin, the active constituent of the suprarenal capsule and its compounds.
Am Physiol Soc 3–4:iii–iiv
Adami M, Coruzzi G, Sotirov E et al (2003) Pharmacological evidence for beta3 adrenoceptors in
the control of rat gastric acid secretion. Dig Dis Sci 48:334–339
Addison T (1855) On the constitutional and local effects of disease of the supra-renal capsules. In:
1st edn. Samuel Highley, London, pp 1793–1860
Ahlquist RP (1948) A study of the ADRENOTROPIC receptors. Am J Physiol Content
153:586–600. https://doi.org/10.1152/ajplegacy.1948.153.3.586
Altman JD, Trendelenburg AU, MacMillan L et al (1999) Abnormal regulation of the sympathetic
nervous system in α 2A -adrenergic receptor knockout mice. Mol Pharmacol 56:154–161
Amobi NIB, Guillebaud J, Kaisary AV et al (2002) Discrimination by SZL49 between contractions
evoked by noradrenaline in longitudinal and circular muscle of human vas deferens. Br J
Pharmacol 136:127–135
Arch JR, Kaumann AJ (1993) Beta 3 and atypical beta-adrenoceptors. Med Res Rev 13:663–729
Arch JRS, Ainsworth AT, Cawthorne MA et al (1984) Atypical β-adrenoceptor on brown
adipocytes as target for anti-obesity drugs. Nature 309:163–165
Awatramani GB, Price GD, Trussell LO (2005) Modulation of transmitter release by presynaptic
resting potential and background calcium levels. Neuron 48:109–121
Babin-Ebell J, Gliese M (1995) Extraneuronal uptake of noradrenaline in human tissue (uptake2).
Heart Vessel 10:151–153
Baez M, Garg D, Jallad N, Weidler D (1986) Antihypertensive effect of doxazosin in hypertensive
patients: comparison with atenolol. Br J Clin Pharmacol 21:63S–67S
Baker JG (2005) The selectivity of β -adrenoceptor antagonists at the human β 1, β 2 and β
3 adrenoceptors. Br J Pharmacol 144:317–322
Baker JG, Gardiner SM, Woolard J et al (2017) Novel selective β 1 -adrenoceptor antagonists for
concomitant cardiovascular and respiratory disease. FASEB J 31:3150–3166
Basil B, Jordan R (1982) Pharmacological properties of diacetolol (M&B 16,942), a major
metabolite of acebutolol. Eur J Pharmacol 80:47–56
Beridze N, Frishman WH (2012) Vascular Ehlers-Danlos syndrome. Cardiol Rev 20:4–7
Berkowitz DE, Nardone NA, Smiley RM et al (1995) Distribution of β3-adrenoceptor mRNA in
human tissues. Eur J Pharmacol Mol Pharmacol 289:223–228
Berlan M, Galitzky J, Bousquet-Melou A et al (1994) Beta-3 adrenoceptor-mediated increase in
cutaneous blood flow in the dog. J Pharmacol Exp Ther 268:1444–1451
Bertrix L, Timour-Chah Q, Lang J et al (1986) Protection against ventricular and atrial fibrillation
by sotalol. Cardiovasc Res 20:358–363
Björklund M, Siverina I, Heikkinen T et al (2001) Spatial working memory improvement by an α2-
adrenoceptor agonist dexmedetomidine is not mediated through α2C-adrenoceptor. Prog
Neuropsychopharmacol Biol Psychiatry 25:1539–1554
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 135

Blier P (2003) The pharmacology of putative early-onset antidepressant strategies. Eur

Neuropsychopharmacol 13:57–66
Boron WF, Boulpaep EL (2003) Medical physiology: a cellular and molecular approach.
W.B. Saunders, Philadelphia
Boyajian CL, Leslie FM (1987) Pharmacological evidence for alpha-2 adrenoceptor heterogeneity:
differential binding properties of [3H]rauwolscine and [3H]idazoxan in rat brain. J Pharmacol
Exp Ther 241:1092 LP–1091098
Brodde OE, Bruck H, Leineweber K, Seyfarth T (2001) Presence, distribution and physiological
function of adrenergic and muscarinic receptor subtypes in the human heart. Basic Res Cardiol
96:528–538
Bruchas MR, Toews ML, Bockman CS, Abel PW (2008) Characterization of the alpha1-
adrenoceptor subtype activating extracellular signal-regulated kinase in submandibular gland
acinar cells. Eur J Pharmacol 578:349–358
Bücheler M, Hadamek K, Hein L (2002) Two α2-adrenergic receptor subtypes, α2A and α2C,
inhibit transmitter release in the brain of gene-targeted mice. Neuroscience 109:819–826
Burgen ASV, Iversen LL (1965) The inhibition of noradrenaline uptake by sympathomimetic
amines in the rat isolated heart. Br J Pharmacol Chemother 25:34–49
Burt RP, Chapple CR, Marshall I (1998) α 1A -adrenoceptor mediated contraction of rat prostatic
vas deferens and the involvement of ryanodine stores and Ca 2+ influx stimulated by
diacylglycerol and PKC. Br J Pharmacol 123:317–325
Bylund DB, Eikenberg DC, Hieble JP et al (1994) International Union of Pharmacology nomen-
clature of adrenoceptors. Pharmacol Rev 46:121–136
Castillo-Meléndez M, McKinley MJ, Summers RJ (2000) Intracerebroventricular administration of
the beta(3)-adrenoceptor agonist CL 316243 causes Fos immunoreactivity in discrete regions of
rat hypothalamus. Neurosci Lett 290:161–164
Cavallo JAKS (2018) Medical aspects of the treatment of lower urinary tract symptoms/benign
prostatic hyperplasia: 5-alpha reductase inhibitors. In: Lower urinary tract symptoms and benign
prostatic hyperplasia. Academic Press, Cambridge, pp 189–206
Chamberlain PD, Jennings KH, Paul F et al (1999) The tissue distribution of the human beta3-
adrenoceptor studied using a monoclonal antibody: direct evidence of the beta3-adrenoceptor in
human adipose tissue, atrium and skeletal muscle. Int J Obes Relat Metab Disord 23:1057–1065
Chapple CR, Yamaguchi O, Ridder A et al (2008) Clinical proof of concept study (blossom) shows
novel β3 adrenoceptor agonist YM178 is effective and well tolerated in the treatment of
symptoms of overactive bladder. Eur Urol Suppl 7:239
Chopin P, Colpaert FC, Marien M (1999) Effects of alpha-2 adrenoceptor agonists and antagonists
on circling behavior in rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal
pathway. J Pharmacol Exp Ther 288:798–804
Cirino G, Sorrentino R, di Villa Bianca RD et al (2003) Involvement of 3-adrenergic receptor
activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum
function. Proc Natl Acad Sci 100:5531–5536
Civantos Calzada B, Aleixandre de Artiñano A (2001a) Alpha-adrenoceptor subtypes. Pharmacol
Res 44:195–208
Civantos Calzada B, Aleixandre De Artiñano A (2001b) Alpha-adrenoceptor subtypes. Pharmacol
Res 44:195–208. https://doi.org/10.1006/phrs.2001.0857
Cleary L, Slattery J, Bexis S, Docherty JR (2004) Sympathectomy reveals α 1A - and α 1D
-adrenoceptor components to contractions to noradrenaline in rat vas deferens. Br J Pharmacol
143:745–752
Collins S, Caron MG, Lefkowitz RJ (1991) Regulation of adrenergic receptor responsiveness
through modulation of receptor gene expression. Annu Rev Physiol 53:497–508
Coman OA, Păunescu H, Ghiţă I et al (2009) Beta 3 adrenergic receptors: molecular, histological,
functional and pharmacological approaches. Romanian J Morphol Embryol 50:169–179
Cotecchia S (2010) The α1-adrenergic receptors: diversity of signaling networks and regulation. J
Recept Signal Transduct Res 30:410–419
136 B. Gorain et al.

Cotecchia S, Schwinn DA, Randall RR et al (1988) Molecular cloning and expression of the cDNA
for the hamster alpha 1-adrenergic receptor. Proc Natl Acad Sci 85:7159–7163
Craig CR, Stitzel RE (2004) Modern pharmacology with clinical applications, 6th edn. Lippincott
Williams & Wilkins, Philadelphia
Daly CJ, Deighan C, McGee A et al (2002) A knockout approach indicates a minor vasoconstrictor
role for vascular α 1B -adrenoceptors in mouse. Physiol Genomics 9:85–91
Daniels DV, Gever JR, Jasper JR et al (1999) Human cloned alpha1A-adrenoceptor isoforms
display alpha1L-adrenoceptor pharmacology in functional studies. Eur J Pharmacol
370:337–343
Danner I, Escande D, Gauthier C (2001) Beta(3)-adrenoceptors control cl() conductance in rabbit
nasal epithelium. Eur J Pharmacol 422:203–207
Daubner SC, Le T, Wang S (2011) Tyrosine hydroxylase and regulation of dopamine synthesis.
Arch Biochem Biophys 508:1–12
Davenport HW (1982) Epinephrin(e). Physiologist 25:76–82
Day HE, Campeau S, Watson SJ, Akil H (1997) Distribution of alpha 1a-, alpha 1b- and alpha
1d-adrenergic receptor mRNA in the rat brain and spinal cord. J Chem Neuroanat 13:115–139
Deng CY, Lin SG, Zhang WC et al (2006) Esmolol inhibits Na(+) current in rat ventricular
myocytes. Methods Find Exp Clin Pharmacol 28:697–702
Di Cesare ML, Micheli L, Crocetti L et al (2017) α2 adrenoceptor: a target for neuropathic pain
treatment. Mini Rev Med Chem 17:95–107
Docherty JR (2010) Subtypes of functional α1-adrenoceptor. Cell Mol Life Sci 67:405–417
Dogrul A, Coskun I, Uzbay T (2006) The contribution of Alpha-1 and Alpha-2 adrenoceptors in
peripheral Imidazoline and adrenoceptor agonist-induced nociception. Anesth Analg
103:471–477
Easson LH, Stedman E (1933) Studies on the relationship between chemical constitution and
physiological action. Biochem J 27:1257–1266
Eltze M (1996) Functional evidence for an α1B-adrenoceptor mediating contraction of the mouse
spleen. Eur J Pharmacol 311:187–198
Emorine L, Marullo S, Briend-Sutren M et al (1989) Molecular characterization of the human beta
3-adrenergic receptor. Science (80-) 245:1118–1121
Fagura MS, Lydford SJ, Dougall IG (1997) Pharmacological classification of α 1 -adrenoceptors
mediating contractions of rabbit isolated ear artery: comparison with rat isolated thoracic aorta.
Br J Pharmacol 120:247–258
Frances Davies M, Tsui J, Flannery JA et al (2004) Activation of α2 adrenergic receptors suppresses
fear conditioning: expression of c-Fos and phosphorylated CREB in mouse amygdala.
Neuropsychopharmacology 29:229–239
Fulton B, Wagstaff AJ, Sorkin EM (1995) Doxazosin. An update of its clinical pharmacology and
therapeutic applications in hypertension and benign prostatic hyperplasia. Drugs 49:295–320
Gaddum JH, Holzbauer M (1957) Adrenaline and noradrenaline. Vitam Horm 15:151–203. https://
doi.org/10.1016/S0083-6729(08)60510-5
Gauthier C, Tavernier G, Charpentier F et al (1996) Functional beta3-adrenoceptor in the human
heart. J Clin Invest 98:556–562
Gingrich JA, Caron MG (1993) Recent advances in the molecular biology of dopamine receptors.
Annu Rev Neurosci 16:299–321
Goldstein DS (2010) Adrenaline and noradrenaline. In: Encyclopedia of life sciences. Wiley,
Chichester, pp 1–9
Gomi T, Ikeda T, Ikegami F (1997) Beneficial effect of alpha-blocker on hemorheology in patients
with essential hypertension. Am J Hypertens 10:886–892
Graaf PH, Shankley NP, Black JW (1996) Analysis of the effects of α1-adrenoceptor antagonists on
noradrenaline-mediated contraction of rat small mesenteric artery. Br J Pharmacol
118:1308–1316
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 137

Graefe K-H, Bönisch H (1988) The transport of amines across the axonal membranes of noradren-
ergic and dopaminergic Neurones. In: Trendelenburg U, Weiner N (eds) Catecholamines
I. Handbook of experimental pharmacology. Springer, Berlin, pp 193–245
Gray K, Short J, Ventura S (2008) The alpha1A-adrenoceptor gene is required for the alpha1L-
adrenoceptor-mediated response in isolated preparations of the mouse prostate. Br J Pharmacol
155:103–109
Griffith RK (2003) Adrenergics and adrenergic-blocking agents. In: Burger’s medicinal chemistry
and drug discovery. Wiley, Chichester
Haapalinna A, Leino T, Heinonen E (2003) The alpha 2-adrenoceptor antagonist atipamezole
potentiates anti-parkinsonian effects and can reduce the adverse cardiovascular effects of
dopaminergic drugs in rats. Naunyn Schmiedeberg’s Arch Pharmacol 368:342–351
Hancock AA, Kyncl JJ, Martin YC, DeBernardis JF (1988) Differentiation of alpha-adrenergic
receptors using pharmacological evaluation and molecular modeling of selective adrenergic
agents. J Recept Res 8:23–46
Hickie JB (1970) Alprenolol (&aptin&) in angina pectoris. A double-blind multicentre trial. Med J
Aust 2:268–272
Hieble JP, Bylund DB, Clarke DE et al (1995) International Union of
Pharmacology. X. Recommendation for nomenclature of alpha 1-adrenoceptors: consensus
update. Pharmacol Rev 47:267–270
Hillarp N-Å, Hokfelt B (1955) Histochemical demonstration of noradrenaline and adrenaline in the
adrenal medulla. J Histochem Cytochem 3:1–5
Hom GJ, Forrest MJ, Bach TJ et al (2001) Beta(3)-adrenoceptor agonist-induced increases in
lipolysis, metabolic rate, facial flushing, and reflex tachycardia in anesthetized rhesus monkeys.
J Pharmacol Exp Ther 297:299–307
Horinouchi T, Tanaka Y, Koike K (2002) [Beta 3-adrenoceptor-mediated relaxation of Guinea-pig
gastric funds smooth muscle: cAMP-independent characteristics and a primary role of
4-aminopyridine-sensitive voltage-dependent K+ (Kv) channels]. Nihon Yakurigaku Zasshi
120:109P–111P
Hosoda C, Koshimizu T-A, Tanoue A et al (2004) Two 1-adrenergic receptor subtypes regulating
the vasopressor response have differential roles in Blood pressure regulation. Mol Pharmacol
67:912–922
Hunter JC, Fontana DJ, Hedley LR et al (1997) Assessment of the role of alpha2-adrenoceptor
subtypes in the antinociceptive, sedative and hypothermic action of dexmedetomidine in
transgenic mice. Br J Pharmacol 122:1339–1344
Ikeshita K, Nishikawa K, Toriyama S et al (2008) Landiolol has a less potent negative inotropic
effect than esmolol in isolated rabbit hearts. J Anesth 22:361–366
Inoue K, Kei Noguchi K, Masumoto M, Wakakura M (2011) Effect of five years of treatment with
nipradilol eye drops in patients with normal tension glaucoma. Clin Ophthalmol 5:1211–1216
Ishide T (2002) Denopamine, a selective beta1-receptor agonist and a new coronary vasodilator.
Curr Med Res Opin 18:407–413
Iversen LL (1971) Role of transmitter uptake mechanisms in synaptic neurotransmission. Br J
Pharmacol 41:571–591
Jaillon P, Drici M (1989) Recent antiarrhythmic drugs. Am J Cardiol 64:65J–69J
Janumpalli S, Butler LS, MacMillan LB et al (1998) A point mutation (D79N) of the alpha2A
adrenergic receptor abolishes the antiepileptogenic action of endogenous norepinephrine. J
Neurosci 18:2004–2008
Jesudason CD, Baker JE, Bryant RD et al (2011) Combination of a Beta adrenoceptor modulator
and a norepinephrine-serotonin uptake inhibitor for the treatment of obesity. ACS Med Chem
Lett 2:583–586
Jinushi K, Kushikata T, Kudo T et al (2018) Central noradrenergic activity affects analgesic effect
of neuropeptide S. J Anesth 32:48–53
Johnston JP (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue.
Biochem Pharmacol 17:1285–1297. https://doi.org/10.1016/0006-2952(68)90066-x
138 B. Gorain et al.

Kanagy NL (2005) Alpha(2)-adrenergic receptor signalling in hypertension. Clin Sci 109:431–437

Kaumann AJ (1996) ()-CGP 12177-induced increase of human atrial contraction through a
putative third beta-adrenoceptor. Br J Pharmacol 117:93–98
Kava MS, Blue DR, Vimont RL et al (1998) Alpha1L-adrenoceptor mediation of smooth muscle
contraction in rabbit bladder neck: a model for lower urinary tract tissues of man. Br J
Pharmacol 123:1359–1366
Khan ZP, Ferguson CN, Jones RM (1999) Alpha-2 and imidazoline receptor agonists. Their
pharmacology and therapeutic role. Anaesthesia 54:146–165
Knepper SM, Buckner SA, Brune ME et al (1995) A-61603, a potent alpha 1-adrenergic receptor
agonist, selective for the alpha 1A receptor subtype. J Pharmacol Exp Ther 274:97–103
Kobilka BK (2011) Structural insights into adrenergic receptor function and pharmacology. Trends
Pharmacol Sci 32:213–218
Kobilka B, Matsui H, Kobilka T et al (1987) Cloning, sequencing, and expression of the gene
coding for the human platelet alpha 2-adrenergic receptor. Science (80-) 238:650–656
Kohout TA, Lefkowitz RJ (2003) Regulation of G protein-coupled receptor kinases and Arrestins
during receptor desensitization. Mol Pharmacol 63:9–18
Lachnit WG, Tran AM, Clarke DE, Ford APDW (1997) Pharmacological characterization of an α
1A -adrenoceptor mediating contractile responses to noradrenaline in isolated caudal artery of
rat. Br J Pharmacol 120:819–826
Lafontan M, Berlan M (1980) Evidence for the α2 nature of the α-adrenergic receptor inhibiting
lipolysis in human fat cells. Eur J Pharmacol 66:87–93
Lafontan M, Barbe P, Galitzky J et al (1997) Adrenergic regulation of adipocyte metabolism. Hum
Reprod 12:6–20
Langlois M, Brémont B, Rousselle D, Gaudy F (1993) Structural analysis by the comparative
molecular field analysis method of the affinity of beta-adrenoreceptor blocking agents for
5-HT1A and 5-HT1B receptors. Eur J Pharmacol 244:77–87
Laurila JMM (2011) α2-adrenoceptors: structure and ligand binding properties at the molecular
level. University of Turku
Lefkowitz RJ (2000) The superfamily of heptahelical receptors. Nat Cell Biol 2:E133–E136
Lemke KA (2004) Perioperative use of selective alpha-2 agonists and antagonists in small animals.
Can Vet J 45:475–480
Lenders JWM, Pacak K, Walther MM et al (2002) Biochemical diagnosis of Pheochromocytoma.
JAMA 287:1427–1434
Liu JH, Dacus AC, Bartels SP (1991) Adrenergic mechanism in circadian elevation of intraocular
pressure in rabbits. Invest Ophthalmol Vis Sci 32:2178–2183
Lomasney JW, Lorenz W, Allen LF et al (1990) Expansion of the alpha 2-adrenergic receptor
family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene
for which is located on chromosome 2. Proc Natl Acad Sci 87:5094–5098
Lund-Johansen P, Omvik P (1991) Acute and chronic hemodynamic effects of drugs with different
actions on adrenergic receptors: a comparison between alpha blockers and different types of beta
blockers with and without vasodilating effect. Cardiovasc Drugs Ther 5:605–615
Ma YC, Huang XY (2002) Novel signaling pathway through the beta-adrenergic receptor. Trends
Cardiovasc Med 12:46–49
Marshall I, Burt RP, Chappie CR (1995) Noradrenaline contractions of human prostate mediated by
α1c-(α1c-) adrenoceptor subtype. Br J Pharmacol 115:781–786
Martí D, Miquel R, Ziani K et al (2005) Correlation between mRNA levels and functional role of
alpha1-adrenoceptor subtypes in arteries: evidence of alpha1L as a functional isoform of the
alpha1A-adrenoceptor. Am J Physiol Heart Circ Physiol 289:H1923–H1932
Martin CA, Advenier C (1995) Beta 3-adrenoceptors and airways. Fundam Clin Pharmacol
9:114–118
Mills K, Hausman N, Chess-Williams R (2008) Characterization of the alpha1-adrenoceptor
subtype mediating contractions of the pig internal anal sphincter. Br J Pharmacol 155:110–117
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 139

Morigny P, Houssier M, Mouisel E, Langin D (2016) Adipocyte lipolysis and insulin resistance.
Biochimie 125:259–266
Morilak DA, Barrera G, Echevarria DJ et al (2005) Role of brain norepinephrine in the behavioral
response to stress. Prog Neuropsychopharmacol Biol Psychiatry 29:1214–1224
Moriyama N, Nasu K, Takeuchi T et al (1997) Quantification and distribution of α 1 -adrenoceptor
subtype mRNAs in human vas deferens: comparison with those of epididymal and pelvic
portions. Br J Pharmacol 122:1009–1014
Morrow AL, Creese I (1986) Characterization of alpha 1-adrenergic receptor subtypes in rat brain: a
reevaluation of [3H]WB4104 and [3H]prazosin binding. Mol Pharmacol 29:321–330
Mottram AR, Erickson TB (2009) Toxicology in emergency cardiovascular care. In: Field JM,
Kudenchuk PJ, O’Connor R, Terry V (eds) The textbook of emergency cardiovascular care and
CPR. Lippincott Williams & Wilkins, Philadelphia, pp 443–452
Müller P, Schier AF (2011) Extracellular movement of signaling molecules. Dev Cell 21:145–158
Muramatsu I, Ohmura T, Kigoshi S et al (1990) Pharmacological subclassification of α1-
adrenoceptors in vascular smooth muscle. Br J Pharmacol 99:197–201
Nagatomo T, Hosohata Y, Ohnuki T et al (2001) Bopindolol: pharmacological basis and clinical
implications. Cardiovasc Drug Rev 19:9–24
Nakajima D, Negoro N, Nakaboh A et al (2006) Effectiveness of low dose denopamine, a β1-
adrenoceptor agonist, in a patient with vasospastic angina refractory to intensive medical
treatment. Int J Cardiol 108:281–283
Nakamura S, Taniguchi T, Suzuki F et al (1999) Evaluation of alpha1-adrenoceptors in the rabbit
iris: pharmacological characterization and expression of mRNA. Br J Pharmacol
127:1367–1374
Nguyen V, Tiemann D, Park E, Salehi A (2017) Alpha-2 Agonists. Anesthesiol Clin 35:233–245
Nicholas AP, Pieribone V, Hökfelt T (1993) Distributions of mRNAs for alpha-2 adrenergic
receptor subtypes in rat brain: an in situ hybridization study. J Comp Neurol 328:575–594
Nishio R, Matsumori A, Shioi T et al (1998) Denopamine, a beta1-adrenergic agonist, prolongs
survival in a murine model of congestive heart failure induced by viral myocarditis: suppression
of tumor necrosis factor-alpha production in the heart. J Am Coll Cardiol 32:808–815
Noble AJ, Chess-Williams R, Couldwell C et al (1997) The effects of tamsulosin, a high affinity
antagonist at functional α 1A - and α 1D -adrenoceptor subtypes. Br J Pharmacol 120:231–238
Nuttall SL, Routledge HC, Kendall MJ (2003) A comparison of the beta1-selectivity of three beta1-
selective beta-blockers. J Clin Pharm Ther 28:179–186
Nyrönen T, Pihlavisto M, Peltonen JM et al (2001) Molecular mechanism for agonist-promoted
alpha(2A)-adrenoceptor activation by norepinephrine and epinephrine. Mol Pharmacol
59:1343–1354
Ohmura T, Oshita M, Kigoshi S, Muramatsu I (1992) Identification of α1-adrenoceptor subtypes in
the rat vas deferens: binding and functional studies. Br J Pharmacol 107:697–704
Okajima M, Takamura M, Taniguchi T (2015) Landiolol, an ultra-short-acting β1-blocker, is useful
for managing supraventricular tachyarrhythmias in sepsis. World J Crit Care Med 4:251–257
Oliver G, Schäfer EA (1895) The physiological effects of extracts of the suprarenal capsules. J
Physiol 18:230–276. https://doi.org/10.1113/jphysiol.1895.sp000564
Palluk R, Hoefke W, Gaida W et al (1986) Interactions of MEN 935 (adimolol), a long acting beta-
and alpha-adrenolytic antihypertensive agent, with postsynaptic alpha-adrenoceptors in differ-
ent isolated blood vessels--influence of angiotensin II. Naunyn Schmiedeberg’s Arch Pharmacol
333:277–283
Parker K, Brunton L, Goodman LS et al (2008) Goodman and Gilman’s manual of pharmacology
and therapeutics. McGraw-Hill Medical, New York, NY
Perez DM, Piascik MT, Graham RM (1991) Solution-phase library screening for the identification
of rare clones: isolation of an alpha 1D-adrenergic receptor cDNA. Mol Pharmacol 40:876–883
Perez DM, Piascik MT, Malik N et al (1994) Cloning, expression, and tissue distribution of the rat
homolog of the bovine alpha 1C-adrenergic receptor provide evidence for its classification as the
alpha 1A subtype. Mol Pharmacol 46:823–831
140 B. Gorain et al.

Philipp M, Brede M, Hein L (2002) Physiological significance of α 2 -adrenergic receptor subtype

diversity: one receptor is not enough. Am J Physiol Integr Comp Physiol 283:R287–R295
Piascik MT, Kusiak JW, Barron KW (1990) Alpha 1-adrenoceptor subtypes and the regulation of
peripheral hemodynamics in the conscious rat. Eur J Pharmacol 186:273–278
Pickering TG, Levenstein M, Walmsley P (1994) Nighttime dosing of doxazosin has peak effect on
morning ambulatory Blood pressure. Am J Hypertens 7:844–847
Pringle TH, Francis RJ, East PB, Shanks RG (1986) Pharmacodynamic and pharmacokinetic
studies on bufuralol in man. Br J Clin Pharmacol 22:527–534
Prokai L, Wu WM, Somogyi G, Bodor N (1995) Ocular delivery of the beta-adrenergic antagonist
alprenolol by sequential bioactivation of its methoxime analogue. J Med Chem 38:2018–2020
Ramsay D, Carr IC, Pediani J et al (2004) High-affinity interactions between human
1A-adrenoceptor C-terminal splice variants produce Homo- and heterodimers but do not
generate the 1L-adrenoceptor. Mol Pharmacol 66:228–239
Rang HP, Dale MM, Ritter J et al (1999) Rang and Dale’s pharmacology, 5th edn. Churchill
Livingstone, Edinburgh
Recio P, Orensanz LM, Martínez MP et al (2008) Noradrenergic vasoconstriction of pig prostatic
small arteries. Naunyn Schmiedeberg’s Arch Pharmacol 376:397–406
Regan JW, Kobilka TS, Yang-Feng TL et al (1988) Cloning and expression of a human kidney
cDNA for an alpha 2-adrenergic receptor subtype. Proc Natl Acad Sci U S A 85:6301–6305
Ross S, Rorabaugh BR, Chalothorn D et al (2003) The α1B-adrenergic receptor decreases the
inotropic response in the mouse Langendorff heart model. Cardiovasc Res 60:598–607
Ruffolo RR, Gellai M, Hieble JP et al (1990) The pharmacology of carvedilol. Eur J Clin Pharmacol
38(Suppl 2):S82–S88
Sakuma T, Hida M, Nambu Y et al (2001) Beta1-adrenergic agonist is a potent stimulator of
alveolar fluid clearance in hyperoxic rat lungs. Jpn J Pharmacol 85:161–166
Schena G, Caplan MJ (2019) Everything you always wanted to know about β3-AR  ( but were
afraid to ask). Cell 8:357
Schramm NL, McDonald MP, Limbird LE (2001) The alpha(2a)-adrenergic receptor plays a
protective role in mouse behavioral models of depression and anxiety. J Neurosci 21:4875–4882
Schwinn DA, Lomasney JW, Lorenz W et al (1990) Molecular cloning and expression of the cDNA
for a novel alpha 1-adrenergic receptor subtype. J Biol Chem 265:8183–8189
Shibata K, Hirasawa A, Moriyama N et al (1996) Alpha 1a-adrenoceptor polymorphism: pharma-
cological characterization and association with benign prostatic hypertrophy. Br J Pharmacol
118:1403–1408
Shorr RGL, McCaslin DR, Strohsacker MW et al (1985) Molecular structure of the beta-adrenergic
receptor. Biochemistry 24:6869–6875
Skeberdis VA (2004) Structure and function of beta3-adrenergic receptors. Medicina (Kaunas)
40:407–413
Smith KM, Macmillan JB, McGrath JC (1997) Investigation of alpha1-adrenoceptor subtypes
mediating vasoconstriction in rabbit cutaneous resistance arteries. Br J Pharmacol 122:825–832
Spence K, Hunter S, Brown C et al (2018) The role of plasma metanephrines and plasma
catecholamines in the biochemical testing for Pheochromocytoma. Endocr Abstr 59:P018
Stanaszek WF, Kellerman D, Brogden RN, Romankiewicz JA (1983) Prazosin update a review of
its pharmacological properties and therapeutic use in hypertension and congestive heart failure.
Drugs 25:339–384
Starke K, Gothert M, Kilbinger H (1989) Modulation of neurotransmitter release by presynaptic
autoreceptors. Physiol Rev 69:864–989
Steinle JJ, Booz GW, Meininger CJ et al (2003) β 3 -adrenergic receptors regulate retinal endothelial
cell migration and proliferation. J Biol Chem 278:20681–20686
Steinle JJ, Zamora DO, Rosenbaum JT, Granger HJ (2005) β3-adrenergic receptors mediate
choroidal endothelial cell invasion, proliferation, and cell elongation. Exp Eye Res 80:83–91
Stone EA, Quartermain D (1999) Alpha-1-noradrenergic neurotransmission, corticosterone, and
behavioral depression. Biol Psychiatry 46:1287–1300
4 Pharmacology of Adrenaline, Noradrenaline, and Their Receptors 141

Strosberg AD (1993) Structure, function, and regulation of adrenergic receptors. Protein Sci
2:1198–1209. https://doi.org/10.1002/pro.5560020802
Summers RJ, Papaioannou M, Harris S, Evans BA (1995) Expression of beta 3-adrenoceptor
mRNA in rat brain. Br J Pharmacol 116:2547–2548
Suryanarayana S, von Zastrow M, Kobilka BK (1992) Identification of intramolecular interactions
in adrenergic receptors. J Biol Chem 267:21991–21994
Szentirmai É, Kapás L (2017) The role of the brown adipose tissue in β3-adrenergic receptor
activation-induced sleep, metabolic and feeding responses. Sci Rep 7:958
Szymonowicz L (1896) Die Function der Nebenniere. Pflüger, Arch 64:97–164. https://doi.org/10.
1007/BF01661663
Tagaya E, Tamaoki J, Takemura H et al (1999) Atypical adrenoceptor-mediated relaxation of
canine pulmonary artery through a cyclic adenosine monophosphate-dependent pathway. Lung
177:321–332
Takamine J (1902) The blood-pressure raising principle of the suprarenal gland. JAMA
XXXVIII:153–155. https://doi.org/10.1001/jama.1902.62480030011001c
Tamaoki J, Yamauchi F, Chiyotani A et al (1993) Atypical beta-adrenoceptor- (beta
3-adrenoceptor) mediated relaxation of canine isolated bronchial smooth muscle. J Appl Physiol
74:297–302
Teng C-M, Guh J-H, Ko F-N (1994) Functional identification of α1-adrenoceptor subtypes in
human prostate: comparison with those in rat vas deferens and spleen. Eur J Pharmacol
265:61–66
Tran TM, Friedman J, Qunaibi E et al (2004) Characterization of agonist stimulation of cAMP-
dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the
2-adrenergic receptor using phosphoserine-specific antibodies. Mol Pharmacol 65:196–206
Trendelenburg U (1991) The TiPS lecture: functional aspects of the neuronal uptake of noradrena-
line. Trends Pharmacol Sci 12:334–337
Vargas HM, Gorman AJ (1995) Vascular alpha-1 adrenergic receptor subtypes in the regulation of
arterial pressure. Life Sci 57:2291–2308
Vaughan Williams EM (1987) Bevantolol: a beta-1 adrenoceptor antagonist with unique additional
actions. J Clin Pharmacol 27:450–460
Villalobos-Molina R, López-Guerrero JJ, Ibarra M (1997) Alpha 1D- and alpha 1A-adrenoceptors
mediate contraction in rat renal artery. Eur J Pharmacol 322:225–227
Vinay HK, Paul A, Goswami SS, Santani D (2002) Effect of SR 58611A, a beta-3 receptor agonist,
against experimental gastro-duodenal ulcers. Indian J Physiol Pharmacol 46:36–44
Wada Y, Aiba T, Tsujita Y et al (2016) Practical applicability of landiolol, an ultra-short-acting β1-
selective blocker, for rapid atrial and ventricular tachyarrhythmias with left ventricular dysfunc-
tion. J Arrhythm 32:82–88
Wallukat G (2002) The β-adrenergic receptors. Herz 27:683–690
Warne T, Serrano-Vega MJ, Baker JG et al (2008) Structure of a beta1-adrenergic G-protein-
coupled receptor. Nature 454:486–491
Warren K, Burden H, Abrams P (2016) Mirabegron in overactive bladder patients: efficacy review
and update on drug safety. Ther Adv drug Saf 7:204–216
Wassall RD, Teramoto N, Cunnane TC (2009) Noradrenaline. In: Encyclopedia of Neuroscience.
Academic Press, Cambridge, pp 1221–1230
Webb DJ, Fulton JD, Leckie BJ et al (1987) The effect of chronic prazosin therapy on the response
of the renin-angiotensin system in patients with essential hypertension. J Hum Hypertens
1:195–200
Wei L, Zhu Y-M, Zhang Y-X et al (2016) The α1 adrenoceptors in ventrolateral orbital cortex
contribute to the expression of morphine-induced behavioral sensitization in rats. Neurosci Lett
610:30–35
142 B. Gorain et al.

Westlund KN, Denney RM, Rose RM, Abell CW (1988) Localization of distinct monoamine
oxidase a and monoamine oxidase b cell populations in human brainstem. Neuroscience
25:439–456
White WB, Moon T (2005) Treatment of benign prostatic hyperplasia in hypertensive men. J Clin
Hypertens 7:212–217
Woodcock EA (2007) Roles of α1A- and α1B-adrenoceptors in heart: insights from studies of
genetically modified mice. In: Clinical and experimental pharmacology and physiology. Wiley,
Chichester
Wurtman RJ, Axelrod J (1966) Control of enzymatic synthesis of adrenaline in the adrenal medulla
by adrenal cortical steroids. J Biol Chem 241:2301–2305
Xie F, Petitti D, Chen W (2005) Prescribing patterns for antihypertensive drugs after the antihyper-
tensive and lipid-lowering treatment to prevent heart attack trial: report of experience in a health
maintenance organization. Am J Hypertens 18:464–469
Yu GS, Han C (1994) Role of alpha 1A- and alpha 1B-adrenoceptors in phenylephrine-induced
positive inotropic response in isolated rat left atrium. J Cardiovasc Pharmacol 24:745–752
Zhao T-J, Sakata I, Li RL et al (2010) Ghrelin secretion stimulated by 1-adrenergic receptors in
cultured ghrelinoma cells and in fasted mice. Proc Natl Acad Sci 107:15868–15873
Zuscik MJ, Chalothorn D, Hellard D et al (2001) Hypotension, autonomic failure, and cardiac
hypertrophy in transgenic mice overexpressing the α 1B -adrenergic receptor. J Biol Chem
276:13738–13743
Pharmacology of Dopamine and Its
Receptors 5
Sunpreet Kaur, Shamsher Singh, Gagandeep Jaiswal,
Sandeep Kumar, Wafa Hourani, Bapi Gorain, and Puneet Kumar

Abstract

Dopamine (DA) is the major catecholamine neurotransmitter in the brain which

regulates multiple functions including the control over voluntary action, reward,
circadian rhythm, consciousness, and cognition. The synthesis of DA involves
two events, i.e. hydroxylation of L-tyrosine to DOPA catalysed by tyrosine
hydroxylase (TH) and further, DOPA gets decarboxylated to final product DA
via aromatic L-amino acid decarboxylase (AADC) enzyme. Metabolism involves
monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT)
enzymes, which degrade dopamine finally into 3,4-Dihydroxyphenylacetic acid
(DOPAC) and homovanillic acid (HVA). DA acts through two different
subclasses of receptors including D1-like (D1 and D5) and D2-like (D2, D3
and D4) dopamine receptors. Dopamine performs various functions through its
receptors like regulation of growth, reward, sleep, locomotion, emotions, renal
functions, gastrointestinal motility, etc. Furthermore, dopaminergic system plays

S. Kaur · S. Singh (*)

Neuroscience Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab,
India
G. Jaiswal · S. Kumar
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India
W. Hourani
Faculty of Pharmacy, Philadelphia University, Amman, Jordan
B. Gorain
School of Pharmacy, Faculty of Health and Medical Science, Taylor’s University, Subang Jaya,
Selangor, Malaysia
P. Kumar
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India

# Springer Nature Singapore Pte Ltd. 2020 143

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_5
144 S. Kaur et al.

an important role in the pathogenesis of various neurological diseases like

Parkinson’s disease (PD), Huntington’s diseases (HD), Alzheimer’s disease
(AD), schizophrenia, anxiety, epilepsy, traumatic brain injury (TBI), and multiple
sclerosis (MS). Also, high pace discoveries that occurred in the research field
pave the way for recent advancements in the dopaminergic system. Currently,
with the help of molecular cloning, two D1-like and three D2-like receptor genes
have been successfully identified. In the current chapter, various roles of dopa-
mine and dopaminergic receptors have been highlighted but there is still a need to
understand a lot of functions and specific roles of the receptors. Hence, the high
pace of research along with newly developed advancements in the field of
neuroscience and pharmacology will be useful to get more knowledge about
dopamine receptor signalling in devastating disorders.

Keywords

Catecholamines · Dopaminergic receptors · Circadian rhythm · Parkinson’s

disease · Huntington’s disease · Alzheimer’s disease · Schizophrenia · Anxiety ·
Epilepsy · Traumatic brain injury · Multiple sclerosis

Abbreviations

5-HT 5-Hydroxytryptamine
AADC Aromatic amino acid decarboxylase
AC Adenyl cyclase
ACC Anterior cingulated
AD Alzheimer’s disease
ADH Alcohol dehydrogenase
ALDH Aldehyde dehydrogenase
ALLO Allopregnanolone
AMP Adenosine monophosphate
AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
BLA Basolateral amygdala
cAMP Cyclic adenosine monophosphate
COMT Catechol-O-methyl-transferase
CREB cAMP response element binding protein
D1R D1 receptor
D5R D5 receptor
DA Dopamine
DARPP-32 Dopamine- and cAMP-regulated neuronal phosphoprotein
DAT Dopamine transporters
DHX Dihydrexidine
DOPA Dihydroxyphenylalanine
DOPAC 3,4-Dihydroxyphenylacetic acid
DOPAL 3,4-Dihydroxyphenylacetaldehyde
5 Pharmacology of Dopamine and Its Receptors 145

DOPET 3,4-Dihydroxyphenylethanol
DSM-IV Diagnostic and Statistical Manual of Mental Disorders
EPS Extrapyramidal symptoms
FAD Flavin adenine dinucleotide
GABA Gamma-aminobutyric acid
GAD Generalized anxiety disorder
GPCRs G-protein-coupled receptors
GPe External globus pallidus
GPi Globus pallidus internal
Gαi Gi alpha subunit
Gαs Gs alpha subunit
HD Huntington’s disease
HVA Homovanillic acid
L-DOPS L-Dihydroxyphenylserine
MAO Monoamino oxidase
MAPK Mitogen-activated protein kinase
MS Multiple sclerosis
MSN Medium spiny neuron
NAc Nucleus accumbens
NMDA N-methyl-D-aspartate receptor
O2 Molecular oxygen
OB Olfactory bulb
OCC Orbitofrontal cortical
OCD Obsessive compulsive disorder
PD Parkinson’s disease
PFC Prefrontal cortex
PKA Protein kinase A
PKC Protein kinase C
PP1 Protein phosphatase
PRMS Progressive relapsing multiple sclerosis
PTSD Post-traumatic stress disorder
RNA Ribose nucleic acid
RRMS Relapsing remitting multiple sclerosis
SCZ Schizophrenia
SNpc Substantia nigra pars compacta
SNpr Substantia nigra pars reticulata
SPD Sensory processing disorder
SPMS Secondary progressive multiple sclerosis
STN Subthalamic nucleus
TBI Traumatic brain injury
TBZ Tetrabenazine
TH Tyrosine hydroxylase
VMAT2 Vesicular monoamine transporter 2
VTA Ventral tegmental area
146 S. Kaur et al.

5.1 Introduction

Dopamine is the major catecholamine neurotransmitter in the brain. It regulates

a variety of functions including the control over voluntary action, reward, circadian
rhythm, consciousness, and cognition (Hasbi et al. 2011). Indeed, it is a neurotrans-
mitter that controls emotions, motor, and mental functions of the brain. The adequate
balance of dopamine assures a state of happiness while overexcitation of dopamine
system leads to manic and psychotic responses. Dopamine plays a major role in
controlling various brain functions including voluntary movement. It not only
participates in movements but also predominantly regulates cognition, conscious-
ness, and addiction. The role of dopamine is unique and complex as dopamine serves
as a key unit in the reward system of the brain. The reward system provides the
evidence regarding drug addiction, dependence, and reinforcement. To understand
the functions of dopamine, it is necessary to understand the widespread distribution
of dopamine neurons and its projections in the brain.
The dopaminergic neurons are specifically localized in the midbrain and its
associated regions including substantia nigra pars compacta, the ventral tegmental
area, and the retrorubral field like regions (Haber 2016). The pathways formed by
these projections are known as nigrostriatal, mesolimbic, mesocortical, and
tuberoinfundibular pathways which individually coordinate distinct functions
like nigro for substantia nigra and striatal for striatum; this pathway initiates from
substantia nigra and terminates in dorsal striatum. The dopaminergic neurons of
these pathways coordinate motor control, thereby implicit in motor coordination
disorders like Parkinson’s disease (PD) and Huntington’s disease (HD). Similarly
mesolimbic pathway as the name suggests originates from the ventral tagmented
area of midbrain and terminates towards ventral striatum and its nearby regions like
nucleus accumbens, olfactory tubercle, amygdala, and hippocampus. The function
of these pathways remains motivation, drug addiction, apathy, and psychiatric
illness, so it is also well known as a reward pathway. Further, the mesocortical
pathway travels from the ventral tegmental area to the prefrontal cortex (PFC) and
other cortical areas and known to coordinate the executive functions including
cognition, motivation, and emotional responses (Jučaitė 2002). This pathway usually
associates with the pathogenesis of schizophrenia and also with behavioural
symptoms in Alzheimer’s disease (AD), HD, and PD. At last, the tuberoinfundibular
pathway originates from the arcuate nucleus of the hypothalamus and ends in
medium eminence of the pituitary gland. It regulates the secretion of prolactin
from anterior pituitary gland so implicit in menstrual disorders and other sexual
disorders. In this way, dopamine is broadly distributed in the brain to mediate a
variety of functions in neurodegenerative and psychiatric disorders.

5.2 History

Historical development in the discovery of dopamine and its receptor is provided in

Table 5.1.
5 Pharmacology of Dopamine and Its Receptors 147

Table 5.1 History of dopamine discovery

Year Important events Reference
1910 Synthesized dopamine at Wellcome labs in London, England Barger and
Dale (1910)
1911 Synthesized L-dopa Hornykiewicz
(2002)
1952 Current name of dopamine suggested Barger and
Dale (1910)
1950 Function of dopamine was discovered as a neurotransmitter at the Carlsson et al.
Laboratory for Chemical Pharmacology of the National Heart (1962)
Institute of Sweden
1956 Effect of dopamine on blood pressure had found Hornykiewicz
(1958)
1957–59 Dopamine as a neurotransmitter in the brain was shown and it Carlsson (1959)
was found that the highest regional concentration existed in the
basal ganglia and in the striatum in high concentrations
1959 It was found that dopamine is responsible for PD Hornykiewicz
(2006)
1960 The enzyme tyrosine hydroxylase that converts L-tyrosine to Nagatsu et al.
L-dopa was discovered (1964)
1965 Dopamine has the potential to excite or inhibit neurons Bloom et al.
(1965)
1966 Dopamine hypothesis of schizophrenia Van Rossum
(1967)
1972 The existence of dopamine receptors was revealed Brown and
Makman
(1972)
1976 Two dopamine receptors proposed: inhibitory and excitatory Cools and Van
Rossum (1976)
1978 Two dopamine receptors: coupled and uncoupled to adenylate Spano et al.
cyclase (1978)
1979 Names of D1 and D2 used Kebabian and
Calne (1979)
1990–91 Dopamine D1 and D5 receptors were cloned Sunahara et al.
(1990)
1990 Dopamine D3 receptor was cloned Sokoloff et al.
(1990)
1991 Dopamine D4 receptor was cloned Van Tol et al.
(1991)
1992 D2 receptor is more than 80% block by the antipsychotics Farde et al.
associated with parkinsonism (1992)
1996 Amphetamine-induced release of dopamine is higher in Laruelle et al.
schizophrenia (1996)
1998 D2 short receptors located mostly in nigral neurones Khan et al.
(1998)
1999 Therapeutic doses of antipsychotics block 60–80% D2 Kapur and
Mamo (2003)
(continued)
148 S. Kaur et al.

Table 5.1 (continued)

Year Important events Reference
2003 Antipsychotics occupy more D2 in limbic areas than striatum Bressan et al.
(2003)
2005 Dopamine supersensitivity correlates with elevated D2 high Seeman et al.
states (2005)
2005 Higher D2 density in healthy identical twins of schizophrenia Hirvonen et al.
patients (2006)
2006 Markedly elevated D2 high receptors in all animal models of Seeman et al.
psychosis (2006)
2008 Dopamine neurons implanted into people with Parkinson’s Mendez et al.
disease survive without pathology for 14 years (2008)
2011 Dopamine neurons derived from human ES cells efficiently Kriks et al.
engraft in animal models of Parkinson’s disease (2011)
2013 Dopamine modulates reward-related vigour Beierholm et al.
(2013)
2014 Protective and toxic roles of dopamine in Parkinson’s disease Segura-Aguilar
et al. (2014)
2016 Unexpected rewards induce dopamine-dependent positive Perry et al.
emotion–like state changes in bumblebees (2016)
2018 Activity of dopamine neurons is more closely associated with the Wei et al.
drug’s reinforcing property (2018)
2019 Dorsal striatum dopamine levels fluctuate across the sleep–wake Dong et al.
cycle and respond to salient stimuli in mice (2019)

5.3 Structure of Dopamine

Chemically, dopamine is a catecholamine consisting of a catechol ring and one

ethylamine group. Various substituted phenethylamine analogues have been formed
leading to the development of many therapeutic drugs including agonists and
antagonists as shown in Fig. 5.1.
Dopamine behaves like an organic base which becomes protonated in the acidic
environment like other amines. Water solubility and stability occur relatively in
protonated form and is degraded when exposed to oxygen or other oxidants. Only in
acidic environment dopamine is protonated but in basic form it is highly reactive and
less soluble in water. Dopamine hydrochloride is a fine colourless powder in dry
form which is used in various pharmaceutical applications.

Fig. 5.1 Structure of

dopamine
5 Pharmacology of Dopamine and Its Receptors 149

5.4 Synthesis, Reuptake, and Metabolism of Dopamine

The classical pathway of DA biosynthesis was initially stated by Blaschko in the late
1930s. It involves two events, proceed with hydroxylation in L-tyrosine to
dihydroxyphenylalanine (DOPA) catalysed by tyrosine hydroxylase (TH) in
the presence of tetrahydrobiopterin, O2, and iron (Fe2+) as cofactors (Juárez Olguín
et al. 2016). Further, DOPA gets decarboxylated to final product DA via enzyme
aromatic amino acid decarboxylase (AADC) where pyridoxal phosphate (PP) is
present as a cofactor. The tyrosine utilized in this pathway formed by the enzyme
phenylalanine hydroxylase in the presence of molecular oxygen (O2) and
tetrahydrobiopterin as cofactors (Meiser et al. 2013) (Fig. 5.2). Two other alternative
pathways of dopamine synthesis can be described in the form of flow charts as
shown in Figs. 5.3 and 5.4.
After formation, dopamine gets readily sequestered inside the synaptic vesicles
through vesicular monoamine transporter 2 (VMAT-2) and remains stored under
slightly acidic pH to prevent oxidation of dopamine. This vesicular transport irre-
versibly inhibited by reserpine and amphetamine like drugs (Meiser et al. 2013).
Further upon excitation of dopaminergic neurons, synaptic vesicles get
degranulated, hence releasing dopamine in the synapse where it interacts with
dopamine receptors for subsequent actions and dopamine transporters (DAT)
remove excess of dopamine from synapse.

Fig. 5.2 Dopamine biosynthesis in the brain

150 S. Kaur et al.

Fig. 5.3 First alternative Tyrosine

pathway
Tyrosinase (converts only during melanin
synthesis)

L-DOPA

Dopamine

Fig. 5.4 Second alternative Tyrosine

pathway

Tyramine

Hydroxylation by
CYP2D

Dopamine (very
low quantity)

Reuptake is mediated either by the dopamine transporter or by the plasma

membrane monoamine transporter. Once it comes back in the cytosol, dopamine
can either be broken down by a monoamine oxidase (MAO) or repackaged into
vesicles by vesicular monoamine transporter 2 (VMAT-2), making it available for
future release.
The remaining excess of dopamine is degraded by enzymatic action. The
enzyme MAO degrades dopamine to hydrogen peroxide and
3,4-dihydroxyphenylacetaldehyde (DOPAL) in the presence of flavin adenine dinu-
cleotide (FAD). This DOPAL either gets frequently oxidized by aldehyde dehydro-
genase (ALDH) or reduced by the action of alcohol dehydrogenase (ADH) to
3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol
(DOPET). The enzyme MAO exists in two types: MAO-A and MAO-B that reside
in the outer mitochondrial membrane in neurons, microglial cells, and astrocytes.
Even glial cells degrade dopamine by MAO or catechol-O methyltransferase
(COMT). COMT, in Mg2+-dependent manner, converts the DOPAC to homovanillic
acid (HVA) but the activity of COMT is seen only in glial cells and not in striatal
neuronal cells (Meiser et al. 2013). This way, well-regulated machinery works to
maintain an adequate level of dopamine in the brain through highly controlled
synthesis, reuptake, and degradation mechanism as shown in Fig. 5.5.
5 Pharmacology of Dopamine and Its Receptors 151

Fig. 5.5 Metabolism of dopamine

5.5 Dopamine (DA) Receptors and Their Localization

The physiological actions of dopamine are mediated by its interaction with dopa-
mine receptors that are localized over dopaminergic synapses. There are five types of
dopamine receptors categorized into two different subclasses including D1-like and
D2-like dopamine receptors (Beaulieu et al. 2015). The D1-like class includes D1
and D5 receptors (D1R and D5R), whereas D2-like class constitutes of D2, D3, and
D4 receptors (D2R, D3R, and D4R). Both classes of receptors differ in their
mechanism of action. The D1-like receptors act by coupling to Gαs that causes
adenylyl cyclase (AC) activation for cAMP production while D2-like couples to
Gαi to negatively regulate AC. Here, the D1-like receptors are found to be expressed
only postsynaptically but D2-like receptors are expressed both presynaptically and
postsynaptically.
The D1 receptors are distributed over nigrostriatal, mesolimbic, and mesocortical
areas, like the caudate-putamen (striatum), substantia nigra, olfactory bulb, amyg-
dala and at small level in the hippocampus, cerebellum, thalamic areas, and hypo-
thalamic areas (Rangel-Barajas et al. 2015). On the other side, D5 receptors are
found to be expressed at a very low level in the prefrontal cortex, premotor cortex,
cingulated cortex, entorhinal cortex, substantia nigra, hypothalamus, hippocampus,
and dentate gyrus. A small proportion is also reported over medium spiny neurons of
the caudate nucleus and nucleus accumbens.
152 S. Kaur et al.

The D2 receptors are expressed on the striatum, the substantia nigra, ventral
tegmental area, hypothalamus, cortical areas, septum, amygdala, hippocampus, the
nucleus accumbens, and the olfactory tubercle like regions (Rangel-Barajas et al.
2015). The distribution of D3 receptor is considered smaller in the limbic areas,
striatum, the substantia nigra, pars compacta, the ventral tegmental area, hippocam-
pus, septal area, and various cortical areas. Low expression of D4 receptor is
reported upon the frontal cortex, amygdala, hippocampus, hypothalamus, globus
pallidus and substantia nigra pars reticulata (Rangel-Barajas et al. 2015). Apart from
this, D1, D2, and D4 receptors have been reported in retina whereas D2 receptors
are detected in pituitary gland.

5.5.1 D1-Like Dopamine Receptor Expression

The striatum or caudo-putamen, nucleus accumbens (NAc), SN pars reticulata

(SNpr), and olfactory bulb (OB) consist of a higher amount of D1-like receptor
family. The D1-like receptor family includes the D1 and D5 receptors that are G-
protein-coupled receptors (GPCRs) (Mishra et al. 2018). Entopeduncular nucleus,
cerebral aqueduct, and ventricles have moderate expression while the dorsolateral
prefrontal cortex, cingulated cortex, and hippocampus show lower expression of D1
receptors (Lud Cadet et al. 2010). This receptor plays a pivotal role in the reward
system regulation, locomotor activity, learning and memory (Arias-Carrión et al.
2010). The adenylyl cyclase (AC) stimulation is induced by D1 receptors and results
in the activation of guanosine nucleotide-binding proteins (G proteins) and produc-
tion of cyclic AMP as a secondary messenger. Typically, signal transduction
pathways are involved in different neuropsychiatric disorders causing activating
the phospholipase C and inducing intracellular calcium release due to the involve-
ment of D1 receptors. Usually, calcium is involved in the modulation of neurotrans-
mitter release by exocytosis and regulating signalling pathway that causes the
activation of proteins, such as calcium-dependent protein kinase C (PKC)
(Ha et al. 2012). The electrochemical gradient is regulated by the Na+K+-ATPase
and activation of this pump is inhibited through D1 receptors by protein kinase A
(PKA) and PKC signalling pathways in the striatum (Pivovarov et al. 2019) and the
kidney (Arnaud-Batista et al. 2016) (Fig. 5.6).

5.5.2 D2-Like Dopamine Receptor Expression

It includes subfamilies of D2, D3, and D4, whereas D2R subtypes possess
2 isoforms: the D2-short and D2-long type receptors. Most brain regions like
striatum, external globus pallidus (GPe), amygdala, cerebral cortex, hippocampus,
and pituitary possess D2 receptor and its subtypes (Mishra et al. 2018). Usually the
prefrontal, temporal, and entorhinal cortex, and the septal region along with the VTA
and SNpc of DAergic neurons show the expression of messenger RNA D2R
(Villanueva 2015). The activity of AC and the production of cAMP levels and
5 Pharmacology of Dopamine and Its Receptors 153

Fig. 5.6 Regulation of the signalling networks by DA in D1-like receptors, D2-like receptors, and
D1-D2 receptor heteromers. DA D1-like family and D2-like family receptor homodimers signal
through Gαs/olf and Gαi/o protein to regulate cyclic AMP through adenylyl cyclase (AC) activity.
D1-like receptors activate AC through Gαs/olf, thereby increasing intracellular cAMP and
stimulating PKA. D2-like receptors inhibit AC through Gαi/o, thereby suppressing cAMP and
inhibiting PKA. The DA D1-D2 receptor heterodimer signals through Gαq, phospholipase C,
enhancing the production of IP3, mobilization of intracellular calcium and of DAG with subsequent
activation of PKC

PKA are inhibited by this class of receptors (Zhang et al. 2006). Behavioural and
extrapyramidal activities are mediated by the D2-type postsynaptic receptor. D2
receptors are known as autoreceptors acting via somato dendritic auto-receptors,
which are known to decrease neuronal excitability (Chiodo and Kapatos 1992;
Lacey et al. 1987) or inhibit dopamine release by terminal auto-receptors, which
mostly reduce DA synthesis and packaging (Onali et al. 1988; Pothos et al. 1998).
DA neuronal development is due to D2 auto-receptor during the embryonic stage
(Baik 2013) and mediate various responses like cell proliferation–related pathways,
such as the mitogen-activated protein kinase (MAPK) (Yoon and Baik 2013) and
Akt (thymoma viral proto-oncogene also knows as protein kinase B) signalling
pathways (Collo et al. 2013) (Table 5.2).

5.6 Function of Dopamine Receptors

The dopamine receptors are widespread across the brain and their crucial localization
makes them play a variety of actions. The presynaptic localization of D2 auto-
receptors inhibits dopamine release that decreases locomotor activity. On the other
 154

Table 5.2 Dopamine receptors

Receptors D1 D2 D3 D4 D5
Types Gs-couple Gi-couple Gi-couple Gi-couple Gs-couple
Mechanism Increased intracellular level of Increased intracellular Adenylate cyclase is Adenylate cyclase is Adenylate
cAMP by activated adenylate level of cAMP by decreased decreased cyclase is increased
cyclase activating adenylate
cyclase
Location Striatum, nucleus accumbens, Striatum, VTA Striatum, VTA Frontal cortex, Cortex, substantia
olfactory bulb, amygdala olfactory bulb, cerebral olfactory bulb, cerebral amygdala, nigra, hypothalamus
hippocampus, substantia nigra, cortex cortex hypothalamus,
hypothalamus, frontal cortex nucleus
accumbens
Function Locomotion, learning and Locomotion, learning Locomotion, cognition, Cognition, impulse Cognition, attention,
memory, attention, impulse and memory, attention, attention, impulse control, control, attention, decision making,
control, sleep, regulation of sleep, reproductive sleep, regulation of food reproductive motor learning, renin
renal function behaviour intake behaviour, sleep secretion
Selective KF-38393 Bromocriptine 5-OH-DPAT A-412997 ABT-670 SKF81297
agonists SKF-81297 Pergolide Pramipexole, Rotigotine, PD-168077
Fenoldopam Cabergoline PD-128907, A 412997
(SKF-82526) Ropinirole ABT-670
PD-168077
Selective SCH-23390 Haloperidol, Raclopride Nafadotride GR-103691, A-381393 SCH39366
antagonists SCH-39166 Sulpiride GR-218231, SB 277011A, FAUC213 L-745870
SKF-83566 Spiperone NGB-2904 PG 01037 L-750667
Risperidone ABT-127
S. Kaur et al.
5 Pharmacology of Dopamine and Its Receptors 155

side, postsynaptic D2 receptors enhance dopamine release to stimulate locomotion.

This way dopamine agonists exhibit biphasic response by activating both presynap-
tic and postsynaptic receptors (Sulzer et al. 2016). Similarly, D3 receptor exhibits
biphasic response but at a moderate level, it regulates reward and reinforcement like
functions of human behaviour. The D4 and D5 are less expressed at motor control
regions so they provide a low contribution to movement control (Beaulieu et al.
2015). Further, D1 and D2 receptors also regulate learning, working memory, and
executive functions due to abundance in prefrontal cortex. But D3, D4, and D5 give
less influence over cognition due to their lower expression in the hippocampus. The
other major action of dopamine receptors is contributed by D2 receptors that regulate
aggression, emotion, motor control, and food intake like human behaviour (Nyberg
et al. 2016). These functions often contribute to psychotic behaviour, so today most
clinical psychotic drugs act by blocking D2 actions for the treatment of schizophre-
nia and bipolar disorder. Other actions of dopamine include D2-mediated prolactin
secretion from the pituitary gland, D1-mediated renin secretion from kidney,
and adrenal gland-mediated D1-directed aldosterone secretion (Lv et al. 2018).
The major function of the dopamine system is to regulate voluntary movements
which are carried out through the direct and indirect pathway in the basal ganglia.
These both pathways are regulated from striatum that carries both D1 and D2
receptors. The D1 receptors are excitatory and send GABAergic inhibitory neuro-
transmission to globus pallidus internal (GPi). As D1 receptor activates GABA, so
globus pallidus get inhibited. Further, the GPi sends GABAergic inhibitory
projections to the thalamus that itself sends excitatory message via glutaminergic
projections to motor cortex for proceeding movement. But as the GPi is already
inhibited here, so cannot inhibit thalamus and voluntary movement generated. The
alternate pathway is indirect pathway coordinated by D2 receptor which itself
is inhibitory in nature. The D2 receptor sets up inhibitory control over glutaminergic
projections going towards the globus pallidus external (GPe), and this GPe remains
unaffected and sends GABAergic projections to the subthalamic nucleus (STN)
which activates the thalamus and motor cortex via glutaminergic projections. In
this way, the inhibited STN cannot activate the thalamus and voluntary movement
stops to initiate. Both direct and indirect pathways act frequently to regulate the
control over voluntary muscles while the entire dopaminergic system that lies in the
basal ganglia is responsible for coordinating the motor activity in human body.
Hence, motor coordination remains a predominant function of this neurotransmitter
and any irregularity can predispose the human body towards movement disorders
(Fig. 5.7).
Apart from this, dopamine receptors also play a vital role in addiction which
reflects from changes in expression of dopamine receptors in the basal ganglia. As
already mentioned, dopamine neurons that project from VTA to NAc are well
known to implicit in reward-associated stimuli and addiction. Usually the abuse
of drugs potentiates dopamine release and mimics the phasic response that results in
fast dopamine release via the activation of D1 receptors (Atcherley et al. 2015).
Changes in dopamine firing patterns are modulated by tonic response rather than
phasic. It results in lowering the dopamine release which is sufficient to activate the
156 S. Kaur et al.

Fig. 5.7 Direct and indirect

pathway for voluntary
movement control

D2 receptor for motivation. Alterations that occur in phasic and tonic responses
result from interaction with cortico-striatal glutaminergic synapses that modulate the
functional signalling of D1 and D2 receptors expressing GABAergic medium spiny
neurons (MSN). The striatal excitatory D1 receptors expressing MSN act through
direct pathway while D2-MSN receptors act in an inhibitory manner through indirect
pathway (Paladini and Roeper 2014). Both the ventral striatal pathways are
evidenced to participate in reward and punishment respectively.
Let cite an example of abused drug which firstly activates the D1 and D2
receptors via enhancing the dopamine release. When dopamine decreases rate
dependently, only D2 receptors remain active and justify the reward phenomenon
in patients (Zweifel et al. 2009). Moreover, the identical pathways also justify
associative learning which brings out the involvement of glutaminergic projections
emerging from the hippocampus, amygdala, and prefrontal cortex. These projections
uniquely regulate activation of D1 receptor that remains responsible for emotion-
associated learning, hippocampal-dependent learning, and cortex associative
learning. Besides this, the role of D2 receptor is confirmed in addiction as the
expression of the receptor in striatum gets downregulated on repeated exposure of
abuse drugs (Volkow and Morales 2015). The downregulation of D2 receptor in
striatum mediates the suppression of the indirect pathway which further distorts the
thalamo-cortical activity in prefrontal cortex. Moreover, decreasing availability of
D2 receptors in both dorsal and ventral regions had been reported in PFC of abused
patients specifically in anterior cingulated (ACC) and orbitofrontal cortical (OCC)
regions. Altered functioning of dopamine receptors in these two regions is observed
to be responsible for impulsive and compulsive behaviour in drug abusers. Hence, in
5 Pharmacology of Dopamine and Its Receptors 157

this way, it is the ratio and imbalance in between the D1- to D2-mediated direct and
indirect pathway that uniquely contribute to addiction, learning, and other
behavioural changes.

5.7 Agonists and Antagonists of Dopamine

Specific agonists and antagonists of dopamine are available for D1 and D2 class of
dopamine. Selective agonists for D1 class include A77636, SKF38393, SKF81297,
and dihydrexidine, while D2 selective compounds include quinpirole and N-0435.
On the other side, antagonists of D1 class involve SCH23390, SCH39166, and
SKF35566, while D2 antagonism is caused by domperidone, nemonapride,
raclopride, and sulpiride (Smee and Overstreet 1976). Moreover, some compounds
show receptor-specific actions and participate in dopamine mediated functioning
as enlisted in Table 5.2.
D1 receptor family agonists and antagonists and their functions:
1. Agonists:
(a) Dihydrexidine: It is a selective D1 agonist. Dihydrexidine gives its action
through stimulating inspiratory motor output and by depressing medullary
expiratory neurons. Dihydrexidine has been used in clinical trials for study-
ing the treatment of SPD, cocaine-related disorders, and schizotypal person-
ality disorder (Lalley 2009).
(b) Fenoldopam: Fenoldopam is a fast-acting vasodilator. It is an agonist for
D1-like dopamine receptors and has a moderate affinity towards binding to
the α2-adrenoceptors. Fenoldopam is a racemic mixture and the R-isomer is
responsible for the biological activity. The R-isomer has approximately
250-fold higher affinity for D1-like receptors than does the S-isomer. It
helps lowering down the blood pressure through arteriolar vasodilation.
Thus, this agonist is also used as an antihypertensive agent (Felder et al.
1993).
(c) SKF38393: It is a selective D1 agonist. It improves motor function in PD
(Robertson et al. 1990).
(d) SKF81297: It is a selective D1/D5 receptor agonist. It is believed to reduce
long-term potentiation to prevent synaptic failure (Reavill et al. 1993).
2. Antagonists:
(a) SCH23390: It is a D1 antagonist and believed to improve motor function in
PD and has anxiolytic effects on the hippocampus, VTA (ventral tegmental
area) (Lidow et al. 1991).
(b) SCH39366: It is a classical benzazepine D1/D5 antagonist. It has been used
in human clinical trials for studying different diseases including schizophre-
nia, cocaine addiction, and obesity (Wu et al. 2005).
158 S. Kaur et al.

D2 receptor family agonists, antagonists, and their functions:

1. Agonists:
(a) Quinpirole: It is a selective D2 agonist. It has been observed that quinpirole
has shown to increase locomotion and sniffing behaviour in mice. One study
has found that quinpirole induces compulsive behaviour symptomatic of
obsessive compulsive disorder in rats (Szechtman et al. 1998).
(b) Bromocriptine: It is a selective D2 agonist. It is used in the treatment of
pituitary tumours, type 2 diabetes, hyperprolactinemia, Parkinson’s disease,
and neuroleptic malignant syndrome (Kimberg et al. 1997).
(c) Cabergoline: It is a long-acting dopamine agonist and also an inhibitor of
prolactin. It is used in the treatment of hyperprolactinemic disorders and
parkinsonian syndrome. Cabergoline possesses potent agonistic activity on
dopamine D2 receptors (Colao et al. 1997).
(d) Carmoxirole: It is a peripherally active selective agonist for D2-like
receptors. It has a role as an antihypertensive agent, a dopamine agonist,
and a platelet aggregation inhibitor (Rump et al. 1992).
(e) Pramipexole: It is a D2-like receptor agonist. Pramipexole is a useful medi-
cament in the treatment of the symptoms of PD. It is a non-ergot dopamine
agonist drug that is effective in treating various symptoms of PD such as
tremor, bradykinesia, and rigidity (slow movement) (Constantinescu 2008).
2. Antagonists:
(a) Clozapine: It is a dopamine antagonist specifically for D4 receptor. Serotonin
antagonist, with strong binding to 5-HT 2A/2C receptor subtype. It is used as
an antipsychotic drug.
(b) Haloperidol: It is a typical D2 antagonist. Haloperidol is an inhibitor of
dopamine neurotransmitters and it increases its turnover. It is a traditional
antipsychotic drug and is primarily used to treat schizophrenia and other
types of psychoses. It is also used for the management of schizoaffective
disorder, ballism, delusional disorders, and Tourette syndrome (a drug of
choice) and used occasionally as an adjunctive therapy for mental retardation
and the chorea associated with Huntington’s disease. It is also a potent
antiemetic and is used in the treatment of intractable hiccups (Seeman
2010) (Table 5.3).

5.8 Role of Dopaminergic System in Parkinson’s Disease

and Potential Therapeutic Drug Targets Under Research

Parkinson’s disease (PD) is the rapidly growing neurodegenerative disorder trou-

bling the ageing population with motor and non-motor complications. The motor
complications in PD include bradykinesia, rigidity, tremor, and postural
abnormalities caused by triggering cell death of dopaminergic neurons in substantia
nigra pars compacta (SNpc), whereas the non-motor complications involve
5 Pharmacology of Dopamine and Its Receptors 159

Table 5.3 Agonists and antagonists of dopamine

Compound Mechanism for dopamine
Dopamine Dopamine agonist
A77636, SKF38393, SKF81297, Selective D1 agonists
dihydrexidine
Quinpirole and N-0437 Selective D2 agonists
Chlorpromazine D2 blocker
Clozapine Dopamine antagonist specifically for D4
receptor
Haloperidol Dopamine antagonist specifically for D2
receptor
Apomorphine Nonselective dopamine agonist
Fenoldopam Selective D1-like partial agonist
Bromocriptine, cabergoline Selective D2-like agonist
Carmoxirole Peripherally active selective agonist for
D2-like receptors
Dihydroergotamine Partial D2-like agonist
Dihydroergocristine Partial dopamine receptor agonist
Piribedil Dopamine agonist
Quinilorane Selective D2-like agonist
Roxindole D2 auto-receptor agonist
Pimozide D2-like antagonist
LE-300, SKF83566, SCH23390, SCH39166, D1 selective antagonists
and SKF35566
Melperone, risperidone, zipseridone, L-741, D2 selective antagonists
L-626, domperidone, nemonapride, raclopride,
and sulpiride
Eticlopride, GR-103691, nafadotride, D3 selective antagonists
NGB-2904, U99194
Fananserin, PNU96415E, L-870, L-742 D4 selective antagonists
Bupropion Nonselective inhibitor for dopamine and
norepinephrine transporters
Cocaine Inhibitor of monoamine transporters
GBR-12783, GBR-12909, GBR-12935, Selective dopamine uptake inhibitors
GBR-13069, indatraline
Reserpine Inhibitor of vesicular monoamine transporter
Rimacazole DAT inhibitor
Tetrabenazine Potent inhibitor of vesicular monoamine
transporter and depletes dopamine store

autonomic disturbances, olfactory dysfunction, and sleep problems which may

originate due to neuron loss in other regions. The aetiology of PD is complex as
multiple factors contribute to it which makes it a multifactorial disease. The
contributing factors in PD are selected on the basis of neuronal loss accompanied
by mitochondrial dysfunction, excitotoxicity, enhanced oxidative stress, abnormal
protein folding and deposition, loss of trophic functions, and neuroinflammation like
pathogenic mechanisms (Maiti et al. 2017).
160 S. Kaur et al.

As PD is a motor disorder and dopamine neurons are major motor coordinating

neurons in the brain, dopamine contribution in PD is not controversial. The loss of
dopaminergic neurons in midbrain remains a prominent feature of
PD. Physiologically, the motor circuit of the basal ganglia constitutes direct and
indirect pathways. The direct pathway regulates the excitatory input over thalamus
via SNpc, SNpr, and GPi like key regions. On the other side, the indirect pathway
regulates the inhibitory control over the thalamus to restrict movement by
modulating GPe and STN. The dopaminergic regulation on SNpc gets defective
due to the loss of dopaminergic neurons, causing PD by inhibiting the direct
pathway, while the indirect pathway gets upregulated because compensatory mech-
anism of D1/D2 disrupts that result in overactivity of STN to mediate over inhibitory
responses (Fig. 5.8).
On this basis, currently most of the approved drugs for PD potentiate dopamine
for neuroprotective approach (Zinger et al. 2011). These drugs include levodopa,
selegiline, bromocriptine, apomorphine, and ropinirole-like molecules that enhance
dopamine levels in the brain. But these drugs exhibit several side effects that limit
their use (Maiti et al. 2017).
This limitation leads to the assessment of receptor-specific drugs for therapeutic
benefit. As discussed above, there are different subclasses of dopamine receptors
belonging to D1 and D2 class. Untreated PD patients having dense D2-like receptors
as compared to D1 receptors. The remaining D2 receptors generally gets super-
sensitive and contribute to tremor-like symptoms of PD. The ongoing research
studies for receptor-specific drugs include evaluation of molecules like D1/D2 full
agonist dihydrexidine; CY-208-243, A86929, and dihydrexidine (DHX) give posi-
tive results over symptoms of PD (Malo et al. 2012). Not only this, several partial
agonists and agonists revealed to have good ligand activity for beneficial effects in
PD (Table 5.4).

5.9 Role of Dopaminergic System in Huntington’s Disease

(HD) and Enlisting Potential Therapeutic Drug Targets
Under Research

HD is a devastating slowly progressive motor disorder that results in chorea (uncon-

trolled dancing movements), cognition loss, and psychiatric problems (Rubinsztein
and Carmichael 2003). The aetiology behind HD associates with autosomal genetic
mutation in Huntingtin gene that causes medial spiny neuron loss in the striatum and
cortex. Other regions affected include hypothalamus, globus pallidus, subthalamic
nucleus, and substantia nigra.
The striatum remains a major regulatory unit for motor control and its degenera-
tive changes contribute to different pathogenic mechanisms in HD. The dopaminer-
gic neurons of striatum principally coordinate movement control via direct and
indirect pathways. The direct pathway expresses D1 receptors that project towards
substantia nigra pars reticulate and internal globus pallidus to regulate disinhibition
of thalamus for motor function, whereas the D2 pathway coordinates its inhibition
5 Pharmacology of Dopamine and Its Receptors 161

Fig. 5.8 Dopamine deficiency and motor symptoms

162 S. Kaur et al.

Table 5.4 Dopaminergic drugs under research for therapeutic benefits in PD

Drugs Mechanism of action Pharmacological effects
Apomorphine Non-selective dopamine agonist Improves motor function in PD
SKF38393 D1 agonist Improves motor function in PD
SCH23390 D1 antagonist Improves motor function in PD
D-512 D2/D3 agonist Improves 6-OHDA-induced
neurotoxicity in PD
Improves motor function in MPTP
induced PD
Quinpirole D2-like receptor agonist Improves motor function in PD
Pramipexole D2-like receptor agonist Improves motor function in PD
Piribedil D2/D3 agonist Reduce symptoms of PD
D-440 Highly D3-receptor-selective agonist Improves motor function in PD
BMY-14802 Sigma-1 receptor antagonist, modulates Improves dyskinesia induced by
dyskinesia like side effects that occur L-DOPA treatment
due to dopamine agonism
SKF81297 D1 receptor agonist Improves motor symptoms of PD

over cortical activity by subthalamic nuclei and globus pallidus external. It has been
reported that this system gets dysregulated in HD and loss over movement control
occurs in the form of chorea. The early symptoms of HD associate with the loss of
D2 receptors which initiate uncontrolled dance-like movements by creating a deficit
in inhibitory regulation of the dopamine system, whereas in later stages of HD, on
the other hand, the lack of D1 receptors also results in thalamus inhibition inducing
dystonia, rigidity, and akinesia-like symptoms. Furthermore, evidence-based justifi-
cation lies behind the degeneration of this system. It has been analysed that dopa-
mine treatment differently modulates NMDA and AMPA receptors. These responses
occur due to the localization of dopamine receptor on glutaminergic terminals. The
involvement of D2 receptors in glutaminergic terminals through excitotoxicity may
exacerbate neurotoxicity in HD, whereas D1 receptor itself mediates potentiation of
NMDA receptor that contributes to neurotoxicity (Chen et al. 2013). Further,
agonists of D1 enhance neurotoxicity via NMDA receptor activation whereas
the activation of D2 receptor reduces NMDA activation for neuroprotection.
Hence, D1 receptors remain neurotoxic whereas D2 receptors prove to be
neuroprotective in HD.
Moreover, cell death of dopamine neurons also proceeds by free radical produc-
tion as they remain more prone to oxidation due to their distinct features. Changes in
dopamine levels also contribute to cognitive loss in earlier HD as cholinergic
neurons reside in striatum also degenerate, thus deteriorating cognitive functions
like attention, execution, learning, and cognition (Wang et al. 2006). The initial
phase of HD reveals to be in a hyperdopaminergic state, so reducing dopamine
remains a potential treatment strategy. The only approved drug in HD is
tetrabenazine (TBZ) that inhibits vesicular monoamine transporter to reduce dopa-
mine in presynaptic vesicles which reduce chorea and other symptoms of
HD. Aripiprazole is a partial D2 receptor agonist that improves symptoms of chorea
5 Pharmacology of Dopamine and Its Receptors 163

Table 5.5 Enlisting the compounds targeting dopamine for therapeutic efficacy in HD
Drug Mechanism of action Status in HD
Tetrabenazine Vesicular monoamine transporter to reduce Clinically approved drug
dopamine level
Haloperidol Typical D2 antagonists Improves symptoms of
disease
Olanzapine, Atypical D2 antagonists Improve chorea and
risperidone behavioural disturbances
Bromocriptine D2 agonist Improves chorea and other
motor symptoms
Lisuride D2 agonist Provides symptomatic relief
Aripiprazole Partial agonist of D2 Improves chorea but
cognition not improved
L-DOPA Dopamine agonist Provides relief over rigidity
Pridopidine DA stabilizer Improves motor dysfunction
SCH23390 D1 antagonist Shows positive result in
animal studies
SKF38393 D1 agonist Shows positive result in
animal studies
Haloperidol D2 antagonist Shows positive result in
animal studies
Quinpirole D2 agonist Shows positive result in
animal studies

in HD whereas dopamine stabilizer pridopidine also provides relief over motor

symptoms of HD (Coppen and Roos 2017). On the other side, different D2
antagonists like olanzapine, risperidone, quetiapine, and ziprasidone improve func-
tional disabilities of HD along with reduced side effects. Thus, numbers of dopamine
agonists, antagonists, and stabilizers of dopamine are going to be evaluated for
therapeutic efficacy in HD to ensure a reliable target (Table 5.5). This way targeting
striatal dopamine dysfunction could be a beneficial approach for therapeutic efficacy
in HD (Fig. 5.9).

5.10 Role of Dopaminergic System in Alzheimer’s Disease

(AD) and Enlisting Potential Therapeutic Targets Under
Research

AD today becomes the most severe neurodegenerative disorder resulting in memory

loss, cognition impairments, and functional abnormalities caused by neuronal loss
in the hippocampus, cerebral cortex, and neocortex inside the brain (Jahn 2013). The
loss of neuron gradually spreads throughout the brain but other regions like striatum,
amygdala, and prefrontal cortex do not remain spared from AD pathology (Braak
and Del Tredici 2015). No doubt, mainly cholinergic neurons get lost in AD but
recent studies showed the loss of other neurons which also contributing in neuro-
chemical alterations in AD.
164 S. Kaur et al.

Fig. 5.9 Excessive dopaminergic activity in HD

The contribution of the dopaminergic system in AD gathers specific attention

for the last few years. Dopaminergic neurons present in ventral tagmental area
regulate functions like memory, cognition, and synaptic plasticity. In early AD,
degeneration of these neurons occurs which may contribute to cognition impairment
and memory loss whereas extrapyramidal symptoms (EPS) occur in late AD which
are associated with the loss of dopaminergic neurons in the striatum (Nobili et al.
2017). About 35–40% of AD patients are affected by EPS in their late age of disease
(Martorana and Koch 2014). On this basis, the treatment strategy adopted to
counteract the loss of dopamine, which includes administration of dopamine and
selegiline, shows to improve memory and neuron loss in the hippocampus (Nobili
et al. 2017). This beneficial effect of dopamine is attributed to the mesocorticolimbic
pathway that contributes to cognition and memory by projecting dopaminergic
projections to hippocampus, cerebral cortex, and nucleus accumbens and symptoms
of late AD associates with mesostriatal pathway that regulates voluntary movements
(Fig. 5.10).
Further loss of dopamine is confirmed by reducing the release of dopamine, its
receptor, decreasing the level of dopamine transporter and enzyme tyrosine hydrox-
ylase that were reported via different research studies. Few studies target receptor-
specific role of dopamine in AD that includes D1 and D2 receptors and their
attributing role towards memory and cognition (Nyberg et al. 2016). Mostly
receptor-specific studies from complex regulation of prefrontal cortex that shows an
abundance of D1 receptors coordinate the executive and working memory. The D2
receptor is distributed in caudate, VTA, and hippocampus to regulate hippocampus
5 Pharmacology of Dopamine and Its Receptors 165

Fig. 5.10 Role of dopaminergic projections in AD

oriented cognitive functions. It also regulates episodic memory and executive

functions in the brain. The decline in D2 and D1 receptors may be caused by
excitotoxicity-mediated degeneration of dopaminergic neurons.
On this basis, different dopamine targeting drugs are going to be evaluated for
therapeutic efficacy in AD. The neuroprotective approach like allopregnanolone
(ALLO) has shown to increase dopaminergic neurons and improve cognitive deficits
in AD studies. Another drug vindeburnol and L-DOPS promote catecholamine
synthesis and found to enhance memory and concentration in preclinical AD studies
(Feinstein et al. 2016). This way, there are a number of dopamine targeting drugs that
exhibit good efficacy in AD studies (Table 5.6).

5.11 Role of Dopaminergic System in Schizophrenia

and Enlisting Potential Therapeutic Drug Targets Under
Research

Schizophrenia (SCZ) is a chronic heterogeneous mental disorder and is characterized

by positive, negative, and cognitive symptoms that make the patient mentally
impaired and socially distorted. The positive symptoms of SCZ include delusions,
hallucinations, distorted thoughts, and abnormal behaviour, while negative
symptoms include social withdrawal and reduced motivation. These symptoms
166 S. Kaur et al.

Table 5.6 Enlisting the compounds targeting dopamine for therapeutic efficacy in AD
Drug Mechanism of action Status in AD
Levodopa DA precursor Improves memory and decreases synaptic
plasticity in hippocampus
Selegiline MAO-B inhibitor Improves memory and decreases synaptic
plasticity in hippocampus
Allopregnanolone Positive modulator of Increases survival of dopaminergic
(ALLO) GABAB neurons and improves cognitive deficits
Vindeburnol Promotes catecholamine Improves memory and concentration in
synthesis AD studies
L-DOPS Promotes catecholamine Improves memory and concentration in
synthesis AD studies
S33138, FP17141 D3 receptor antagonist Reverse cognitive decline
SKF81297 Selective D1/D5 receptor Reduces long-term potentiation to prevent
agonist synaptic failure
PD168077 D4 receptor agonist Enhances working memory and attention
Dimebon Increases dopamine level in Effective in mild to moderate AD
the brain
Rimonabant CB1 receptor antagonist Improves social and working memory
Increases dopamine level
Methylphenidate Dopamine reuptake Improves neuropsychiatric symptoms
inhibitor
ITI-007 Dopamine receptor Improves neuropsychiatric symptoms
modulator
Rasagiline Monoamine oxidase B Enhances cognition
inhibitor
Rotigotine Dopamine agonist Improves executive function in AD

proceed along with cognitive impairments like working memory and attention
deficits (Robertson et al. 1990). The pathogenesis of SCZ strikes the neurochemical
alterations occurring in patients with SCZ. It includes dopamine theory, glutamate
theory, and other neurochemicals alterations like serotonin dysfunction (Laruelle
2014). As dopamine theory always remains a major contributor in pathogenesis, its
role in SCZ does not remain controversial.
Initially proposed dopamine hypothesis postulates that hyperactivity of dopamine
neurotransmission results in schizophrenic symptoms. This theory was put forward
on the basis of evidence that the administration of dopamine releaser amphetamine
and dopamine enhancer levodopa potentially exacerbate symptoms of schizophrenia
(Brisch et al. 2014). On the other side, treatment drugs against enhanced dopa-
mine levels decrease the activity of dopamine and improve therapeutic reliability on
antipsychotic drugs including D2 antagonists. The mesostriatal dopamine system
remains a key target of these drugs confirmed by blockage of high concentrations of
D2 receptors that also contribute to extrapyramidal side effects. Later revisions to
classical dopamine theory provided a new finding (da Silva Alves et al. 2008). This
new evidence suggests that amphetamine does not exhibit all symptoms of schizo-
phrenia but only exacerbates positive symptoms along with the improvement in
5 Pharmacology of Dopamine and Its Receptors 167

negative symptoms. Thus, it becomes confirmed that hypoactivity in the dopaminer-

gic system contributes to negative symptoms of schizophrenia. This leads to the uti-
lization of atypical antipsychotics including clozapine and typical antipsychotics like
haloperidol that exhibit antipsychotic activity with no extrapyramidal side effects.
This both typical and atypical antipsychotics exhibit selectivity for the mesolimbic
dopamine system along with weak D2 blocking striatal activity (Brisch et al. 2014).
Thus, DA hypothesis gets much more revised and postulates that hyperactivity of
dopamine in the mesostriatal region contributes to positive symptoms whereas
hypoactivity of dopamine in the prefrontal cortex results in negative symptoms
of SCZ.
Moreover, subcortical dopamine dysfunction also remains a prominent patho-
genic mechanism in schizophrenia. It includes dysfunction of cortical areas, func-
tional abnormality in the frontal cortex, and structural changes in the prefrontal
cortex, entorhinal cortex, hippocampus, amygdala frontal and temporal brain regions
(Weiner and Joel 2002). Further, the degenerating changes in the mesolimbic path-
way and targeting region of treatment drugs reveal nucleus accumbens that encounter
glutaminergic projections from cortical areas whose dysfunction led to different
symptoms of schizophrenia. It has been proposed that dysfunction of the fronto-
temporo limbic-mesolimbic DA pathway contributes to reduced cortical input to
mesolimbic region (Weiner and Joel 2002).
On the basis of the above discussion, pharmacotherapy of schizophrenia includes
second-generation atypical antipsychotics and first-generation typical antipsychotics
(Patel et al. 2014). Here, the typical antipsychotics remain less preferable due to their
extra pyramidal side effects but even atypical antipsychotics give metabolic adverse
effects like obesity, hypercholesterolaemia, and diabetes mellitus. Further, in pre-
clinical and clinical research, several dopamine modulating drugs are targeting
schizophrenia (Table 5.7).

Table 5.7 Enlisting the compounds targeting dopamine for therapeutic efficacy in
schizophrenia (SCZ)
Typical antipsychotics Atypical antipsychotics
(first-generation (second-generation Drugs under preclinical and
antipsychotics) antipsychotics) clinical trials
Chlorpromazine Aripiprazole ITI-007 (presynaptic partial D2
agonist and postsynaptic D2
antagonist)
Fluphenazine Clozapine RP5063 (partial D2, D3, and D4
agonist)
Haloperidol Lurasidone SKF-38393, SKF-83959, SPD-451
(partial D1 agonist)
Perphenazine Olanzapine DAR-0100A
Thioridazine Paliperidone SKF-81297
Thiothixene Quetiapine A-77636
Risperidone ABT-431
168 S. Kaur et al.

5.12 Role of Dopaminergic System in Anxiety and Enlisting

Potential Therapeutic Drug Targets Under Research

Anxiety is considered to be the most prevalent psychiatric disorder today. It could be

defined as an abnormal state of mind with excessive fear associated with sympathetic
hyperactivity, apprehension, and hypervigilance that interfere with daily life. The
Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) classifies anxiety
disorders that include panic disorder, phobias, generalized anxiety disorder
(GAD), post-traumatic stress disorder (PTSD), and obsessive compulsive disorder
(OCD) (Thibaut 2017). Often anxiety associates with mood disorder, severe neuro-
degenerative condition, and depression-like disorders. The contributing mechanism
for anxiety in the mammalian brain includes the neurochemical and neuropeptide
changes occurring at cortical and subcortical levels. Usually the regions affected in
anxiety include the hippocampus, amygdala, septum, and prefrontal cortex. Altered
neurochemistry remains a confirmed mechanism behind anxious state of mind, so
different neurochemicals have been targeted for decades regarding their contribution
to anxiety (Martin et al. 2009).
In recent years, altered dopamine levels in the brain emerge as potential thera-
peutics in anxiety disorders. Different studies have confirmed the role of dopamine
in social behaviour, fear processing, and avoidance learning like phenomenon. The
decrease in dopamine is considered as a possible cause of anxiety and depression as
L-DOPA treatment improves its symptoms (Stansley and Yamamoto 2015). Fur-
thermore, one study has confirmed that stress activates the mesolimbic pathway that
causes increase in dopamine over synapse with reduced reuptake. Thus, dopamine
results in the activation of dopamine receptors which contributes to stress. Dopami-
nergic neurons are abundant not only in the striatum but the ventral tagmental area
also remains a major dopamine modulatory unit in the striatum. There are about 60%
of dopamine neurons present in VTA. These neurons constitute D1 receptors in
moderate to low density along with a high level of D2 receptors (Zarrindast and
Khakpai 2015). Here, activation of D2 receptors contributes to stress and anxiety
like condition by inhibiting dopamine release in VTA. On administering an antago-
nist of D2 receptor, D1 gets activated, thus favouring the release of dopamine to
relieve anxiety. The contributory function of the amygdala in anxiety never remains
controversial; it regulates anxiety, stress, fear conditioning, and emotional memory
(Forster et al. 2012). The structural changes in the basolateral amygdala (BLA)
including stress induced hypertrophy contributes to symptoms of anxiety. The BLA
activity normally remains suppressed by PFC but on stress dopaminergic projections
relieves inhibitory stimulus and triggers anxiety. Thus, mesolimbic dopaminergic
projections highly contribute to fear-associated anxiety. Here, activation of D1
receptors of amygdala contributes to anxiety whereas D2 receptors associate with
vestibular nuclei associated anxiety (de la Mora et al. 2010). Septum remains another
region that lies in the basal forebrain that regulates fear, stress, anxiety, emotion, and
aggression. Activation of septum generally contributes to anxiety as it gets dopami-
nergic projections from VTA that contributes to anxiety (Zarrindast and Khakpai
5 Pharmacology of Dopamine and Its Receptors 169

Table 5.8 Enlisting the compounds targeting dopamine for therapeutic efficacy in anxiety
Mechanism of
Drugs action Effects observed (region specific)
Apomorphine D1/D2 receptor Anxiolytic effects (amygdala), anxiogenic effects
agonist (hippocampus)
SCH23390 D1 antagonist Anxiolytic effects (BLA, VTA, NAc), anxiogenic effects
(hippocampus, amygdala)
Quinpirole D2 agonist Anxiolytic effects (VTA), anxiogenic effects
(hippocampus, BLA)
Sulpiride D2 antagonist Anxiolytic effects (BLA), anxiogenic effects (amygdala)
Raclopride D2 receptor Anxiolytic effects (BLA), anxiogenic effects (CeA)
antagonist
Eticlopride D2 receptor Anxiolytic effects (CeA)
antagonist
SKF38393 D1 agonist Anxiogenic effects (BLA, hippocampus)

2015). Thus, VTA remains a main unit in regulating anxiety and it is regulated by
excitatory and inhibitory inputs.
Moreover, the hippocampus not only modulates learning and memory but also
involved in fear and anxiety-like behavioural disorders. There are two different
regions of the hippocampus including ventral hippocampus and dorsal hippocampus
that differentially precede their functions. The ventral hippocampus involves fear
and anxiety due to its projections to the prefrontal cortex whereas the dorsal just
regulates memory and learning-like functions (Bannerman et al. 2004). This contri-
bution is further confirmed by lesions of the ventral hippocampus that contributes to
anxiety-like symptoms. The mesolimbic dopaminergic projections from ventral
tagmental area and SNc when gets affected from plasticity also contribute to anxiety.
On this basis several agonists and antagonists are going to target the treatment of
anxiety; some of them are enlisted in Table 5.8.

5.13 Role of Dopaminergic System in Epilepsy and Enlisting

Potential Therapeutic Drug Targets Under Research

Epilepsy is the most widespread neurological disorder that initiates with uncontrol-
lable excitatory neurotransmission. It affects 1–2% of the world’s population and
features recurrent epileptic seizures. Seizures may affect localized areas (“focal” or
“partial” seizures) or spread throughout the whole cerebral hemisphere
(“generalized” seizures). The predisposing factor behind epilepsy is the hyperactiv-
ity persisting in different brain regions that involve imbalance between excitatory
and inhibitory impulses (Stafstrom and Carmant 2015). Currently most of
the approved drugs in epilepsy target this imbalance by upregulating the GABAergic
system in the brain. Whether, GABA/Glutamate remains a main target for the
development of drug therapy of epilepsy but recently growing evidence strengthens
the role of other neurotransmitters and neuropeptide in the epileptic brain.
170 S. Kaur et al.

Dopamine, acetylcholine, serotonin, and noradrenalin have gathered specific atten-

tion for their contribution to epilepsy pathogensis (Werner and Coveñas 2015).
Among this, dopamine seems to be a prominent neurotransmitter that could
modulate GABA/glutamate ratio in epilepsy (Howes et al. 2015). Plenty of evi-
dence has shown that dopamine crucially controls the activity of seizure. Even the
epileptic brain suggested alterations that occur in the dopaminergic system reflected
from altered release, synthesis, and metabolism of dopamine.
Moreover, different studies evaluated the role of dopamine receptors in seizure
specifically in limbic epilepsy. The D1-like receptors are reported for their epilepto-
genic activity while D2-like receptors provides antiepileptic effects (Clinckers et al.
2004). The D1 receptors in limbic regions on activation stimulate glutamate release
that contributes to seizure. The role of dopamine in epilepsy also depends upon the
region involved in seizure generation. As the temporal lobe epilepsy is reported to
be triggered from the hippocampus (Kandratavicius et al. 2012). Here, D2 receptors
density is more than D1 receptors. So, dopamine provides inhibitory effects on
seizure generation in the hippocampus. Similarly in the dentate gyrus, the rich
distribution of D2 receptors offers positive effects for the control of epilepsy. The
expression of dopamine receptors also gets varied in epilepsy as shown in animal
and human studies. The decrease in D2/3 receptor binding is reported in the epileptic
brain specifically in the temporal lobe, thalamus, and basal ganglia region (Paredes
et al. 2015). Even the density of dopamine transporters gets reduced in the basal
ganglia.
Compounds like apomorphine, amphetamine, L-DOPA, and anti-parkinson drugs
including pergolide and bromocriptine (stimulate D2 receptor) provide antiepileptic
activity in different studies. On the other side, antipsychotic drugs (D2-like
antagonists) have demonstrated to decrease the seizure duration; however similar
drugs are also reported to provoke seizures in previously unaffected individuals
(Rezaei et al. 2017). The SKF38393 agonist of D1 receptor causes convulsions
through G-protein signalling (Table 5.7). The D1-like receptor enhances the cAMP
levels and activity of protein kinase A to mediate the adenylyl cyclase activation.
Here, the downstream protein DARPP-32 (DA and cAMP-regulated phosphoprotein
of 32 kDa) gets activated by PKA to regulate neuronal excitability. On the another
side, D2 receptor provides antiepileptic activity due to its opposite action to D1. The
D2 receptor activates Gi protein to decrease cAMP production and counteract the
DARPP-32 signalling (Bozzi and Borrelli 2013). Even after a lot of positive results,
dopaminergic drugs do not target therapeutic benefits in epilepsy as they exhibit
a variety of side effects. The old findings suggested that the implication of bromo-
criptine D2 agonist provides antiepileptic action in animal studies (Table 5.9).
5 Pharmacology of Dopamine and Its Receptors 171

Table 5.9 Dopamine targeting drugs in epilepsy

Drugs Mechanism of action Therapeutic effects
Raloxifene Selective oestrogen receptor modulator Antiepileptic effects
(SERM)
Fluoxetine Selective serotonin reuptake inhibitor Antiepileptic effects, antipsychotic
(SSRI) effects
Bromocriptine Dopamine agonist Antiepileptic effects
Apomorphine Dopamine agonist Antiepileptic effects,
antiparkinsonian effects
L-DOPA Dopamine agonist Antiepileptic effects
Pergolide Dopamine agonist Antiepileptic effects
Liraglutide GLP-1 agonist Antiepileptic effects
Haloperidol DA antagonist Decrease seizure threshold

5.14 Role of Dopaminergic System in Traumatic Brain Injury

and Enlisting Potential Therapeutic Drug Targets Under
Research

Traumatic brain injury (TBI) is a neurological disorder attributed by head injury. It

could occur due to a violent blow, injury, or aggressive shakiness to head which
produces functional and structural changes in the brain. The functional changes
include behavioural cognition and motor abnormalities while structural changes
include cerebral damage and neuron loss (Madikians and Giza 2006). The most
common TBI-associated cases are prevalent in traffic accidents, military, sports,
violence, construction, industrials sites, etc. These cases also occur in infants when
sudden shake to babies body causes a violent impact on their heads. The common
pathogenic features of TBI involve oxidative stress, mitochondrial dysfunction,
excitotoxicity, and cerebral ischaemia (Quillinan et al. 2016). The damage to
neurons results in neurotransmitter alterations in TBI. Among the different
neurotransmitters, alterations in dopamine levels in TBI prominently contribute to
abnormalities associated with posttraumatic brain injury. The breakdown of dopa-
mine in the striatum and frontal cortex is reported in the TBI brain. Reduction in 25%
of dopaminergic neurons of the basal ganglia is reported after cortical injury. The
frontal cortex, striatum, and hippocampus are the major regions affected in TBI
that reflect abnormalities in motor, behavioural, attention, execution, and memory.
However, the dysregulation in the catecholaminergic system is strongly evidenced in
TBI. Increased dopamine metabolism markers including DOPAC and increased
expression of COMT are indicative of the dysregulation in the dopaminergic system
(Chen et al. 2017). The initial increase in the release of dopamine after TBI may
occur to combat excitotoxicity but when the disease gets severe, enhanced
excitotoxicity and oxidative stress damage the cellular function of dopamine neurons
that cause a deficit in its release in TBI brain.
172 S. Kaur et al.

Table 5.10 Enlisting the compounds targeting dopamine for therapeutic efficacy in TBI
Drugs Mechanism of action Pharmacological effects in TBI
Methylphenidate Increases dopamine synthesis Improves cognition, working memory,
and attention
Bromocriptine D2 receptor agonist Improves cognition
Atomoxetine Increases dopamine Improves cognition and attention
Guanfacine Increases dopamine Improves cognition and attention
Levodopa Dopamine agonist Improves cognition and attention
Methamphetamine Increases dopamine Improves memory and cognition
Amantadine Increases dopamine Improves depression symptoms
Selegiline Monoamine-oxidase-B Prevents excitotoxicity
(MAO-B) inhibitor
Rasagiline Monoamine-oxidase-B Prevents excitotoxicity
(MAO-B) inhibitor
Pramipexole Dopamine agonist Prevents excitotoxicity
Ropinirole Dopamine agonist Prevents excitotoxicity
Buproprion Increases dopamine Reduces neuroinflammation

Different reports have reported the dysfunction in nigrostriatal and mesolimbic

pathways after TBI. Either it could result from excitotoxicity or exacerbated
neuroinflammation inside the TBI brain. The damage of dopaminergic neurons
could also result from enhanced oxidative stress-mediated mitochondrial dysfunc-
tion in TBI. The alterations in glutamate release, sodium/potassium ATPase (Na/K
ATPase) function, and enhancing the production of reactive oxidative species
remain prominent pathogenic events in TBI. These further raise the metabolic
needs of neuronal cell and cause depletion of adenosine triphosphate (ATP) that
results in ischaemia/hypoxia-like conditions. Hypoxia increases phosphorylation of
NMDA subunits including NR1 and NR2 that enhance phosphorylation of dopa-
mine cAMP regulated phosphoprotein 32 kDa (DARPP-32) to alter downstream
protein phosphatase activity (PP1). Further this PP1 causes transcription of nuclear
cAMP response element binding protein (CREB) that further phosphorylates the
Na/K ATPase. DARPP-32 functioning is tightly regulated by the release of dopa-
mine, glutamate, and adenosine (Kochanek et al. 2015). Any abnormal alteration in
their release hinders the neuron survival. Moreover, aggregation of α-synuclein
protein and Lewy body formation are considered as the major pathological event
of TBI (Irwin and Trojanowski 2013). The altered dopamine neurotransmission
subsequently causes depression, anxiety, and substance abuse like common
behavioural changes after TBI. Such evidence leads to utilize dopamine targeting
drug for therapeutic benefits in TBI (Table 5.10). The low-dose methamphetamine
improves memory and cognition after TBI evidenced by histopathological and
neurochemical studies. The bromocriptine-like D2 agonists possess antioxidant
properties that are proven to be protective against memory and cognition deficits
(Jenkins et al. 2016). Another drug named as L-deprenyl enhanced dopamine and
norepinephrine to restore cognition and memory deficits due to synaptic plasticity.
5 Pharmacology of Dopamine and Its Receptors 173

On the other side, dopamine antagonists including antipsychotics improve the

agitation and psychotic symptoms occurring after injury. Whether haloperidol and
risperidone provide positive symptoms over psychiatric illness, side effects like
akinesia and pseudoparkinsonism limit their efficacy. But with the administration
of olanzapine, with low D2 antagonism, no side effects appear. It has also
been observed that haloperidol and risperidone recover motor symptoms but does
not affect cognition and memory. Further, it is justified that D2 receptor inhibition is
important as anti-inflammatory and immunological responses so may be the blockage
of it gives negative effects. The D1 inhibition provides positive results in TBI and the
utilization of dopamine agonist remains a beneficial neuroprotective approach.

5.15 Role of Dopaminergic System in Multiple Sclerosis

and Enlisting Potential Therapeutic Drug Targets Under
Research

Multiple sclerosis (MS) is a severe neurological disorder associated with autoimmune

demyelination of the central nervous system. It affects the genetically susceptible
younger adults ranging from 15 to 45 years old. Other factors like exposure to viruses
like Epstein-Barr virus, smoking, and low serum vitamin D levels remain major
environmental contributors in MS. Indeed, there is no exact cause and cure for the
disease but the prevalence of MS is increasing day by day. Clinically, four forms of MS
are there which are well known as relapsing remitting MS (RRMS), primary progres-
sive MS (PPMS), secondary progressive MS (SPMS), and progressive relapsing MS
(PRMS) (Loma and Heyman 2011). The combined characteristic features of these
forms of MS include impairments of functions in motor, visual, and sensory systems.
Moreover, some undiagnosed features including cognitive dysfunction, fatigue, and
mood disturbances contribute to cortical damage. The treatment therapy in MS
includes interferons, monoclonal antibodies, and cytotoxic drugs which provide symp-
tomatic relief but none of them are able to halt or cure the disease (Derwenskus 2011).
The pathogenic mechanism behind these factors includes demyelination, axonal dam-
age, dysfunction of glial cell, and inflammation like neurodegenerative features that are
under research for therapeutic approach in MS (Loma and Heyman 2011).
The dysregulated dopamine in MS is reported as dopamine fatigue hypothesis
postulated by Chaudhuri and Behan (2000). They suggested that abnormal dopa-
mine release contributes to both mental and physical weakness and feebleness in MS
patients. Chronic exhaustion that occurs in MS-affected individuals may result
in abnormal functioning of neurons in the basal ganglia. Moreover, structural
impairments in the brain due to white matter loss may damage dopaminergic
projections including striatal and mesocorticolimbic pathway (Dobryakova et al.
2015).
The expression of D1 receptors gets reduced while D2 receptors increased to
cause abnormal catecholamine release for inhibition in apoptosis. This way dopa-
mine initiates abnormal autoimmune response that severe MS. The administration of
dopaminergic agonists bromocriptine and methylphenidate to the mesocorticolimbic
174 S. Kaur et al.

Table 5.11 Enlisting the compounds targeting dopamine for therapeutic efficacy in MS
Drugs Mechanism of action Pharmacological effects in MS
Bromocriptine Dopamine agonist Improves chronic fatigue and other symptoms
Methylphenidate Dopamine agonist Improves chronic fatigue symptoms
Amantadine Dopamine agonist Improves chronic fatigue symptoms
Risperidone D2 receptor like Decrease spinal cord lesions and autoimmunity
antagonist
Phenelzine MAO-B inhibitor Improves MS symptoms
Fenoldopam D1-like receptor agonist Decreases autoimmunity and improves MS
symptoms
Dopamine Dopamine agonist Decreases autoimmunity and improves MS
symptoms
L-DOPA Dopamine precursor Decreases autoimmunity
Pergolide Dopamine agonist Decreases autoimmunity
Haloperidol D2 antagonist Decreases autoimmunity
Pimozide D2 antagonist Decreases autoimmunity
Fluoxetine D2 blocker Decreases autoimmunity and neuroinflammation
Domperidone D2 antagonist Decreases autoimmunity and neuroinflammation

region improves chronic fatigue symptoms. The administration of modafinil and

amantadine like dopaminergic agonists in MS patients effectively treats chronic
fatigue. Thus, chronic fatigue occurring in MS seems to be a function of
mesocorticolimbic dopaminergic projections and targeting it could provide thera-
peutic benefits in MS (Table 5.11).
Moreover, the abundance of dopamine not only distorts the dopaminergic neuro-
transmission but also damages the key players of the immune system like peripheral
monocytes, lymphocytes T cells, B cells, and macrophages (Levite 2016). The
stressful conditions induce dopamine release from immune cells in extracellular
space that are further uptaken by lymphocytes to initiate phagocytosis and for
activation of T cell and B cell lymphocytes to regulate adhesion, migration, survival,
proliferation, and communication with other cells that express dopamine receptors.
But in the case of MS, the dopamine receptors on immune cells get altered.

5.16 Clinical Trials and Investigational Drugs

The recent advancements in dopamine are fully contributed by eventual set of high
pace discoveries occurred in the research field. The presence of dopamine in the brain
was first reported in the 1960s but gets revolutionized when the mystery of parkin-
sonism got unlocked. Some Swedish works found it to be responsible for extrapyra-
midal symptoms of PD. Afterward scientific discoveries were mostly devoted to
possible roles of catecholamine synthesis and dopamine until levodopa was
introduced. The following years were remarkably well known for dopaminergic
pathways and their contributory role in various disorders. In the 1980s,
advancements in techniques and modern concepts of discovery highly evolved the
5 Pharmacology of Dopamine and Its Receptors 175

neurotransmitter studies through the introduction of dopamine receptors (D1 and

D2). Later, a number of different substances were made to explore their specificity
towards dopamine receptors including toxins for dopaminergic systems and different
MAO and COMT inhibitors. A large number of molecules were evaluated for their
therapeutic potential towards the dopaminergic system. Some of them are approved
as a therapy for Parkinson’s disease while few well known as typical and atypical
antipsychotics which are utilized for psychosis and schizophrenia-like behavioural
disorders. Recent outbreaks have suggested the role of dopamine in memory coordi-
nation and cognition. May the most reliable neurotransmitter dopamine also govern
the mechanism to execute memory-oriented functions. Moreover, the localized
administration of receptor-specific dopamine compounds to different regions of
the brain has given unexpected results to control anxiety. Several new molecules
like SCH23390, sulpiride, and eticlopride modulate the dopamine system to provide
the anxiolytic action, while levodopa itself and other molecules like bromocriptine,
sulpiride, and aripiprazole have improved the symptoms of chorea and rigidity in
Huntington’s disease. Other dopamine targeting drugs like BMY-14802,
SCH23390, and piribedil have significantly reduced the motor complications of
PD. Other recent outbreaks of dopamine in neurodegenerative disorders are enlisted
in Table 5.12.
Currently, with the help of molecular cloning, two D1-like and three D2-like
receptor genes have been successfully identified. The first dopamine receptor cloned
was reported by Bunzow et al. (1988) and it was the D2 receptor that was cloned.
Low stringency screening of a rat brain cDNA library helped in the isolation of this
receptor. The rat D3 and D4 receptors, i.e. two additional members of this family,
have been cloned by low stringency hybridization using probes derived from the D2
receptor. The first functional D1-like receptor (referred to herein as the D1A
dopamine receptor) was cloned simultaneously in several laboratories (Dearry
et al. 1990; Gerfen et al. 1990; Sunahara et al. 1990; Grandy et al. 1990). Similar
strategies based on the sequences of the cloned D1A receptor were used for the
cloning of the second member of the D1-like receptor family. This second D1-like
receptor clone was isolated nearly simultaneously by several groups and has been
referred to as the D5, the D1b, or the D1β receptor (Grandy et al. 1990; Sunahara
et al. 1991; Tiberi et al. 1991; Gingrich and Caron 1993) (Table 5.12).

5.17 Conclusion

Dopamine regulates many functions through its receptor signalling which are com-
plex and may also depend on cellular protein like kinases or other enzymes. The role
of dopamine has been well studied extensively in Parkinson’s, Alzheimer’s, and
Huntington’s disease but its role is also widely expressed in various other neurologi-
cal disorders like epilepsy, multiple sclerosis, schizophrenia, anxiety, and traumatic
brain injury. In this chapter, various roles of dopamine and dopaminergic receptors
have been brightly highlighted. Several disorders demand a lot of research on
the dopaminergic system for various therapeutic approaches. Hence, the high pace
176 S. Kaur et al.

Table 5.12 Investigational drugs in clinical trials

Drug candidates Therapeutic target Clinical phase Therapeutic intervention
Pridopidine Stabilizes dopamine Phase III Improves motor symptoms
of HD
Atypical D2 receptor antagonist Randomized Improve motor symptoms
antipsychotics controlled trials of HD
Bromocriptine DRD2 agonist Rodent studies Enhances spatial memory
and survival rate of
hippocampal neuron in TBI
L-deprenyl Enhances dopamine Rodent studies Enhances cognition and
level improves neuroplasticity in
TBI
Bromocriptine, DA agonist Observational Improve positive and
lisuride, and studies and negative symptoms of SCZ
methylphenidate randomized
trials
RP5063 Partial D2, D3, and D4 Phase III Improves positive and
agonist completed negative symptoms of SCZ
ITI-007 Presynaptic partial D2 Phase III Improves positive and
agonist and negative symptoms of SCZ
postsynaptic D2
antagonist
Methylphenidate Increases dopamine Randomized Improves cognition and
release trials neuropsychiatric symptoms
Atomoxetine Improves dopamine Observational Improves symptoms of TBI
signalling study
Levodopa Potentiates dopamine Observational Improves symptoms of TBI
release study
Rasagiline Potentiate dopamine Phase II Improves cognition and
memory in AD
Piromelatine DA agonist Phase II Improves cognition and
memory in AD
Aripiprazole, DA agonist Phase I Improve cognition and
brexpiprazole, memory in AD
and
methylphenidate
Risperidone D2 receptor antagonist Accepted for Improvement in psychiatric
human use disturbances and
stabilization of motor score
L-Dopa Precursor of DA Accepted for Reduction in chorea in some
human use HD patients
Zonisamide Facilitates dopamine Accepted for Generalized epilepsy
human use
5 Pharmacology of Dopamine and Its Receptors 177

of research along with newly developed advancements in the field of neuroscience

and pharmacology will be useful to get more knowledge about dopamine receptor
signalling in devastating disorders.

References
Arias-Carrión O, Stamelou M, Murillo-Rodríguez E, Menéndez-González M, Pöppel E (2010)
Dopaminergic reward system: a short integrative review. Int Arch Med 3(1):24
Arnaud-Batista FJ, Peruchetti DB, Abreu TP, do Nascimento NR, Malnic G, Fonteles MC, Caruso-
Neves C (2016) Uroguanylin modulates (Na++ K+) ATPase in a proximal tubule cell line:
interactions among the cGMP/protein kinase G, cAMP/protein kinase a, and mTOR pathways.
Biochim Biophys Acta 1860(7):1431–1438
Atcherley CW et al (2015) The coaction of tonic and phasic dopamine dynamics. Chem Commun
51(12):2235–2238. Royal Society of Chemistry
Baik JH (2013) Dopamine signaling in food addiction: role of dopamine D2 receptors. BMB Rep 46
(11):519
Bannerman DM et al (2004) Regional dissociations within the hippocampus—memory and anxiety.
Neurosci Biobehav Rev 28(3):273–283. Elsevier
Barger G, Dale HH (1910) Chemical structure and sympathomimetic action of amines. J Physiol 41
(1–2):19–59. Wiley Online Library
Beaulieu J, Espinoza S, Gainetdinov RR (2015) Dopamine receptors–IUPHAR review 13. Br J
pharmacol 172(1):1–23. Wiley Online Library
Beierholm U et al (2013) Dopamine modulates reward-related vigor. Neuropsychopharmacology.
38(8):1495. Nature Publishing Group
Bloom FE, Costa E, Salmoiraghi GC (1965) Anesthesia and the responsiveness of individual
neurons of the caudate nucleus of the cat to acetylcholine, norepinephrine and dopamine
administered by microelectrophoresis. J Pharmacol Exp Ther 150(2):244–252. ASPET
Bozzi Y, Borrelli E (2013) The role of dopamine signaling in epileptogenesis. Front Cell Neurosci
7:157. Frontiers
Braak H, Del Tredici K (2015) The preclinical phase of the pathological process underlying
sporadic Alzheimer’s disease. Brain 138(10):2814–2833. Oxford University Press
Bressan RA et al (2003) Optimizing limbic selective D2/D3 receptor occupancy by risperidone: a
[123I]-epidepride SPET study. J Clin Psychopharmacol 23(1):5–14. LWW
Brisch R et al (2014) The role of dopamine in schizophrenia from a neurobiological and evolution-
ary perspective: old fashioned, but still in vogue. Front Psychiatry 5:47. Frontiers
Brown JH, Makman MH (1972) Stimulation by dopamine of adenylate cyclase in retinal
homogenates and of adenosine-30 : 50 -cyclic monophosphate formation in intact retina. Proc
Natl Acad Sci U S A 69(3):539–543. National Acad Sciences
Bunzow JR et al (1988) ‘Cloning and expression of a rat D 2 dopamine receptor cDNA’, Nature
336(6201):783–787
Carlsson A (1959) The occurrence, distribution and physiological role of catecholamines in the
nervous system. Pharmacol Rev 11(2):490–493. ASPET
Carlsson A, Falck B, Hillarp N-Å (1962) Cellular localization of brain monoamines. Acta Physiol
Scand Suppl 56(196):1
Chaudhuri A, Behan PO (2000) Fatigue and basal ganglia. J Neurol Sci 179(1–2):34–42. Elsevier
Chen JY et al (2013) Dopamine imbalance in Huntington’s disease: a mechanism for the lack of
behavioral flexibility. Front Neurosci 7:114. Frontiers
Chen Y-H et al (2017) Impact of traumatic brain injury on dopaminergic transmission. Cell Transpl
26(7):1156–1168. SAGE Publications Sage CA: Los Angeles, CA
Chiodo LA, Kapatos G (1992) Membrane properties of identified mesencephalic dopamine neurons
in primary dissociated cell culture. Synapse 11(4):294–309. Wiley Online Library
178 S. Kaur et al.

Clinckers R et al (2004) Anticonvulsant action of hippocampal dopamine and serotonin is indepen-

dently mediated by D2 and 5-HT1A receptors. J Neurochem 89(4):834–843. Wiley Online
Library
Colao A et al (1997) Prolactinomas resistant to standard dopamine agonists respond to chronic
cabergoline treatment. J Clin Endocrinol Metab 82(3):876–883. Oxford University Press
Collo G, Bono F, Cavalleri L, Plebani L, Mitola S, Pich EM, Millan MJ, Zoli M, Maskos U,
Spano P, Missale C (2013) Nicotine-induced structural plasticity in mesencephalic dopaminer-
gic neurons is mediated by dopamine D3 receptors and Akt-mTORC1 signaling. Mol Pharmacol
83(6):1176–1189
Constantinescu R (2008) Update on the use of pramipexole in the treatment of Parkinson’s disease.
Neuropsychiatr Dis Treat. 4(2):337
Cools AR, Van Rossum JM (1976) Excitation-mediating and inhibition-mediating dopamine-
receptors: a new concept towards a better understanding of electrophysiological, biochemical,
pharmacological, functional and clinical data. Psychopharmacologia 45(3):243–254. Springer
Coppen EM, Roos RAC (2017) Current pharmacological approaches to reduce chorea in
Huntington’s disease. Drugs 77(1):29–46. Springer
da Silva Alves F et al (2008) The revised dopamine hypothesis of schizophrenia: evidence from
pharmacological MRI studies with atypical antipsychotic medication. Psychopharmacol Bull 41
(1):121–132
de la Mora MP et al (2010) Role of dopamine receptor mechanisms in the amygdaloid modulation
of fear and anxiety: structural and functional analysis. Prog Neurobiol 90(2):198–216. Elsevier
Dearry A et al (1990) Molecular cloning and expression of the gene for a human D1 dopamine
receptor. Nature 347(6288):72. Nature Publishing Group
Derwenskus J (2011) Current disease-modifying treatment of multiple sclerosis. Mt Sinai J Med 78
(2):161–175. Wiley Online Library
Dobryakova E et al (2015) The dopamine imbalance hypothesis of fatigue in multiple sclerosis and
other neurological disorders. Front Neurol 6:52. Frontiers
Dong H et al (2019) ‘Dorsal striatum dopamine levels fluctuate across the sleep-wake cycle and
respond to salient stimuli in mice. Front Neurosci 13:242. Frontiers
Farde L et al (1992) Positron emission tomographic analysis of central D1 and D2 dopamine
receptor occupancy in patients treated with classical neuroleptics and clozapine: relation to
extrapyramidal side effects. Arch Gen Psychiatry 49(7):538–544. American Medical
Association
Feinstein DL, Kalinin S, Braun D (2016) Causes, consequences, and cures for neuroinflammation
mediated via the locus coeruleus: noradrenergic signaling system. J Neurochem 139:154–178.
Wiley Online Library
Felder CC et al (1993) cAMP-independent, G protein-linked inhibition of Na+/H+ exchange in
renal brush border by D1 dopamine agonists. Am J Physiol 264(6):F1032–F1037. American
Physiological Society Bethesda, MD
Forster GL et al (2012) The role of the amygdala in anxiety disorders. In: The amygdala-a discrete
multitasking manager. IntechOpen, London
Gerfen CR et al (1990) D1 and D2 dopamine receptor-regulated gene expression of striatonigral and
striatopallidal neurons. Science 250(4986):1429–1432. American Association for the Advance-
ment of Science
Gingrich JA, Caron MG (1993) Recent advances in the molecular biology of dopamine receptors.
Annu Rev Neurosci 16(1):299–321. Annual Reviews 4139 El Camino Way, PO Box 10139,
Palo Alto, CA 94303-0139, USA
Grandy DK et al (1990) A human D1 dopamine receptor gene is located on chromosome 5 at q35.
1 and identifies an EcoRI RFLP. Am J Hum Genet. 47(5):828. Elsevier
Ha CM et al (2012) Calcyon forms a novel ternary complex with dopamine D1 receptor through
PSD-95 protein and plays a role in dopamine receptor internalization. J Biol Chem 287
(38):31813–31822. ASBMB
5 Pharmacology of Dopamine and Its Receptors 179

Haber SN (2016) Corticostriatal circuitry. In: Neuroscience in the 21st century. Springer,
New York, NY, pp 1–21
Hasbi A, O’Dowd BF, George SR (2011) Dopamine D1-D2 receptor heteromer signaling pathway
in the brain: emerging physiological relevance. Mol Brain 4(1):26. BioMed Central
Hirvonen J et al (2006) Striatal dopamine D1 and D2 receptor balance in twins at increased genetic
risk for schizophrenia. Psychiatry Res 146(1):13–20. Elsevier
Hornykiewicz O (1958) The action of dopamine on the arterial blood pressure of the guinea-pig. Br
J Pharmacol Chemother 13(1):91–94. Wiley Online Library
Hornykiewicz O (2002) L-DOPA: from a biologically inactive amino acid to a successful therapeu-
tic agent. Amino Acids 23(1–3):65–70. Springer
Hornykiewicz O (2006) The discovery of dopamine deficiency in the Parkinsonian brain. In:
Parkinson’s disease and related disorders. Springer, New York, NY, pp 9–15
Howes O, McCutcheon R, Stone J (2015) Glutamate and dopamine in schizophrenia: an update for
the 21st century. J Psychopharmacol 29(2):97–115. Sage Publications Sage UK: London,
England
Irwin DJ, Trojanowski JQ (2013) Many roads to Parkinson’s disease neurodegeneration: head
trauma-a road more traveled than we know? Mov Disord 28(9):1167. NIH Public Access
Jahn H (2013) Memory loss in Alzheimer’s disease. Dialogues Clin Neurosci 15(4):445. Les
Laboratoires Servier
Jenkins PO, Mehta MA, Sharp DJ (2016) Catecholamines and cognition after traumatic brain
injury. Brain 139(9):2345–2371. Oxford University Press
Juárez Olguín H et al (2016) The role of dopamine and its dysfunction as a consequence of
oxidative stress. Oxid Med Cell Longev 2016. https://doi.org/10.1155/2016/9730467. Hindawi
Jučaitė A (2002) Dopaminergic modulation of cerebral activity and cognitive functions. Medicina
38:357–362
Kandratavicius L et al (2012) Psychiatric comorbidities in temporal lobe epilepsy: possible
relationships between psychotic disorders and involvement of limbic circuits. Braz J Psychiatry
34(4):454–466. SciELO Brasil
Kapur S, Mamo D (2003) Half a century of antipsychotics and still a central role for dopamine D2
receptors. Prog Neuropsychopharmacol Biol Psychiatry 27(7):1081–1090. Elsevier
Kebabian JW, Calne DB (1979) Multiple receptors for dopamine. Nature 277(5692):93. Nature
Publishing Group
Khan ZU et al (1998) Prominence of the dopamine D2 short isoform in dopaminergic pathways.
Proc Natl Acad Sci U S A 95(13):7731–7736. https://doi.org/10.1073/pnas.95.13.7731. The
National Academy of Sciences
Kimberg DY, D’esposito M, Farah MJ (1997) Effects of bromocriptine on human subjects depend
on working memory capacity. Neuroreport 8(16):3581–3585. LWW
Kochanek PM et al (2015) Emerging therapies in traumatic brain injury. In: Seminars in neurology.
Thieme Medical Publishers, New York, NY, pp 83–100
Kriks S et al (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal
models of Parkinson’s disease. Nature 480(7378):547. Nature Publishing Group
Lacey MG, Mercuri NB, North RA (1987) Dopamine acts on D2 receptors to increase potassium
conductance in neurones of the rat substantia nigra zona compacta. J Physiol 392(1):397–416.
Wiley Online Library
Lalley PM (2009) D1/D2-dopamine receptor agonist dihydrexidine stimulates inspiratory motor
output and depresses medullary expiratory neurons. Am J Physiol Regul Integr Comp Physiol
296(6):R1829–R1836. American Physiological Society
Laruelle M (2014) Schizophrenia: from dopaminergic to glutamatergic interventions. Curr Opin
Pharmacol 14:97–102. Elsevier
Laruelle M et al (1996) Single photon emission computerized tomography imaging of
amphetamine-induced dopamine release in drug-free schizophrenic subjects. Proc Natl Acad
Sci U S A 93(17):9235–9240. National Acad Sciences
180 S. Kaur et al.

Levite M (2016) Dopamine and T cells: dopamine receptors and potent effects on T cells, dopamine
production in T cells, and abnormalities in the dopaminergic system in T cells in autoimmune,
neurological and psychiatric diseases. Acta Physiol. 216(1):42–89. https://doi.org/10.1111/
apha.12476. John Wiley & Sons, Ltd (10.1111)
Lidow MS et al (1991) Distribution of dopaminergic receptors in the primate cerebral cortex:
quantitative autoradiographic analysis using [3H] raclopride,[3H] spiperone and
[3H] SCH23390. Neuroscience 40(3):657–671. Elsevier
Loma I, Heyman R (2011) Multiple sclerosis: pathogenesis and treatment. Curr Neuropharmacol 9
(3):409–416. Bentham Science Publishers
Lud Cadet J, Jayanthi S, McCoy MT, Beauvais G, Cai NS (2010) Dopamine D1 receptors,
regulation of gene expression in the brain, and neurodegeneration. CNS Neurol Disord Drug
Targets 9(5):526–538
Lv C et al (2018) Dopamine D2-like receptors (DRD2 and DRD4) in chickens: tissue distribution,
functional analysis, and their involvement in dopamine inhibition of pituitary prolactin expres-
sion. Gene 651:33–43. Elsevier
Madikians A, Giza CC (2006) A clinician’s guide to the pathophysiology of traumatic brain injury.
Indian J Neurotrauma 3(01):9–17. Thieme Medical and Scientific Publishers Private Ltd.
Maiti P, Manna J, Dunbar GL (2017) Current understanding of the molecular mechanisms in
Parkinson’s disease: targets for potential treatments. Transl Neurodegener 6:28. https://doi.org/
10.1186/s40035-017-0099-z. England
Malo M et al (2012) Investigation of D1 receptor–agonist interactions and D1/D2 agonist selectivity
using a combination of pharmacophore and receptor homology modeling. Chem Med Chem 7
(3):483–494. Wiley Online Library
Martin EI et al (2009) The neurobiology of anxiety disorders: brain imaging, genetics, and
psychoneuroendocrinology. Psychiatr Clin North Am 32(3):549–575. https://doi.org/10.1016/
j.psc.2009.05.004. United States
Martorana A, Koch G (2014) Is dopamine involved in Alzheimer’s disease? Front Aging Neurosci
6:252. Frontiers
Meiser J, Weindl D, Hiller K (2013) Complexity of dopamine metabolism. Cell Commun Signal 11
(1):34. BioMed Central
Mendez I et al (2008) Dopamine neurons implanted into people with Parkinson’s disease survive
without pathology for 14 years. Nat Med 14(5):507. Nature Publishing Group
Mishra A, Singh S, Shukla S (2018) Physiological and functional basis of dopamine receptors and
their role in neurogenesis: possible implication for Parkinson’s disease. J Exp Neurosci
12:1179069518779829
Nagatsu T, Levitt M, Udenfriend S (1964) Tyrosine hydroxylase the initial step in norepinephrine
biosynthesis. J Biol Chem 239(9):2910–2917. American Society for Biochemistry and Molec-
ular Biology
Nobili A et al (2017) Dopamine neuronal loss contributes to memory and reward dysfunction in a
model of Alzheimer’s disease. Nat Commun 8:14727. Nature Publishing Group
Nyberg L et al (2016) Dopamine D2 receptor availability is linked to hippocampal–caudate
functional connectivity and episodic memory. Proc Natl Acad Sci 113(28):7918–7923. National
Acad Sciences
Onali P, Olianas MC, Bunse B (1988) Evidence that adenosine A2 and dopamine autoreceptors
antagonistically regulate tyrosine hydroxylase activity in rat striatal synaptosomes. Brain Res
456(2):302–309. Elsevier
Paladini CA, Roeper J (2014) Generating bursts (and pauses) in the dopamine midbrain neurons.
Neuroscience 282:109–121. Elsevier
Paredes VEB et al (2015) Reduced D2/D3 receptor binding of extrastriatal and striatal regions in
temporal lobe epilepsy. PLoS One 10(11):e0141098. Public Library of Science
Patel KR et al (2014) Schizophrenia: overview and treatment options. Pharm Ther 39(9):638.
MediMedia, USA
5 Pharmacology of Dopamine and Its Receptors 181

Perry CJ, Baciadonna L, Chittka L (2016) Unexpected rewards induce dopamine-dependent

positive emotion–like state changes in bumblebees. Science 353(6307):1529–1531. American
Association for the Advancement of Science
Pivovarov AS, Calahorro F, Walker RJ (2019) Na+/K+-pump and neurotransmitter membrane
receptors. Invert Neurosci 19(1):1
Pothos EN et al (1998) D2-like dopamine autoreceptor activation reduces quantal size in PC12
cells. J Neurosci 18(15):5575–5585. Soc Neuroscience
Quillinan N, Herson PS, Traystman RJ (2016) Neuropathophysiology of Brain Injury. Anesthesiol
Clin 34(3):453–464. https://doi.org/10.1016/j.anclin.2016.04.011. United States
Rangel-Barajas C, Coronel I, Florán B (2015) Dopamine receptors and neurodegeneration. Aging
Dis 6(5):349
Reavill C et al (1993) Pharmacological characterization of the discriminative stimulus properties of
the dopamine D1 agonist, SKF 81297. Behavioural pharmacology 4:135–146. Lippincott
Williams & Wilkins
Rezaei M, et al (2017) Epilepsy and dopaminergic system. Physiol Pharmacol 21:1–14
Robertson GS, Vincent SR, Fibiger HC (1990) Striatonigral projection neurons contain D1 dopa-
mine receptor-activated c-fos. Brain Res 523(2):288–290. Elsevier
Rubinsztein DC, Carmichael J (2003) Huntington’s disease: molecular basis of neurodegeneration.
Expert Rev Mol Med 5(20):1–21. https://doi.org/10.1017/S1462399403006549. England
Rump LC et al (1992) Effects of the novel dopamine DA 2-receptor agonist carmoxirole (EMD
45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney. Naunyn-
Schmiedeberg’s Arch Pharmacol 345(3):300–308. Springer
Seeman P (2010) Dopamine D2 receptors as treatment targets in schizophrenia. Clin Schizophr
Relat Psychoses 4(1):56–73
Seeman P et al (2005) Dopamine supersensitivity correlates with D2High states, implying many
paths to psychosis. Proc Natl Acad Sci U S A 102(9):3513–3518. National Acad Sciences
Seeman P et al (2006) Psychosis pathways converge via D2high dopamine receptors. Synapse 60
(4):319–346. Wiley Online Library
Segura-Aguilar J et al (2014) Protective and toxic roles of dopamine in Parkinson’s disease. J
Neurochem 129(6):898–915. Wiley Online Library
Smee ML, Overstreet DH (1976) Alterations in the effects of dopamine agonists and antagonists on
general activity in rats following chronic morphine treatment. Psychopharmacology 49
(2):125–130. Springer
Sokoloff P et al (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as
a target for neuroleptics. Nature. 347(6289):146. Nature Publishing Group
Spano P, Govoni S, Trabucchi M (1978) Studies on the pharmacological properties of dopamine
receptors in various areas of the central nervous system. Adv Biochem Psychopharmacol
19:155–165
Stafstrom CE, Carmant L (2015) Seizures and epilepsy: an overview for neuroscientists. Cold
Spring Harb Perspect Med 5(6):a022426
Stansley B, Yamamoto B (2015) L-dopa and brain serotonin system dysfunction. Toxics 3
(1):75–88. Multidisciplinary Digital Publishing Institute
Sulzer D, Cragg SJ, Rice ME (2016) Striatal dopamine neurotransmission: regulation of release and
uptake. Basal Ganglia 6(3):123–148. Elsevier
Sunahara RK et al (1990) Human dopamine D 1 receptor encoded by an intronless gene on
chromosome 5. Nature 347(6288):80. Nature Publishing Group
Sunahara RK et al (1991) Cloning of the gene for a human dopamine D5 receptor with higher
affinity for dopamine than D1. Nature 350(6319):614. Nature Publishing Group
Szechtman H, Sulis W, Eilam D (1998) Quinpirole induces compulsive checking behavior in rats: a
potential animal model of obsessive-compulsive disorder (OCD). Behav Neurosci 112(6):1475.
American Psychological Association
Thibaut F (2017) Anxiety disorders: a review of current literature. Dialogues Clin Neurosci 19
(2):87–88. Les Laboratoires Servier
182 S. Kaur et al.

Tiberi M et al (1991) Cloning, molecular characterization, and chromosomal assignment of a gene

encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain
compared with the D1A receptor. Proc Natl Acad Sci U S A 88(17):7491–7495. National Acad
Sciences
Van Rossum JM (1967) The significance of dopamine-receptor blockade for the action of neuro-
leptic drugs. Neuropsychopharmacology. Proceedings 5th Collegium Internationale
Neruropsychopharmacologicum, 1967. Excerpta Medica
Van Tol HHM et al (1991) Cloning of the gene for a human dopamine D4 receptor with high
affinity for the antipsychotic clozapine. Nature. 350(6319):610. Nature Publishing Group
Villanueva IE (2015) Dopamine signaling is essential in cognitive process and motor performance.
Universidad Complutense de Madrid, p. 1
Volkow N, Morales M (2015) The brain on drugs: from reward to addiction. Cell 162(4):712–725.
Elsevier
Wang Z et al (2006) Dopaminergic control of corticostriatal long-term synaptic depression in
medium spiny neurons is mediated by cholinergic interneurons. Neuron 50(3):443–452.
Elsevier
Wei C et al (2018) Response dynamics of midbrain dopamine neurons and serotonin neurons to
heroin, nicotine, cocaine, and MDMA. Cell Discov 4:60. https://doi.org/10.1038/s41421-018-
0060-z. Nature Publishing Group UK
Weiner I, Joel D (2002) Dopamine in schizophrenia dysfunctional information processing in basal
ganglia—thalamocortical split circuits. In: Dopamine in the CNS II. Springer, New York, NY,
pp 417–471
Werner FM, Coveñas R (2015) Neuropeptides and neurotransmitters involved in generalized
epilepsy: how can the antiepileptic effect be improved. J Neurol Neurophysiol 6(303):2
Wu W-L et al (2005) Dopamine D1/D5 receptor antagonists with improved pharmacokinetics:
design, synthesis, and biological evaluation of phenol bioisosteric analogues of benzazepine
D1/D5 antagonists. J Med Chem 48(3):680–693. ACS Publications
Yoon S, Baik JH (2013) Dopamine D2 receptor-mediated epidermal growth factor receptor
transactivation through a disintegrin and metalloprotease regulates dopaminergic neuron devel-
opment via extracellular signal-related kinase activation. J Biol Chem 288(40):28435–28446
Zarrindast M-R, Khakpai F (2015) The modulatory role of dopamine in anxiety-like behavior. Arch
Iranian Med. 18(9):591–603
Zhang X, Candas M, Griko NB, Taussig R, Bulla LA (2006) A mechanism of cell death involving
an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus
thuringiensis. Proc Natl Acad Sci 103(26):9897–9902
Zinger A et al (2011) The involvement of neuroinflammation and kynurenine pathway in
Parkinson’s disease. Parkinson’s Dis 2011:716859. Hindawi
Zweifel LS et al (2009) Disruption of NMDAR-dependent burst firing by dopamine neurons
provides selective assessment of phasic dopamine-dependent behavior. Proc Natl Acad Sci U
S A 106(18):7281–7288. National Acad Sciences
Pharmacology of Serotonin and Its
Receptors 6
Satyendra Deka, Ratnali Bania, Pobitra Borah, Sanjib Das,
and Pran Kishore Deb

Abstract

Serotonin (5-hydroxytryptamine) is found in platelets, neuronal bodies, and with

higher concentrations in GIT enterochromaffin cells and lesser amount in the
brain. Serotonin is responsible for various secondary actions as it is one of the
most important neurotransmitters in the CNS. It comprises seven families, namely
5-HT1 to 5-HT6, which are further divided into different subfamilies. 5-HT is
associated with the pathophysiology of many diseases including vomiting, IBS,
anxiety, schizophrenia, depression, hypertension, migraine, obsessive-
compulsive panic disorders, eating disorders, and carcinoid diarrhea. The present
chapter gives emphasis on the action of serotonin on different physiological
systems via the serotonin receptors along with their receptor pharmacology,
including the agonists, antagonists, and SSRIs.

Keywords
Serotonin · 5-hydroxytryptamine (5HT) · Serotonin receptors · SSRIs

Abbreviations

5-CT 5-Carboxamidotryptamine
5-HIAA 5-Hydroxyindoleacetic acid

S. Deka (*) · R. Bania (*) · P. Borah

Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam, India
S. Das
Department of Pharmaceutical Sciences, Assam University, Silchar, Assam, India
P. K. Deb
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman,
Jordan

# Springer Nature Singapore Pte Ltd. 2020 183

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_6
184 S. Deka et al.

5-HTT 5-Hydroxytryptophan transporter

5-HTTP 5-Hydroxytryptophan
8-OH DPAT 8-Hydroxy-di-N-propylamino tetralin
AC Adenylyl cyclase
Ach Acetylcholine
ACTH Adrenocorticotrophic hormone
cAMP Cyclic adenosine monophosphate
CNS Central nervous system
CSF Cerebrospinal fluid
DAT Dopamine transporter
DOB 2,5-Dimethyl-4-bromoamphetamine
DOI 2,5-Dimethoxy-4-iodoamphetamine
GABA Gamma-aminobutyric acid
GIT Gastrointestinal tract
IBS Irritable bowel syndrome
IL Intracellular
IM Intramuscular
IV Intravenous
MAO Monoamine oxidase
MAT Monoamine transporter
MH Malignant hyperthermia
NE Norepinephrine
NET Norepinephrine transporter
NO Nitric oxide
PAH Pulmonary arterial hypertension
PLA Phospholipase A
PLC Phospholipase C
PNS Peripheral nervous system
PPH Primary pulmonary arterial hypertension
SERT Serotonin transporter
SSRI Selective serotonin reuptake inhibitor
TCA Tricyclic antidepressant
TM Transmembrane
UTI Urinary tract infection

6.1 Introduction

Serotonin is a chemical messenger that is available in each tissue of the human body
as well as identified in plants and aerobic organisms like bacteria. Serotonin is a
ubiquitous monoamine acting as a neurotransmitter and hormone (Mohammad-
Zadeh et al. 2008), which is also recognized as 5-hydroxytryptamine (5-HT) (Shad
2017). Serotonin was first independently recognized in the late 1940s in the USA
and Italy. At that time it was known as “Serotonin” in the USA and “Enteramine” in
Italy. In 1950, it was confirmed that the structure of both the compounds were same.
In the mid-1950s, serotonin was identified in the brain of animals (Glennon and
6 Pharmacology of Serotonin and Its Receptors 185

NH2
CH2 C COOH H2 NH2 H2 H2
H HO C CH COOH C C NH2
HO
N Tryptophan
H N Decarboxylase N
hydroxylase H H
Tryptophan 5-Hydroxytryptophan
Serotonin (5-Hydroxytryptamine)

MAO+Aldeh
ydrogenase

H2 O
HO C C OH

N
H

5-Hydroxyindoleacetic acid

Fig. 6.1 Synthesis and degradation of serotonin (5-hydroxytryptamine) (Sources: Pytliak et al.
2011)

Dukat 2002). It is also found in the platelets, neurons, and GIT (enterochromaffin
cells of the GIT mucosa). Over 90% (Shajib and Khan 2015) of the total amount in
the body is present in the enterochromaffin cells of the gut and a lesser amount in the
brain and retina (Pytliak et al. 2011; Peterlin and Rapoport 2007). In the brain, cell
bodies of serotonergic neurons are mostly located in the brainstem midline with
broad axonal ridge to all areas of the CNS (Peterlin and Rapoport 2007). Serotonin is
associated with various diseases like schizophrenia, anxiety, depression, hyperten-
sion, migraine, carcinoid diarrhea, vomiting, irritable bowel syndrome, pulmonary
hypertension, and eating disorders (Pauwels 2003; De Ponti 2004).
Serotonin secreted by the nuclei in the median raphe of CNS is transferred toward
the spinal cord and other parts of the brain including the hypothalamus. Serotonin
discharged from the enterochromaffin cells of GIT finds it own way out of the tissues
into the blood, where it is taken up by the blood platelets (Zarrindast et al. 2014).
Serotonin synthesis depends on two factors: (1) accessibility of tryptophan which is an
amino acid obtained on or after nutritional ingestion, and (2) the action of rate-limiting
enzymes tryptophan hydroxylase (Peterlin and Rapoport 2007). Serotonin synthesis
starts through active uptake of tryptophan in neurons and enterochromaffin cell by use
of amino acid transporters (Pytliak et al. 2011; Upadhyay 2003). Initially, in the
presence of tryptophan hydroxylase, tryptophan is converted into
5-hydroxytryptophan, which is finally converted into 5-hydroxytryptamine (serotonin)
with the help of aromatic L-amino acid decarboxylase (Fig. 6.1) (Berger et al. 2009).
Serotonin is mainly degraded through oxidative deamination, catalyzed by mono-
amine oxidase A (MAO A), and followed by oxidation to 5-hydroxyindoleacetic acid
(5-HIAA) (Ni and Watts 2006). In the brain, it is protected from enzymatic degrada-
tion by storing in synaptic vesicles of neurons. When 5-HT is released from the storage
vesicles, it is either metabolized into 5-HIAA or reuptaken into the presynaptic
186 S. Deka et al.

Fig. 6.2 Steps involved in Presynaptic Neurons Tryptophan

synthesis and degradation of
Tryptophan Hydroxylase
serotonin
Serotonin
synthesis
Storage Breakdown

MAO
Release
Reuptake

Postsynaptic Neurons

neurons by the serotonin transporter (5-HTT or SERT) (Fig. 6.2) (Hamel and Currents
2007).
Serotonin possesses trophic factors in the early stages of pregnancy in humans.
Serotonin secreted in mother’s enterochromaffin cells of the GIT moves to the
platelets in mother’s blood. In the meantime from early stage of pregnancy, the
fetus additionally begins its own 5-HT secretion process in the nuclei of the
midbrain. After distribution of 5-HT by serotonergic neurons through the body
and brain of the fetus, it expands division, relocation, and development of peripheral
and central tissues (Shad 2017). Serotonin has vast numbers of physiological activity
like modulation of platelet aggregation, contraction of vascular and nonvascular
smooth muscle, regulation of appetite, mood, anxiety, controlling of body tempera-
ture, wakefulness, perception of sexual behavior, and hormone secretion (Upadhyay
2003; Kroeze et al. 2002). Serotonin and its receptors play a significant role in the
functioning of the brain and therefore, dysregulation of serotonin system leads to
various psychiatric and neurological disorders (Berger et al. 2009).
Serotonin, a notable neurotransmitter in the CNS, also plays a significant role in
peripheral tissues as well as in immunity. The growing human body is the proof of
recommending that a wide range of immune cells express the mechanism to create,
store, react, and transport serotonin, including mast cells, T cells, macrophages,
platelets, and dendritic cells. Moreover, there is rising evidence of a possible
association between mood disorder, serotonin, and T cells. However, it is not clear
how serotonin associates with immunity (Wu et al. 2019).
6 Pharmacology of Serotonin and Its Receptors 187

6.2 Action of Serotonin in Physiological Systems

6.2.1 Cardiovascular System

Acting on 5-HT2 receptors, 5-HT causes narrowing of vascular smooth muscle.

Although serotonin acts as a vasoconstrictor, in the heart and skeletal muscle it acts
as a vasodilator. Serotonin releases adrenaline from the adrenal medulla, which
affects ganglionic transmission and elicits cardiovascular reflexes. Serotonin has
positive inotropic and chronotropic activities on the heart, which may be blunted by
concurrent incitement of afferent nerves from baroreceptors and chemoreceptor. On
vagus nerve endings, activation of 5-HT3 receptors evokes the Bezold-Jarisch reflex
which causes hypotension and bradycardia. On arterial blood vessels serotonin
shows inhibitory action, which causes endothelial nitric oxide production, synthesis
of prostaglandin, and obstruction of NE release from sympathetic nerves (Sibley
et al. 2018; Tripathi 2013; Katzung 2018).

6.2.2 GIT

The direct action of serotonin on 5-HT2 smooth muscle receptors of GIT causes
enhancing tone and facilitates peristalsis movement. In the enteric nervous system,
activation of 5-HT4 receptors causes increased release of acetylcholine. Serotonin
inhibits gastric secretion but increases mucous production. Overproduction of sero-
tonin in carcinoid tumors causes severe diarrhea (Katzung 2018). Serotonin is also
involved in digestion right after food enters the body. Actuation of taste bud cells on
the tongue causes serotonin release onto sensory afferent nerves that move the taste
information to the CNS. When food enters in the GIT, serotonin regulates the
peristaltic movement and secretions. Modified 5-HT signaling has been concerned
in bowel disorders like irritable bowel syndrome (IBS) (Gershon and Tack 2007) and
is efficiently treated by the medication targeting both 5-HT3 and 5-HT4 receptors.
Excessive serotonin secretion in GIT can also activate the 5-HT3 receptors present in
afferent vagal nerves that innervate vomiting center present in the brainstem, which
also justifies why 5-HT3 antagonists like ondansetron are valuable antiemetic
(Berger et al. 2009).

6.2.3 Nervous System

Serotonin shows a significant feature in the development of the brain (Nordquist and
Oreland 2010). In the CNS, serotonergic neurons secrete serotonin which influences
various functions like temperature regulation, appetite, mood, hormone secretion,
cognition, sensory perception, and motor activity (Shajib and Khan 2015). A
decrease in serotonin levels leads to various psychiatric disorders like depression,
suicidal tendency, and violence. Notably, females are more prone to depressive
disorders than males, because the rate of serotonin synthesis is only about half of
188 S. Deka et al.

that in males. Depressive disorders are prominent in aged person which may
be because of less serotonin synthesis (Sibley et al. 2018).

6.2.4 Respiratory System

Serotonin has stimulant effect on smooth muscles of bronchiole through 5-HT2A

receptors. It enhances acetylcholine secretion from bronchial vagal nerve endings.
Usually, serotonin possesses hyperventilation but excess doses cause transient apnea
through coronary chemoreflex. Serotonin causes relaxation and constriction of
bronchi and bronchioles (Katzung 2018). Augmenting the release of Ach or releas-
ing Ach from activated cholinergic nerves causes bronchoconstriction (Cazzola and
Matera 2000).

6.2.5 Platelets

In platelets, the synthesis of serotonin does not occur but expresses the mechanisms
for serotonin uptake, storage, and exocytotic release. Serotonin promotes platelet
aggregation by binding to the platelet 5HT2A receptors. Serotonin released from
adherent platelets causes vasodilation, when the endothelium is undamaged, aiding
proper blood flow, whereas in damaged endothelium serotonin causes constriction
and further impairs the blood flow. These impacts of platelet-derived 5-HT are
believed to be significant in vascular disease (Sibley et al. 2018; Rang et al. 2016).

6.3 Role of Serotonin

The various roles of serotonin are highlighted in Fig. 6.3.

6.3.1 Neurotransmitter

Serotonin is a neurotransmitter present in different parts of the brain. Serotonin is

concerned with the regulation of sleep, temperature, mood, behavior etc., and the
imbalance of serotonin causes different diseases like schizophrenia, depression,
and anxiety (Tripathi 2013).

6.3.2 Malignant Hyperthermia (MH)

The episodes of MH can be activated by ecological stress such as heating, exercise,

excitement, and anxiety. Some investigations performed on pigs specify that the
sympathetic nervous system is concerned in MH as a minor response. Stresses like
environmental, exercise, and heating also increase serotonin release in the CNS and
distribute serotonin levels in blood, which induces MH (Wappler et al. 2001).
6 Pharmacology of Serotonin and Its Receptors 189

Serotonin
Syndrome Obesity Depression

Anxiety Schizophrenia

ROLE OF SEROTONIN

Migraine
GI Disorder

MH PAH Nausea Vomiting

Fig. 6.3 Role of serotonin

6.3.3 Pulmonary Arterial Hypertension (PAH)

Serotonin increases pulmonary vascular smooth muscle cell proliferation, which

causes pulmonary arterial vasoconstriction. Changes of serotonin yield cause
increasing amount of free serotonin in the surrounding area of the pulmonary artery
wall, which is an important pathophysiological process for the development of PAH.
There are some conditions where changes in serotonin amount cause PAH such as
increasing plasma concentration of free serotonin by lesser serotonin platelet storage,
PPH patients with transplantation of the heart or lung, genetic defects in serotonin
platelet storage, and low platelet count with PPH patients (Maclean et al. 2000).

6.3.4 Nausea and Vomiting

Serotonin plays an important role in nausea and vomiting induced by chemothera-

peutic agents or antineoplastic agents. Vagal-dependent pathway is considered as the
main mechanism to initiate vomiting after administration of antineoplastic agents
(Hesketh 2008). Vomiting is activated when afferent impulses from the cerebral
cortex to the vomiting center (located in medulla) travel through pharynx and vagal
afferent nerves of the GIT. Efferent nerve impulses at that point make a journey from
the vomiting center to the stomach muscles, salivary gland, cranial nerves, and
respiratory system which cause vomiting. Chemotherapeutic agents cause vomiting
by activation of neurotransmitters like serotonin, Ach, GABA situated in the che-
moreceptor trigger zone, GIT, and vomiting center (Navari 2013).
190 S. Deka et al.

6.3.5 Cancer

Recently it has been found that serotonin plays an important role in human tumor
cells of different origins like glioma, carcinoid, and carcinomas. Serotonin is
involved in the migration of cancer cells, tumor angiogenesis, and metastatic
dissemination. Different studies show that the levels of serotonin play a major role
in cancer cell growth (Sarrouilhe and Mesnil 2019).

6.3.6 Intestinal Motility and IBS

Serotonin has an important role in normal and dysfunctional GIT motility. Serotonin
receptors especially 5-HT3 and 5-HT4 receptors are targeted to treat various GIT
motility disorders. Different studies have been performed on rat to investigate
the relevance of serotonin with colonic motility (Kendig and Grider 2015). Serotonin
is a significant flagging particle in the gut particularly in enterocytes, smooth
muscles, and enteric neurons. Most of the body serotonin is available in enterochro-
maffin cells. Serotonin promotes the activation of extrinsic and intrinsic afferent
neurons to start the peristaltic and secretary reflexes and convey information to the
CNS. Serotonin is reuptaken by the serotonin transporter (SERT) in the enterocytes
or neurons. Exogenous serotonin application inspires such a large number of
responses that it is hard to figure out which is physiologically relevant. This impact
is to a great extent because of the nearness of multiple receptor subtypes, which
seem, by all accounts, to be available on a few classes of myenteric neurons, on
smooth muscle cells, and on epithelial cells. IBS is an unpredictable issue that is
associated with altered gastrointestinal motility, discharge, and sensation. Changed
serotonin signaling may prompt both intestinal and extraintestinal frameworks in
IBS (Sikander et al. 2009).

6.3.7 Obesity

Serotonin receptors present in the CNS are related with the parameter of food
ingestion leading to obesity (Thomsen et al. 2008). The capacity to store and prepare
vitality is essential for physiologic capacity. Overabundance vitality is put away in
fat tissue as triglycerides, which is discharged as free unsaturated fats when required,
through cell forms firmly managed by insulin. Fat cell functioning is required to
regulate the metabolism of lipid and body glucose level. Studies have been conducted
to understand the effect of 5-HT on lipid digestion and glucose homeostasis and
cytokine emission (Fex and Stenkula 2019). Obesity increases the risk of various
diseases like diabetes mellitus, congestive heart failure, coronary heart diseases,
stroke, hypertension, and osteoarthritis (Smith et al. 2009).
6 Pharmacology of Serotonin and Its Receptors 191

6.3.8 Migraine

Migraine pain is the most continuous neurological issue in the growing population
around the world, influencing up to 12% of the all inclusive community and more
common in women about 25%. It highly affects the general public because of its
crippling nature and in that, decreased personal satisfaction and expanded absence
from work. Cerebral pain is the essential clinical sign and it has been related with
hereditary affectability of neurovascular responses to specific improvements or to
cyclic changes in the central nervous system. Among the numerous synapses in the
brain, the serotonergic system (5-HT) from the brainstem raphe core has been most
convincingly ensnared in migraine pathophysiology. The changes in metabolism of
serotonin and serotonin-mediated responses during the migraine pain recommend
that migraine pain is a result of a central neurochemical imbalance that includes a
low serotonergic character, although the correct flow between serotonergic neuro-
transmission to the sign and symptoms of migraine pain is still not completely
understood (Hamel and Currents 2007).

6.4 Classification of Serotonin Receptors

Gaddum and Picarelli proposed the arrangement of 5-HT receptors in 1957, when it
was exhibited that practical reactions of the guinea pig ileum could be mostly
obstructed by morphine (M); at the same time the rest of the reaction can be
obstructed by dibenzyline (D) and named them as M and D receptors, respectively
(Göthert 2013).
Presently serotonin receptor families are classified into seven families (Table 6.1)
naming 5-HT1 to 5-HT6. All classes are G-protein-coupled receptors (GPCRs)

Table 6.1 5-HT families and mechanisms

Signaling
Family Type Mechanism of action effect
5-HT1 Gi/G0-protein Decreasing intracellular concentration of cAMP #AC
coupled
5-HT2 Gi/o-protein Increasing intracellular concentration of "PLC,
coupled phospholipase A2 and arachidonic acid PLA
5-HT3 Ligand-gated Na+/ Depolarization of cell plasma membrane Cations
K+ channel
5-HT4 Gs-protein Increasing intracellular concentration of cAMP "AC
coupled
5-HT5 Gi/G0-protein Decreasing intracellular concentration of cAMP #AC
coupled
5-HT6 Gs-protein Increasing intracellular concentration of cAMP "AC
coupled
5-HT6 Gs-protein Increasing intracellular concentration of cAMP "AC
coupled
Sources: Pytliak et al. (2011), Leysen (2004), Sibley et al. (2018)
192 S. Deka et al.

5-HT

5-HT1 5-HT2 5-HT3 5-HT4 5-HT5 5-HT6 5-HT7

5-HT1A 5-HT2A 5-HT3A 5-HT4A 5-HT5A

5-HT1B 5-HT2B 5-HT3B 5-HT4B 5-HT5B

5-HT1D 5-HT2C 5-HT3C 5-HT4C

5-HT1E 5-HT3D 5-HT4D

5-HT1F 5-HT3E 5-HT4E

5-HT4F

5-HT4G

5-HT4H

Fig. 6.4 Families and subfamilies of 5-HT

except 5-HT3 receptor which is a ligand-gated ion channel (Kroeze et al. 2002).
Based on sequence and pharmacological activity, all 5-HT receptors are further
subdivided (Fig. 6.4) through alternative splicing, RNA editing, etc. (Barnes and
Sharp 1999).

6.5 5-HT Receptors and Subfamilies

6.5.1 5-HT1 Receptors Subfamily

The 5-HT1 receptor is the largest class of 5-HT receptor, which has five subtypes
5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F (Fig. 6.4). All the subfamilies of
5-HT1 receptor show high affinity for the synthetic agonist,
5-carboxamidotryptamine (5-CT), except 5-HT1E and 5-HT1F receptors (Lanfumey
and Hamon 2004). The mechanism of these receptors is to decrease adenylyl cyclase
(AC) and cAMP levels by pairing to Gi/o protein. Additionally signal transduction
mechanisms also have been described (Pytliak et al. 2011).

6.5.1.1 5-HT1A Receptors

Mostly 5-HT1A receptors are dispersed in the CNS, like in axon hillock of neurons,
soma, dendrites, and the cell body. 5-HT1A receptors control the production of
adrenocorticotrophic hormone (ACTH) but they do not regulate prolactin discharge
(Wang et al. 2009). In humans, 5-HT1A receptor possesses a number of physiological
and behavioral effects like aggression, anxiety, addiction, and appetite. They also
regulate cardiovascular functions, heart rate, blood pressure, pupil dilation, and
6 Pharmacology of Serotonin and Its Receptors 193

Table 6.2 5-HT subtypes, their agonists and antagonists

Subtype Localization Agonists Antagonists
5-HT1A Raphe nuclei, Buspirone, 8-OH-DPAT WAY 100135,
hippocampus Spiperone
5-HT1B Subiculum, globus Sumatriptan, GR-55562
pallidus, substantia nigra, Methysergide, Zolmitriptan
basal ganglia
5-HT1D Cranial vessels,globus Sumatriptan, Eletriptan SB714786
pallidus, substantia nigra
5-HT1E Cortex, striatum - -
5-HT1F Dorsal raphe, Eletriptan, Naratriptan -
hippocampus, periphery
5-HT2A Platelets, smooth muscle, α – CH3-5HT, DOI Ketanserin,
cerebral cortex Cyproheptadine,
Methysergide, LY53857
5-HT2B Stomach fundus α – CH3-5HT, DOI LY53857
5-HT2C Choroid plexus, α-Methyl-5-HT, LY53857,
substantia nigra, basal Aripiprazole, Cyproheptadine,
ganglia Ergonovine, Lorcaserin Methysergide,
Mesulergine
5-HT3 Parasympathetic nerves, 2-CH3-5HT,Quipazine Ondansetron,
solitary tract, area Tropisetron
postrema, GI tract
5-HT4 Hippocampus, striatum, Cisapride, Tegaserod, GR113808
GI tract Prucalopride, Renzapride,
Metoclopramide
5-HT5A Cortex, hippocampus - SB-699551
5-HT6 Hippocampus, striatum, WAY-181187 SB-271046
nucleus accumbens
5-HT6 Hypothalamus, 5-CT, LP-12 SB-269970, Clozapine
hippocampus, GI tract
Source: Pytliak et al. (2011), Katzung (2018), Sibley et al. (2018)

vasoconstriction (Glennon and Dukat 2002). The quantity of 5-HT1A receptors is

high in hippocampus, limbic area, lateral septum, cortical area, and also in raphe
nuclei. But in cerebellum and basal ganglia, binding sites are rarely found. Some
selective agonists of 5-HT1A are Gepirone, Dipropyl-5-CT, 8-OH-DPAT, etc.
(Table 6.2) (Barnes and Sharp 1999).

6.5.1.2 5-HT1B Receptors

The 5-HT1B receptors are available on axon terminal of non-serotonergic and
serotonergic neurons. They reduce the discharge of neurotransmitters, including
gamma aminobutyric acid (GABA), serotonin, acetylcholine, glutamate, and nor-
adrenaline. 5-HT1B receptors are available in different parts of the brain but mostly
found in the frontal cortex, basal ganglia, and striatum. 5-HT1B receptors are also
194 S. Deka et al.

found on cerebral arteries and additional vascular tissues mediate vasomotor

properties of 5-HT (Lanfumey and Hamon 2004).

6.5.1.3 5-HT1D Receptors

By using the radioligand technique, 5-HT1D receptors were identified in the CNS and
broadly dispersed in the brain. They inhibit AC and paired with G-protein. They are
involved in locomotion and anxiety. However, clinical significance of 5-HT1D
receptors remains still largely unknown (Glennon and Dukat 2002).

6.5.1.4 5-HT1E Receptors

The role of 5-HT1E receptor is still unidentified because of the deficiency of selective
animal models, specific antibodies, and pharmacological tools. Hypothetically based
on their distribution in the brain like olfactory bulb, frontal cortex, and hippocam-
pus, 5-HT1E receptor regulates memory. The gene of 5-HT1E receptor is composed
of 365 amino acids and located in chromosome position 6q14-q15. Higher amount
of receptor distribution is found in cortex, claustrum, and caudate putamen but lesser
amount is observed in amygdala and hippocampus (Barnes and Sharp 1999).

6.5.1.5 5-HT1F Receptors

The 5-HT1F receptors are mostly mRNA recognized in the cortex, dorsal raphe,
hippocampus, hypothalamus, striatum, and thalamus of the human brain. From its
distribution it suggests that it may act as a 5-HT autoreceptor (Hoyer et al. 2002).
There were no selective ligands for the 5-HT1F receptors, but recently two agonists
are found, i.e., LY344864 and LY334370. Depending on the anatomical location
where the 5-HT1F receptors are present, they play an important role in cognitive and
visual functions (Barnes and Sharp 1999).

6.5.2 The 5-HT2 Receptor

The 5-HT2 receptor is a G-protein-coupled receptor and shows a typical heptahelical

structure (Leysen 2004). The 5-HT2 receptors have three subtypes, i.e., 5-HT2A,
5-HT2B, and 5-HT2C receptors (Fig. 6.4); they are coupled to Gi/o. The three 5-HT2
receptor subtypes trigger phospholipase A2 and promote the discharge of
arachidonic acid (Leysen 2004). The families of 5-HT2 receptor have different
regional distributions within the brain. Therefore each subtype may be capable of
mediating definite physiological functions (Knight et al. 2004). The 5-HT2 receptors
have showed various roles in CNS disorders like eating patterns, and influence sleep,
migraine, schizophrenia, depression, and anxiety (Knight et al. 2004).

6.5.2.1 5-HT2A Receptor

Both centrally and peripherally 5-HT2A receptors occur. They are found in post-
synaptic nonserotonergic neurons in the claustrum, olfactory nuclei, basal ganglia,
and neocortex of the CNS. They inhibit the release of neurotransmitter like dopa-
mine, glutamate, noradrenaline, and acetylcholine (Naughton et al. 2000). They also
6 Pharmacology of Serotonin and Its Receptors 195

regulate the endocrine responses like secretion of ACTH, corticosterone, oxytocin,

renin, and prolactin (Bortolozzi et al. 2005; Feng et al. 2001). 5-HT2A receptors are
peripherally found in bronchial, urinary, platelets, and vascular smooth muscle
tissues. They cause platelet aggregation, bronchoconstriction, and vasoconstriction.
5-HT2A receptors are concerned in various CNS diseases like migraine, anxiety,
depression, schizophrenia, psychosis, and hypertension (Naughton et al. 2000).

6.5.2.2 5-HT2B Receptors

The mRNA of 5-HT2B receptors is found in the human brain. In the brain, 5-HT2B
receptors are observed in dorsal hypothalamus, lateral septum, cerebellum nuclei,
medial amygdala, and also in the nucleus of dorsal raphe. Along with the brain, these
receptors are also found in the intestine, stomach, myocardium, and pulmonary
smooth muscles (Leysen 2004). On cerebral arteries of endothelial cells, stimulation
of 5-HT2B receptors leads to the secretion of nitric oxide, which causes vascular
relaxation in arteries. These vascular relaxations of cerebral arteries lead to migraine
headache (Schmuck et al. 1996). Activation of 5-HT2B receptors produces pulmo-
nary hypertension on pulmonary arteries (Launay et al. 2002). In GIT, 5-HT2B
receptors cause hypersensitivity induced by serotonin in colonic smooth muscle.
These receptors also involved in the IBS, cardiac valvulopathy, and neuroendocrine
malignancy (Leysen 2004).

6.5.2.3 5-HT2C Receptors

The mRNA and protein of 5-HT2C receptors are observed in striatum, thalamic
nuclei, brain stem nuclei, spinal cord, hippocampal formation, septal nuclei, mid-
brain nuclei in high amount (Leysen 2004). 5-HT2C is quite similar to 5-HT2A
receptors; they show high homologous series of amino acids and also coupled to
phosphoinositol hydrolysis. Some studies reported that in migraine, 5-HT2C
receptors have greater role than 5-HT2A receptors. 5-HT2C receptors are applied in
the treatment of schizophrenia, drug abuse, urinary incontinence, Parkinson’s dis-
ease, obesity, anxiety, depression, etc. Lorcaserin and WAY-163909 are the
examples of 5-HT2C receptor agonists, which are used in the treatment of obesity
(Glennon and Dukat 2002).

6.5.3 5-HT3 Receptors

The 5-HT3 receptor is only 5-HT receptor which is a part of ionotropic ligand-gated
ion channel. The structure and functions of 5-HT3 receptors resemble the members
of the cys-loop ligand-gated ion channel family (Thompson and Lummis 2007). The
receptors are recognized on neurons of both central and peripheral origin, where they
produce quick depolarization because of a transient internal current, resulting in the
opening of nonselective cation channels (Na+, Ca++ influx, K+ efflux). 5-HT3
receptors cause vomiting and nausea during radiotherapy and chemotherapy process,
which are treated with 5-HT3 antagonist like ondansetron, granisetron, tropisetron,
palonosetron, and dolasetron (Table 6.2) (Hoyer et al. 2002; De Ponti 2004). At
196 S. Deka et al.

higher concentration 5-HT3 receptors are present in the brainstem especially in

nucleus tractus solitarius and postrema, which are involved in the process of
vomiting. The 5-HT3 receptors are available in postsynaptic and presynaptic
neurons and activation can regulate the release of neurotransmitters like GABA,
dopamine, acetylcholine, and substance P (Lummis 2012). These receptors also
control GIT secretion, motility, and peristalsis in enteric nervous system and also
are involved in information transfer in the GIT (Galligan 2002). 5-HT3 receptors
have five subunits, i.e., 5-HT3A to 5-HT3E (Fig. 6.4). The structure of subunits differ
from each other like 5-HT3A varies with 32 amino acids, 5-HT3B varies with three
translational variants, and 5-HT3E with five isoforms. 5-HT3 receptor antagonists are
used not only to treat chemotherapy-induced nausea and vomiting but also useful in
the treatment of IBS, ischemic colitis, bipolar disorder, anorexia, anxiety etc.
(Lummis 2012).

6.5.4 5-HT4 Receptors

In the CNS, 5-HT4 receptors alter neurotransmitter (dopamine, acetylcholine,

GABA, and serotonin) discharge and increase synaptic diffusion. They may likewise
assume a role in memory upgrade; in any case, positive clinical investigations are
still energetically anticipated (Hoyer et al. 2002). 5-HT4 receptors are subdivided
into seven subtypes, i.e., 5-HT4A-5-HT4H (Fig. 6.4), which are coupled to Gs to
activate AC and increase cAMP production (Sibley et al. 2018), though all subtypes
contain analogous pharmacological activity and connected with AC activity
(Pauwels 2003). 5-HT4 receptors are found in the septum, hippocampus, prefrontal
cortex, and basal ganglia, which are related with cholinergic, glutamatergic, and
GABAergic neuro-transmission (King et al. 2008). 5-HT4 receptors are also avail-
able in the heart, bladder, enteric neurons, and smooth muscle cells of GIT. On
activation of the receptor, acetylcholine releases from motor neurons and inter
neurons; this increases the motility of GIT (Tack et al. 2012). Some examples of
5-HT4 receptor agonists are cisapride, mosapride, renzapride, naropride, clebopride,
and metoclopramide (Table 6.2) belonging to the benzamide groups. Besides
benzamide group agonists, other 5-HT4 agonists are tegaserod, velusetrag
(TD-5108), prucalopride, etc. (Tack et al. 2012).

6.5.5 5-HT5 Receptors

These receptors have two subfamilies, i.e., 5-HT5A and 5-HT5B (Fig. 6.4). They are
coupled to Gi/o to inhibit AC. Humans only express functional 5-HT5A receptors.
From their regions, it has been theorized that they might be engaged with feeding,
anxiety, motor control, depression, adaptive behavior, learning, memory consolida-
tion, and brain expansion (Thomas 2006). Disturbance of 5-HT neuron-glial
associations might be engaged with the advancement of certain CNS pathologies
including Alzheimer’s disease, Down’s syndrome, and some drug-actuated
6 Pharmacology of Serotonin and Its Receptors 197

development deficits. After analyzing the location of chromosome, it was found that
for human 5-HT5A receptors: position 7q36 and chromosome 7; 5-HT5B receptors:
position 2q11–13, chromosome 2. 5-HT5A consists of 357 amino acids but in
5-HT5B receptor, end codons are present in the gene. Studies on 5-HT5A and
5-HT5B are very less among the other serotonin receptors (Nelson 2004).

6.5.6 5-HT6 Receptors

It is the first receptor of 5-HT which is coupled to AC (Woolley et al. 2004) and
increases cAMP intracellular level by pairing to Gs protein. 5-HT6 receptors are
located predominantly inside limbic and extrapyramidal cerebral zones in the CNS,
which suggest that it is important for motor control and cognition (Sibley et al.
2018). The accurate scientific importance of 5-HT6 receptors remains still indistin-
guishable. 5-HT6 receptors have a role in learning and memory process. It also plays
a role in obesity. Additionally, the 5-HT6 receptors have been recommended to be
concerned in psychotic disease such as epilepsy and anxiety (Kitson 2007). The
5-HT6 receptor was first discovered in rats and humans in 1993 and 1996, respec-
tively with the help of molecular biology. The 5-HT6 receptor shows a unique
pharmacological activity of high affinity toward antipsychotic molecules and tricy-
clic and atypical antidepressant molecule like clozapine, loxapine, amitriptyline, and
mianserin. In 1998, first selective 5-HT6 receptor antagonist was reported in humans,
i.e., Ro04–6790; chemically it is 4-amino-N-(2,6-bis-methyl-amino-pyrimidin-4-
yl)-benzene sulphonamide. Ro04–6790 has less penetration through BBB, but
when 30 mg/kg dose intraperitoneal was given it showed 70% receptor occupancy
from CSF levels (Woolley et al. 2004).

6.5.7 5-HT7 Receptors

In human beings, 5-HT7 receptors enhance the activation of AC by pairing to Gs

protein. It is mainly distributed in the CNS (Sibley et al. 2018). 5-HT7 receptors
incite relaxation in human colonic smooth muscle and in the guinea pig ileum. 5-HT7
receptors have a task in reducing the peristalsis by 5-HT. 5-HT7 receptors are also
expressed in extravascular GIT smooth muscles and CNS (De Ponti 2004).
Recent studies have provided evidences on the modulation of 5-HT7 receptors for
the treatments of various CNS disorders like anxiety and depression. Also, the 5-HT7
receptor agonist facilitates memory and has antiamnesic effects (Meneses 2015).
AS-19, LP-44, and LP-211 are recently available as highly selective 5-HT7 agonists
(Ciranna 2006). The mRNA and protein of 5-HT7 receptors, which are present in
hypothalamic nuclei play a role in regulation of body temperature. 5-HT7 receptors
also regulate circadian rhythms and REM sleep (Thomas and Hagan 2004).
198 S. Deka et al.

6.6 Clinical Pharmacology of Serotonin

6.6.1 5-HT Receptor Agonist

Azapirones like buspirone, gepirone, and ipsapirone are agonists of 5-HT1A receptor
which is partially active. These are used as anxiolytic drugs and may work on the
autoreceptors to reduce serotonergic activity (Katzung 2018).
Dexfenfluramine (Fig. 6.5) is another agonist of 5-HT receptors, which is a
selective agonist. It may be broadly used as a hunger suppresser but it was with-
drawn due to cardiac valvulopathy (Katzung 2018).
Lorcaserin (Fig. 6.5) is approved for weight loss medication which is a 5-HT2C
agonist (Fig. 6.7) (Katzung 2018). The drug is thought to reduce food consumption
and increase satiety by selectively activating 5HT2C receptors on hypothalamus.
Chemically lorcaserin is 1R-8-chloro-2,3,4,5-tetrahydro-1-methyl-1H-3-benzapine
(Smith et al. 2009). In humans, lorcaserin shows 100-fold selectivity for 5-HT2C
versus the closely related 5-HT2B receptor and 17-fold selectivity over the 5-HT2A
receptor (Thomsen et al. 2008; Smith et al. 2008). There is no useful action at
additional 5-HT receptors when the concentration of lorcaserin is less than
1 micromol per liter. This leads to the minimal adverse effects than that of nonselec-
tive 5-HT receptor agonist (Smith et al. 2009). The clinical dose recommendation for
18 years and older patient of lorcaserin is 10 mg, two times in a day. On oral
administration, lorcaserin absorbed rapidly greater than 90%. The Cmax is about
1.5–2 h and t1/2 is about 11 h. About 70% drugs are bound to plasma protein.
Lorcaserin is metabolized in the liver and the main metabolite is lorcaserin
sulfamate. It is excreted by the kidney through urine and the main metabolite is N-
carbamoylglucuronides (Bai and Wang 2011). Common adverse effects are
vomiting, nausea, diarrhea, constipation, fatigue, UTI, upper respiratory tract
infections, rashes, headache, dizziness and back pain; memory and attention defi-
ciency are also seen in 1.9% of patients (Brashier et al. 2014).
The 8-hydroxydipropylamino tetraline (8-OH DPAT) is an aminotetralin deriva-
tive and highly selective 5-HT1A agonist. It is used as an experimental tool and not
used therapeutically because of its low oral bioavailability (Tripathi 2013).
Sumatriptan and other triptans are selective 5-HT1B/1D agonist and effective to
treat acute migraine pain attacks (Fig. 6.7). Migraine pain is a disease characterized
by pulsating pain in the head lasting for 4–8 h and often associated with vomiting,
nausea, sensitivity to sound and light, flashes of light, and other symptoms. Dilata-
tion of certain cranial vessels causes migraine pain; selective 5-HT1B/1D agonists
(triptans) constrict the dilated cerebral blood vessels. Triptans have three
mechanisms of action which relieve the migraine pain. The mechanisms are by
direct effect on vascular smooth cells which causes vasoconstriction of cranial
vessels, reduction of nociceptive neurotransmission, and reduction of discharge of
vasoactive neuropeptides (Tepper et al. 2002).
In 1984, Sumatriptan (Fig. 6.5) was discovered. It is the first selective 5-HT1B/1D
agonist among the triptans. At first, it was introduced as injectable, followed by
tablet, nasal spray, and suppositories (Dahlöf 2001). Sumatriptan demonstrates its
6 Pharmacology of Serotonin and Its Receptors 199

N CH3
NH H2 CH3
O H2 H2 N N CH2 CH3
C C N OH H2C O S O H2C C N
C C H2C CH3
N N CH2
H2 H2 CH2 CH3
O N
H
Buspirone 8-Hydroxy-DPAT
Sumatriptan

N CH3
CH3
H3C CH3 N NH2
N H2 HC CH CH3
H2 H2C CH2 H2 CH2 C H2C
C O C H2C H3CO
S CH2 O S O N
NH O N H
O N H NH OCH3
H
CH3 I
O
Eletriptan Naratriptan DOI
Zolmitriptan

NH2
H2C
CH2
HO N R
NH CH3
Cl N N N
H
H3C

2-Methyl 5-HT Quipazine

Lorcaserin CH3
F HN
OCH3 O N
N
N
H3CO H
O H
C N N
N
H2N H OCH3 H
Cl
Tegaserod
Cisapride

H3C
N CH3
O
O N
H Cl
N N
N N H
N N H2N O
H
CF3

Rizatriptan Prucalopride
Dexfenfluramine

Fig. 6.5 5-HT receptor agonists (Sources: Glennon and Dukat 2002)

remedial impact by incitement of the 5HT1B receptor on cranial vascular smooth

muscle that causes vasoconstriction, which relieves the headache (Fuseau et al.
2002; Dahlöf 2001). Sumatriptan has definite limits like high headache reappear-
ance, contraindications in patients with coronary artery disease, and low oral bio-
availability (Villaln et al. 2003).
200 S. Deka et al.

The bioavailability of sumatriptan is about 15% in intranasal, 14% after oral, and
96% after subcutaneous administrations. Incomplete absorption and presystemic
metabolism cause low bioavailability. The therapeutic dose is 8–66 ng/ml. After
subcutaneous administration Cmax is 25 min and after oral or intranasal administra-
tion the Cmax is 60–90 min. The t1/2 is 1.5–2.6 h (Femenia-Font et al. 2005). Adverse
effects of sumatriptan are difficulty and abnormal thinking, tiredness, dizziness,
agitation, fatigue, tremor, vertigo, etc. (Dodick and Martin 2004).
Zolmitriptan (Fig. 6.5) is another Triptans which have higher oral bioavailability
than sumatriptan, about 40% (Tepper et al. 2002). Chemically zolmitriptan is (S)4-
[3-[2-dimethylamino-ethyl]-1H-indol-5-yl]methyl-2-oxazolidinone (Goads and
Boes 2001). It is a selective 5-HT1B/1D receptor agonist. It is used effectively in
the treatment of migraine and adolescent migraine (Lewis et al. 2007). It is also used
in the treatment of cluster headache (Bahra et al. 2000). The t1/2 is 3 h and Tmax is 2 h.
Zolmitriptan is metabolized in the liver and eliminated with cytochrome P450
pathways (Goads and Boes 2001).
Naratriptan (Fig. 6.5) has 60% more bioavailability than sumatriptan, greater
lipophilicity, less readily metabolized, and better CNS penetration (Tepper et al.
2002). It belongs to triptans, a second-generation antimigraine drug. Naratriptan is
5-HT1B/1D receptor agonists. Chemically naratriptan is (N-methyl-3-1-methyl-4-
piperidinyl)-1-H-indole-5-ethanesulphonamide hydrochloride. Among the orally
administered triptans, naratriptans have highest bioavailability. The approximate
dose is 35 microgram per Kg. The pharmacokinetics of naratriptan is that it has
longest t1/2 of about 5–6 h after oral administration. 70% of naratriptan eliminates
unchanged; only a considerable part is subjected to P450 metabolism. Naratriptan
has fewer side effects (Lambert 2005).
Rizatriptan (Fig. 6.5) has high bioavailability than sumatriptan, is more potent,
and has a rapid onset of action (Tepper et al. 2002). Chemically rizatriptan is N,N-
dimethyl-2-[5-(1H-1,2,4-triazol-1-ylmethyl)-1H-indol-3-yl]ethanamine. Metabo-
lism of rizatriptan occurs in the liver by MAO-A. Rizatriptan-N-oxide and
triazolomethyl indole-3-acetic acid are two major metabolites of rizatriptan; both
are inactive. N-monodesmethyl-rizatriptan is a minor active metabolite of rizatriptan.
After oral administration, bioavailability is 45%, Cmax is 1–1.5 h, t1/2 is about 2–3 h,
and 14% bound to plasma protein. In 1998, the FDA approved rizatriptan and it is
available as oral disintegrating tablets in a dose of 5 mg or 10 mg (Wellington and
Jarvis 2002; Kacperski and O’brien 2012). Rizatriptan is used in the migraine with
aura and migraine without aura treatment in adult patients (Wellington and Jarvis
2002).
Cisapride (Fig. 6.5) is a benzamide derivative (Crowell 2004) and it agonizes the
5-HT4, which is used to treat GIT disorders like gastroesophageal motility and reflux
(Fig. 6.7) (Katzung 2018). Cisapride speeds up the process of stomach emptying and
intestinal passage but it has limitations on varying bowel functions. Some studies
confirmed that it is used in chronic constipation. But nowadays it is available only
for considerate use in the USA because of its toxicity like patient deaths and cardiac
dysrhythmias (Crowell 2004).
Tegaserod (Fig. 6.5) is a partial agonist highly selective for the 5-HT4 receptor
(Camilleri 2001), which is used for IBS and constipation. It is an
6 Pharmacology of Serotonin and Its Receptors 201

indolecarbazimidamide (Tack et al. 2012), a new class of compound which shows

GIT prokinetic effects (Crowell 2004). The chemical name of tegaserod is
[3-(5-methoxy-1H-indole-3-ylmethylene)-N-pentyl-carboximidamide]hydrogen
maleate (Müller-Lissner et al. 2001). After oral administration, tegaserod quickly
absorbed and widely distributed in tissues. Presystemically, it is metabolized and
excreted through bile as N-gluconides. From toxicity studies, it is confirmed that
tegaserod is safe and there were no drug-drug interactions found clinically (Camilleri
2001). It is effective for the treatment of gastroesophageal reflux disease, lower
bowel motility disorders, constipation, and predominant irritable bowel syndrome
(Camilleri 2001).
Prucalopride (Fig. 6.5) is a benzofurancarboxamide (Tack et al. 2012), a newer
5-HT4 agonist, stimulates peristalsis movement, and accelerates colonic transit
(De Ponti 2004). It shows more potent laxative efficacy than tegaserod (Spiller
2002). Chemically prucalopride is (4-amino-5-chloro-2,3-dihydro-N-
[1-(3-methoxypropyl)-4-piperidinyl]-7 benzofurancarboxamidemonohydrochloride
(Briejer et al. 2001). Prucalopride is quickly absorbed from GIT after oral adminis-
tration. It is excreted by urine about more than 60% and through feces about 6%. The
dose recommendation for elderly person is started with 1 mg daily and it may be
increased to 2 mg daily if required (Frampton 2009).
Velusetrag (TD-5108) is 5-HT4 agonist which has high affinity and selectivity.
Velusetrag is a derivative of dihydroquinoline carboxylic acid. It is an effective
stimulant of GIT motility. It is more potent than other 5-HT4 agonist in stimulating
colonic transit (Tack et al. 2012).

6.6.2 5-HT Antagonist

These are a variety of compounds, which block serotonergic receptors and antago-
nize the action. Some newly developed antagonists are described below:
Cyproheptadine (Fig. 6.6) has potent 5-HT2 blocking action. The major clinical
uses include management of the smooth muscle manifestations, carcinoid tumor, and
cold-induced urticaria. It is used in children for increasing appetite and poor eater for
weight gaining. Cyproheptadine also reduces muscle spasms following spinal cord
(Hoyer et al. 2002) injury, in which constitutive activity of 5-HT2C receptors is
connected with rising Ca2+ currents leading to spasms. It has some side effects like
dry mouth, drowsiness, weight gain, and confusion ataxia (Tripathi 2013).
Methysergide (Fig. 6.6) antagonizes some action of 5-HT on smooth muscles
including blood vessels. It is a 5-HT2A/2C antagonist. It is used in migraine prophy-
laxis and in the treatment of carcinoid and postgastrectomy dumping syndrome.
Long-term use causes abdominal, pulmonary, and endocardial fibrosis (Tripathi
2013). Clinically, methysergide is available in tablet form of 1 mg. Long-term
uses of methysergide has adverse effects like pleura pulmonary fibrosis, fibrotic
thickening, and retroperitoneal fibrosis (Dahlöf and Maassen Van Den Brink 2012).
Ketanserin (Fig. 6.6) is a 5-HT2 receptor antagonist. Among 5-HT2 receptors,
ketanserin shows stronger blockade in 5-HT2A than 5-HT2C. It antagonizes
202 S. Deka et al.

CH3
O N
F CH3
CH3
NH HO
NH C CH3 N
N N H
N CH3 N
O O
OCH3
O

N
WAY100135 Spiperone GR-55562

O
HN S CH3
O O
H2 H2 O
O N
O N C C N C F

N
H
N
CH3

Ketanserin
GR113808

H
N
N N
O H2C
Cl N
H3C N
N CH3
N
N
CH3

Clozapine Cyproheptadine
Ondansetron

CH3

NH
HO CH3
O N
H

N
CH3

Methysergide

Fig. 6.6 5-HT receptor antagonists (Sources: Glennon and Dukat 2002)

vasoconstriction, platelet aggregation, and contraction of airway smooth muscles

which are induced by serotonin. It is an effective antihypertensive drug (Katzung
2018). Another 5-HT2 antagonist is ritanserin; it has no α-blocking action. It alters
the bleeding time and reduces thromboxane formation (Katzung 2018).
Ondansetron (Fig. 6.6) is a newer selective 5-HT3 antagonist, which has shown
efficacy in controlling nausea and vomiting process of chemotherapy and radiother-
apy of cancer (Fig. 6.7) (Katzung 2018). In the CNS region, ondansetron inhibits
6 Pharmacology of Serotonin and Its Receptors 203

Drug Example

Buspirone, Ipsapirone 5-HT1A Anxiety, depression

Sumatriptan, Rizatriptan,
Zolmitriptan, Naratriptan 5-HT1D Migraine

Lorcaserin 5-HT2C Obesity

Ketanserin, Methysergide, Migraine, depression,

Trazodone, Risperidone 5-HT2A/2C Schizophrenia

Chemotherapy induced
Ondansetron, Tropisetron 5-HT3 emesis

Cisapride, Mosapride,
Renzapride 5-HT4 Gastrointestinal disorder

Fig. 6.7 Example of drugs with clinical uses

5-HT3 receptors or antagonizes including the region of nucleus tractus solitarius,

amygdala, postrema, and dorsal raphe nucleus. In the PNS, by antagonizing 5-HT3
receptors, ondansetron blocks the myenteric neurons and depolarization of vagal
afferent nerves, which cause a decrease of 5-HT3 receptor-mediated nociceptive
reaction. The bioavailability of oral ondansetron is low due to hepatic first-pass
metabolism. The highest plasma concentration of ondansetron is about 1.5 h after
oral administration (Ye et al. 2001). Plasma protein binding capacity of ondansetron
is about 70–76%. It has 140 L volume of distribution after IV or IM or oral routes of
administration. The t1/2 is about 3–3.5 h. Metabolism occurs in the liver by cyto-
chrome P450 enzyme pathway. Ondansetron is excreted about 5% through urine.
Ondansetron is used to treat nausea and vomiting induced by postoperative, chemo-
therapy, radiation therapy and bone marrow transplantation etc. (Culy et al. 2001).
Palonosetron is another newer 5-HT3 antagonist, which has been shown more
efficacy than first-generation 5-HT3 antagonist (ondansetron or granisetron) in
clinical trials phase III to treat emesis during chemotherapy. Palonosetron has
different chemical structure from the other drugs in its class. It is a compound
tricyclic ring system attached to quinuclidine moiety. It shows longer activity
(Rojas et al. 2008). Palonosetron has long t1/2 which is about 40 h and slower
elimination. Due to its long t1/2, it shows better efficacy about 48 h after surgery
(Srivastava et al. 2016).
204 S. Deka et al.

6.7 Serotonin Transporter

Serotonin transporter (SERT or 5-HTT) is a member of monoamine transporter

(MAT) (Xue et al. 2019). SERT terminates the actions of serotonin by various
ways like enzymatic degradation, restriction of diffusion in synapse, and reuptake
to presynaptic neuron from extracellular site (Glennon and Dukat 2002). The SERT
is located in the cytoplasm of neuron; the protein consists of amino acids (in humans
630 amino acids) and 12 reversed topological transmembrane (TM) spanning helices
with COOH and NH2 group at end point (Fig. 6.8) (Beecher et al. 2019). Trans-
membrane domain has been interlined by 2 intracellular (IL1 and IL5) and 3 extra-
cellular (EL2, EL3, and El5) hydrophilic loops (Beecher et al. 2019). SERT is 50%
homologous to norepinephrine transporter (NET) and dopamine transporter (DAT)
of MAT (Glennon and Dukat 2002). The SERT protein binds with sodium ion and
chloride ion to form a complex for transportation of serotonin (Glennon and Dukat
2002; Barnes and Neumaier 2011). SERT has vital roles in homeostasis in the CNS,
GIT, and blood platelets. SERT has in affective role in many disorders like stress,
obsessive compulsive disease, poor mood, anxiety, sexual dysfunction, and depres-
sion (Beecher et al. 2019).
Some tricyclic antidepressants (TCA) and selective serotonin reuptake inhibitors
(SSRIs) are used to block the SERT, which causes inhibition of uptake and increas-
ing the amount of neurotransmitter in the synapse (Schloss and Williams 1998).
Some examples of SERT inhibitors are fluoxetine, fluvoxamine, paroxetine,
citalopram, sertraline, imipramine, and desipramine (Fig. 6.9) (Rudnick 2006;
Glennon and Dukat 2002).
The bioavailability of fluoxetine is less than 90% due to hepatic first-pass
metabolism after oral administration. It has long t1/2 about 1–4 days. Fluoxetine is
excreted through urine. More than 90% of fluvoxamine is absorbed after oral
administration. About 100% of drug excreted through urine. Metabolism of
fluvoxamine occurs in the liver. Clinically paroxetine is a potent chiral SSRI. It is

Extracellular

Intracellular

NH2 COOH

Fig. 6.8 Serotonin transporter

6 Pharmacology of Serotonin and Its Receptors 205

H
H N
O N
CH3 H2 H2 H2 H2 O
F3C C C C C C O CH3 CH2
F3C O
N H H O
2 2
O C C NH2
Fluoxetine Fluvoxamine Paroxetine
F F

CH3
CH3 N
Cl N CH3 N
H O
Cl NC
CH3
N
Sertraline Citalopram Imipramine NH2

NH
CH3
Desipramine

Fig. 6.9 Structure of some SERT inhibitors (Sources: Glennon and Dukat 2002)

absorbed from GIT and metabolized in the liver. Complete absorption takes place
from the GIT in case of sertraline. The Cmax of sertraline is 6–8 h (Hiemke and
HÄrtter 2000).

6.8 Serotonin Toxicity or Serotonin Syndrome

Serotonin toxicity or syndrome is due to excess concentrations of 5-HT in the brain

or CNS (Birmes et al. 2003). It is characterized by different dose-related adverse
effects. Generally, it includes a group of symptoms like autonomic instability,
neuromuscular excitability, and cognitive behavioral changes, called serotonin syn-
drome (Ener et al. 2003). Depending on the increased level of serotonin in the CNS,
the toxic effect ranges from mild to severe. Overdose of drug and rising therapeutic
doses cause moderate toxicity. The combination of two or more serotonergic drugs
causes severe toxicity (Buckley et al. 2014). For analysis of serotonin syndrome, the
patient’s complete history is required including serotonergic drug uses, its signs and
206 S. Deka et al.

symptoms, and the elimination of extra situation (Birmes et al. 2003). On clinical
ground severe toxicity is diagnosed and characterized by a quickly rising tempera-
ture and inflexibility. Mild toxicity is difficult to recognize from numerous ailments
or other unfriendly medication impacts (Buckley et al. 2014). Sternbach criteria are
used for the analysis of serotonin syndrome, but it is still complicated in cases of
benign symptoms or normal neurological test results (Birmes et al. 2003). Serotonin
syndrome or toxicity is treated primarily by caring, charcoal lavage, consisting of
external cooling with blankets, and dialysis in the case of lithium overdose (Ener
et al. 2003). Generally, discontinuation of serotonergic medications is done to
overcome from toxicity (Buckley et al. 2014). Benzodiazepines are used in some
neurological symptoms, including serious myoclonus and hyperreflexia. In severe
toxicity cases cyproheptadine is recommended (Frank 2008).

6.9 Clinical Studies on Serotonin and Its Receptors

Serotonin agonists and mood: A randomized phase I study was initiated in

40 participants by the University of Chicago to determine the efficacy of very low
doses serotonergic agonist on a human volunteer with depression. The study will
measure the changes from the baseline in the Profile of Mood States (POMS) in the
subjects. This study was initiated in May 2018 and estimated to be completed by
May 2020 (ClinicalTrials.gov Identifier: NCT03790358).
Serotonin 1A agonists and cognition in schizophrenia: A phase III study was
performed in 60 participants by Vanderbilt University to observe the cognitive
functioning of schizophrenia patient with antipsychotic treatment. For 6 weeks
patients were assigned to take their antipsychotic drugs with active medication
buspar or placebo. Before and after treatment, memory and problem-solving abilities
were evaluated. The study was initiated in January 2003 and completed in October
2004 (ClinicalTrials.gov Identifier: NCT00178971).
5-HT4 agonist and chronic constipation: A phase II study was performed in
360 participants by Theravance Biopharma to compare the effectiveness and safety
in chronic constipation of TD-5108 drug (Investigational drug) with a sugar pill
(placebo). The study was performed to determine the efficacy and safety of a 5-HT4
agonist in chronic constipation. The study was initiated in October 2006 and
completed in May 2007 (ClinicalTrials.gov Identifier: NCT00391820).
Lorcaserin hydrochloride and smoking cessation: A randomized phase II study
was performed in 603 participants by Arena Pharmaceuticals to evaluate the effect of
lorcaserin hydrochloride in smokers for smoking cessation. The study was
performed by giving treatment with lorcaserin and placebo. The effect was measured
on the last 4 weeks of treatment. The study was initiated in March 2014 and
completed in November 2014 (ClinicalTrials.gov Identifier: NCT02044874).
Sarpogrelate and coronary artery disease: A randomized phase IV study was
performed in 40 participants by Seoul National University Bundang Hospital to
determine the effect of sarpogrelate, a serotonin receptor antagonist, on coronary
artery disease. The study was performed in Korean type 2 diabetic patients with
6 Pharmacology of Serotonin and Its Receptors 207

atherosclerosis disease and to review the comparisons of sarpogrelate with aspirin.

The study was initiated in July 2015 and completed in April 2016 (ClinicalTrials.gov
Identifier: NCT02607436).
Granisetron and sympathomimetics during cesarean section: An observational
study was performed in 240 participants by Johann Wolfgang Goethe University
Hospital to determine the effect of granisetron on usage of sympathomimetics during
cesarean section. The study was performed in patient with cesarean section to
quantify the usage and dose of sympathomimetics used. The study was initiated in
October 2017 and completed in February 2016 (ClinicalTrials.gov Identifier:
NCT03318536).
Ramosetron Plus DX, Dexamethasone, Granisetron plus DX, and vomiting and
nausea: A randomized phase III study was performed in 287 participants by Astellas
Pharma Inc. to compare Ramosetron Plus DX and Dexamethasone to Granisetron
plus DX. The comparisons were measured on safety and efficiency parameter for the
treatment of vomiting and nausea induced by chemotherapy. The study was initiated
in January 2006 completed in March 2006 (ClinicalTrials.gov Identifier:
NCT00272285).
5-HT3 antagonists and cardiac safety: An observational study was performed in
250 participants by the University of British Columbia to evaluate cardiac safety of
commonly used antiemetic drugs like ondansetron. The study was performed to
determine the increased cardiac safety of ondansetron. The study was initiated in
June 2014 and estimated to be completed by July 2020 (ClinicalTrials.gov Identifier:
NCT02436798).
Emend and emetogenic chemotherapy: A non-randomized phase II study was
performed in 22 participants by the University of Illinois at Chicago to review the
effect of emend or aprepitant on chemotherapy-induced nausea and vomiting for
multiple days. The drugs used in the study were aprepitant, dexamethasone, and
ondansetron. The study was initiated in November 2005 and completed in January
2009 (ClinicalTrials.gov Identifier: NCT00711555).
5-HT6 antagonist and bipolar disorder: A randomized study was initiated in
68 participants by King’s College London to determine the efficacy of
JNJ-18038683, a 5-HT6 antagonist on cognitive impaired people with bipolar
disorder and healthy volunteer using functional MRI. The study was also designed
to validate 5-HT6 antagonist for the treatment of bipolar disorders and effects of
5-HT6 antagonist for brain functioning in healthy volunteers. The study was initiated
in August 2018 and estimated to be completed by July 2020 (ClinicalTrials.gov
Identifier: NCT03633357).
5-HT3 antagonist and opioid withdrawal: A randomized study was performed in
133 participants by Stanford University to examine the uses of ondansetron on
reducing the withdrawal symptoms of opioid and to prevent the sequence of physical
dependence of opioid. The drugs used in the study were ondansetron, naloxone, and
morphine. The study was initiated in April 2011 and completed in October 2016
(ClinicalTrials.gov Identifier: NCT01549652).
Ondansetron and lactulose-induced diarrhea: A randomized phase IV study was
initiated in 16 participants by the University of Nottingham to determine the efficacy
208 S. Deka et al.

of ondansetron (5-HT3 antagonist) on the quantity of water in large and small bowel.
The study will measure the mode of action of ondansetron in lactulose-induced
diarrhea. The study was initiated in October 2018 and estimated to be completed by
August 2019 (ClinicalTrials.gov Identifier: NCT03833999).
Aprepitant in preventing nausea and vomiting: A phase II study was performed in
22 participants by Wake Forest University Health Sciences to evaluate the effect of
aprepitant in preventing nausea and vomiting in patients undergoing chemotherapy
and radiation therapy for pancreatic cancer. The drugs used in the study were
aprepitant, gemcitabine hydrochloride, capecitabine, and fluorouracil. The study
was initiated in August 2006 and completed in August 2012 (ClinicalTrials.gov
Identifier: NCT01534637).
Granisetron and myofascial pain: A randomized phase IV study was performed
in 40 participants by Karolinska Institute to evaluate the effect of granisetron on
myofascial pain in the orofacial muscles. The study was initiated in March 2007 and
completed in July 2015 (ClinicalTrials.gov Identifier: NCT02230371).
ONZETRA® Xsail® and episodic migraine: A randomized phase III study was
undertaken in 420 participants by Avanir pharmaceuticals to evaluate the safety and
efficacy of ONZETRA® Xsail® (sumatriptan nasal powder) for the acute treatment
of episodic migraine with or without aura in adolescents. The study was designed to
examine the efficacy and safety of sumatriptan nasal powder for treating migraine in
12–17 years old patients. The study was initiated in November 2017 and completed
by November 2019 (ClinicalTrials.gov Identifier: NCT03338920).
Ondansetron and CNS distribution: A non-randomized phase I study has been
performed in 18 participants by Washington University School of Medicine to
determine the time course of plasma and CSF concentrations of IV ondansetron in
healthy subjects, with and without selective inhibition of Pgp efflux transporter. The
study was initiated in May 2019 and estimated to be completed by June 2020
(ClinicalTrials.gov Identifier: NCT03809234).
Ondansetron and hypotension: A randomized phase IV study has been performed
in 100 participants by Hospital de base to verify the hypothesis that ondansetron IV
(5-HT3 receptor antagonist) decreases the occurrence of hypotension induced by
spinal anesthesia. The study was initiated in March 2019 and estimated to be
completed by March 2020 (ClinicalTrials.gov Identifier: NCT03973411).

6.10 Conclusion

Among the monoamines, serotonin or 5-hydroxytryptamine (5-HT) is unique and its

effects are served by one ligand-gated ion channel and G-protein-coupled receptors.
Seven families and different subfamilies of 5-HT receptor have been recognized. In
recent two decades, a tremendous research work has been carried out to recognize
the different 5-HT receptor families and subfamilies, and their uniqueness, reveal-
ing various connections between 5-hydroxytryptamine receptors and diseases. How-
ever, further studies would provide better insight about functions, effect of some
receptors and their subtypes in health or disease.
6 Pharmacology of Serotonin and Its Receptors 209

References
Bahra A, Gawel M, Hardebo J-E, Millson D, Breen S, Goadsby P (2000) Oral zolmitriptan is
effective in the acute treatment of cluster headache. Neurology 54:1832–1839
Bai B, Wang Y (2011) The use of lorcaserin in the management of obesity: a critical appraisal. Drug
Des Devel Ther 5:1
Barnes NM, Neumaier JF (2011) Neuronal 5-HT receptors and SERT. Tocris Biosci Sci Rev Ser
34:1–16
Barnes NM, Sharp T (1999) A review of central 5-HT receptors and their function. Neuropharma-
cology 38:1083–1152
Beecher K, Belmer A, Bartlett SE (2019) Anatomy of the serotonin transporter. Serotonin. Elsevier,
Amsterdam
Berger M, Gray JA, Roth BL (2009) The expanded biology of serotonin. Annu Rev Med
60:355–366
Birmes P, Coppin D, Schmitt L, Lauque D (2003) Serotonin syndrome: a brief review. CMAJ
168:1439–1442
Bortolozzi A, Díaz-Mataix L, Scorza MC, Celada P, Artigas F (2005) The activation of 5-HT2A
receptors in prefrontal cortex enhances dopaminergic activity. J Neurochem 95:1597–1607
Brashier DB, Sharma A, Dahiya N, Singh S, Khadka A (2014) Lorcaserin: a novel antiobesity drug.
J Pharmacol Pharmacother 5:175–178
Briejer MR, Bosmans J-P, Van Daele P, Jurzak M, Heylen L, Leysen JE, Prins NH, Schuurkes JA
(2001) The in vitro pharmacological profile of prucalopride, a novel enterokinetic compound.
Eur J Pharmacol 423:71–83
Buckley NA, Dawson AH, Isbister GK (2014) Serotonin syndrome. BMJ 348:g1626
Camilleri M (2001) tegaserod. Aliment Pharmacol Ther 15:277–289
Cazzola M, Matera MG (2000) 5-HT modifiers as a potential treatment of asthma. Trends
Pharmacol Sci 21:13–16
Ciranna Á (2006) Serotonin as a modulator of glutamate-and GABA-mediated neurotransmission:
implications in physiological functions and in pathology. Curr Neuropharmacol 4:101–114
Crowell MD (2004) Role of serotonin in the pathophysiology of the irritable bowel syndrome. Br J
Pharmacol 141:1285–1293
Culy CR, Bhana N, Plosker GL (2001) Ondansetron. Paediatr Drugs 3:441–479
Dahlöf CG (2001) Sumatriptan: pharmacological basis and clinical results. Curr Med Res Opin 17:
s35–s45
Dahlöf C, Maassen Van Den Brink A (2012) Dihydroergotamine, ergotamine, methysergide and
sumatriptan–basic science in relation to migraine treatment. Headache 52:707–714
De Ponti F (2004) Pharmacology of serotonin: what a clinician should know. Gut 53:1520–1535
Dodick DW, Martin V (2004) Triptans and CNS side-effects: pharmacokinetic and metabolic
mechanisms. Cephalalgia 24:417–424
Ener RA, Meglathery SB, Decker WAV, Gallagher RM (2003) Serotonin syndrome and other
serotonergic disorders. Pain Med 4:63–74
Femenia-Font A, Balaguer-Fernandez C, Merino V, Rodilla V, Lopez-Castellano A (2005) Effect
of chemical enhancers on the in vitro percutaneous absorption of sumatriptan succinate. Eur J
Pharm Biopharm 61:50–55
Feng J, Cai X, Zhao J, Yan Z (2001) Serotonin receptors modulate GABAA receptor channels
through activation of anchored protein kinase C in prefrontal cortical neurons. J Neurosci
21:6502–6511
Fex M, Stenkula KG (2019) Serotonin and adipocyte function. Serotonin. Elsevier, Amsterdam
Frampton JE (2009) Prucalopride. Drugs 69:2463–2476
Frank C (2008) Recognition and treatment of serotonin syndrome. Can Fam Physician 54:988–992
Fuseau E, Petricoul O, Moore KH, Barrow A, Ibbotson T (2002) Clinical pharmacokinetics of
intranasal sumatriptan. Clin Pharmacokinet 41:801–811
210 S. Deka et al.

Galligan J (2002) Ligand-gated ion channels in the enteric nervous system. Neurogastroenterol
Motil 14:611–623
Gershon MD, Tack J (2007) The serotonin signaling system: from basic understanding to drug
development for functional GI disorders. Gastroenterology 132:397–414
Glennon RA, Dukat M (2002) Serotonin receptors and drugs affecting serotonergic neurotransmis-
sion. In: Foye’s Textbook of Medicinal Chemistry. Lippincott Williams & Wilkins,
Philadelphia, pp 315–337
Goads PJ, Boes CJ (2001) Zolmitriptan: differences from sumatriptan. Curr Med Res Opin 17:s46–
s50
Göthert M (2013) Serotonin discovery and stepwise disclosure of 5-HT receptor complexity over
four decades. Part I. general background and discovery of serotonin as a basis for 5-HT receptor
identification. Pharmacol Rep 65:771–786
Hamel E, Currents H (2007) Serotonin and migraine: biology and clinical implications. Cephalalgia
27:1293–1300
Hesketh PJ (2008) Chemotherapy-induced nausea and vomiting. N Engl J Med 358:2482–2494
Hiemke C, HÄrtter S (2000) Pharmacokinetics of selective serotonin reuptake inhibitors.
Pharmacol Ther 85:11–28
Hoyer D, Hannon JP, Martin GR (2002) Molecular, pharmacological and functional diversity of
5-HT receptors. Pharmacol Biochem Behav 71:533–554
Kacperski J, O’brien HL (2012) Triptan use in pediatric migraine: focus on rizatriptan. Future
Neurol 7:385–394
Katzung BG (2018) Basic & clinical pharmacology. McGraw Hill Education (India) Private
Limited, New Delhi
Kendig DM, Grider JR (2015) Serotonin and colonic motility. Neurogastroenterol Motil
27:899–905
King MV, Marsden CA, Fone KC (2008) A role for the 5-HT1A, 5-HT4 and 5-HT6 receptors in
learning and memory. Trends Pharmacol Sci 29:482–492
Kitson SL (2007) 5-hydroxytryptamine (5-HT) receptor ligands. Curr Pharm Des 13:2621–2637
Knight AR, Misra A, Quirk K, Benwell K, Revell D, Kennett G, Bickerdike M (2004) Pharmaco-
logical characterisation of the agonist radioligand binding site of 5-HT 2A, 5-HT 2B and 5-HT
2C receptors. Naunyn Schmiedeberg’s Arch Pharmacol 370:114–123
Kroeze WK, Kristiansen K, Roth BL (2002) Molecular biology of serotonin receptors-structure and
function at the molecular level. Curr Top Med Chem 2:507–528
Lambert GA (2005) Preclinical neuropharmacology of naratriptan. CNS Drug Rev 11:289–316
Lanfumey L, Hamon M (2004) 5-HT1 receptors. Curr Drug Targets CNS Neurol Disord 3:1–10
Launay J-M, Herve P, Peoc’H K, Tournois C, Callebert J, Nebigil C, Etienne N, Drouet L,
Humbert M, Simonneau G (2002) Function of the serotonin 5-hydroxytryptamine 2B receptor
in pulmonary hypertension. Nat Med 8:1129–1135
Lewis DW, Winner P, Hershey AD, Wasiewski WW (2007) Efficacy of zolmitriptan nasal spray in
adolescent migraine. Pediatrics 120:390–396
Leysen J (2004) 5-HT2 receptors. Current Drug Targets CNS Neurol Disord 3:11–26
Lummis SC (2012) 5-HT3 receptors. J Biol Chem 287:40239–40245
Maclean MR, Herve P, Eddahibi S, Adnot S (2000) 5-hydroxytryptamine and the pulmonary
circulation: receptors, transporters and relevance to pulmonary arterial hypertension. Br J
Pharmacol 131:161–168
Meneses A (2015) Serotonin, neural markers, and memory. Front Pharmacol 6:143
Mohammad-Zadeh L, Moses L, Gwaltney-Brant S (2008) Serotonin: a review. J Vet Pharmacol
Ther 31:187–199
Müller-Lissner S, Fumagalli I, Bardhan K, Pace F, Pecher E, Nault B, Rüegg P (2001) Tegaserod, a
5-HT4 receptor partial agonist, relieves symptoms in irritable bowel syndrome patients with
abdominal pain, bloating and constipation. Aliment Pharmacol Ther 15:1655–1666
Naughton M, Mulrooney JB, Leonard BE (2000) A review of the role of serotonin receptors in
psychiatric disorders. Hum Psychopharmacol Clin Exp 15:397–415
6 Pharmacology of Serotonin and Its Receptors 211

Navari RM (2013) Management of chemotherapy-induced nausea and vomiting. Drugs 73:249–262

Nelson D (2004) 5-HT5 receptors. Curr Drug Targets CNS Neurol Disord 3:53–58
Ni W, Watts SW (2006) 5-hydroxytryptamine in the cardiovascular system: focus on the serotonin
transporter (SERT). Clin Exp Pharmacol Physiol 33:575–583
Nordquist N, Oreland L (2010) Serotonin, genetic variability, behaviour, and psychiatric disorders-
a review. Ups J Med Sci 115:2–10
Pauwels PJ (2003) 5-HT receptors and their ligands. Neuropharmacology 1083:38
Peterlin BL, Rapoport AM (2007) Clinical pharmacology of the serotonin receptor agonist,
zolmitriptan. Expert Opin Drug Metab Toxicol 3:899–911
Pytliak M, Vargová V, Mechírová V, Felsöci M (2011) Serotonin receptors-from molecular biology
to clinical applications. Physiol Res 60:15
Rang HP, Ritter J, Flower R, Henderson G (2016) Rang & Dale’s pharmacology. Elsevier,
Amsterdam
Rojas C, Stathis M, Thomas AG, Massuda EB, Alt J, Zhang J, Rubenstein E, Sebastiani S,
Cantoreggi S, Snyder SH (2008) Palonosetron exhibits unique molecular interactions with the
5-HT3 receptor. Anesth Analg 107:469–478
Rudnick G (2006) Serotonin transporters–structure and function. J Membr Biol 213:101–110
Sarrouilhe D, Mesnil M (2019) Serotonin and human cancer: a critical view. Biochimie 161:46–50
Schloss P, Williams DC (1998) The serotonin transporter: a primary target for antidepressant drugs.
J Psychopharmacol 12:115–121
Schmuck K, Ullmer C, Kalkman HO, Probst A, Lübbert H (1996) Activation of meningeal 5-HT2B
receptors: an early step in the generation of migraine headache? Eur J Neurosci 8:959–967
Shad KF (2017) Introductory chapter: serotonin-the Most ancient neurotransmitter, hormone and
trophic factor. In: Serotonin-a chemical messenger between all types of living cells. IntechOpen,
London
Shajib M, Khan W (2015) The role of serotonin and its receptors in activation of immune responses
and inflammation. Acta Physiol 213:561–574
Sibley DR, Hazelwood LA, Amara SG (2018) Goodman & Gilman’s the pharmacological basis of
therapeutics. McGraw Hill Education, New York, NY
Sikander A, Rana SV, Prasad KK (2009) Role of serotonin in gastrointestinal motility and irritable
bowel syndrome. Clin Chim Acta 403:47–55
Smith BM, Smith JM, Tsai JH, Schultz JA, Gilson CA, Estrada SA, Chen RR, Park DM, Prieto EB,
Gallardo CS, Sengupta D, Dosa PI, Covel JA, Ren A, Webb RR, Beeley NRA, Martin M,
Morgan M, Espitia S, Saldana HR, Bjenning C, Whelan KT, Grottick AJ, Menzaghi F, Thomsen
WJ (2008) Discovery and structure activity relationship of (1R)-8-Chloro-2,3,4,5-tetrahydro-1-
methyl-1H-3-benzazepine (Lorcaserin), a selective serotonin 5-HT2C receptor agonist for the
treatment of obesity. J Med Chem 51(2):305–313
Smith SR, Prosser WA, Donahue DJ, Morgan ME, Anderson CM, Shanahan WR, Group AS (2009)
Lorcaserin (APD356), a selective 5-HT2C agonist, reduces body weight in obese men and
women. Obesity 17:494–503
Spiller R (2002) Serotonergic modulating drugs for functional gastrointestinal diseases. Br J Clin
Pharmacol 54:11–20
Srivastava A, Raghavendra KP, Parate LH (2016) A comparative study of palonosetron versus
palonosetron and dexamethasone for the prevention of postoperative nausea and vomiting in
subjects undergoing laparoscopic surgeries: a randomized double-blind control study.
Karnataka Anaesth J 2:19–24
Tack J, Camilleri M, Chang L, Chey W, Galligan J, Lacy B, Müller-Lissner S, Quigley E,
Schuurkes J, De Maeyer J (2012) Systematic review: cardiovascular safety profile of 5-HT
4 agonists developed for gastrointestinal disorders. Aliment Pharmacol Ther 35:745–767
Tepper SJ, Rapoport AM, Sheftell FD (2002) Mechanisms of action of the 5-HT1B/1D receptor
agonists. Arch Neurol 59:1084–1088
Thomas DR (2006) 5-ht5A receptors as a therapeutic target. Pharmacol Ther 111:707–714
Thomas DR, Hagan JJ (2004) 5-HT7 receptors. Curr Drug Targets CNS Neurol Disord 3:81–90
212 S. Deka et al.

Thompson AJ, Lummis SC (2007) The 5-HT3 receptor as a therapeutic target. Expert Opin Ther
Targets 11:527–540
Thomsen WJ, Grottick AJ, Menzaghi F, Reyes-Saldana H, Espitia S, Yuskin D, Whelan K,
Martin M, Morgan M, Chen W (2008) Lorcaserin, a novel selective human
5-hydroxytryptamine2C agonist: in vitro and in vivo pharmacological characterization. J
Pharmacol Exp Ther 325:577–587
Tripathi K (2013) Essential of medical pharmacology, Jaypee Brothers Medical Publishers Pvt. Ltd,
New Delhi
Upadhyay S (2003) Serotonin receptors, agonists and antagonists. Indian J Nucl Med 18:1–11
Villaln C, Centurin D, Valdivia L, De Vries P, Saxena PR (2003) Migraine: pathophysiology,
pharmacology, treatment and future trends. Curr Vasc Pharmacol 1:71–84
Wang H-T, Han F, Shi Y-X (2009) Activity of the 5-HT1A receptor is involved in the alteration of
glucocorticoid receptor in hippocampus and corticotropin-releasing factor in hypothalamus in
SPS rats. Int J Mol Med 24:227–231
Wappler F, Fiege M, Am Esch JS (2001) Pathophysiological role of the serotonin system in
malignant hyperthermia. Br J Anaesth 87:794–798
Wellington K, Jarvis B (2002) Spotlight on rizatriptan in migraine. CNS Drugs 16:715–720
Woolley ML, Marsden CA, Fone KC (2004) 5-ht6 receptors. Curr Drug Targets CNS Neurol
Disord 3:59–79
Wu H, Denna TH, Storkersen JN, Gerriets VA (2019) Beyond a neurotransmitter: the role of
serotonin in inflammation and immunity. Pharmacol Res 140:100–114
Xue W, Fu T, Zheng G, Tu G, Zhang Y, Yang F, Tao L, Yao L, Zhu F (2019) Recent advances and
challenges of the drugs acting on monoamine transporters. Curr Med Chem. https://doi.org/10.
2174/0929867325666181009123218
Ye JH, Ponnudurai R, Schaefer R (2001) Ondansetron: a selective 5-HT3 receptor antagonist and its
applications in CNS-related disorders. CNS Drug Rev 7:199–213
Zarrindast M-R, Nasehi M, Hoseinpourfard M (2014) A mini review of serotonin and its receptors.
Int J Med Rev 1:39–45
Pharmacology of Histamine, Its Receptors
and Antagonists in the Modulation 7
of Physiological Functions

Bapi Gorain, Pallav Sengupta, Sulagna Dutta, Manisha Pandey,

and Hira Choudhury

Abstract

Histamine is an important monoamine consisting of an imidazole ring, which is

connected to the amino group by an ethylene group. It is biosynthesized through
histidine decarboxylase-catalysed decarboxylation of the amino acid L-histidine.
It acts through four different G-protein-coupled receptors in different locations of
the body to exert its pharmacological response. There are different structural
analogues that have been introduced to activate such receptors, whereas specific
antagonists are also introduced to inhibit their effect. This chapter highlights the
pharmacology of histamine along with structural biology, biological distribution
and physiological role of the different histaminergic receptors within the
biological system. Further, this chapter also includes different moderators to the
specific histamine receptors and projection for their pharmacological response.

Keywords
Histamine · Histamine receptor · Pharmacology · Antagonists · Agonists ·
Biological role · Structural biology · Distribution

B. Gorain (*)
School of Pharmacy, Faculty of Health and Medical Science, Taylor’s University, Subang Jaya,
Selangor, Malaysia
P. Sengupta
Department of Physiology, Faculty of Medicine, Bioscience and Nursing, MAHSA University,
Kuala Lumpur, Malaysia
S. Dutta
Department of Oral Biology and Biomedical Sciences, Faculty of Dentistry, MAHSA University,
Kuala Lumpur, Malaysia
M. Pandey · H. Choudhury
School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia

# Springer Nature Singapore Pte Ltd. 2020 213

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_7
214 B. Gorain et al.

Abbreviations

5-HT 5-hydroxy tryptamine

AC Adenylyl cyclase
ADHD Attention deficit hyperactivity disorder
BBB Blood-brain barrier
BMMC Bone marrow-derived mast cells
Ca2+ Calcium
CCL C-C motif chemokine ligand
cDNA Complementary deoxyribonucleic acid
cGMP Cyclic guanosine monophosphate
CNS Central nervous system
DNA Deoxyribonucleic acid
GPCR G-protein-coupled receptors
GSK Glycogen synthase kinase
IgE Immunoglobulin E
IP3 Inositol triphosphate
IPs Inositol phosphates
kDa Kilo Dalton
kg Kilogramme
L Litre
MAPK Mitogen-activated protein kinases
MDR1 Multidrug resistance protein 1
MHRA Medicines and Healthcare products Regulatory Agency
mRNA Messenger ribonucleic acid
PI3K Phosphatidylinositol 3-kinase
PLC Phospholipase C
RA Rheumatoid arthritis
REM Rapid eye movement
TM Transmembrane

7.1 Introduction

Histamine, a biologic amine, was isolated from a mould ergot by Sir Henry Dale and
his group at the Wellcome Laboratories more than a century ago. Since then, this
amine was studied enormously to explore associated physiological roles within the
biological system (Parsons and Ganellin 2009). This amine is considered as the most
imperative antique mediator within the biologic system, and it had been studied
extensively among the chemical mediators, including catecholamine and others
derived from amino acids for its role in the biological system (MacGlashan 2003).
Officially histamine was synthesized and characterized as a potential biologic amine
in 1907 and 1910, respectively (Barger and Dale 1910). During initial experiments
with this amine, its capabilities of constricting guinea pig ileum and its convincing
vasopressor potential had been reported. Further studies revealed the stimulatory
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 215

effect of this biologic amine on different smooth muscles of the respiratory or

gastrointestinal tract, and injecting this amine into animal system prompted shock-
like condition and cardiac contractility (Dale and Laidlaw 1910, 1919). In 1920, one
of the scientist, Popielski, depicted the stimulatory role of histamine on secretion of
gastric juice from the dog stomach. Later in 1924, Lewis demonstrated the “triple
response” of histamine, which consisted of vasodilatation and altered vascular
permeability leading to the formation of red spots, fluid extravasation and swelling
effect. Together with incidence of cutaneous flare also results as a consequence of
axon reflex (Lewis and Grant 1924). However, it took 17 years from its characteri-
zation to establish the existence of histamine in normal tissues as a natural constitu-
ent. In 1927, Best and his team had isolated this amine from lungs and liver samples
(Best et al. 1927; Shahid et al. 2009). It was also rumoured that the presence of
histamine in normal cells might be due to breakdown of histidine (Parsons and
Ganellin 2009). Subsequently, the role of histamine in connection to anaphylactic
reaction was established quickly in 1929 through analysis of histamine content
within the lung prior and after the shock, and thereafter in 1932 this histamine was
demonstrated as an important mediator of anaphylactic shock (Dale 1929; Shahid
et al. 2009). However, the connection of histamine with mast cells was established in
1952 (Riley 1953), whereas linking with basophil was recognized in 1972 (Ishizaka
et al. 1972).

7.2 Pharmacology of Histamine

To explain the biological role of histamine, it displays two basic and vital
functionalities, viz. imidazole (pKa 9.4) and primary aliphatic amine (pKa 5.8). In
aqueous environment, histamine establishes various acid-base equilibrium. Actually,
the neutral histamine or the free-base is able to receive one or two protons to form the
monocationic or dicationic fraction of histamine, respectively. The formation of
cationic fraction of histamine depends on the pH of the media, where the major
fraction (96%) of histamine forms the monocationic form at physiological pH (7.4),
with 3% dicationic and a small remaining fraction of neutral form. Alternatively, the
predominant form at less than pH 5 is the dicationic, and neutral at pH above
10 (Ramírez et al. 2003; Vianello and Mavri 2012).
Figure 7.1 represents the chemical structure of histamine with nomenclature of
the positions. This nomenclature process of histamine represents its tautomeric
forms, which is important to denote synthesis, storage, release, and metabolism of
histamine, including derivatives of this biological amine.

Fig. 7.1 Structure and Nα

Nπ β
nomenclature for histamine α
2
4
Nτ
216 B. Gorain et al.

7.2.1 Biosynthesis of Histamine

Histamine is a low molecular weight amine, which is synthesized from the amino
acid L-histidine by the presence of histidine decarboxylase (Huang et al. 2018). Any
other pathway has not been reported to achieve the transformation of histamine
(Parsons and Ganellin 2009). Following purification of histidine decarboxylase
protein from different cells of mouse and rat, a complementary DNA (cDNA) was
cloned that encodes this protein. The gene responsible for histidine decarboxylase
synthesis is Hdc gene, which prepares the proenzyme for the protein. The proenzyme
consists no enzymatic activity encompassing a molecular mass of 74 kDa. Probably
by Caspase-9, the proenzyme is cleaved to form two subunits, 20 kDa C-terminus
and 53 kDa N-terminus subunits. This N-terminal subunit then forms a homodimer
to form the active enzyme, which takes part in catalyse histamine synthesis (Furuta
et al. 2007; Komori et al. 2012). The expression of histidine decarboxylase enzyme
can be observed in several living cells in human body system, including mast cells,
basophils, parietal cells, gastric mucosa, neurons, and in central nervous system
(CNS). Therefore, this enzyme catalyses conversion of histamine from histidine in
different cells and tissues to exert its physiological roles via acting on four different
types of receptors (Oda et al. 2000; Akdis and Blaser 2003; MacGlashan 2003).
Recently, several lymphoid and myeloid cell types have been established with
higher histidine decarboxylase response, which are also capable of synthesizing high
quantities of histamine (Szeberényi et al. 2001). This type of amine is known as
“neo-synthesized histamine”, the presence of which has been located in
haematopoietic progenitors, T cells, dendritic cells, platelets, macrophages, and
neutrophils (Shiraishi et al. 2000; Tanaka et al. 2004; Dy and Schneider 2004;
Shahid et al. 2009).
The preparation of histidine decarboxylase knockout mouse model generates a
system devoid of histamine, where such models are more beneficial to establish the
role of endogenous histamine in a broad range of diseased and normal living system.
Due to lack of histamine, simultaneously a number of mast cells storage granules are
found to decrease in such knockout system, which could be correlated with the
action of histamine on production of mast cells granule proteins (Ohtsu et al. 2001).
It had been shown that in the absence of specific antigens, immunoglobulin E (IgE)
clones activate interleukin-3-dependent BMMC (bone marrow-derived mast cells),
leading towards histamine production, cytokine secretion, adhesion, migration and
improvement of survival time (Kawakami and Kitaura 2005). Simultaneous research
by Tanaka and group added that the transient induction of histidine decarboxylase by
the stimulation of IgE in the BMMC was approximately 200-fold higher than the
production of histidine decarboxylase in the presence of any antigen (Tanaka et al.
2002). Therefore, such induction further increases in the quantity of stored histamine
at the site.
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 217

7.2.2 Storage and Release of Histamine

It is evident from the earlier section that the production of histamine may occur in
several tissues within the body. However, binding capabilities to different areas of
the body and subsequent physiological roles are different because of the types of
receptor where it binds to bring the conformational change and physiological
response. Even though the heterogeneity of the histamine receptor had been
established in the 1940s, much information was on histamine-related activities
based on its release, storage and metabolism. Most of the related scientists of British
pharmacology were in agreement, including West GB, Trendelenburg U, Schild HO,
Riley JF, Perry WLM, Paton WDM, Mongar JL, Gaddum JH, Feldberg W and
Blaschko H (Corcoran 1957). Much of the research related to histamine was
compiled together and brought to the researchers worldwide jointly by the British
Pharmacological Society and the Physiological Society in a CIBA Foundation
symposium in honour of Sir Henry Dale (Schayer 1956). The mast cell as a storage
location for histamine was recognized by Riley and West, and it had been established
that certain components are responsible for inducing the release of histamine via
disruption of the mast cells and thereby results in decrease in tissue histamine
content (Riley and West 1952). Therefore, there was a strong positive correlation
established between mast cell population and histamine content in a variety of
tissues. Most of the studies performed initially to release histamine was compound
48/80, a polymer that induces histamine release. It was first described in 1951 by
Paton (Feldberg and Mongar 1954). However, simultaneous comments by Riley and
West made potential changes to the concept that some of the body tissues contained
certain cells other than mast cells to store histamine to account their histamine
content. Thus, subsequent research had brought forward towards those other cells
that store histamine, including blood basophils and platelets in some species
(Parsons and Ganellin 2006). Therefore, mast cells and basophils are the storage
location of histamine in the haematopoietic system, where this histamine could be
found within the specific granules. In mast cells, this amine is closely accompanying
anionic proteoglycans heparin, whereas in basophils it is associated with chondroi-
tin-4-sulphate. The release of a large quantity of histamine is potentiated during
degranulation of the storage granules by the action of immunological response by
IgE or cytokines, or by non-immunological response by calcium ionophore, com-
pound 48/80, substance P, etc. (Dy and Schneider 2004).
The production of histamine within enterochromaffin-like cells also has been
proven and its role in the secretion of gastric juice is established (Dy and Schneider
2004). Therefore, the presence of histamine can be found in all types of tissues
within the biological system of mammalian species; however, the range of concen-
tration of histamine may vary from 1 to over 100 μg/g. Moreover, major storage of
histamine was found in the skin, lung, connective tissues and most of the gastroin-
testinal tract (Parsons and Ganellin 2006).
218 B. Gorain et al.

7.2.3 Metabolism of Histamine

So far, we have discussed the synthesis of histamine from L-histidine followed by

storage within the granules over different tissues within the body. If the granules are
stimulated to release histamine, it follows metabolic pathways to be excreted from
the system. Small quantities of released histamine (2–3%) can be excreted from the
system unchanged, whereas the larger fraction (>97%) follows major pathways of
excretion. Two metabolizing enzymes control the major metabolic pathway for this
biological amine, which includes histamine Nτ methyl-transferase (Fig. 7.1 to
identify Nτ position) and diamine oxidase (Hill et al. 1997; Ogasawara et al.
2006). Among these two metabolizing enzymes, histamine Nτ methyl-transferase
takes part in the metabolism of a major portion of released histamine (50–80%).
Following methylation of histamine in this process, the product further undergoes
oxidation by monoamine oxidase to form the primary urinary metabolite M-
methylimidazole acetic acid. The other metabolizing enzyme, diamine oxidase,
metabolizes around 15–30% of histamine to imidazole acetic acid (Akdis and Blaser
2003). Metabolic pathway of histamine involving histamine Nτ methyl-transferase
can be prominently expedited through the application of a highly specific and potent
inhibitor of diamine oxidase, aminoguanidine. Simultaneously, blockade histamine
Nτ methyl-transferase using SKF 91488 has shown to result in in vitro and in vivo
increase in bronchoconstriction potential of histamine, suggesting this an important
enzyme in the metabolism of histamine, because inhibition of diamine oxidase did
not affect any (Sekizawa et al. 1993). The location of histamine Nτ methyl-
transferase was detected in the airway epithelial cells, and it has been hypothesized
that the released histamine from the airway mast cells gets metabolized locally.
Thereby, mechanical removal of the epithelium cells from the airway increases
in vitro bronchoconstriction potential of histamine, which can be demonstrated by
the removal of local metabolizing enzyme present on the epithelial cells (Barnes
et al. 1985; Knight et al. 1990). Because of the immediate metabolism of histamine
by two major enzymes, pharmacologically active doses of histamine has a very short
half-life in experimental rats (10 s), whereas it is 20–30 s in dogs (Parsons and
Ganellin 2006). It was also reported that the induction of infection by the virus
reduces the activity of histamine Nτ methyl-transferase; thereby the responsiveness
of inhaled histamine could be increased (Nakazawa et al. 1994).

7.3 Histamine Receptors

Being an important biological amine, histamine mediates several physiological effects

via acting on specific receptors to the amine on different target cells. Four different
types of histamine receptors have been identified so far, to produce histaminergic
responses upon binding of histamine or any structural analogue to it. Based on the
physiological role upon conformational change of the receptors, two distinct histamine
receptors, H1 and H2, were identified in 1966 (Ash and Schild 1966). Further, it was
reported that some of the histamine responses were inhibited by the use of low doses of
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 219

mepyramine, while other responses were indifferent. Just before two decades, another
two types of histamine receptors, H3 and H4, were identified in consecutive years,
1999 and 2000, respectively (Oda et al. 2000; Nieto-Alamilla et al. 2016). The
structural biology, biological distribution, physiological roles, and available
antagonists are summarized in successive sections of this chapter.

7.3.1 Histamine H1 Receptor

The histamine H1 receptor belongs to rhodopsin-like G-protein-coupled receptors

(GPCRs) family and is activated by histamine (biogenic amine) (Monczor and
Fernandez 2016). The GPCR belongs to the largest family of membrane proteins
and is being encoded by more than 800 genes in humans. As H1 receptor it is a
GPCR (coupled to Gq); it works by activating phospholipase C and inositol triphos-
phate (IP3) signalling pathway (Church 2017). H1 receptors are found in smooth
muscle, in the CNS and on vascular epithelial cells in the heart (Criado et al. 2010).
Histamine release is mostly responsible for allergic reactions against various
allergens (Church 2017). Histamine binding to the extracellular domain of H1
receptor induces conformational changes of the transmembrane section resulting in
alterations in the C terminal area (Church 2017). The C terminal in turn, via its
interaction with G proteins and activation of the Gq signalling pathway, elicits
allergic reactions (Church 2017). Several antihistaminergic drugs are used against
allergy, which bind to H1 receptor without causing activation of the receptor and
thus preventing any response (Monczor and Fernandez 2016).

7.3.1.1 Structural Biology

The structure of H1 histamine receptor has been elucidated via binding of antihista-
mine, doxepin (Shimamura et al. 2011). Binding of doxepin in the transmembrane
(TM) alpha helices of H1 receptor is stabilized by its interactions with amino acids.
The binding domain consists of a conserved tryptophan residue, which is common
among the GPCRs. The second-generation antihistamines have been shown to
access an anion-binding site comprised of two lysine residues, and they interrelate
with a phosphate (Shimamura et al. 2011).
Since histamine H1 receptor is a GPCR, it includes a conserved DRY (aspartate
(D), arginine (R), tyrosine (Y)) motif adjacent to its cytosolic face, in its seven-helix
TM surface. This motif is specialized for “ionic lock” interaction between an
aspartate and arginine in some G protein receptors that stabilizes the inactive state.
But the inactive state of the histamine H1 receptor is stabilized by the hydrogen bond
between arginine 125 and glutamine 416 (Shimamura et al. 2011).

7.3.1.2 Biological Distribution

A number of research have been carried out to demonstrate the distribution of
histamine H1 receptors in different mammalian tissues, using specific radio-ligands
(Shahid et al. 2009). Back in 1997, the development of a selective radio-ligand,
mepyramine, aided the identification of the H1 receptors in a wide array of tissues,
220 B. Gorain et al.

such as in the CNS, gastrointestinal tract, respiratory system and vascular smooth
muscle, hepatocytes, endothelial cells, T and B lymphocytes, dendritic cells,
chondrocytes, monocytes, neutrophils, cardiovascular system, genitourinary system
and adrenal medulla (Chand and Eyre 1975; Hill et al. 1977; Matsuda et al. 2004;
Sander et al. 2006; Shahid et al. 2009). A greater density of H1 receptors has been
shown in the hippocampus, posterior hypothalamus, neocortex, nucleus accumbens
and thalamus. These receptors are found in lower density in the cerebellum and basal
ganglia (Shahid et al. 2009; Mahdy and Webster 2014).

7.3.1.3 Role in Biological System

Signalling Pathway
H1 receptors’ established signalling mechanisms include Gq/11 protein activation
along with subsequent activation of phospholipase C (PLC). This leads to increased
intracellular inositol phosphates (IPs) and calcium levels, followed by activation of
small G proteins, RhoA and Rac (Notcovich et al. 2010). Alternatively, in heterolo-
gous native H1 receptors expression systems, the pathway is mediated via Gi/o,
phospholipase A2 activation and production of cyclic guanosine monophosphate
(cGMP). This is followed by nitric oxide production to trigger inflammatory
conditions (Monczor and Fernandez 2016). In certain tissues like the brain and
adrenal glands in mammals, and ovary cells (as seen in Chinese hamster), H1
receptors may also lead to the activation of adenylyl cyclase (AC) followed by
increased intracellular cAMP (30 ,50 -cyclic adenosine monophosphate) production
(Notcovich et al. 2010; Monczor and Fernandez 2016). Besides the signalling
activations by ligand binding, H1 receptors have also been reported to show sponta-
neous receptor activities even when agonists do not bind them. They are able to
activate both IP production and alter gene expression via nuclear factor-κβ
(Fitzsimons et al. 2004; Notcovich et al. 2010; Monczor and Fernandez 2016).

Neurophysiology
Endogenous histamine from neurons that have their cell bodies in the hypothalamic
tuberomammillary nucleus is able to activate the histamine H1 receptors (Haas et al.
2008). These neurons turn active during the “wakefulness” cycle (firing at about
2 Hz), while at the time of slow wave sleep the neurons firing rate reduces to as low
as 0.5 Hz and finally ceases during REM sleep (Thakkar 2011). Thus, the
tuberomammillary nucleus (histaminergic nucleus) plays the major role in regulating
sleep-wakefulness cycle (Sherin et al. 1998). Therefore, these histamine H1
receptors are one of the most important receptors that mediate internal clock. As
histamine acts upon these H1 receptors, it modulates the neurochemistry to trigger
wakefulness and a state of alertness. H1-antihistamines that are able to breach the
blood-brain barrier (BBB) are reported to inhibit H1 receptor activity on the hista-
minergic neurons arising from the tuberomammillary nucleus. This explains the
effect of drowsiness associated with these drugs. In the cerebellum and hippocam-
pus, plentiful histamine H1 receptors have been found in the Purkinje and pyramidal
cells dendrites (Haas et al. 2008). Hippocampal activation of histamine H1 receptors
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 221

leads to inhibition of hyperpolarization in hippocampal neurons. This reflects upon

intracellular calcium ions release (Hill 1990). However, neurons associated with
cortex, thalamus, brainstem and supraoptic hypothalamus are excited by the hista-
mine H1 receptor via blockade of potassium (K) conductance (Huang et al. 2006;
Haas et al. 2008; Thakkar 2011; Sundvik et al. 2011).

Inflammation
Stimulation of H1 receptors (other than in the nervous system) attributes to allergic
reactions such as motion sickness, bronchoconstriction, separation of the blood
vessels cell-lining, vasodilatation, redness of skin, hives (skin rashes) and smooth
muscle relaxation. Excessive stimulation of these receptors thus leads to aggravated
allergic progression, such as hay fever and other seasonal allergies. H1 receptor
induced inflammation is mostly mediated via expression of the transcription factor,
NF-κβ. Histamine H1 receptors activation in vascular endothelial cells triggers the
production and release of many neuromodulators, such as platelet-activating factor,
prostacyclin and nitric oxide (Sharma 2004). H1 receptor activation can also cause
vascular permeability alterations, specifically in the postcapillary venule by contrac-
tion of endothelial cells (Sharma 2004; Thurmond et al. 2008; Criado et al. 2010).
H1-antihistamines have been reported to downregulate NF-κβ expression, thereby
attenuating certain inflammatory processes (Church and Church 2011; Church
2017).

7.3.1.4 Antagonists of H1 Receptor

Treatment of allergic diseases mostly involves induction of antihistaminic action via
competitive antagonism of histamine binding to cellular receptors, commonly to the
H1 receptors, found on nerve endings, glandular cells and smooth muscles. Numer-
ous in vitro and animal experiments suggest several pharmacological properties of
the recognized H1 receptor antagonists. The first-generation H1-antihistamines are
found to have local anaesthetic, anticholinergic, sedative and anti-5-HT effects that
may mitigate symptoms of the allergic response, but are associated with various side
effects (Church and Church 2011) (Fig. 7.2).

First-Generation H1-Antihistamines
The first-generation H1-antihistamines share similar chemical constituents with
cholinergic muscarinic antagonists, antipsychotics, tranquilizers and antihyperten-
sive drugs (Mahdy and Webster 2014). These similarities render them poorly
selective leading to undesired cross-talks with other receptors and often causing
anti-α-adrenergic, antimuscarinic, as well as antiserotonin effects. They are potent to
cross the BBB and thereby can adversely affect histaminergic transmission (Church
and Church 2011).
Physiologically, histamine release during the daytime leads to wakefulness or
state of alert while the reduced levels at night account for passive decrease in arousal
response. The first-generation H1-antihistamines during the day often result in
daytime drowsiness and impaired concentration. At night, these drugs delay the
onset of REM sleep and decrease its duration (Rojas-Zamorano et al. 2009). The lack
222 B. Gorain et al.

Fig. 7.2 Conformational changes in histamine H1 receptor on binding with agonists and
antagonists

of sleep often impairs attention, working memory, as well as overall sensory-motor

activities for prolonged duration (Church and Church 2011; Mahdy and Webster
2014). The first generation H1-antihistamines have been extensively studied for their
detrimental effects upon the CNS, memory and learning, and in children, they may
also affect the performance in examinations (Church and Church 2011). These reports
have resulted in banning of these groups of drugs in several developed countries. For
example, the Medicines and Healthcare products Regulatory Agency (MHRA), in the
United Kingdom, in February 2009 (Anon 2009) has suggested stopping using of
cough and cold remedial drugs, which contain particular components including the
first-generation H1-antihistamines, while they are strictly prohibited for children
younger than 6 years. The adverse effect of these drugs accounts much for their safety.
According to published reports, more than 3000 people suffered from adverse effects
of these drugs. Importantly, diphenhydramine and chlorpheniramine had accounted
for 27 and 11 deaths, respectively (Church and Church 2011).

Second-Generation H1-Antihistamines
The 1980s had witnessed a major advance in antihistaminergic drug development
with the introduction of the second-generation H1-antihistamines (Criado et al.
2010). These possess limited access across the BBB rendering them less sedating
as compared to the first-generation H1-antihistamine. Moreover, they are more
ligand specific and have no anticholinergic effects (Church and Church 2011).
Usually, two factors determine the net efficacy of H1-antihistaminer agents, one
being its receptor affinity or absolute potency and the other is the drug concentration
at the receptors site. It has been reported that desloratadine is the most effective
antihistamine (Ki 0.4 nM), followed by levocetirizine (Ki 3 nM) and fexofenadine
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 223

(Ki 10 nM). These drugs attain higher potency in lower concentration. Physical
conditions like temperature and pH may modulate the efficacy of these drugs in
physiologic and pathologic conditions. For example, the H1 receptor affinity of
fexofenadine and levocetirizine is increased even by fivefold in inflammatory
conditions when tissue pH drops (Church and Church 2011; Mahdy and Webster
2014).
Several factors influence the free drug concentrations in the tissue compartments
and extracellular fluids. In this regard, their absorption into the systemic circulation
followed by ingestion of tablet or capsule or other oral dosage form containing the
drug is vital. H1-antihistamines are properly absorbed, with the exception of
fexofenadine which has variable absorption due to the effects of active transporter
proteins (Devillier et al. 2008). Another important factor is the plasma binding of the
drugs, which in case of this group is quite high (~65% with desloratadine, ~90%
with levocetirizine) (Church and Church 2011). Finally, the volume of distribution
of the drugs throughout the body influences its plasma levels after the distribution.
For example, levocetirizine (0.4 L/kg) has low body distribution potency, while
fexofenadine (5.4–5.8 L/kg) and desloratadine (~49 L/kg) are highly distributed
throughout the body tissues (Molimard et al. 2004). Since H1-antihistamines
concentrations in specific extracellular fluids are difficult to obtain, an indirect
estimate of their efficacy can be calculated using the data on its receptor occupancy
(absolute potency) and its peak plasma concentrations generally at ~4 h followed by
one oral dose:
Receptor occupancy (%) ¼ Bmax  L + Ki, where Bmax: maximal number of
binding sites (set to 100%); L: plasma free drug concentration; Ki: equilibrium
inhibition constant or absolute potency (Gillard et al. 2005).
The time required for onset of drug action correlates with its oral absorption rate,
but this straight relation on speed of onset of antihistamine actions with its oral
absorption rate is often breached by several confounding factors. In a study, it has
been shown that plasma concentration of levocetirizine reached after 30 min from its
injection in children with histamine-induced flare response. However, the drug had
taken more than one and half-hour for its diffusion into the extravascular space for
utmost clinical effect. Most of the H1-antihistamines take around 4 h to inhibit such
flare responses to the maximal limits in adults (Tashiro et al. 2009).
Certain anti-H1-histamines, like levocetirizine and desloratadine, possess longer
systemic action in mitigating histamine-induced flare responses than their predicted
duration of action from their plasma concentrations (Tashiro et al. 2009). This
presumably occurs owing to drug “trapping” by the H1 receptors by strong and
enduring binding (Church and Church 2011). Alternatively, fexofenadine
possesses comparatively shorter duration action. The primary reason behind this less
prolonged action of fexofenadine is that it is actively secreted into the intestine and
urine by P-glycoprotein (Tannergren et al. 2003).
A moderate quantity of research on H1-antihistamines has been centred on the
early phase allergic responses of histamines, but recent studies focus upon the anti-
inflammatory effects of these drugs (Boyle et al. 2006; Church and Church 2011).
H1-antihistamines have been seen to attenuate inflammations owing to nasal
224 B. Gorain et al.

congestion and hyper-reactivity, while continued antihistamine therapy is needed for

complete relief from such inflammatory diseases (Day et al. 2004).
The H1-antihistamines, cetirizine and levocetirizine, do not undergo any metab-
olism and are eliminated in the urine in unchanged condition (Simons et al. 2007).
Desloratadine has been demonstrated to undergo metabolism in the liver but has no
major drug interactions. Fexofenadine is minimally metabolized and follows faecal
excretion in almost unchanged version (Mahdy and Webster 2014).

7.3.2 Histamine H2 Receptor

Ash and Schild proposed the existence of two classes of histamine receptors, H1 and
H2, upon introduction of selective ligands for α- and β-adrenergic receptors and
subsequently Black proved the concept following research outcomes with selective
ligands for β-adrenergic receptors and later accepted upon synthesis of selective H2
blockers. The histamine H2 receptor is a Gs-coupled GPCRs and was firstly defined
pharmacologically by Sir James (Church 2009; Timmerman and van der Goot
2009).

7.3.2.1 Structural Biology

The structural studies of histamine H2 receptor suggested that it has a molecular
weight of 59 kDa in guinea pig striatum and hippocampus (Ruat et al. 1990). The
presence of glycosylated native histamine H2 receptor with N-glycosylation sites in
the N-terminus region in the guinea pig brain was evident with molecular weights
40.2–40.5 kDa of cloned H2 receptors (Fukushima et al. 1995; Shahid et al. 2009).
However, N-glycosylation of the receptor is not vital for cell surface localization.
For the first time, Gantz and colleagues cloned H2 receptor to strengthen a partial
length H2 receptor sequence via polymerase chain reaction from canine gastric
parietal cDNA and used it to identify a full-length H2 receptor clone following
screening of a canine genomic library (Gantz et al. 1991). For human, canine and
guinea pig the intronless gene (DNA) sequences encode 359 amino acids, whereas
358 amino acids for the rat H2 receptors. Histamine H2 receptor gene in humans was
localized to human chromosome 5, which was evident in chromosomal mapping
study (Traiffort et al. 1995). Birdsall demonstrated that an aspartate residue in TM
domain 3 and an aspartate and threonine residue in TM domain 5 were accountable
for histamine binding (Birdsall 1991). Pharmacological specificity of the H2 recep-
tor is associated with specific key amino acid residues (Shahid et al. 2009).

7.3.2.2 Biological Distribution

Histamine H2 receptors are distributed on several morphologically diverse cell types
and further it varies from species to species. It is distributed in rat uterus, gastric mucosa
of rats, guinea pigs, dogs, humans, bronchi and bronchioles of sheep and mast cells of
mice (Chand and Eyre 1975). Widespread distribution of H2 receptor mRNA expres-
sion was observed in layers III and V of cerebral cortex, granular cell layer and olfactory
bulb of guinea pig brain. H2 receptors are highly distributed in the basal ganglia,
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 225

amygdala, hippocampus and cerebral cortex, whereas a low distribution is observed in

cerebellum and thalamic and hypothalamic nuclei of human brain and guinea pig brain.
The highest ligand binding with H2 receptors was evident with layers II of prefrontal
cortex in the human brain. The H2 receptors expression is not only limited to neurons
but also evident to be present in astrocytes and in cultured brain endothelial cells. The
expression of H2 receptors is manifested in parietal cells of the rat gastric mucosa,
mainly in the apical side as compared to basal parts (Panula et al. 2015).

7.3.2.3 Role in Biological System

Activation of H2 receptors is responsible for the production of key regulators
responsible for neuronal physiology and plasticity. Therefore, the excitation of H2
receptor leads to increased production of protein kinase A, cAMP and the transcrip-
tion factor cAMP response element-binding protein (Haas and Konnerth 1983;
Pedarzani and Storm 1995; Atzori et al. 2000). In consequence, cognitive deficits
and abnormalities in nociception were reported in mice deficient of H2 receptor
functions (Panula et al. 2015). Investigation on the role of histamine in the CNS via
H2 receptor had revealed inhibition of nerve cells; then again the most exciting
action involves blockade of the long-lasting after-hyperpolarization along with
accommodation of firing, which ultimately leads to the excitation of human and
rodent brain (Haas and Konnerth 1983; Haas and Panula 2003). A slow excitation of
the H2 receptor is also observed, as in oriens-alveus interneurons, where the fast
spiking frequency is reduced by activation of Kv3.2-containing K-channel (Atzori
et al. 2000; Haas et al. 2008). Further, long-lasting potentiation of synaptic trans-
mission in the hippocampus is reported to be markedly increased or induced
(Kostopoulos et al. 1988; Haas and Panula 2003). Further, depolarization of the
thalamic relay neurons leads to opening up the doors of consciousness through an
increase in hyperpolarization-activated inward current (Panula et al. 2015).
Furthermore, the activation of the H2 receptor is also found to inhibit phospholi-
pase A2 along with arachidonic acid release, which interprets the contrast physio-
logical role of stimulated H1 or H2 receptors in different other tissues (Traiffort et al.
1992). The prediction of the central role of histamine, including behavioural
changes, through H2 receptor is quite difficult as the specific ligands of H2 receptors
hardly cross the BBB biological barrier. However, H2 receptor deficit mice were
found to display cognitive deficits, diminishing long-term potentiation of the hippo-
campal region, abnormalities in immune function, nociception and gastric function
(Panula et al. 2015). The immune response suppression via H2 receptors is also
evident in human antigen presenting cells, where stimulation of H2 receptors
revealed downregulation of different cytokines (Glatzer et al. 2013).

7.3.2.4 Antagonists of H2 Receptor

H2 receptor antagonists are found to be useful in treating hyper acid-related
disorders in the gastrointestinal system, such as gastroesophageal reflux disease
and peptic ulcer (Parsons and Ganellin 2006; Rojas-Zamorano et al. 2009). Sir
James Black won the Nobel Prize for his work on β-receptor antagonists and on
H2 receptor antagonists (Parsons and Ganellin 2006).
226 B. Gorain et al.

Nα-guanylhistamine is a weak partial agonist to H2 receptors acting on the heart

and on GI tract leading to gastric secretion. Around 1964 to 1972, burimamide was
discovered, which is a highly specific and competitive H2 receptor antagonist with
100 times more potency than Nα-guanylhistamine (Black et al. 1972). However,
further exploration of its clinical potential did not succeed due to the lack of oral
efficacy of burimamide. Metiamide was the second potent H2 receptor antagonist,
which showed good oral bioavailability with therapeutic efficacy against duodenal
ulcer disease, however precluded commercialization due to the presence of thiourea
group in the structural moiety, which caused toxicity. Afterwards, cimetidine, the
first marketed H2 receptor antagonist, introduced by Sir James Black, was developed
by replacing thiourea group with the highly polar cyanoguanidine group, effective in
the treatment of peptic ulcer and gastro-oesophageal reflux disease (Black et al.
1972; Brimblecombe et al. 1975; Parsons and Ganellin 2006). Subsequently, H2
receptor antagonists became very popular medicines for the treatment of gastric and
duodenal ulcers (Fig. 7.3) (Church 2009).
The development of H2 receptor antagonists located at the GI tract brought the lead
in the physiological control of gastric acid secretion, which could inhibit histamine,
gastrin and acetylcholine, and vagally stimulated acid secretion. Subsequently, various
H2 receptor antagonists were developed and marketed; however, specially ranitidine
become very popular among them due to the lack of drug-drug interaction potential,
unlike cimetidine. Due to high attrition in drug development considering safety and
efficacy in patients, there are only five H2 receptor antagonists (cimetidine and
ranitidine, famotidine, roxatidine and nizatidine) marketed, although several
candidates have undergone clinical study (Panula et al. 2015).

7.3.3 Histamine H3 Receptor

Histamine H3 receptor expressions are mostly predominant in the CNS rather than in
the peripheral nervous system. These receptors function as autoreceptors in presyn-
aptic histaminergic neurons (Nieto-Alamilla et al. 2016). H3 receptors also mediate
histamine turnover via negative feedback to inhibit the synthesis and release of
histamine. These also lead to feedback inhibition of several other neurotransmitters
released by functioning as an inhibitory heteroreceptor. These neurotransmitters
include gamma amino-butyric acid (GABA), dopamine, noradrenaline, acetylcho-
line, serotonin and histamine. The H3 receptors gene sequences possess as less as
about 22% and 20% homology with H1 and H2 histamine receptors, respectively.
The H3 receptor is a potential therapeutic target due to its regulatory functions in
several neuronal mechanisms (Nieto-Alamilla et al. 2016).

7.3.3.1 Structural Biology

Histamine H3 receptors also belong to the GPCR group with the core seven-TM
domain, an extracellular amino terminus (NT), an intracellular carboxyl terminus
(CT), three extracellular (ECL) and three intracellular (ICL) loops (Nieto-Alamilla
et al. 2016). The H3 histamine receptors consist of a DRF motif instead of common
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 227

Fig. 7.3 Different ligands acting on H2 receptor (Panula et al. 2015)

GPCRs DRY sequence in the interface of TM domain and intracellular loops.

Similar to NPXXY motif in all GPCRs, the histamine H3 receptor possesses a
NPVLY motif in the TM domain (Bongers et al. 2007). The intracellular carboxy
terminal of the receptor possesses a palmitoylation site, which aids the formation of
helix 8. The Cys107 and Cys188 of the first and second extracellular loops form
disulphide bonds. The receptor contains a short amino terminal of 39 amino acids,
which have a glycosylation site on Asn11, while the third intracellular loop is long
consisting of 142 amino acids. These sequences serve as loci for naturally occurring
mutations that give rise to several isoforms of H3 histamine receptors (Nieto-
Alamilla et al. 2016).
228 B. Gorain et al.

7.3.3.2 Biological Distribution

The histamine H3 receptor is a popular presynaptic autoreceptor acting upon
histamine-containing neurones. These receptors are immensely distributed in the
CNS, mostly in the cortex, thalamus, the caudate nucleus, hippocampus, hypothala-
mus and in the olfactory tubercle (Arrang et al. 1983, 1987). High expression of the
histamine H3 receptors throughout the cortex suggests their significant role in the
modulation of a wide range of neurotransmitters, such as GABA, acetylcholine,
norepinephrine, dopamine and serotonin in the CNS and peripheral tissues. The
distribution of histamine H3 receptors is quite low in the periphery including in the
heart, small intestine, prostate and testis in humans, while their expressions in tissues
like the lung and spleen are not reported (Lovenberg et al. 1999).

7.3.3.3 Role in Biological System

The histamine H3 receptors’ potency in neurotransmitters modulation renders them a
novel therapeutic target in the treatment of innumerable disorders. These include a
number of inflammations and allergic reactions. In addition, these also account for
various neurological disorders, leading to innumerable conditions. Obesity is caused
by histamine cross-talks with orexinergic system, movement disorders owing to
dopamine and GABA modulations by H3 receptor in the basal ganglia, schizophre-
nia and attention deficit hyperactivity disorder (ADHD) due to dopamine modula-
tion, as well as irregular sleep-wakefulness cycle by alterations in noradrenaline,
glutamate and histamine (Lovenberg et al. 1999; Morisset et al. 2000; Leurs et al.
2005, 2012). They also play a role in the regulation of satiety (Passani et al. 2011).
Histamine H3 receptors are coupled to G-protein Gi, inhibiting intracellular
cAMP generation (Lovenberg et al. 1999). The interactions between the dissociated
βγ and N-type voltage-gated calcium channels lead to decreased action potential-
induced calcium influx. This ultimately results in reduced neurotransmitter release,
thereby acting as presynaptic autoreceptors on histamine containing neurons (Arrang
et al. 2007; Feuerstein 2008). The Gαi/o protein-dependent histamine H3 receptor
signalling is usually mediated by both the Gα subunits and the Gβγ complexes,
acting through the subsequent pathways that include one or more of the following:
arachidonic acid, adenylyl cyclases; cAMP; Na+/H+ exchanger; mitogen-activated
protein kinases; phosphatidylinositol 3-kinase; phospholipase A2; phospholipase C;
protein kinase A (Fig. 7.4) (Morisset et al. 2000; Nieto-Alamilla et al. 2016).

Inhibition of Na+/H+ Exchanger

Histamine H3 receptors have been reported to inhibit Na+/H+ exchanger activities in
sympathetic nerve terminals in myocardial ischemia, thereby inhibiting noradrena-
line release. The mechanism may involve a direct interaction of Gαi/o subunit with
the Na+/H+ exchanger (Bongers et al. 2007).

Inhibition of Voltage-Gated Calcium Channels

Histamine H3 receptors mediate inhibition of neurotransmitter through action
potential-induced Ca2+ entry by Gβγ complexes binding to pore-forming α1-subunit
of calcium channels (N- and P/Q-type voltage-gated) (Zamponi and Currie 2013).
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 229

Fig. 7.4 Molecular signalling pathway of activated histamine H3 receptor (H3R) located in
presynaptic and postsynaptic neuron to mediate biological functions. AC adenylyl cyclase; MAPK
mitogen-activated protein kinases; PI3K phosphoinositide 3-kinase; PLC phospholipase C; PLA2
phospholipase A2; GIRK G-protein-coupled inwardly rectifying potassium channel

Accordingly, the histamine H3 receptor activation downregulates calcium influx in

dissociated histaminergic neurons of hypothalamus, in the transfected human neu-
roblastoma cells, striatal synaptosomes, and transfected pheochromocytoma cells
(Nieto-Alamilla et al. 2016).

Activation of G-Protein-Gated Potassium Channels

Gβγ subunits of the histamine H3 receptors bind and activate G-protein-gated
inwardly rectifying potassium K+ channels (GIRK) (Sahlholm et al. 2012), which
can downregulate synaptic transmission (Meneses et al. 2015) and modulate neuro-
transmitter release. This occurs at the postsynaptic neurons secreting melanin-
concentrating hormone (MCH) (Parks et al. 2014).

Activation of Phospholipase C
Histamine H3 receptors may activate phospholipase C (PLC), thereby leading to
increased intracellular calcium ions concentration via IP3 pathway to mediate
physiological functions (Bongers et al. 2007).
230 B. Gorain et al.

Activation of the MAPK Pathway

Histamine H3 receptors are able to mediate MAPK phosphorylation in heterologous
systems as well as native tissues (Flores-Clemente et al. 2013). Gβγ complexes have
been suggested to play a key role in this action (Lai et al. 2016).

Activation of the Phosphatidylinositol 3-Kinase (PI3K) Pathway

Histamine H3 receptor activation triggers PI3K pathway via Gβγ complexes and
protein kinase B (Akt) phosphorylation, which by subsequent actions inhibits
glycogen synthase kinase 3-β (GSK3β) activities (Bongers et al. 2007).

Stimulation of Phospholipase A2
Histamine H3 receptor-induced phospholipase A2 activation leads to the release of
docosahexaenoic acid, arachidonic acid and lysophospholipids. These mediate
physiological functions such as relaxation of bronchioles via endothelium-derived
relaxing factor, which is an arachidonic acid metabolite (Nieto-Alamilla et al. 2016).

7.3.3.4 Antagonists of H3 Receptor

The histamine H3 receptor, as previously discussed, is an autoreceptor, which also
regulates the release of several other neurotransmitters, including noradrenaline,
dopamine and 5-HT acetylcholine. Thus, it serves as a popular therapeutic target
for CNS disorders, as agents with multiple and complementary modes of action.

Early Pharmacophore
The initial focus of H3 receptor ligands development was upon the agonists with an
imidazole ring within its structure (Celanire et al. 2005). The imidazole ring in these
drugs leads to undesired inhibition of cytochrome P450 isoenzymes and this results
in adverse drug interactions (Celanire et al. 2005). Moreover, they fail to cross the
BBB and also proved to have a certain degree of toxicity (Schwartz 2011). Another
problem with these drugs is less specificity and action upon other receptors such as
on the histamine H4 receptor (Sadek et al. 2016).
Thioperamide represents the first imidazole-based histamine H3 antagonist. It
was quite potent as well as selective in action but could cause hepatotoxicity. It was
first designed in order to enhance cognition functions and wakefulness (Schwartz
2011). A potential study has reported the efficacy of thioperamide treatment in
ameliorating circadian rhythm Parkinson patient (Masini et al. 2017).

New Pharmacophore
To mitigate the limitations of histamine H3 receptor imidazole based antagonists,
non-imidazole H3 receptor antagonists emerged. These drugs can easily cross the
BBB and reach the CNS. However, few problems also cropped up in the use of these
drugs, for example phospholipidosis, its strong binding to potassium channel, and
problems with P-glycoprotein also known as multidrug resistance protein 1 (MDR1)
substrate (Gemkow et al. 2009). Pitolisant, a highly selective antagonist for the H3
receptor, was the first H3 receptor antagonist to proceed to clinical trials. Presently, it
is the sole drug that is approved in the USA and Europe.
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 231

Histamine H3 receptor antagonists are immensely introduced in clinical trials for

the treatment of cognitive impairments in Alzheimer’s disease, narcolepsy, attention
deficit hyperactivity disorder (ADHD), Parkinson’s disease and schizophrenia
(Benarroch 2010). A striking property of H3 receptors is their high degree of
constitutive activity in vivo (Rouleau et al. 2002). This discovery is important for
drug development, since the ability to compete with constitutively active H3 recep-
tor states (inverse agonism) has important therapeutic implications. H3 antagonists
are useful in treating sleep disorders (Lin et al. 2011). They are highly potent drugs
for addressing cognition disorders as pharmacological blockade of H3 receptors has
been reported to elicit procognitive outcomes in different preclinical models of
Alzheimer’s disease, ADHD and schizophrenia (Brioni et al. 2011). At present,
the treatment modulation involves a single neurotransmitter system
(e.g. cholinesterase inhibitors to treat Alzheimer’s disease, dopaminergic stimulants
for ADHD). However, multiple neural circuits and neurotransmitter systems are
associated with such treatments as well. The histamine H3 receptor antagonism can
increase the concentrations of noradrenaline, dopamine, acetylcholine, serotonin and
histamine in the cortex resulting in alterations in cognitive processes (Esbenshade
et al. 2008).

7.3.4 Histamine H4 Receptor

The gene of H4 receptor was discovered in 1999 from the Human Genome Project,
which had a feature of class A GPCR. This receptor is found to have similarity with
other histamine receptors in its homology; thus it became the fourth receptor in the
histamine family. After the huge success of H1 and H2 targeting, H4 was also
underwent investigation for its targetability and functions. A vast literature review
revealed that some of the histaminic activities were not mediated by H1, H2 and H3
receptors. In this context, pruritus and asthma came up with the major area of
investigation as histamine plays an important role in the pathogenesis of disease.
However, H1 and H2 targeted histamines does not respond effectivity for these
disease conditions. Later this gap was filled with the concept of H4 receptor media-
tion in inflammation and lung functions. Likewise, patients with atopic dermatitis
were not responding to H1 receptor antagonists. Subsequently, the generation of H4
antagonist has shown tremendous effect on disease conditions (Leurs et al. 2012;
Thurmond 2015; Kiss and Keserű 2016).

7.3.4.1 Structural Biology

Structural biology of a receptor is key to understanding the binding site of a receptor
and to develop new ligands for receptors. After the discovery of the histamine H4
receptor in 1999, several studies were conducted to predict the structure; it was
found that the organization of H4 is in homology with H3 receptors. In perspective to
discover the binding site Shin et al. revealed the involvement of the Glu1825.4.6 and
Asp943.3.2 pockets in H4 receptors in the histamine binding site. It was reported that
nitrogen atom of histamine imidazole ring interacts with Glu1825.4.6 pocket; how-
ever, other identified pockets, such as Asn147 and Ser306, were involved in receptor
232 B. Gorain et al.

binding but play significant roles in receptor activation (Shin et al. 2002). Another
study for homology model development confirmed Asp943.3.2 and Glu1825.4.6 as the
major anchoring point for H4 receptor binding and has shown participation in GPCR
ligand binding. Moreover, Asp943.3.2 is the major site for ligand interaction and
situated closer to Glu1825.4.6 (Evers and Klabunde 2005; Kiss et al. 2008a; Pontiki
and Hadjipavlou-Litina 2017). The binding mode of antagonists and agonists was
also investigated by using histamine as agonist and JNJ7777120 as antagonist.
Experimental results revealed that antagonists interact with Asp943.3.2 and
Glu1825.4.6, whereas agonists interact with Thr3236.5.5, Asp943.3.2 and Glu1825.4.6
which suggest the involvement of Thr3236.5.5 in agonist binding and activation of
receptors (Jablonowski et al. 2003; Kiss et al. 2008b; Jójárt et al. 2008).

7.3.4.2 Biological Distribution

H4 receptor is a pertussis-toxin-sensitive GPCR, mainly expressed on the cells of
peripheral tissue (thymus, spleen, colon, bone marrow and blood leukocytes) and
immune system (eosinophils, monocytes, T cells, dendritic cells and natural killer
cells). The affinity of H4 receptor for inflammation via Gαi/o proteins is through
activation of leukocyte chemotaxis and increases in intracellular Ca2+ concentration.
Moreover, LEC/CCL16, which is a liver-expressed chemokine, is identified as a
non-histamine endogenous H4 receptor agonist and involved in eosinophil traffick-
ing (Nakayama et al. 2004).

7.3.4.3 Role in Biological System

Histamine H4 plays a significant role in immunomodulation due to high expression
in several types of immune cells (Fig. 7.5). H4 receptors are reported to have
eosinophil chemotaxis and control pro-inflammatory responses and leukocyte traf-
ficking by induced activation of eosinophils (Raible et al. 1994; Barnard et al. 2008).
The presence of H4 receptor on eosinophils came out after investigation on pharma-
cology of various histamine ligands. Researchers found that H1 and H2 receptor
ligands are ineffective and the potency of H3 receptor ligands was also not appro-
priate in some disease. Therefore, they have evaluated the H4 receptor ligands and
results revealed the presence of H4 receptor on eosinophils and showed effectiveness
(Thurmond et al. 2009; Reher et al. 2012). Moreover, H4 receptor also governs
paracrine histamine-induced processes and autocrine (Lippert et al. 2004).
The presence of histamine H4 receptor was noticed in mast cells, which is
associated with high secretion of histamine. Activation of H4 receptor on mast
cells causes intracellular Ca2+ mobilization and chemotaxis leading to chronic
allergic inflammation (Hofstra et al. 2003). It was evident from the study on animal
that absence of histamine induced calcium response in mast cell block by H4
receptor antagonist (Thurmond et al. 2004, Yu et al. 2010; Jemima et al. 2014).
This receptor was also involved in zymosan-induced recruitment of neutrophils via
regulation of leukotriene B4 release from mast cells (Takeshita et al. 2003;
Thurmond et al. 2004; Thurmond 2015). Additionally, cytokine and chemokine
production was also controlled by H4 receptor on T cells. Immunomodulatory
function of this receptor was also associated with the release of IL-16 from CD8+
T lymphocytes (Gantner et al. 2002). It had been reported that H4 receptors
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 233

Fig. 7.5 Indicative immunomodulatory actions of histamine that are mediated through histamine
H4 receptors (H4) predominately expressed in immune cells. Gαi/o, G-protein; TH, helper T cell
(Zampeli and Tiligada 2009)

downregulate the production of ILs and that of CCL2 (C-C motif chemokine ligand
2) in human monocyte-derived inflammatory dendritic epidermal cells, the later
leading to decreased monocyte migration (Dijkstra et al. 2007, 2008).
Diseases like asthma and atopic dermatitis are associated with mast cells and
eosinophils. Histamine H4 receptors act as a mediator in lung disorders by
controlling the migration of inflammatory cells, production of chemokine and
cytokine. The expression of histamine H4 receptor in the lung is low; however,
high expression in smooth cells and bronchial epithelial contributes to disease
phenotype (Gantner et al. 2002). As discussed earlier, it also mediated the recruit-
ment and distribution of mast cells in bronchial epithelium, thus leading to allergic
inflammation of the airways (Thurmond et al. 2004). This was evident by a study on
guinea pig model, where the H4 receptor antagonist JNJ7777120 showed reduction
in inflammation, improved lung function and inflammation mediators synthesis in
the lungs after allergen challenge (Somma et al. 2013). Histamine H4 receptor
involvement is also recognized in autoimmune disease, such as rheumatoid arthritis
(RA). Further, RA severity could be related to the expression of H4 receptor along
with H1 and H2. This concept was supported by the presence of H4 receptor in
vascular wall cells and synovial tissue of the patients suffering with RA and
osteoarthritis (Ohki et al. 2007; Kiss et al. 2008a).
234 B. Gorain et al.

7.3.4.4 Antagonists of H4 Receptor

Thioperamide was developed as a H3 receptor antagonist and has affinity towards
H4 receptor as well. However, it is not the ideal tool for proper understanding of H4
receptor. Jablonowski and team identified the first potent antagonist (JNJ 7777120)
of H4 receptor, which was introduced to understand the physiological role of H4
receptors. Inflammatory properties of JNJ 7777120 was evident from the H4 recep-
tor transfected SK-N-MC cell model and animal model (Jablonowski et al. 2003).
Moreover, the reduction of neutrophil efflux in the mouse periodontal model was
observed after pre-treatment with H4 antagonists (Varga et al. 2005). JNJ 7777120
and its analogue (JNJ 10191584) were found effective in the animal model of several
diseases, such as colitis, asthma, pain and atopic dermatitis (Hsieh et al. 2010;
Cowden et al. 2010). Potential issue was identified for JNJ 7777120 compound as
it was reported that it had shown H4 receptor agonistic activity in some ani-
mal models and cell models due to arresting activation of receptor (Seifert et al.
2011). Additionally, short half-life and hypoadrenocorticism toxicity of JNJ
7777120 limit its clinical application. Thus another analogue of JNJ 7777120, JNJ
39758979, was discovered by Thurmond et al. with low toxicity and has shown
effective anti-pruritic and anti-inflammatory activities in animals. However, this
compound was withdrawn from study due to occurrence of drug-induced agranulo-
cytosis. This leads to the development of toreforant, an antagonist of histamine H4
receptor, which did not possess any serious side effect and clinically tested on
patients with RA, dermatitis and asthma (Thurmond 2015; Thurmond et al. 2016).
Many histamine H4 antagonists have been developed, but only a few were
investigated in humans for their effectiveness. In conclusion, still it is difficult to
find the proper ligand for H4 receptor having human underscoring with all necessary
properties.

7.4 Conclusion

Histamine is a well-preserved autacoid, found in most of the tissues in vertebrates.

The histaminergic system is recognized by the action of histamine on four 7-TM
GPCRs. These histamine H1, H2, H3 and H4 receptors showed molecular heteroge-
neity and constitutive activity. The physiological role of different modulators on
these receptors varies largely, from antiallergic, sedating, antiulcer, wake promoting,
anti-inflammatory actions to improvement of memory. Moreover, novel compounds
for these receptors and exploration of their physiological role are still under investi-
gation to bring new compounds in the treatment of complicated disorders.

References
Akdis CA, Blaser K (2003) Histamine in the immune regulation of allergic inflammation. J Allergy
Clin Immunol 112:15–22
Anon (2009) Joint formulary committee. In: British national formulary, 57rd edn. British Medical
Association and Royal Pharmaceutical Society of Great Britain, London
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 235

Arrang J-M, Garbarg M, Schwartz J-C (1983) Auto-inhibition of brain histamine release mediated
by a novel class (H3) of histamine receptor. Nature 302:832–837
Arrang J-M, Garbarg M, Lancelo J-C et al (1987) Highly potent and selective ligands for histamine
H3-receptors. Nature 327:117–123
Arrang J-M, Morisset S, Gbahou F (2007) Constitutive activity of the histamine H3 receptor.
Trends Pharmacol Sci 28:350–357
Ash AS, Schild HO (1966) Receptors mediating some actions of histamine. Br J Pharmacol
Chemother 27:427–439
Atzori M, Lau D, Tansey EP et al (2000) H2 histamine receptor-phosphorylation of Kv3.2
modulates interneuron fast spiking. Nat Neurosci 3:791–798
Barger G, Dale H (1910) The presence of ergot and physiological activity of Beta-
imidazolylethylamine. J Physiol 40:xxxviii–xl
Barnard R, Barnard A, Salmon G et al (2008) Histamine-induced actin polymerization in human
eosinophils: an imaging approach for histamine H 4 receptor. Cytom Part A 73A:299–304
Barnes PJ, Cuss FM, Palmer JB (1985) The effect of airway epithelium on smooth muscle
contractility in bovine trachea. Br J Pharmacol 86:685–691
Benarroch EE (2010) Histamine in the CNS: multiple functions and potential neurologic
implications. Neurology 75:1472–1479
Best CH, Dale HH, Dudley HW, Thorpe WV (1927) The nature of the vaso-dilator constituents of
certain tissue extracts. J Physiol 62:397–417
Birdsall NJ (1991) Cloning and structure-function of the H2 histamine receptor. Trends Pharmacol
Sci 12:9–10
Black JW, Duncan WA, Durant CJ et al (1972) Definition and antagonism of histamine H
2 -receptors. Nature 236:385–390
Bongers G, Bakker RA, Leurs R (2007) Molecular aspects of the histamine H3 receptor. Biochem
Pharmacol 73:1195–1204
Boyle J, Eriksson M, Stanley N et al (2006) Allergy medication in Japanese volunteers: treatment
effect of single doses on nocturnal sleep architecture and next day residual effects. Curr Med Res
Opin 22:1343–1351
Brimblecombe RW, Duncan WA, Durant GJ et al (1975) The pharmacology of cimetidine, a new
histamine H2-receptor antagonist. Br J Pharmacol 53:435P–436P
Brioni JD, Esbenshade TA, Garrison TR et al (2011) Discovery of histamine H3 antagonists for the
treatment of cognitive disorders and Alzheimer’s disease. J Pharmacol Exp Ther 336:38–46
Celanire S, Wijtmans M, Talaga P et al (2005) Keynote review: histamine H3 receptor antagonists
reach out for the clinic. Drug Discov Today 10:1613–1627
Chand N, Eyre P (1975) Classification and biological distribution of histamine receptor sub-types.
Agents Actions 5:277–295
Church MK (2009) Histamine and its receptors. In: Allergy Frontiers: classification and
Pathomechanisms. Springer, Tokyo, pp 329–356
Church MK (2017) Allergy, histamine and antihistamines. In: Handbook of experimental pharma-
cology. Springer, New York, NY, pp 321–331
Church DS, Church MK (2011) Pharmacology of antihistamines. World Allergy Organ J 4:S22–
S27
Corcoran AC (1957) Histamine. Ciba foundation symposium jointly with the Physiological Society
and the British pharmacological Society in Honor of sir Henry Dale. Arch Intern Med
99:157–158
Cowden JM, Zhang M, Dunford PJ, Thurmond RL (2010) The histamine H4 receptor mediates
inflammation and pruritus in Th2-dependent dermal inflammation. J Invest Dermatol
130:1023–1033
Criado PR, Criado RFJ, Maruta CW, Machado FC (2010) Histamine, histamine receptors and
antihistamines: new concepts. An Bras Dermatol 85:195–210
Dale HH (1929) Some chemical factors in the control of the circulation. Lancet 1:167–223
236 B. Gorain et al.

Dale HH, Laidlaw PP (1910) The physiological action of beta-iminazolylethylamine. J Physiol

41:318–344
Dale HH, Laidlaw PP (1919) Histamine shock. J Physiol 52:355–390
Day JH, Briscoe MP, Rafeiro E, Ratz JD (2004) Comparative clinical efficacy, onset and duration of
action of levocetirizine and desloratadine for symptoms of seasonal allergic rhinitis in subjects
evaluated in the environmental exposure unit (EEU). Int J Clin Pract 58:109–118
Devillier P, Roche N, Faisy C (2008) Clinical pharmacokinetics and pharmacodynamics of
Desloratadine, fexofenadine and Levocetirizine. Clin Pharmacokinet 47:217–230
Dijkstra D, Leurs R, Chazot P et al (2007) Histamine downregulates monocyte CCL2 production
through the histamine H4 receptor. J Allergy Clin Immunol 120:300–307
Dijkstra D, Stark H, Chazot PL et al (2008) Human inflammatory dendritic epidermal cells express a
functional histamine H4 receptor. J Invest Dermatol 128:1696–1703
Dy M, Schneider E (2004) Histamine–cytokine connection in immunity and hematopoiesis.
Cytokine Growth Factor Rev 15:393–410
Esbenshade TA, Browman KE, Bitner RS et al (2008) The histamine H 3 receptor: an attractive
target for the treatment of cognitive disorders. Br J Pharmacol 154:1166–1181
Evers A, Klabunde T (2005) Structure-based drug discovery using GPCR homology modeling:
successful virtual screening for antagonists of the Alpha1A adrenergic receptor. J Med Chem
48:1088–1097
Feldberg W, Mongar JL (1954) Comparison of histamine release by compound 48/80 and
octylamine in perfused tissues. Br J Pharmacol Chemother 9:197–201
Feuerstein TJ (2008) Presynaptic receptors for dopamine, histamine, and serotonin. In: Handbook
of experimental pharmacology. Springer, New York, NY, pp 289–338
Fitzsimons CP, Monczor F, Fernández N et al (2004) Mepyramine, a histamine H 1 receptor inverse
agonist, binds preferentially to a G protein-coupled form of the receptor and sequesters G
protein. J Biol Chem 279:34431–34439
Flores-Clemente C, Osorio-Espinoza A, Escamilla-Sánchez J et al (2013) A single-point mutation
(Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine
H 3 receptor stably expressed in CHO-K1 cells. Br J Pharmacol 170:127–135
Fukushima Y, Oka Y, Saitoh T et al (1995) Structural and functional analysis of the canine
histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action.
Biochem J 310(Pt 2):553–558
Furuta K, Nakayama K, Sugimoto Y et al (2007) Activation of histidine decarboxylase through
post-translational cleavage by Caspase-9 in a mouse Mastocytoma P-815. J Biol Chem
282:13438–13446
Gantner F, Sakai K, Tusche MW et al (2002) Histamine H4 and H2 receptors control histamine-
induced Interleukin-16 release from human CD8+ T cells. J Pharmacol Exp Ther 303:300–307
Gantz I, Munzert G, Tashiro T et al (1991) Molecular cloning of the human histamine H2 receptor.
Biochem Biophys Res Commun 178:1386–1392
Gemkow MJ, Davenport AJ, Harich S et al (2009) The histamine H3 receptor as a therapeutic drug
target for CNS disorders. Drug Discov Today 14:509–515
Gillard M, Benedetti MS, Chatelain P, Baltes E (2005) Histamine H1 receptor occupancy and
pharmacodynamics of second generation H1-antihistamines. Inflamm Res 54:367–369
Glatzer F, Gschwandtner M, Ehling S et al (2013) Histamine induces proliferation in keratinocytes
from patients with atopic dermatitis through the histamine 4 receptor. J Allergy Clin Immunol
132:1358–1367
Haas HL, Konnerth A (1983) Histamine and noradrenaline decrease calcium-activated potassium
conductance in hippocampal pyramidal cells. Nature 302:432–434
Haas H, Panula P (2003) The role of histamine and the tuberomamillary nucleus in the nervous
system. Nat Rev Neurosci 4:121–130
Haas HL, Sergeeva OA, Selbach O (2008) Histamine in the nervous system. Physiol Rev
88:1183–1241
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 237

Hill SJ (1990) Distribution, properties, and functional characteristics of three classes of histamine
receptor. Pharmacol Rev 42:45–83
Hill SJ, Young JM, Marrian DH (1977) Specific binding of 3H-mepyramine to histamine H1
receptors in intestinal smooth muscle. Nature 270:361–363
Hill SJ, Ganellin CR, Timmerman H et al (1997) International Union of Pharmacology. XIII.
Classification of histamine receptors. Pharmacol Rev 49:253–278
Hofstra CL, Desai PJ, Thurmond RL, Fung-Leung W-P (2003) Histamine H4 receptor mediates
chemotaxis and calcium mobilization of mast cells. J Pharmacol Exp Ther 305:1212–1221
Hsieh GC, Chandran P, Salyers AK et al (2010) H4 receptor antagonism exhibits anti-nociceptive
effects in inflammatory and neuropathic pain models in rats. Pharmacol Biochem Behav
95:41–50
Huang Z-L, Mochizuki T, Qu W-M et al (2006) Altered sleep-wake characteristics and lack of
arousal response to H3 receptor antagonist in histamine H1 receptor knockout mice. Proc Natl
Acad Sci 103:4687–4692
Huang H, Li Y, Liang J, Finkelman FD (2018) Molecular regulation of histamine synthesis. Front
Immunol 9:1392
Ishizaka T, De Bernardo R, Tomioka H et al (1972) Identification of basophil granulocytes as a site
of allergic histamine release. J Immunol 108:1000–1008
Jablonowski JA, Grice CA, Chai W et al (2003) The first potent and selective non-imidazole human
histamine H4 receptor antagonists. J Med Chem 46:3957–3960
Jójárt B, Kiss R, Viskolcz B, Keseru GM (2008) Activation mechanism of the human histamine H4
receptor--an explicit membrane molecular dynamics simulation study. J Chem Inf Model
48:1199–1210
Jemima EA, Prema A, Thangam EB (2014) Functional characterization of histamine H4 receptor on
human mast cells. Mol Immunol 62:19–28. https://doi.org/10.1016/j.molimm.2014.05.007
Kawakami T, Kitaura J (2005) Mast cell survival and activation by IgE in the absence of antigen: a
consideration of the biologic mechanisms and relevance. J Immunol 175:4167–4173
Kiss R, Keserű GM (2016) Structure-based discovery and binding site analysis of histamine
receptor ligands. Expert Opin Drug Discov 11:1165–1185
Kiss R, Kiss B, Könczöl A et al (2008a) Discovery of novel human histamine H4 receptor ligands
by large-scale structure-based virtual screening. J Med Chem 51:3145–3153
Kiss R, Noszál B, Rácz A et al (2008b) Binding mode analysis and enrichment studies on homology
models of the human histamine H4 receptor. Eur J Med Chem 43:1059–1070
Knight DA, Adcock JA, Phillips MJ, Thompson PJ (1990) The effect of epithelium removal on
human bronchial smooth muscle responsiveness to acetylcholine and histamine. Pulm
Pharmacol 3:198–202
Komori H, Nitta Y, Ueno H, Higuchi Y (2012) Structural study reveals that Ser-354 determines
substrate specificity on human histidine decarboxylase. J Biol Chem 287:29175–29183
Kostopoulos G, Psarropoulou C, Haas HL (1988) Membrane properties, response to amines and to
tetanic stimulation of hippocampal neurons in the genetically epileptic mutant mouse tottering.
Exp Brain Res 72:45–50
Lai X, Ye L, Liao Y et al (2016) Agonist-induced activation of histamine H3 receptor signals to
extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent
mechanisms. J Neurochem 137:200–215
Leurs R, Bakker RA, Timmerman H, de Esch IJP (2005) The histamine H3 receptor: from gene
cloning to H3 receptor drugs. Nat Rev Drug Discov 4:107–120
Leurs R, Hough LB, Blandina P, Haas HL (2012) Histamine. In: Basic neurochemistry, 8rd edn.
Elsevier, pp 323–341
Lewis T, Grant RT (1924) Vascular reactions of the skin to injury. Part 11. The liberation of
histamine-like substance in the injured skin, the underlying cause of factitious urticaria and of
wheals produced by burning: and observations upon the nervous control of certain skin
reactions. Heart 11:209–265
238 B. Gorain et al.

Lin J-S, Sergeeva OA, Haas HL (2011) Histamine H3 receptors and sleep-wake regulation. J
Pharmacol Exp Ther 336:17–23
Lippert U, Artuc M, Grützkau A et al (2004) Human skin mast cells express H2 and H4, but not H3
receptors. J Invest Dermatol 123:116–123
Lovenberg TW, Roland BL, Wilson SJ et al (1999) Cloning and functional expression of the human
histamine H3 receptor. Mol Pharmacol 55:1101–1107
MacGlashan D (2003) Histamine: a mediator of inflammation. J Allergy Clin Immunol 112:S53–
S59
Mahdy AM, Webster NR (2014) Histamine and antihistamines. Anaesth Intensive Care Med
15:250–255
Masini D, Lopes-Aguiar C, Bonito-Oliva A et al (2017) The histamine H3 receptor antagonist
thioperamide rescues circadian rhythm and memory function in experimental parkinsonism.
Transl Psychiatry 7:e1088–e1088
Matsuda N, Jesmin S, Takahashi Y et al (2004) Histamine H1 and H2 receptor gene and Protein
levels are differentially expressed in the hearts of rodents and humans. J Pharmacol Exp Ther
309:786–795
Meneses D, Mateos V, Islas G, Barral J (2015) G-protein-coupled inward rectifier potassium
channels involved in corticostriatal presynaptic modulation. Synapse 69:446–452
Molimard M, Diquet B, Benedetti MS (2004) Comparison of pharmacokinetics and metabolism of
desloratadine, fexofenadine, levocetirizine and mizolastine in humans. Fundam Clin Pharmacol
18:399–411
Monczor F, Fernandez N (2016) Current knowledge and perspectives on histamine H1 and H2
receptor pharmacology: functional selectivity, receptor crosstalk, and repositioning of classic
histaminergic ligands. Mol Pharmacol 90:640–648
Morisset S, Rouleau A, Ligneau X et al (2000) High constitutive activity of native H3 receptors
regulates histamine neurons in brain. Nature 408:860–864
Nakayama T, Kato Y, Hieshima K et al (2004) Liver-expressed chemokine/CC chemokine ligand
16 attracts eosinophils by interacting with histamine H4 receptor. J Immunol 173:2078–2083
Nakazawa H, Sekizawa K, Morikawa M et al (1994) Viral respiratory infection causes airway
hyperresponsiveness and decreases histamine N-methyltransferase activity in Guinea pigs. Am J
Respir Crit Care Med 149:1180–1185
Nieto-Alamilla G, Marquez-Gomez R, Garcia-Galvez A-M et al (2016) The histamine H3 receptor:
structure, pharmacology, and function. Mol Pharmacol 90:649–673
Notcovich C, Diez F, Tubio MR et al (2010) Histamine acting on H1 receptor promotes inhibition
of proliferation via PLC, RAC, and JNK-dependent pathways. Exp Cell Res 316:401–411
Oda T, Morikawa N, Saito Y et al (2000) Molecular cloning and characterization of a novel type of
histamine receptor preferentially expressed in leukocytes. J Biol Chem 275:36781–36786
Ogasawara M, Yamauchi K, Satoh Y-I et al (2006) Recent advances in molecular pharmacology of
the histamine systems: organic cation transporters as a histamine transporter and histamine
metabolism. J Pharmacol Sci 101:24–30
Ohki E, Suzuki M, Aoe T et al (2007) Expression of histamine H4 receptor in synovial cells from
rheumatoid arthritic patients. Biol Pharm Bull 30:2217–2220
Ohtsu H, Tanaka S, Terui T et al (2001) Mice lacking histidine decarboxylase exhibit abnormal
mast cells. FEBS Lett 502:53–56
Panula P, Chazot PL, Cowart M et al (2015) International Union of Basic and Clinical Pharmacol-
ogy. XCVIII. Histamine receptors. Pharmacol Rev 67:601–655
Parks GS, Olivas ND, Ikrar T et al (2014) Histamine inhibits the melanin-concentrating hormone
system: implications for sleep and arousal. J Physiol 592:2183–2196
Parsons ME, Ganellin CR (2006) Histamine and its receptors. Br J Pharmacol 147:S127–S135
Parsons ME, Ganellin CR (2009) Histamine and its receptors. Br J Pharmacol 147:S127–S135
Passani MB, Blandina P, Torrealba F (2011) The histamine H3 receptor and eating behavior. J
Pharmacol Exp Ther 336:24–29
7 Pharmacology of Histamine, Its Receptors and Antagonists in the Modulation of. . . 239

Pedarzani P, Storm JF (1995) Protein kinase A-independent modulation of ion channels in the brain
by cyclic AMP. Proc Natl Acad Sci U S A 92:11716–11720
Pontiki E, Hadjipavlou-Litina D (2017) QSAR models on H 4 receptor antagonists associated with
inflammation and anaphylaxis. J Biomol Struct Dyn 35:968–1005
Raible DG, Lenahan T, Fayvilevich Y et al (1994) Pharmacologic characterization of a novel
histamine receptor on human eosinophils. Am J Respir Crit Care Med 149:1506–1511
Ramírez FJ, Tunon I, Collado JA, Silla E (2003) Structural and vibrational study of the Tautomer-
ism of histamine Free-Base in solution. J Am Chem Soc 125:2328–2340
Reher TM, Neumann D, Buschauer A, Seifert R (2012) Incomplete activation of human eosinophils
via the histamine H4-receptor: evidence for ligand-specific receptor conformations. Biochem
Pharmacol 84:192–203
Riley JF (1953) Histamine in tissue mast cells. Science (80-) 118:332–333
Riley JF, West GB (1952) Histamine in tissue mast cells. J Physiol 117:72P–73P
Rojas-Zamorano JA, Esqueda-Leon E, Jimenez-Anguiano A et al (2009) The H1 histamine receptor
blocker, chlorpheniramine, completely prevents the increase in REM sleep induced by immobi-
lization stress in rats. Pharmacol Biochem Behav 91:291–294
Rouleau A, Ligneau X, Tardivel-Lacombe J et al (2002) Histamine H3-receptor-mediated [35S]
GTPγ[S] binding: evidence for constitutive activity of the recombinant and native rat and human
H3 receptors. Br J Pharmacol 135:383–392
Ruat M, Bouthenet ML, Schwartz JC, Ganellin CR (1990) Histamine H1-receptor in heart: unique
electrophoretic mobility and autoradiographic localization. J Neurochem 55:379–385
Sadek B, Saad A, Sadeq A et al (2016) Histamine H3 receptor as a potential target for cognitive
symptoms in neuropsychiatric diseases. Behav Brain Res 312:415–430
Sahlholm K, Nilsson J, Marcellino D et al (2012) Voltage sensitivities and deactivation kinetics of
histamine H3 and H4 receptors. Biochim Biophys Acta Biomembr 1818:3081–3089
Sander LE, Lorentz A, Sellge G et al (2006) Selective expression of histamine receptors H1R, H2R,
and H4R, but not H3R, in the human intestinal tract. Gut 55:498–504
Schayer RW (1956) The origin and fate of histamine in the body. In: Wolstenholme GEW,
O’Connor CM (eds) Ciba foundation symposium on histamine. J and A Churchill Ltd.,
London, pp 183–188
Schwartz J-C (2011) The histamine H3 receptor: from discovery to clinical trials with pitolisant. Br
J Pharmacol 163:713–721
Seifert R, Schneider EH, Dove S et al (2011) Paradoxical stimulatory effects of the “standard”
histamine H4-receptor antagonist JNJ7777120: the H4 receptor joins the Club of 7 transmem-
brane domain receptors exhibiting functional selectivity. Mol Pharmacol 79:631–638
Sekizawa K, Nakazawa H, Ohrui T et al (1993) Histamine N-methyltransferase modulates hista-
mine- and antigen-induced bronchoconstriction in Guinea pigs In Vivo. Am Rev Respir Dis
147:92–96
Shahid M, Tripathi T, Sobia F et al (2009) Histamine, histamine receptors, and their role in
immunomodulation: an updated systematic review. Open Immunol J 2:9–41
Sharma HS (2004) Histamine influences the blood-spinal cord and brain barriers following injuries
to the central nervous system. Blood Spinal Cord Brain Barriers Health Dis:159–189. https://
doi.org/10.1016/B978-012639011-7/50017-6
Sherin JE, Elmquist JK, Torrealba F, Saper CB (1998) Innervation of histaminergic
tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral
preoptic nucleus of the rat. J Neurosci 18:4705–4721
Shimamura T, Shiroishi M, Weyand S et al (2011) Structure of the human histamine H1 receptor
complex with doxepin. Nature 475:65–70
Shin N, Coates E, Murgolo NJ et al (2002) Molecular modeling and site-specific mutagenesis of the
histamine-binding site of the histamine H4 receptor. Mol Pharmacol 62:38–47
Shiraishi M, Hirasawa N, Oikawa S et al (2000) Analysis of histamine-producing cells at the late
phase of allergic inflammation in rats. Immunology 99:600–606
240 B. Gorain et al.

Simons KJ, Benedetti MS, Simons FER et al (2007) Relevance of H1-receptor occupancy to
H1-antihistamine dosing in children. J Allergy Clin Immunol 119:1551–1554
Somma T, Cinci L, Formicola G et al (2013) A selective antagonist of histamine H 4 receptors
prevents antigen-induced airway inflammation and bronchoconstriction in Guinea pigs: involve-
ment of lipocortin-1. Br J Pharmacol 170:200–213
Sundvik M, Kudo H, Toivonen P et al (2011) The histaminergic system regulates wakefulness and
orexin/hypocretin neuron development via histamine receptor H1 in zebrafish. FASEB J
25:4338–4347
Szeberényi JB, Pállinger E, Zsinkó M et al (2001) Inhibition of effects of endogenously synthesized
histamine disturbs in vitro human dendritic cell differentiation. Immunol Lett 76:175–182
Takeshita K, Sakai K, Bacon KB, Gantner F (2003) Critical role of histamine H4 receptor in
leukotriene B4 production and mast cell-dependent neutrophil recruitment induced by Zymosan
in vivo. J Pharmacol Exp Ther 307:1072–1078
Tanaka S, Takasu Y, Mikura S et al (2002) Antigen-independent induction of histamine synthesis
by immunoglobulin E in mouse bone marrow–derived mast cells. J Exp Med 196:229–235
Tanaka S, Deai K, Konomi A et al (2004) Expression of L-histidine decarboxylase in granules of
elicited mouse polymorphonuclear leukocytes. Eur J Immunol 34:1472–1482
Tannergren C, Petri N, Knutson L et al (2003) Multiple transport mechanisms involved in the
intestinal absorption and first-pass extraction of fexofenadine. Clin Pharmacol Ther 74:423–436
Tashiro M, Kato M, Miyake M et al (2009) Dose dependency of brain histamine H1 receptor
occupancy following oral administration of cetirizine hydrochloride measured using PET with
[11 C]doxepin. Hum Psychopharmacol Clin Exp 24:540–548
Thakkar MM (2011) Histamine in the regulation of wakefulness. Sleep Med Rev 15:65–74
Thurmond RL (2015) The histamine H4 receptor: from orphan to the clinic. Front Pharmacol 6:65
Thurmond RL, Desai PJ, Dunford PJ et al (2004) A potent and selective histamine H4 receptor
antagonist with anti-inflammatory properties. J Pharmacol Exp Ther 309:404–413
Thurmond RL, Gelfand EW, Dunford PJ (2008) The role of histamine H1 and H4 receptors in
allergic inflammation: the search for new antihistamines. Nat Rev Drug Discov 7:41–53
Thurmond R, Wolin RL, Wei J et al (2009) Pharmacological characterization of oxime agonists of
the histamine H4 receptor. J Receptor Ligand Channel Res 3:37
Thurmond RL, Greenspan A, Radziszewski W et al (2016) Toreforant, a histamine H4 receptor
antagonist, in patients with active rheumatoid arthritis despite methotrexate therapy: results of
2 phase II studies. J Rheumatol 43:1637–1642
Timmerman H, van der Goot H (2009) Histamine receptors and their ligands: mechanisms and
applications. In: Encyclopedia neuroscience. Springer, New York, NY, pp 1149–1166
Traiffort E, Ruat M, Arrang JM et al (1992) Expression of a cloned rat histamine H2 receptor
mediating inhibition of arachidonate release and activation of cAMP accumulation. Proc Natl
Acad Sci 89:2649–2653
Traiffort E, Vizuete ML, Tardivellacombe J et al (1995) The Guinea pig histamine H2 receptor:
gene cloning, tissue expression and chromosomal localization of its human counterpart.
Biochem Biophys Res Commun 211:570–577
Varga C, Horvath K, Berko A et al (2005) Inhibitory effects of histamine H4 receptor antagonists on
experimental colitis in the rat. Eur J Pharmacol 522:130–138
Vianello R, Mavri J (2012) Microsolvation of the histamine monocation in aqueous solution: the
effect on structure, hydrogen bonding ability and vibrational spectrum. New J Chem 36:954
Yu F, Wolin RL, Wei J et al (2010) Pharmacological characterization of oxime agonists of the
histamine H4 receptor. J Receptor Ligand Channel Res 3:37–49. https://doi.org/10.2147/
JRLCR.S6468
Zampeli E, Tiligada E (2009) The role of histamine H4 receptor in immune and inflammatory
disorders. Br J Pharmacol 157:24–33
Zamponi GW, Currie KPM (2013) Regulation of ca(V)2 calcium channels by G protein coupled
receptors. Biochim Biophys Acta 1828:1629–1643
Pharmacology of GABA and Its Receptors
8
Sunpreet Kaur, Shamsher Singh, Anchal Arora, Parladh Ram,
Sachin Kumar, Puneet Kumar, and Sara Nidal Abed

Abstract

GABA is an important neurotransmitter in vertebrates where it acts at synapses of

the CNS; in nematodes GABA acts primarily at neuromuscular synapses. Specif-
ically, GABA acts to relax the body muscles during locomotion and foraging and
to contract the enteric muscles during defecation. Following the recognition of
GABA as an inhibitory neurotransmitter, the discovery of high-affinity GABA
uptake and the characterization of GABA receptors have made great progress in
developing GABA pharmacology. Tiagabine, the first marketed GABA uptake
inhibitor, may be followed by new and more selective uptake inhibitors. This
chapter centers on the discoveries made during more than six decades of neuro-
science research on the role of GABA as a neurotransmitter. In doing so, special
emphasis is placed on the significant involvement of GABA in the normal
physiology of the human body such as sleep, reproductive system, heart, learning
and memory. GABA dysregulation also categorizes various neurological
disorders and enlisted their potential therapeutic drug targets under research
that encompass Parkinson’s disease, Alzheimer’s disease, epileptic disorders,
traumatic brain injury (TBI), Huntington’s disease, anxiety, multiple

S. Kaur · S. Singh
Neuroscience Division, Department of Pharmacology, ISF College of Pharmacy, Moga,
Punjab, India
A. Arora · P. Ram · S. Kumar
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India
P. Kumar (*)
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India
S. N. Abed
Faculty of Pharmacy, Philadelphia University, Amman, Jordan

# Springer Nature Singapore Pte Ltd. 2020 241

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_8
242 S. Kaur et al.

sclerosis (MS), and schizophrenia. This chapter further highlights the recent and
previously conducted clinical trials and future investigational targets of GABA.
Now it is readily accepted as a vital spinal and supra-spinal inhibitory transmitter
and we know many details regarding its molecular structure and trafficking
around neurons. The chapter highlights the synthesis, reuptake, and metabolism
of GABA. Thereafter, mechanisms of action of various GABA subtypes
(GABAA, GABAB, GABAC) have been discussed which make the prospects
for further research very exciting.

Keywords
Basal ganglia · GABA · Hyperpolarization · Neurological disorders · Inhibition

Abbreviations

AC Adenylyl cyclase
AD Alzheimer’s disease
AEDs Antiepileptic drugs
BGT Betaine-GABA transporter
BZD Benzodiazepine
cAMP Cyclic adenosine monophosphate
CB Cannabinoid receptor
CNS Central nervous system
D Dopamine
DSM Diagnostic and Statistical Manual of Mental Disorder
FSH Follicle stimulating hormone
GABA Gamma aminobutyric acid
GAD Glutamic acid decarboxylase
GAD Generalized anxiety disorder
GATs GABA transporters
GDNF Glial cell derived neurotrophic factor
GHB Gamma-hydroxybutyrate
Gln Glutamine
GnRH Gonadotropin releasing hormone
GPCR G-protein-coupled receptor
Gpe Globus pallidus external
Gpi Globus pallidus internal
HD Huntington’s disease
IN Interneuron
IPSPs Inhibitory postsynaptic potentials
mBDNF mature Brain-derived neurotrophic factor
MnPO Median preoptic nuclei
MNTB Medial nucleus of the trapezoid body
MPN Methylpyridoxine
8 Pharmacology of GABA and Its Receptors 243

MS Multiple sclerosis
NMDA N-methyl-D-aspartate
nNOS Neuronal nitric oxide synthase
OCD Obsessive compulsive disorder
PAG Phosphate-activated glutaminase
PD Panic disorder
PD Parkinson’s disease
PFC Prefrontal cortex
PLTS Persistent low-threshold spiking
PPMS Primary progressive multiple sclerosis
PRMS Progressive relapsing multiple sclerosis
PTSD Post-traumatic stress disorder
RIMS Rapid eye movement sleep
RRMS Relapsing remitting multiple sclerosis
SACs Starburst amacrine cells
SAD Social anxiety disorder
SCZ Schizophrenia
SNPc Substantia nigra pars compacta
SPMS Secondary progressive multiple sclerosis
SSADH Succinate semialdehyde dehydrogenase
STN Subthalamic nuclei
TBI Traumatic brain injury
TGB Tiagabine
THDOC Tetrahydrodeoxycorticosterone
THIP Tetrahydroisooxazolopyridinol
VGB Vigabatrin
VLPO Ventrolateral preoptic nuclei
VTA Ventral tegmental area

8.1 Introduction

In the brain, neuronal excitation and inhibition is responsible for the basis of
information transfer within the CNS. Amino acid neurotransmitters are the
messengers which exert excitatory and inhibitory actions, which include glutamate
and GABA, respectively. The balance between these two neurotransmitters ensures
normal functioning of neurons showing rhythmic activity. GABA is the main
inhibitory neurotransmitter in the brain and is made up of amino acid sequences.
The primary action of GABA is exerted by binding to the postsynaptic receptor,
which stimulates the Cl
ion channels, opens Cl
ions channels resulting in hyper-
polarization of the cell, and thus exerts the inhibitory response, i.e., conductance of
action potential is completely blocked. Clinically, the significance of GABA has not
been estimated yet, but mainly it controls the transmission of neurotransmitters in the
synapses and excitability of the neurons within the CNS. The ubiquitous distribution
of GABA in the brain helps to exhibit a variety of physiological functions including
244 S. Kaur et al.

calmness, sleep, motor functions, cognition, and memory like behavioral phenome-
non (Wu and Sun 2015). No doubt, it has an inhibitory control over mammalian brain
but in neonatal brain it also exhibits some excitatory functions. Here, it participates in
a variety of functions including neurogenesis, synapse formation, and synaptic plas-
ticity. This indicates that GABA is well implicated in normal neural development and
physiology of the brain. The inhibitory nature of GABA helps to execute over the
excitatory neurons for maintaining the homeostasis between GABA and glutamate
which is known as GABA/glutamate cycle. Any asymmetry in this balance creates the
pathological situations associated with different disorders like epilepsy, schizophrenia,
anxiety, dementia, and motor disorders like Parkinson’s disease. Such pathological
associations make GABAergic dysfunction as a major target for significant therapeutic
approach in CNS disorders (Wu and Sun 2015). GABA signaling modulation or
enhancing GABAergic inhibition is the way for the treatment of many pharmacologic
drugs in various pathological situations.
GABA is present in different parts of the mammalian body, but its various roles
are still unknown. Primarily it shows inhibitory responses in the CNS as well as in
the spinal cord. Usually, the main function of the beta cells of the pancreas is to
produce insulin but is also able to produce GABA. Thus GABA produced from the
beta cell can stimulate the growth of beta cells, formation of beta cells from alpha
cells, and inhibiting alpha cells. Within the body, the other parts contain low
amounts of GABA, though the proper function and relevant clinical significance
of this remain unknown, but it mainly counterbalances the excitatory responses.
Firing of the neuronal cell is too fast, in the absence of GABA. When GABA
activity is significantly decreased in the CNS, various disorders like anxiety,
convulsions, panic attacks, cognitive dysfunction, Parkinson’s disease, drug addic-
tion, etc. occur due to excitatory discharge. Transmission of nerve impulses is
significantly hindered from one neuron to another by GABA, due to inhibitory
responses; it has calming or quieting effect. In the brain, the activity of growth
hormone, synthesis of protein, and plasma concentration are improved by GABA,
but diminishes small airway-derived lung adenocarcinoma. Other responses include
hypotensive, tranquilizing, diuretic, and antidiabetic effects.
Inhibitory neurotransmission is a complex process but it can be achieved through
the activation of different types of GABA receptors. Historically only two types of
GABA receptors (i.e., GABAA and GABAB) were identified, but now GABA
receptors are classified into three types (GABAA, GABAB, GABAC) based on phar-
macological and electrophysiological properties. Recently, GABAA and GABAC are
classified as ligand-gated chloride channel receptors and GABAB receptors are
known as metabotropic, i.e., G-protein-coupled receptors.
The therapeutic reliability of neurotransmitter GABA could be analyzed from
the success of benzodiazepines (BZD). BZDs are a class of drugs which act by
interacting with GABA receptors and utilized as the most commonly prescribed
drugs clinically. BZDs are the positive allosteric modulator of GABAA receptor to
potentiate the inhibitory neurotransmission derived calming effect in the brain
(Griffin et al. 2013). The drugs under BZDs include diazepam, flurazepam, alprazo-
lam, chlordiazepoxide, etc. which are commonly prescribed for anxiety, insomnia,
8 Pharmacology of GABA and Its Receptors 245

muscle relaxation, and epilepsy. Furthermore, several new possible indications of

these drugs emerge under newly revealed mechanisms. New possibilities for classic
medications working through GABA modulation may provide suitable therapeutic
benefits in different neurodegenerative diseases. To study the possibilities for spe-
cific disorders, first it is essential to discuss the physiology of GABA inside the brain
which includes synthesis, metabolism, and reuptake including the structure and
localization of receptors mediating its actions.

8.2 History and Discovery of GABA; Agonists and Antagonists

In 1883, it was known only as a plant and microbial metabolic product. There are
evidences which establish that in the mammalian CNS, γ-aminobutyric acid
(GABA) is the most important inhibitory neurotransmitter which was fully accepted
at the end of the 1960s/early 1970s by its role. In 1910, it was shown to be present in
biological tissues but at that time its presence in the mammalian CNS was not
confirmed. Only after 1950, i.e., 40 years later, the presence of free amino acid
was identified in the brain that arose the neurochemical significance of GABA.
During the ensuing decade, how neuronal activity is affected by GABA and related
compounds has been reported to define its role within the CNS.
Additionally, the role of excitatory and inhibitory activities of GABA in crusta-
cean is defined by concurrent findings of Kuffler (1954) (Jijón-Lorenzo et al. 2018).
The initial finding related to GABA was muted. After 5 years of discovery by
Roberts and Frankel, only two articles related to GABA activity within the brain
were reported in the Journal of Biological Chemistry and PubMed had only four
articles related to GABA activity. Nevertheless, in those days due to the lack of
methods that define its presence and function, research community remained silent.
The inkling properties of GABA were not shown by anyone in the brain. In 1957,
researchers in Canada confirmed the activity of GABA due to the inhibitory activity
of an unknown compound on crayfish neuron. GABA transporters were studied by
Baruch Kanner of Hebrew University, Hadassah Medical School in Israel, who is a
member of the Journal of Biological Chemistry editorial board.
Kanner said, “GABA receptors have an inhibitory input which is the major
inhibitory action within the brain.” Roberts and Frankel formed the basis and provided
evidence; then only it was clear about how neurotransmitters control the brain activity.
Roberts analyzed various extracts and reported the existence of a free amine com-
pound by migration of ninhydrin reactive compounds on chromatograms. This amino
acid is accumulated in higher concentrations in the brain than in other tissues.
In 1987, Eric A Barnard in Cambridge (UK) who is a molecular biologist and
Peter H Seeburg at Gene tech in San Francisco (CA, USA) succeeded to define
GABAA receptor, which is then classified as ligand-gated ion channels. After a
decade, airway epithelium containing GABAergic system which is excitatory was
described in 2007. On exposure to allergens the system becomes activated
and participates in the mechanisms of asthma. In the testis and eye lens GABAergic
system is present as shown in Table 8.1.
246 S. Kaur et al.

Table 8.1 Historical aspects of GABA receptors

Year Important event Reference
1883 GABA synthesized, known as a product of microbial and plant Cooper et al. (2003)
metabolism
1950 GABA identified as a normal constituent of mammalian CNS Roberts and
Frankel (1950)
1959 GABA was extracted from mammalian brain Florey and
McLennan (1959)
1962 Baclofen, a lipophilic derivative of GABA, was synthesized Keberle et al.
(1968)
1967 It was recognized as an inhibitory neurotransmitter Hall and Kravitz
(1967)
1975 Benzodiazepines the important modulator of GABA was Haefely et al.
described (1975)
1977 Benzodiazepine binding site discovered in the brain Tallman et al.
(1977)
1978 The distribution of forming enzyme for GABA (GAD) in the Barber et al. (1978)
mammalian spinal cord was described
1981 GABAA and GABAB receptors were pharmacologically Bowery et al.
distinguished (1982)
1984 GABA receptors that were insensitive to both bicuculline and Chebib and
baclofen known as GABAC receptor Johnston (2000)
1987 GABAA receptors were cloned Schofield et al.
(1987)
1990 Molecular composition of GABAA receptors was elucidated Martin and Olsen
(2000)
1997 Structure of GABAB receptors was identified Kaupmann et al.
(1997)
1991 ρ1 subunit of GABAC receptors was cloned Polenzani et al.
(1991)
1992 ρ2 subunit was cloned Cutting et al.
(1992)
1996 ρ3 subunit was cloned Shingai et al.
(1997)
1997 GABAB1a GABAB1b cloned Bettler et al. (2004)
1998 GABAB2 Jones et al. (1998)
2001 Positive allosteric modulators of GABAB receptors were cloned Martin et al. (2001)
2004 Glutamic acid decarboxylase activator, pregabalin Silverman (2008)
2009 Malfunction in GABA and glutamate described as a pathway to Sharpley (2009)
cure depression
2010 Acute psychological stress on prefrontal GABA concentration Hasler et al. (2010)
plays an important role in the pathophysiology of anxiety
disorders
2011 Modulation of GABA system by Passiflora Appel et al. (2011)
2012 Role of GABA in primary insomnia Plante et al. (2012)
2013 GABA exerts anti-inflammatory and immunosuppressor effects Prud’homme et al.
(2013)
(continued)
8 Pharmacology of GABA and Its Receptors 247

Table 8.1 (continued)

Year Important event Reference
2014 Inhibiting ARG1 or agonizing the GABAA receptor treated Hackett et al.
neuroblastoma (2014)
2015 GABA administration helps in improving action selection Steenbergen et al.
processes (2015)
2016 Obstructive sleep apnea is associated with low GABA and high Macey et al. (2016)
glutamate in the insular cortex
2017 Somatostatin-positive GABAergic interneuron: new targets for Zhang et al. (2017)
depression
2018 GABA modulating bacteria of the human gut microbiota Strandwitz et al.
(2019)
2019 GABAA receptor signaling mechanisms revealed by structural Masiulis et al.
pharmacology (2019)

8.3 Synthesis, Storage, and Release of GABA

The synthesis, storage, release, reuptake, and metabolism of GABA occur due to
signaling of the GABA system. Both ionotropic and metabotropic receptors
expressed in the plasma membranes of cells and GABA exert their action through
these receptors (Olsen and Sieghart 2009). GABA is formed by various metabolic
pathways, also called the GABA shunt, and operates via closed loop process. The
purpose of this pathway is to produce and conserve the supply of GABA. Brain
regions contain high amount of GABA and the amount is 1000 times greater than
other monoamine neurotransmitters. The endogenous synthesis of GABA proceeds
via α- decarboxylation of L-glutamate by action of the enzyme glutamic acid
decarboxylase (GAD) as shown in Fig. 8.1. The functioning of GAD depends
upon the presence of cofactor pyridoxal-50 phosphate to catalyze the single-step
irreversible reaction for production of GABA (Roth and Draguhn 2012). Here, GAD
exists in two forms; GAD 65 (65 kDa) was found to reside in axons and
synaptosomes along with plasma membrane whereas GAD67 (67 kDa) was found
to be localized in the cytosol of neuronal cells. These both mediate the synthesis of
GABA by vesicular mechanism and cytoplasmic production, respectively.
On production, GABA gets recruited to synaptic vesicles via vesicular GABA
transporter (vGAT), which releases GABA in synapse on membrane depolarization
of neurons. After the release, GABA interacts with its receptors for mediating its
action. The small amount of GABA release is re-uptaken by membrane-bound
GABA transporters (GATs), localized on neurons and astrocytes. The reuptake of
GABA is temperature and ion dependent. The GABA transporters are usually
GABA/Na+/Cl symporters that constitute four different types including GABA
transporter 1 (GAT1), GABA transporter 2 (GAT2), GABA transporter 3 (GAT3),
and the betaine-GABA transporter (BGT1). These transporters are engaged over
synapses to clear GABA from the synaptic cleft (Roth and Draguhn 2012). Further,
 hetase
248

Presynaptic Terminal Glu Synt Kreb

Glutamine Glutamine cycle
Glutaminase
Glutamate
GAD 65/67 GABA
GABA GABAAR
GABA T,
G SSADH
AB
AB GAT
vGAT R Succinate

Ca2+
chann
el
Astrocyte
GAT

K+ channel
GABAAR

BR
GABA
Synaptic receptor Extrasynaptic receptor
Postsynaptic Terminal

Fig. 8.1 Synthesis, storage, release, reuptake, and metabolism of GABA

S. Kaur et al.
8 Pharmacology of GABA and Its Receptors 249

GABA

GABA – Transaminase (GABA-T)

+ Vit. B6

Succinic semialdehyde

Succinic Semialdehyde NAD Succinic Semialdehyde

Reductase NADH Dehydrogenase (SSADH)
NAD NADH

Succinic acid

γ- Hydroxybutyric acid (GHB)

Kreb’s cycle

Fig. 8.2 The metabolic pathway of GABA

there is tight regulation over GABA metabolism maintained by astroglial processes

through glutamate/GABA-glutamine cycle as shown in Fig. 8.2.
The astrocytes keep checking over GABA release in synapses and reuptake
GABA, where it is further catabolized to succinate by mitochondrial enzymes,
GABA transaminase (GABA-T), and succinate semialdehyde dehydrogenase
(SSADH) in two steps reaction (Rowley et al. 2012). Succinate remains a sequential
component of the Krebs cycle that gets frequently converted to glutamine (Gln) via
the action of glutamine synthase. Estimation states that GABA metabolism
constitutes 8–10% integral part of Krebs cycle and the produced glutamine is then
delivered into neurons where it converts to glutamate (Glu) through neuron localized
enzyme phosphate activated glutaminase (PAG). In this way, glutamate again gets
readily available for the production of GABA.

8.4 Structure and Regulation of GABA

Neurotransmitter has the ability to affect the synaptic transmission by binding to the
postsynaptic receptors. In the past, based on different structural and pharmacological
classes, GABAergic receptors were divided into two distinct classes, i.e., GABAA
and GABAB. But now the third type of GABA is also recognized, i.e., GABAC. At
the end of 1980, the structure elucidation of the GABAA receptor was done after the
clarification of the receptor subunits; it is the member of the superfamily to which
various other types of receptors are included such as nicotinic acetylcholine and
glycine. GABAρ receptor which was initially known as GABAC receptor is a
subclass of GABAA receptor and is found in the retina (Johnston et al. 2003).
The GABAA receptor is the ligand-gated ion channel (anion selective) that
induces inhibitory responses and is made up of different subunits of 8 subfamilies
250 S. Kaur et al.

Fig. 8.3 Different subunits

of GABAA receptor

α (1–6), β (1–4), γ (1–3), δ, ε, θ, π, and ρ (1–3) (Sigel and Steinmann 2012) as shown
in Fig. 8.3.
Various combinations are possible when different 19 subunits are combined in
the pentameric structure. However, the nerve cells contain around 25–30 GABAA
receptor combinations (Birnir and Korpi 2007; Olsen and Sieghart 2009). Most
common subunits are α, β, and γ which assemble to form a sequence of unique
functionality, while δ, ε, and ρ remains less common and elusive for its functions
(Sigel and Steinmann 2012). The most common ones are enlisted as α1β2, α3β3γ2,
and α2β3γ2. The available research data provides evidence regarding the role and
precise locations of GABA subunits. Immunocytochemistry studies reveal the wide
distribution of α1, β1, β2, β3, and γ2 throughout the brain (Olsen and Sieghart 2009).
The expression of β2 is specifically confined to the cerebellum, thalamus, hypothal-
amus, olfactory bulb, amygdala, and midbrain, whereas β3 is highly localized in
cerebellum and midbrain regions like substantia nigra pars compacta and the ventral
tegmental area. The expression of α2, α3, α4, α5, and α6 remains more region
specific as can be studied from an example of α5 which is highly localized in
olfactory bulb, hippocampus, and hypothalamus (Rudolph and Möhler 2014), and
the α4 subunit which is highly localized in the thalamus, striatum, hippocampus, and
outer cortex. Apart from specific regional findings, it is neuron specificity that
reveals the presence of GABAergic subunits using immunofluorescence staining.
The result of these studies postulates the pallidum neuron as α1 specific while
brainstem as α1 and α3 specific. Moreover, their widespread distribution of seroto-
ninergic neurons inside the raphe nuclei has α1 subunit of GABA while GABAergic
neurons here express specificity for α1 and α3 subunits (Wu and Sun 2015). This
way the widespread distribution of subunits and localized expression all over the
CNS performs a number of various actions as shown in Table 8.2.
The GABA network is highly distributed in the CNS and plays a role in neuronal
excitability. Different brain regions contain different expression of the receptor and
distribution based on the function of the subunit as shown in Table 8.3.
8 Pharmacology of GABA and Its Receptors 251

Table 8.2 The role of GABAA receptor subunits in the CNS

Effect of benzodiazepines α1 α2 α3 α5 γ2 β2 β3 δ
Sedation +


+
Anxiolysis
+
/+

Amnesia + +
Myorelaxation
+
Motor impairment



Anticonvulsant +



Ethanol reinforcement
+
Effects of anesthetics
+ + +
Anxiety +
Learning/memory + +

Table 8.3 Different subunits in different regions with concentrations of GABAA receptors
Concentration
Subunits (%) Regions
α1β2γ2 60 In most brain areas and it is localized in interneuron in
hippocampus and cortex Purkinje cells
α2β2/ 15–20 Forebrain especially hippocampus/striatum
3γ2
α3βnγ2/ 10–15 Primarily in cortex
γ3
α2βnγ1 8 Bergman glia
α4βnδ 5 Thalamus and hippocampal dentate gyrus
α4βγ2 5 Thalamus and hippocampal dentate gyrus
α4βnγ 5 Hippocampal pyramidal cells, deep cortical layers, amygdala,
olfactory bulb, hypothalamus, superior colliculus, spinal
trigeminal nucleus
α5β3γ2/ 4 Hippocampus, cortex, and olfactory bulb
γ3
α5β2γ2 5 Hippocampus
α6β2/ 5 Cerebellum and dorsal cochlear nucleus
3γ2
α6βγ2 2 Cerebellar granule cell
α6βnδ 2–3 Cerebellar granule cell

Heteropentameric chloride channel formed from different five subunits is assem-

bled to form functional GABAA receptor (Olsen and Sieghart 2009) and comprises a
variety of different combinations of protein subunits. Here, the ionotropic receptor
belongs to nicotinicoid superfamily which consists of a pentameric structure around
a central pore for ion flow that mediates chloride ions influx. Different subunits of
GABA have the same membrane topology, having four transmembrane α-helices
(M1, M2, M3, & M4) consisting of long extracellular N-terminus and C-terminus
regions with an intracellular loop between the M3 and M4 regions. The channel pore
wall is formed from 5 subunits of the M2 domain. The functional properties such as
252 S. Kaur et al.

Fig. 8.4 Multiplicity of ionotropic GABAA receptors

Fig. 8.5 Various binding sites at GABA

ion conductance of the receptor and fate of GABA are affected by intracellular loop
because it contains the binding site of intracellular proteins and phosphorylation
proteins (Olsen and Sieghart 2009) as shown in Fig. 8.4.
There are various binding sites located in the complex but the GABA binding site
is between the interface of α and β subunits and other binding sites can modulate the
action of GABA. Various drugs such as benzodiazepines, barbiturates,
neurosteroids, and ethanol (Sieghart et al. 2012) positively modulate the GABAA
receptors where as GABAA receptor blocked by bicuculline and picrotoxin
(Semyanov 2002) as shown in Fig. 8.5. Different brain regions contain a combina-
tion of various subunits of GABAA receptor performing different functions at the
same time. Each class of units has different pharmacological and electrical properties
and performs specific functions.
On the other side, the metabotropic receptor (GABAB) belongs to G-protein-
coupled receptors (GPCRs) or metabotropic receptors for GABA and acts by
coupling with G-proteins to mediate the intracellular signaling pathway. The
8 Pharmacology of GABA and Its Receptors 253

Fig. 8.6 Multiplicity of metabotropic GABAB receptors

GABAB receptors were determined by Norman Bowery and their team in 1981 when
they are distributed in the CNS (Blackburn 2010). The GABAB receptors are
activated by GABA which is the main inhibitory neurotransmitter present in
the CNS. The GABAB receptors presynaptically inhibit voltage-activated Ca2+
channel to reduce the release of GABA and other neurotransmitters. On the other
side, the GABAB receptor postsynaptically activates Kir3 channels and inhibits the
neuronal activity by generating hyperpolarizing postsynaptic potentials, which over-
all minimize the excitatory neurotransmission of GABAB receptors by interacting
with their effectors through Gα and Gβγ subunits of the G protein; the first effector of
GABAB remains adenylate cyclase (AC) via activation of an inhibitory subunit
(Gαis/Gα/Gβγ) that further inhibits voltage-dependent Ca2+ channels to limit the
neurotransmitter release (Pin and Prézeau 2007). Here, GABAB receptors act as
autoreceptors which therefore will inhibit the release of GABA and also
the excitability of neurons. The GABAB receptors stimulate the opening of K+
channels which brings the neuron closer to the equilibrium potential of K+ thereby
reducing the frequency of action potential and the release of neurotransmitters.
Hence, GABAB receptors are inhibitory receptors. These receptors also reduce the
activity of adenylyl cyclase and Ca2+ channels (Orts-Del’Immagine and Pugh 2018).
Another important mechanistic regulation of GABAB involves activating the Kir3
channels which maintain slow inhibitory postsynaptic potentials (IPSPs) by inducing
efflux of K+ ions for hyperpolarization of the membrane (Pin and Prézeau 2007) as
shown in Fig. 8.6.
Further, GABAB is of two subtypes: GABAB1 which activates neurons and
GABAB2 that mediates specific sequential signaling. The GABAB1 further consists
254 S. Kaur et al.

Fig. 8.7 Multiplicity of

ionotropic GABAρ receptors

Table 8.4 Difference between three types of GABA receptors

Receptor GABAA receptor GABAB receptor GABAC receptor
Category Ligand-gated channel G-protein- Ligand-gated channel
coupled receptor
Subunits α (1–6), β (1–4), γ (1–3), δ, ε, GBR1, GBR2 ρ
θ, π, and ρ (1–3)
Agonists Muscimol, THIP Baclofen cis-4-aminocrotonic
acid (CACA)
Antagonists Bicuculline, Picrotoxin Phaclofen TPMPA, Picrotoxin
Desensitization Yes No No
Modulator Benzodiazepines barbiturates Rac-BHFF Zinc

of GABAB1α and GABAB1β like subtypes; here the GABAB1α carries a specific
domain named “sushi,” to control the physiological release of glutamate at presyn-
aptic terminals. The GABAB1α is encoded by the GABAR1 gene. But, GABAB1β
receptors are located on postsynaptic terminals, encoded by GABBR2 gene and
cause inhibition there. The GABAB1α and GABAB1β are isoforms and combine with
GABA to form a functional GABAB(1a,2) and GABAB(1b,2).
Another receptor for GABA includes GABAC which shows similarity to GABAA
receptor. The GABAC receptors were first described in interneurons of the spinal
cord. It remains ionotropic in nature and their pentameric chloride channels are com-
posed only of ρ (1–3) subunits. This receptor is found to be expressed in the retina,
observed by Miledi from bovine retina in Xenopus oocytes, and unlike GABAA it
remains insensitive to bicuculline and baclofen like compounds (Johnston et al.
2003). GABA has high sensitivity for GABAC receptors. The pharmacological
profile of GABAC receptors is different from GABAA and GABAB receptors. In
rats, tiger salamander, and hybrid bass, GABAC receptors were found on bipolar
cells (Du and Yang 2000). It is evidenced that GABAC receptors are composed of ρ
subunits (ρ1,2 in humans; ρ1,2 in rat; ρ2,3 in rat; ρ1,2 in chicken) (Zhang et al. 2001)
as shown in Fig. 8.7 and Table 8.4.
8 Pharmacology of GABA and Its Receptors 255

8.5 Agonists and Antagonists

GABA receptors exert inhibitory responses after binding to the neurotransmitter, i.e.,
gamma-aminobutyric acid (GABA).
GABAA receptors are ligand-gated ion channels which are pentameric proteins in
nature and constitute 5 subunits belonging to different families (α1–6, β1–3, γ1–3, δ,
π, ε, ρ, θ). They contain an integral chloride channel and hyperpolarize the receptor,
and have modulatory sites for barbiturates, neurosteroids, ethanol, and
benzodiazepines.
GABAB receptors are metabotropic in nature, i.e., G-protein-coupled receptors
and exist as heterodimers of GABAB1 and GABAB2 subunits. The GABAB1 subunit
is able to bind with GABA, and the GABAB2 subunit shows interactions as shown in
Table 8.5.

8.6 Role of GABA in Normal Physiology

8.6.1 Sleep

Sleep in human beings and animals has been partitioned into rapid eye movement
sleep (REMS) and non-REMS (NREMS). REMS keeps up the house-keeping
function of the brain and its loss influences the vast majority of the psycho-physical
physiological processes (Yadav et al. 2019). Another report proposes the sleep
promoting neurons (SPNs) incorporate GABAergic neurons in the ventrolateral
preoptic nuclei (VLPO)/intermediate nuclei, middle preoptic nuclei (MnPO), and
brainstem parafacial zone (Ma et al. 2019; Anaclet and Fuller 2017).
Outstanding among other best-studied co-transmission system is the co-release of
glutamate by primary GABAergic/glycinergic neuron terminals in the medial
nucleus of the trapezoid body (MNTB). For instance, starburst amacrine cells
(SACs) in the retina discharge GABA through vesicles in a Ca2+ dependent process
(Lee et al. 2010). This enables few SACs to encode both movement and course
sensitivities using GABA signaling, respectively. Glutamate is co-released from
dopaminergic neurons in the ventral tegmental zone (VTA) (Chuhma et al. 2009).
Dopamine acts upon a moderate timescale by binding to G-protein-coupled
receptors, while glutamate acts upon a fast timescale when bound to ionotropic
glutamate receptors and conveys temporarily exact signals. Glutamate co-release is
helpful for exact prediction error signals, allowing the reward to be encoded in the
firing rates of dopaminergic neurons and mediating dopamine-dependent behaviors
(Mingote et al. 2017).

8.6.2 Female Reproductive System

GABA also appears to play a role in female reproductive functions, perhaps because
hormonal cycles may synchronize with the body clock. GABA shows an increase in
256 S. Kaur et al.

Table 8.5 Selective agonist and antagonist of GABAA, GABAB, GABAC receptors
Name of compound Description
GABAA
Muscimol Competitive GABAA receptor agonist
GABA Endogenous agonist
Isoguvacine hydrochloride Selective GABAA agonist
L-838,417 GABAA partial agonist; displays subtype selectivity
MK 0343 GABAA partial agonist; displays subtype selectivity
Muscimol Potent GABAA agonist; also GABAA-ρ partial agonist
TACA GABAA agonist; also GABA-T substrate and GABA uptake
inhibitor
TCS 1205 GABAA agonist; displays subtype selectivity
THIP hydrochloride GABAA agonist
ZAPA sulfate “Low affinity” GABAA agonist; also GABAA-ρ antagonist
SR 95531 hydrobromide Potent, selective, competitive GABAA receptor antagonist
(Gabazine)
(-)-Bicucullinemethiodide Competitive GABAA receptor antagonist
Picrotoxin Noncompetitive GABAA receptor antagonist/glycine receptor
inhibitor
Flumazenil GABAA receptor antagonist
MRK 016 α5-selective GABAA inverse agonist
TB 21007 α5-selective GABAA inverse agonist
Furosemide GABAA antagonist; also Na+/2Cl-/K+ cotransporter blocker
PHP 501 trifluoroacetate Potent GABAA antagonist
Picrotoxin GABAA antagonist
SCS Potent GABAA antagonist; β1-subunit selective
SR 95531 hydrobromide Competitive and selective GABAA antagonist
Suramin hexasodium salt Competitive α1β2γ2 GABAA antagonist; also nonselective P2
antagonist
GABAB
Baclofen Selective GABAB receptor agonist
SKF 97541 Potent GABAB receptor agonist
(RS)-Baclofen Selective GABAB agonist
GABA Endogenous agonist
SKF 97541 Highly potent GABAB agonist; also GABAA-ρ antagonist
CGP 55845 Potent, selective GABAB receptor antagonist
Saclofen Selective, competitive GABAB receptor antagonist
SCH 50911 Selective, competitive GABAB receptor antagonist
CGP 35348 Selective GABAB antagonist; brain penetrant
CGP 36216 hydrochloride GABAB antagonist; displays activity at presynaptic receptors
2-Hydroxysaclofen Selective GABAB antagonist; more potent than Saclofen
Phaclofen Selective GABAB antagonist
SCH 50911 Selective and competitive GABAB antagonist
GABAA-ρ receptors/GABAC
GABA Endogenous agonist
(continued)
8 Pharmacology of GABA and Its Receptors 257

Table 8.5 (continued)

Name of compound Description
Muscimol GABAA-ρ partial agonist; also GABAA agonist
RuBi GABA Caged GABA; inhibits neural activity
trimethylphosphine
TACA GABAA-ρ agonist; also GABAA agonist, GABA-T substrate, and
GABA uptake inhibitor
SKF 97541 GABAA-ρ antagonist; also highly potent GABAB agonist
THIP hydrochloride GABAA-ρ antagonist; also GABAA agonist
TPMPA Selective GABAA-ρ antagonist
ZAPA sulfate GABAA-ρ antagonist; also GABAA agonist

the levels of luteinizing hormone, estrogen, and progesterone while decrease in the
level of FSH. GnRH neurons express both GABA and GABA receptors and receive
GABAergic input that expresses estrogen receptors; therefore GABA has been
implicated as a major player in the regulation of GnRH neuron activity and secretion
(Maffucci and Gore 2009).

8.6.3 Learning and Memory

Hippocampus is the region of the brain which is known to be involved in learning

and memory functions and is prominent in GABA. It helps in reshaping the neural
connections of the brain. GABA is reported to help and harm the formation of
memories (Jay 2003). Ethanol stimulates transmission of GABA by enhancing its
action. Therefore it relieves both anxiety and unwanted effects on memory and
learning. Pentobarbital has strong memory inhibition in spatial discrimination
activities in rats (Chapouthier and Venault 2002). Barbiturates show the positive
modulation of GABAergic receptors that have been analyzed in various memory
tasks. Kim and coworkers hypothesized that the enhancement of memory consoli-
dation caused by blockade of GABAA receptors within a limited time window
depends on the increase of BDNF levels, induced by GABAA receptor blockade.
The results of their study suggest that the enhancement of the level of mBDNF and
its function during a restricted time window after training are required for the
enhancement of memory consolidation induced by GABAA receptor blockade. It
is well known that hippocampal GABAA and NMDA receptors play an important
role in spatial memory (Kim et al. 2012). Calcineurin signaling pathway involved in
the interaction between GABAA and NMDA receptors, to justify this
relationship Saito et al. 2010 explored the effects of cyclosporin A, a calcineurin
inhibitor, along with muscimol, on the retention of a delayed SWSh task in a radial
arm maze. The results of these studies indicated that hippocampal NMDA receptors
regulate the effect of spatial working memory induced by muscimol. In addition, the
calcineurin signaling pathway may be involved in muscimol-induced impairment of
memory retention. The downregulation of GABA modulates memory storage by
facilitating the release of norepinephrine, which, after binding to β-ARs, initiates an
258 S. Kaur et al.

intracellular cascade culminating in the synthesis of new proteins, which are utilized
for the synaptic changes required to stabilize the new, reactivated, or inhibitory
memory trace (Makkar et al. 2012).

8.6.4 Blood Pressure

In the mammalian central nervous system, GABA is the main inhibitory neurotrans-
mitter. GABAA and GABAB receptors are activated by aminobutyric acid in
neurocytes, which promote the dilation of blood vessels and also reduce blood
pressure (Ma et al. 2015). GABA reduces the renal sympathetic nerve secretion in
parallel with the fall in blood pressure. Several studies reveal that stimulation of the
GABA receptor can centrally reduce sympathetic nerve activity, causing a drop in
blood pressure. Although the physiological significance and location of these
receptors are unknown, the pharmacological manipulations of these receptors can
cause extreme functional changes and involve GABA as a possible central
modulator of cardiovascular control.

8.6.5 Heart

GABA slows heart rate by activating GABA receptor. GABA inhibits the central
norepinephrine neurotransmitter system and produces the tension booster effect and
may increase the heart rate. Experimental study suggests that muscimol decreased
heart rate in insulated heart experiments and may represent an effect on intrinsic
cardiac activity (Bentzen and Grunnet 2011). Bicuculline, a GABA antagonist,
shows a negative chronotropic effect which can be described by the excitatory
actions of vagal neurons. Reports suggest that GABA has an excitatory effect in
early development due to high chlorine intracellular concentrations. The change has
inhibitory GABA phenotype due to the increased aspect of the cotransporter KCC2
chlorine output (Ben-Ari 2002). Lin et al. reported that there is a correlation between
the reduction in HR and temperature and state that the temperature could influence
ion channels, hence extending the duration of a cardiac tissue action potential, with a
greater effect on atrial tissue (Lin et al. 2014). Rana et al. reported a decrease in HR
with exposure to caffeine and caused cardiac arrest in some cases. The reports
suggested that the negative chronotropic effects could be due to the presence of
adenosine receptors, which modify the activity of potassium channels, sustain
cardiomyocyte membrane hyperpolarization and therefore reduce heart rate (Rana
et al. 2010).
8 Pharmacology of GABA and Its Receptors 259

8.7 Mechanism of Action of the Natural Neurotransmitter

GABA and Benzodiazepines on Nerve Cells (Neurons)
in the Brain

Benzodiazepines show their activity by increasing the activity of GABA. It is an

agent that exchanges messages starting with one neuron then onto the next. The
messages that GABA exchanges are inhibitory: they show neurons coming in
contact, to slow down or stop firing. About 40% of the millions of neurons in the
brain respond to GABA, which implies that GABA has a calming hypnotic effect in
the brain. This regular activity of GABA starts with benzodiazepines, which thus
exerts an additional extra (often excessive) inhibitory activity (Bowery and Smart
2006; Bateson 2004) as shown in Fig. 8.8. Its response with special sites (GABA-
receptors) on the outside of the accepting neuron opens a channel, permitting
negatively charged particles (chloride ions) to pass within the neuron. These nega-
tive ions “supercharge” the neuron making it less receptive to other
neurotransmitters which would typically excite it. Benzodiazepines additionally
react at their very own unique sites (benzodiazepine receptors), situated on the
GABA-receptor. A combination of a benzodiazepine at this site acts as a booster
to the activities of GABA, enabling more chloride ions to enter the neuron, making it
considerably progressively resistant to excitation. Different subtypes of benzodiaze-
pine receptors have slightly various activities. One subtype (alpha 1) is responsible
for sedative effects, another (alpha 2) for anti-anxiety effects, and both alpha 1 and
alpha 2, as well as alpha 5, for anticonvulsant effects. All benzodiazepines combine
to a greater or lesser extent, with all these subtypes and all enhance GABA action in
the brain (Rudolph et al. 2000). Enhancement of GABA’s inhibitory activity caused
by benzodiazepines, the brain’s output of excitatory neurotransmitters, including
norepinephrine (noradrenaline), serotonin, acetylcholine, and dopamine, is

Fig. 8.8 Mechanism of action of GABA and BZD on nerve cells in the brain
260 S. Kaur et al.

diminished. Such excitatory neurotransmitters are important for normal alertness,

memory, muscle tone and coordination, emotional responses, endocrine gland
secretions, heart rate and blood pressure control, and a group of different functions,
all of which may be impaired by benzodiazepines (Ciranna 2006).

8.7.1 Mechanism of Action of GABA Receptor as Shown in Fig. 8.9

Inhibitory synapses are the most abundant synapses in the CNS and include neuro-
transmitter GABA. Aspects of health, especially emotional and physical stability, are
affected by GABA. During locomotion specifically, it relaxes the body muscles and
foraging and helps in the process of defecation by contracting the enteric muscle.
The important functions of this neurotransmitter are included in various natural
processes and pathological processes as it is required for basic motor function.
Various types of neurological and psychiatric disorders including anxiety, depres-
sion, insomnia, and epilepsy occurred due to the deficiency of GABA and also due to
the obstruction of electrical impulses. GABA has been shown to affect many
biological functions in the brain, e.g., cognition, learning, emotions, locomotion,
circadian rhythms, and sleep. Furthermore, GABA also has roles in cellular events
such as differentiation, proliferation, migration, axonal growth, synapse formation,
and neuronal death (Kilb 2012; Birnir and Korpi 2007).
Pharmacological actions are responsible for the GABAA effects including
excitability of the neuron (Carver and Reddy 2013), anxiety modulation (Nuss

Fig. 8.9 Different subunits with different activity on receptor

8 Pharmacology of GABA and Its Receptors 261

2015), behavior (Caldji et al. 2004), circadian rhythms (Albus et al. 2005), and
learning and memory abilities (Chapouthier and Venault 2002).

8.7.1.1 GABAA Receptor Action

Activation of GABAA receptor leads to the opening of the central pore to 0.7 nm,
enough to allow passage of partially hydrated Cl
ions. Due to this, hyperpolariza-
tion of neuronal membrane occurs. Hence, decreased occurrence of action potential
results in inhibition of neurotransmission as shown in Fig. 8.10.

8.7.1.2 GABAB Receptor Action

Binding of GABA to the extracellular domain of B1 leads to allosteric changes in B2
subunit (coupled to G protein) which results in inhibition of adenylyl cyclase,
activation of K+ channels, and a decrease in calcium conductance and hence
decrease in neurotransmitter release and action potential as shown in Fig. 8.11.

8.7.1.3 GABAC Receptor Action

GABA is more selective to GABAC. Binding of GABA to the extracellular domain
of C results in allosteric changes in the receptor structure. It leads to an influx of
chloride ions, ultimately hyperpolarization which results in the inhibition of neurons
as shown in Fig. 8.12.

Fig. 8.10 GABAA receptor

action
262 S. Kaur et al.

Fig. 8.11 GABAB receptor

action

Fig. 8.12 GABAC receptor

action

Pharmacology and Function of GABAA Receptor (Recent Update

on Involvement of GABAA in Normal and Pathophysiological States)
GABAA trafficking and clustering are regulated by different intracellular and trans-
membrane proteins that form GABAA receptor (Birnir and Korpi 2007). The brain
possesses rare expression of other subunits but α1, β2, γ2 subunits are highly
8 Pharmacology of GABA and Its Receptors 263

expressed according to their respective families (Bormann 2000). Hippocampus has

a high expression of the α5 subunit and can combine with β and γ2 subunit in the
other region of the brain mediating tonic inhibition by forming extrasynaptic
channels (Korol et al. 2015). Similarly, forebrain and cerebellum contain α4 and
α6 subunits mediating tonic inhibition by combining with β and δ subunit to form
extrasynaptic channels (Farrant and Nusser 2005).
GABA acts via the ligand-gated ionotropic receptor and is a ligand for the
receptor and N-terminus having a binding site between the interface of α and β
subunits. Selective agonists such as muscimol and THIP activate the receptor
and competitive antagonists such as bicuculline and gabazine (SR95531) block the
receptor. At low concentrations steroids such as progesterone, pregnenolone, and
their derivative neurosteroids such as allopregnanolone, THDOC, act as positive
allosteric modulators of GABAA receptors but at a higher concentration alone they
activate the GABAA channels (Uusi-Oukari and Kopri 2010).
The effects of diazepam that were mediated by respective receptors are lost and
comparison does not occur. So it was demonstrated that various effects such as
sedative, anterograde amnesic, and partly the anticonvulsant actions of diazepam
(Crestani et al. 2000) are mediated by α1 subunit. GABAA receptor containing α5
subunits showed enhanced cognition properties without exerting convulsant,
proconvulsant, or anxiogenic activity (Chambers et al. 2004). The function of β
subunit of GABAA receptor is identified by a similar approach that was used to
define α subunit. Different subunits such as α1, β1, β2, β3, and γ2 have different
distribution throughout the brain suggested by immunocytochemistry data. Majorly,
the GABAA receptors are formed from the high proportion of α1 and γ2 subunits and
are widely expressed subunits. Other subunits such as α2, α3, α4, α5, and α6 have
confined distribution.

Pharmacology and Function of GABAB Receptor (Recent Update

on Involvement of GABAB in Normal and Pathophysiological States)
The highest concentration of GABAB is found in the molecular layer of the cerebel-
lum, the frontal cortex, and certain thalamic nuclei (Billinton et al. 2000). GABAB
receptors are generally divided into auto and hetero-receptors. GABAB receptors are
composed of the two subunits GABAB1 and GABAB2, which convey particular
capacities. GABAB1 harbors the binding site for GABA to the large extracellular
found in the N-terminal domain, though GABAB2 is required for high-affinity
agonist binding to GABAB1. The clinical significance of GABAB receptors
contributed to ongoing research, and their particular role in the overall regulation
of gastric function is being discovered (Collares and Vinagre 2005). The receptor is
coupled to adenylyl cyclase (AC) through Giα and Goα proteins which inhibit
adenylyl cyclase types I, III, V, and VI, while Gβγ stimulates adenylyl cyclase
types II, IV, and VII. GABAB receptors were shown to mediate presynaptic inhibi-
tion on some nerve endings and postsynaptic inhibition on some cell bodies or
dendrites. The presynaptic GABAB receptors activation inhibits GABA and gluta-
mate release through a decrease in Ca2+ conductance by inhibiting voltage-sensitive-
P, N, and L-type Ca2+ channels, whereas postsynaptic initiation of GABAB receptors
264 S. Kaur et al.

resulting in an increase in membrane conductance through G-protein-coupled inter-

nally rectifying potassium channels (GIRK orKir3) and inhibition of adenylate
cyclase (Emson 2007). Conversely, autoreceptors facilitate the LTP by limiting
postsynaptic inhibition (Pinard et al. 2010). GABAB heteroreceptors mediate
heterosynaptic depression of glutamate discharge at hippocampal neurotransmitters
(Guetg et al. 2009). This was appeared to modulate the expression of LTP at neural
connections in the hippocampus and the lateral amygdala (Shaban et al. 2006).
GABAB receptors are predominantly localized at extrasynaptic sites to control the
excitability of neurons by inhibiting transmitter release as well as hyperpolarizing
the neuronal membrane (Gassmann and Bettler 2012). The GABAB receptors are
also responsible for most effects of the drug of abuse gamma-hydroxybutyrate
(GHB) which acts as a GABAB partial agonist at high doses (Pin and Prézeau
2007; Drasbek et al. 2006). GABAB agonists also established several beneficial
effects both in animals and in humans (Bowery and Smart 2006). GABAB receptors
were pharmacologically recognized in the early 1980s and were selectively activated
by baclofen (β-p-chlorophenyl-GABA) (McDonnell et al. 2007), the molecule that is
clinically used for the treatment of spasticity in patients with multiple sclerosis,
cerebral palsy, and spinal cord injury (Froestl 2010). Baclofen was synthesized by
Heinrich Keberle in 1962, 30 years before a GABAB receptor was cloned (Froestl
2010). Baclofen is a lipophilic derivative of GABA and was designed to enhance the
penetration of the blood-brain barrier. The R isomer of baclofen shows a three times
greater affinity/efficacy for GABAB receptors than the racemate (Filip and
Frankowska 2008). However the use of baclofen has been associated with serious
side effects such as sedation, muscle relaxation, and marked hypothermia (Cryan
et al. 2004).
Two recently discovered GABAB receptor positive allosteric modulators are
COR627 and COR628 (Castelli et al. 2012). The advantage of these positive
allosteric modulators (PAMs) is that they are free from side effects which are
associated with GABAB receptor agonists such as sedation, muscle relaxation, and
marked hypothermic potential (Cryan et al. 2004). Compounds showed high recep-
tor selectivity but their low affinity and weak brain penetration after peripheral
administration limited their use in pharmacological studies (Kerr and Ong 1996).
More potent and selective GABAB receptor antagonists (phosphinic acid
derivatives) like CGP36742 (also known as SGS742), CGP55845A and
CGP56433A, CGP46381, CGP51176) (Froestl et al. 1995) and SCH50911 were
developed. These GABAB receptor antagonists are used widely in research, and
several compounds have shown promising results in preclinical behavioral models
of depression and cognitive disorders (Nowak et al. 2006).

Pharmacology and Function of GABAC Receptor (Recent Updates

on Involvement of GABAC in Normal and Pathophysiological States)
GABAC is a subtype and specially considered as part of GABAA receptor due to
the presence of ρ subunit (Olsen and Sieghart 2009). Retina possess high expression
of GABA receptor with ρ subunit which is insensitive to bicuculline- and baclofen-,
showing pharmacology distinct features from the classical GABAA or GABAB
8 Pharmacology of GABA and Its Receptors 265

receptors, known as GABAC. Initially, the receptor comprised only three subunits
(ρ1, ρ2, and ρ3), but later it was revealed that five (ρ4 and ρ5) subunits form the
ionotropic GABAC receptors necessary to form a functional channel (Zhang et al.
2001). Also, the brain regions contain the expression of ρ subunits with function in
the visual pathways (Lukasiewicz et al. 2004) as well as in the local GABA circuit of
developing visual cortex (Morales et al. 2002), so expression is not only restricted to
the retina and does not indicate a separate ionotropic receptor subfamily. It was
confirmed that the human retina contains ρ2 subunits with significant abundance
(Naffaa et al. 2017) and other regions of the brain such as hippocampus, cerebellum,
and pituitary were detected with lower expression levels (López-Chávez et al. 2005).
The sensitivity of GABA to GABAC receptors is more as compared to GABAA
and GABAB receptors. Ionotropic GABAC receptors desensitization and opening
time of channel are also longer than in the other receptors. Excellent models are
available to characterize GABAc receptors in the retina, i.e., rod-driven horizontal
cells and availability of GABAc receptors on other types of retinal neurons.
The influence of GABAC receptors on sleep-waking processes is limited.
GABAA-modulatory drugs such as benzodiazepines, barbiturates, and neurosteroids
do not affect GABAC receptors. GABAC receptors unlike the GABAA receptors
show different electrophysiological responses and is additionally sensitive to the
physiological agonist while the Hill slopes are steeper reflecting the presence of five
ligands binding site whereas two sites appear to be present on the GABAA receptor.
GABAA receptors (such as SR95531 and hydrastine) or GABAB receptors (such as
phaclofen and saclofen) antagonists are not sensitive to GABAC receptors on retinal
neurons. Detailed studies indicated that GABAC receptor binding preferences
depend on different conformation of GABA molecule.
Recently, a novel target for analgesia is ρ2 receptors subunit implicated in pain
perception found presynaptically in the spinal dorsal horn (Tadavarty et al. 2015).
Sleep-waking behavior of rats demonstrated by behavioral pharmacological studies
(Arnaud et al. 2001), learning and memory in chicks and rats (Chebib et al. 2009),
the inhibitory modulation of the olfactory bulb (Chen et al. 2007) involves ρ1
receptors.

8.8 Therapeutic Potential and Recent Advances of GABA

Receptor Agonist and Antagonist in Various Diseases

8.8.1 Role of GABAergic System in AD and Potential Therapeutic

Targets Under Research

Alzheimer’s disease is the most prevalent neurological disorder associated with

the loss of neurons in the hippocampus, neocortex, and cerebral cortex. It is
characterized by memory loss, cognition impairments, and behavioral abnormalities
triggered by synaptic dysfunction (Kumar and Singh 2015). Whether acetylcholine
remains a major neurotransmitter in memory formation and processing, the role of
other neurotransmitters like GABA and glutamate becomes essential to discuss
266 S. Kaur et al.

due to their contribution to excitotoxicity-mediated cell death. Neuron loss in AD

contributes to abnormal production and accumulation of protein misfolds over the
synapses. Excitotoxicity, hypercholesterolemia, insulin resistance, oxidative stress,
and genetic susceptibility remain major contributors in AD (Kumar and Singh 2015).
Here, excitotoxicity-mediated neuron loss seems to be a predominant pathogenic
mechanism in AD. GABAergic neurotransmission affected in excitotoxicity as the
GABA and glutamate balance altered. Further, an in- depth analysis of GABAergic
neurotransmission suggests the reliable therapeutics in AD. The amyloid beta
formation upregulates the calcium release in hippocampal neuronal cultures which
distorts neuronal function via overexcitability (Lazzari et al. 2015). Initially, NMDA
gets activated and further GABA gets upregulated to counterbalance it. Abnormal
elevation in GABAergic neurotransmission alters synapse function which results in
long-term potentiation in dentate gyrus that is reversed by picrotoxin-like GABA
antagonist. In hippocampal astrocytes, expression of bestrophin-1 (Best-1) has
been reported to have decreased, which in turn confirms the altered GABA and
glutamate in AD (Wu et al. 2014). It could be hypothesized that initial enhanced
release of neurotransmitters to combat the enhanced excitotoxicity may damage the
functionality of Best-1. Moreover, the enhanced astrocytic GABA resultant LTP
deficits in the dentate gyrus have been proposed as the possible biomarker in AD. On
the other side, the apoE-4 secreted from GABAergic neurons disrupts the function-
ality of GABAergic neurons which impairs cognitive function (Li et al. 2016).
It is fundamentally established that an adequate balance of GABA and glutamate
is necessary for neuronal function and justifies the excitotoxicity-mediated inhibition
of GABA expression in AD. Excitotoxicity-mediated overactivation of NMDA
receptor causes degradation of subunits of GABAB in AD, as shown in Fig. 8.13.
The GABAB consists of GABAB1 and GABAB2 like subunits that get
phosphorylated by calcium calmodulin complex on ser867 and ser783, respectively.
This will mediate endocytosis of GABAB through lysosomal degradation and disrupt
neural signaling via overexcitation of neurons (Kantamneni et al. 2008). Thus, a
number of evidence strongly prosecute the deregulated GABAergic neurotransmis-
sion in AD. Many implicit the GABA agonists for neuroprotective action in AD-
associated amyloid beta neurotoxicity. Etazolate is one of the GABAergic agonists
that gave reliable effects in phase II clinical trials under AD research (Li et al. 2016).
Other drugs like muscimol and propofol provide beneficial effects in AD studies.
Further, there is more precise data available for the subunits of GABAA. It has been
reported that α5 subunit negatively regulates learning and spatial memory. The
deletion of α5 enhances the hippocampus-dependent learning and spatial memory
in mice. This led to the utilization of α5 subunit-specific inverse agonists in AD for
beneficial effects over cognition.
Several inverse agonists like Ro-4938581 and Ro-4882224 are proven to be
beneficial for cognition in clinical studies. Moreover, there are antagonists of
GABAB which were also tested in AD studies for neuroprotection. It has been
hypothesized that the astrocytic release of GABA may interfere with the synaptic
activity of neurons which results in cognition impairment. On this basis, the com-
pound SGS742 which is a novel GABAB antagonist provides beneficial effects in
8 Pharmacology of GABA and Its Receptors 267

Fig. 8.13 GABAergic transmission and its receptor activation

clinical studies under AD research (Li et al. 2016). This way evidence-based studies
over a number of pathogenic mechanisms made implicit different agonists. Inverse
agonists and antagonists for GABA for therapeutic approach in AD are shown in
Table 8.6.

8.8.2 Role of GABAergic System in Parkinson’s Disease

and Potential Therapeutic Targets Under Research

Parkinson’s disease is the second most devastating neurodegenerative disorder

affecting elderly people with motor and non-motor complications. The motor
complications in PD include bradykinesia, rigidity, tremor, and postural
abnormalities caused by damage in the dopaminergic neurons of substantia nigra
pars compacta (SnPc), whereas the non-motor complications involve autonomic
disturbances, olfactory dysfunction, and sleep problems which may originate due
to loss of other neurons in specific regions of the brain (Maiti et al. 2016).
The voluntary control in the mammalian brain is coordinated by signaling of
basal ganglia. The basal ganglia including the striatum, substantia nigra pars
compacta (SNPc), subthalamic nuclei (STN), globus pallidus external and internal
(GPe and GPi) like regions constitute to form a circuit to regulate voluntary
movement control (Dézsi and Vécsei 2011). This circuit is incorporated by
268 S. Kaur et al.

Table 8.6 Drugs targeting GABA for therapeutic benefit in AD

Compound Mechanism of action Pharmacological action
Muscimol GABAA receptor agonist Protective against overproduction of amyloid
beta
Propofol GABAA receptor agonist Protective against amyloid beta-induced
neurotoxicity
Improves cognition
MRK-016 Inverse agonists of GABAA at Improved cognition in preclinical studies
α5 subunit
CGS9896 Inverse agonists of GABAA at Enhances memory in different tasks
α5 subunit
Ro- Inverse agonists of GABAA at Reverses memory deficit in different AD
4938581 α5 subunit studies
Ro- Inverse agonists of GABAA Reverses memory deficit in different AD
4882224 α5 subunit studies
SGS742 GABAB receptor antagonist Enhances memory and attention in human and
animal studies
Etazolate GABAA receptor agonist Protects neurons against amyloid beta-induced
neurotoxicity
CGP55845 GABAB receptor antagonist Improves cognition in animal studies

GABAergic and glutaminergic neuronal projections that maintain inhibitory and

excitatory control over movement through the availability of dopamine. Dopamine
neurons are highly abundant in striatum along with D1 (excitatory) and D2 (inhibi-
tory) receptors. In this circuit, the direct pathway includes D1 excitatory control over
GABAergic inhibition of GPi that removes inhibitory control from thalamus which
results in movement whereas the indirect pathway mediates D2-mediated inhibition
over inhibitory control of GPe that further excites STN to mediate glutaminergic
excitation of GPi to inhibit movement. This way the direct pathway acts as the
inhibitory switch on GPi and the indirect pathway excites the GPi to control over
thalamus which sends excitatory projections to the motor cortex which results in
adequate movement. The loss of dopaminergic neurons (specifically D1) in PD
results in interference to direct pathway, which causes hindrance in movement that
results in hypokinesia, rigidity, and gait abnormalities as shown in Fig. 8.14.
The role of GABAergic system in motor complications is generally assessed on
the basis of inhibitory GABAergic control over voluntary movements regulating
brain regions. Dopamine loss in the striatum leads to excessive stimulation of
GABAergic inhibitory control over movement which causes symptoms of PD
(Alexander 2004). Another prospective target is the excitotoxicity-mediated loss of
GABAergic neurons that results in a loss of adequate control over movement
(Błaszczyk 2016). This way both the agonists and antagonists of GABA have
made implicit for the treatment of PD. On one side progabide, a positive allosteric
modulator of gamma-aminobutyric acid B, and Zolpidem like GABA facilitators
improve the symptoms of PD, and on the other side antagonists for GABA also get
evaluated for beneficial effects.
8 Pharmacology of GABA and Its Receptors 269

Fig. 8.14 Altered GABA

and glutamate functioning
in PD

Not only this, non-motor symptoms are believed to be brought out by GABAergic
dysfunction. Here, the 80% of cases of PD reports for abnormal olfactory functions.
The distorted olfactory function in PD is associated with the loss of dopaminergic
neurons in olfactory bulbs and olfactory nuclei which depends upon glial cell-
derived neurotrophic factors (GDNF) for survival. The availability of GDNF
depends upon neuro-glial interactions effected by GABA/Ca2+ mechanism in both
midbrain and olfactory nuclei. During normal conditions, the physiological release
of GABA causes hyperpolarization of neurons for long-term inhibition of synaptic
transmission. This hyperpolarization phase blocks the calcium channel and protects
neurons from excitotoxicity (Błaszczyk 2016). The removal of calcium from
mitochondria and cytoplasm requires a high amount of energy; therefore neuronal
cells in the midbrain have energy requirements to cope with calcium overload. On
the other side, calcium overloads lead to oxidative stress-mediated dysfunction of
mitochondria. This way subsequent decline in ATP production, increase in oxidative
stress, and calcium load result in mitochondrial dysfunction-mediated neuronal loss
in SnPc in PD (Błaszczyk 2016). The excitotoxicity mediated initial release of
GABA from astrocytic neurons but on exceeding the limits density of synaptic
GABA receptors gets decreased which leads to an increase of GABA in synaptic
cleft and initiate self-mediates apoptosis in neuronal cell. Furthermore, the defect in
GABA-glutamate cycle (neuron-astrocyte interaction) for GABA recycling could
also result in neuronal damage as shown in Table 8.7.
270 S. Kaur et al.

Table 8.7 Drugs targeting GABA for therapeutic benefit in PD

Compound Mechanism of action Pharmacological action
Tiagabine Inhibitor of GABA transporter 1 • Neuroprotective over
Blocks microglia cell activation in PD dopaminergic neuron loss in
PD
• Protective against
inflammation in PD
Muscimol GABAA receptor agonist Neuroprotective in PD
Baclofen GABAB receptor agonist Neuroprotective in PD
Flumazenil GABA antagonist that binds to the Treat hyper-GABAergic
benzodiazepine (BZ) binding site of the GABAA associated motor symptoms in
receptor PD
Zolpidem • Facilitates GABAergic neurotransmission Improvement in motor
• Positive allosteric modulation of GABAA symptoms of PD
receptors
Lycopene • Enhances density of GABA receptors in SNPc Neuroprotective in PD
• Antioxidant

8.8.3 Role of GABAergic System in Huntington’s Disease

(HD) and Enlisting Potential Therapeutic Drugs Under
Research

HD is the hereditary predisposed neurodegenerative disorder that is characterized by

a progressive neuronal loss in the striatum that gradually spreads throughout the
brain. Patients with HD suffer from motor, cognitive, and psychiatric disabilities
(Rubinsztein 2006). The motor dysfunction-mediated loss over voluntary muscles
remains major symptoms of the disease. The loss of striatal pathogenic neurons
terminates their projections to different brain regions, which is assumed to be a major
contributor in HD. The GABAergic projections are major abundant neurons in the
striatum and their loss remains a predominant step in HD (Garret et al. 2018). The
GABA neurons in indirect pathway loss first, that leads to overactivation of GPe and
results in diminishing the activity of STN-mediated glutaminergic projections.
The net result of this leads to an increase in thalamo-cortical activity that causes
dance-like movements in HD. Simultaneously, the effects of disease on the direct
pathway results in the activation of the subthalamic region as inhibitory tone is lost
via deterioration of GABAergic neurons. This first effect SNr then proceeds to GPi
that is seen via symptoms progression from dancing to akinesia. Here, the deteriora-
tion of GABAergic projections confirms that the brains of HD patients lose their
capacity to synthesize and release the required amount of GABA. The remaining
projections in the striatum are found to have increased GABA receptors whereas few
studies report the loss of GABAergic neurons as an early event of disease. On this
basis, different studies have implicit GABA replacement therapy and agonists of
GABA for the treatment of HD disease (Mason and Barker 2009). The compounds
like exogenous gangliosides, hesperidin, and quercetin ameliorate symptoms of HD
by showing neuroprotective effect over alter neurochemistry as shown in Table 8.8.
8 Pharmacology of GABA and Its Receptors 271

Table 8.8 Drugs targeting GABA for therapeutic benefit in HD

Compound Mechanism of action Pharmacological action
Baclofen GABA agonist Neuroprotective in HD
Exogenous Enhance synthesis of • Protective over alter neurochemistry
ganglioside GM1 gangliosides • Decrease striatal atrophy and neuron
Microglia protective loss
• Protective over cognition and
psychiatric symptoms
WIN55,212-2 Cannabinoid (CB1) agonist • Protective over alter neurochemistry
• Prevent excitotoxicity in HD
Spermidine Enhances polyamines level Protective over alter neurochemistry
in the brain
Modulates NMDAR
function
Muscimol GABAmimetic • Protective over alter neurochemistry
• Neuroprotective in HD

Among different receptors for GABA, the receptor GABAA remains a potential
target for therapeutics in HD. The distribution of GABAA in striatum mediates tonic
(α5 by α 1–3, δ or by γ 2 forming extrasynaptic receptor) and phasic inhibition (α1,
α2, α3 in combination with β and γ2) via its different subunits (Waldvogel and Faull
2015). Here, α1 facilitates the fast inhibitory current and α2 subunit gets altered and
hence confirms the decrease in GABAA in HD brain. The α1 participates in the
co-release of GABA and dopamine in medium spiny neurons of the striatum and its
decreased expression contributes to motor symptoms of HD.
Further, there is a spatial distribution of GABAergic interneurons (IN) in HD,
different classes of IN including fast-spiking INs (FSIN) disposing parvalbumin,
persistent low-threshold spiking INs (PLTS) expressing somatostatin or nNOS, and
INs expressing calretinin. Specific degeneration of fast-spiking neurons specifically
contributes to HD (Du et al. 2017). Moreover, the role of cholinergic IN remains
essential to discuss as they are widely distributed in the striatum which bears
localization of GABA receptors. The increase in α3 subunits of GABAA receptor
on cholinergic IN mediates inhibition over acetylcholine release in HD. Decrease in
Tonic inhibition also a prominent pathogenic mechanism in HD, which is confirmed
by a decrease in tonic inhibition regulatory subunits like α5 and δ (Allen et al. 2009).
Moreover, the reduced tonic current mediates the movement and psychiatric
symptoms in patients affected with HD.
The GABAergic neurons remain major inhibitory control above overexcited GPe,
so it is essential to discuss the subunits that are expressed there. The α1, β2, and
γ2 subunits get reduced along with decreased expression of vesicular GABA
transporter in GPe, which is also reported in HD. Moreover, the subunits like α2,
α3, and γ1 remain expressed in GPe, which is reported to get altered in the HD brain
(Du et al. 2017). So, in this way specific agonists and antagonists for different
subunits could be more reliable towards therapeutics in HD.
272 S. Kaur et al.

8.8.4 Role of GABAergic System in Anxiety and Enlisting Potential

Therapeutic Drug Targets Under Research

Anxiety is the most prevalent stress-associated behavioral disorder that brings

an abnormal state of mind which is characterized by excessive anxiousness, fear,
and nervousness. The neurobiological terms utilized to define anxiety include
overactivation of the sympathetic nervous system, amygdala-linked fear response,
and neurochemical alterations in associated brain regions (Shin and Liberzon 2010).
Furthermore, anxiety brings about somatic, cognitive, and behavioral changes that
impair the normal functioning of an individual. The Diagnostic and Statistical
Manual of Mental Disorders, Fourth Edition (DSM-4), and Diagnostic and Statistical
Manual of Mental Disorders, Fifth Edition (DSM-5), establish the diagnostic criteria
for anxiety which states that anxiety should be treated properly when “the anxiety,
worry, or physical symptoms cause clinically significant distress or impairment in
social, occupational, or other important areas of functioning” (Andrews et al. 2010).
The different classifications specified the five different subtypes of anxiety disorders
which include generalized anxiety disorder (GAD), panic disorder (PD), obsessive
compulsive disorder (OCD), social anxiety disorder (SAD), different phobias, and
post-traumatic stress disorder (PTSD). The neurochemical alterations occurring in
the brain evidence the involvement of GABA, serotonin, opioid peptides,
endocannabinoids, neuropeptides-Y oxytocin, and corticotropin-releasing hormone
in the pathogenesis of anxiety. These alterations provide significant therapeutic
approaches for the treatment of anxiety.
About 50 years’ time passage confirmed the successful clinical reliability of
BZDs for the therapy of anxiety-associated disorders. The BZDs target the
GABAergic neurotransmission to provide significant therapeutics in anxiety
disorders (Möhler 2012). Moreover, the GABAergic neurotransmission in amygdala
is shown to be a reliable therapeutic candidate for anxiety treatment. Preclinical
studies show that the administration of GABA into amygdala relieves the fear
associative anxiety symptoms whereas the antagonists of GABA give anxiogenic
effect (Aroniadou-Anderjaska et al. 2007). Moreover, the human studies also give
reliable results by the administration of BZDs which abolish the negative symptoms
through amygdala activation. There are different subunits of GABAA; the BZDs
bind to the interface of α and γ subunits for inhibitory and calming effects in the brain
but these drugs show sedative effects rather than calming. Therefore, this side effect
of BZDs leads to the discovery of much more specific agonists of different subunits
which are today under research as shown in Table 8.9.
Later the selective drugs for α1 given by compounds like zolpidem, zopiclone,
(S)-zopiclone, and zaleplon show clinical reliability for sedation and hypnotic
effects. The α2 selective compound with no other subunit binding might contribute
to ideal anxiolytic properties. L-838417 which has partial agonist activity towards
α2, α3, α5 subunits with α1 antagonistic activity shows good reliability for anxio-
lytic effect with no sedative side effects but its unfavorable pharmacokinetic restricts
further possibilities (Nuss 2015). Another compound ocinaplon exhibits good
nonsedative anti-anxiety profile even in humans but its hepatotoxicity limits its
8 Pharmacology of GABA and Its Receptors 273

Table 8.9 Drugs targeting GABA for therapeutic benefit in anxiety

Pharmacological
Compound Mechanism of action effects
NS 11394 Partial agonist on α2 and α3 subunits and full agonist on Anxiolytic effect
α5 subunit ons GABAA
L-838417 Partial agonist towards α2, α3, and α5 subunits but shows Anxiolytic effect
affinity even also towards α1 on GABAA
Ocinaplon Partial agonists towards α2, α3, and α5 subunits and full Anxiolytic effect
agonist for α1on GABAA
SL 651498 Agonist for α2, α3 and partial agonist for α1 and α5 Anxiolytic effect
subunits on GABAA
TPA023 Partial agonist on α2 and α3 subunits on GABAA Anxiolytic effect
TPA023B Partial agonist on α2, α3, and α5 subunits on GABAA Anxiolytic effect
TPA123 Partial agonist on α1, α2, α3, and α5 subunits on GABAA Anxiolytic effect
TPA003 Agonist at α3 with high selectivity on GABAA Anxiolytic effect
ELB-139 Unknown selectivity Anxiolytic effect
XBD173 Translocator protein (TSPO) ligand (TSPO is a Anxiolytic effect
benzodiazepine binding site protein that acts on GABAA with no sedation
receptor)
CGP Selective Anxiolytic effect
56433A GABAB receptor antagonists Antidepressant
effect
CGP Selective Anxiolytic effect
55845A GABAB receptor antagonists Antidepressant
effect
CG 39783 GABAB receptor positive allosteric modulator Anxiolytic effect
Antidepressant
effect
Compound Mechanism of action Pharmacological
effects
Eszopiclone Highly selective for α5, medium binding to α2, α3 but Anxiolytic effect
lower towards α1 on GABAA Antidepressant
effect
NS 11394 Partial agonist on α2, α3 subunits and full agonist on α5 Anxiolytic effect
subunit on GABAA
L-838 417 Partial agonist towards α2, α3, and α5 subunits but show Anxiolytic effect
affinity even also towards α1 on GABAA
Ocinaplon Partial agonists towards α2, α3, and α5 subunits and full Anxiolytic effect
agonist for α1 on GABAA
SL 651498 Agonist for α2, α3 and partial agonist for α1 and α5 Anxiolytic effect
subunits on GABAA
TPA023 Partial agonist on α2 and α3 subunits on GABAA Anxiolytic effect
TPA023B Partial agonist on α2, α3, and α5 subunits on GABAA Anxiolytic effect
TPA123 Partial agonist on α1, α2, α3, and α5 subunits on GABAA Anxiolytic effect
TPA003 Agonist at α3 with high selectivity on GABAA Anxiolytic effect
ELB-139 Unknown selectivity Anxiolytic effect
(continued)
274 S. Kaur et al.

Table 8.9 (continued)

Pharmacological
Compound Mechanism of action effects
XBD173 Translocator protein (TSPO) ligand (TSPO is a Anxiolytic effect
benzodiazepine binding site protein that acts on GABAA with no sedation
receptor)
CGP Selective Anxiolytic effect
56433A GABAB receptor antagonists Antidepressant
effect
CGP Selective Anxiolytic effect
55845A GABAB receptor antagonists Antidepressant
effect
Eszopiclone Highly selective for α5, medium binding to α2, α3 but Anxiolytic effect
lower towards α1 on GABAA Antidepressant
effect

further reliability. This way several different subunits-specific molecules are under
investigation for lesser side effects with improved anxiolytic profile for therapeutic
benefits.
Not only GABAA but GABAB also provides some remarkable therapeutic effects
in anxiety. There are some endogenous steroids which also show specific binding
towards the GABAA for anxiolytic activity with lesser side effects than BZDs.
Animal studies revealed the anxiolytic effects of different neurosteroids including
pregnenolone, dehydroepiandrosterone, and progesterone even at low
concentrations (Nuss 2015). Etifoxine, which is a small structurally unrelated mole-
cule to BZDs, promotes neurosteroids synthesis in the brain that shows anxiolytic
effects in both animals and humans. The anxiolytic effects of etifoxine are
contributed by positive allosteric modulating effect on GABAA by binding to β
subunit specifically β2 and β3 sites. Therefore, GABAergic system potentially
participates in the regulation of anxiety and could be a novel approach for future
studies.

8.8.5 Role of GABAergic System in Multiple Sclerosis and Enlisting

Potential Therapeutic Drug Targets Under Research

Multiple sclerosis (MS) is a severe neurological disorder characterized by an auto-

immune demyelination of the CNS. It affects the genetically susceptible younger
adults from 15 to 45 years old. Other factors like exposure to viruses like Epstein-
Barr virus, smoking, and low serum vitamin D levels remain major environmental
contributors in MS. Indeed, there is no exact cause and cure for the disease but the
prevalence of MS is increasing day by day. Clinically, there are four forms of MS
which are seen as relapsing remitting MS (RRMS), secondary progressive MS
(SPMS), primary progressive MS (PPMS), and progressive relapsing MS (PRMS)
(Loma and Heyman 2011). The combined characteristic features of these forms of
8 Pharmacology of GABA and Its Receptors 275

MS include impairments of functional symptoms in motor, visual, and sensory

systems. Moreover, some undiagnosed features including cognitive dysfunction,
fatigue, and mood disturbances contribute to the cortical damage. The treatment
therapy in MS includes interferons, monoclonal antibodies, and cytotoxic drugs
which provide symptomatic relief but none of them are able to halt or cure the
disease (Derwenskus 2011). The pathogenic mechanism behind these factors
includes demyelination, axonal damage, dysfunction of glial cell, and inflammation
like neurodegenerative features which are under research for therapeutic approach in
MS (Loma and Heyman 2011).
The role of GABAergic pathway in MS is uncertain but the structural and
metabolic damage to neuronal and glial cells provides the evidence regarding the
role of neurochemical alterations. It estimates the reduced level of GABA with
a concomitant increase in the level of glutamate in the brain of MS patients. The
adequate balance of glutamate and GABA is essential for motor performance and its
alterations in MS cause sensorimotor dysfunction. Evidences remarkably report the
deficits in GABA levels in the hippocampus, sensorimotor cortex, and prefrontal
cortex that contribute to motor and cognitive dysfunction in MS (Cawley et al.
2015). These remarks get strengthened by excitotoxicity, inflammation, and demye-
lination like factors. This includes acute lesions enhancing the glutamate level
implicit in cell death mechanisms for MS. The neuronal cell terminal resides in the
GABAA receptors which could inhibit the calcium and glutamate release via depo-
larization. This way, the GABAA receptor could be a potential therapeutic target in
MS. Further, the neurotransmitter GABA is also synthesized in astrocytes; hence it
remain a major regulator of immunity. Here, it is important to notify that exogenous
GABA could be deteriorating in MS-like conditions as it exacerbates the disease by
worsening the immune system. It upregulates the cytokine release-mediated neuro-
nal cell death (Mandolesi et al. 2015). Therefore, if drugs could enhance the
endogenous GABA levels then it could be much more beneficial. For example,
drugs like vigabatrin that inhibits GABA-transaminase give neuroprotective effect
on systemic administration. Similar effects are gained from sodium valproate
whereas diazepam and phenobarbitone sodium give mixed results.
Moreover, the presence of cannabinoid receptor CB1 over the GABAergic and
glutaminergic neuronal terminals makes implicit endocannabinoids targeting drugs
in MS (Scotter et al. 2010). Here, agonists of CB1 inhibit the activity of adenylyl
cyclase that decreases the levels of cyclic adenosine monophosphate (cAMP) to
decrease neurotransmitters release from synaptic vesicles as shown in Table 8.10.
It also inhibits the calcium channels by binding to Gi/o proteins that contribute to
the inhibition of the neurotransmitter release. This mechanism could make possible
successful implications of endocannabinoids targeting drugs like nabilone, HU210,
URB597, and WIN 55,212-2 to protect GABAergic neurons from autoimmunity and
excitotoxicity like factors in MS (Scotter et al. 2010).
276 S. Kaur et al.

Table 8.10 Drugs targeting GABA for therapeutic benefit in MS

Pharmacological
Compound Mechanism of action effects
Phenobarbitone • GABA facilitatory as well as GABA mimetic effect Neuroprotective
sodium • Blocks calcium channels to inhibit glutamate release
Sodium valproate • Inhibitor of GABA transaminase and increases Neuroprotective
GABA levels
• Inhibits glutamate-mediated excitotoxicity
Allopregnanolone Agonist for GABA-A receptors on neurons and glial Neuroprotective,
cells Anti-
inflammatory
Vigabatrin Inhibitor of GABA transaminase and increases GABA Reduces disease
levels severity
Diazepam GABA agonist Reduces disease
severity
Topiramate Blocks sodium and calcium channel for enhancing Reduces disease
action of GABA severity
Exogenous Worsen immune system Increases disease
GABA severity
Gabapentin Inhibits calcium channel to enhance concentration of Improves disease
GABA over synapse symptoms
Baclofen GABA agonist Mixed effect
Ganaxolone Synthetic allopregnanolone analogue, an agonist for Improves disease
GABAA receptors on neurons and glial cells symptoms
Etifoxine TSPO agonists, enhance endogenous GABA Improves disease
symptoms
XBD-173 TSPO agonists, enhance endogenous GABA Improves disease
symptoms

8.8.6 Role of GABAergic System in Schizophrenia and Enlisting

Potential Therapeutic Drug Targets Under Research

Schizophrenia (SCZ) is a heterogeneous disorder that markedly causes neuropsy-

chiatric illness in patients and accompanies negative and positive symptoms along
with cognitive abnormalities. The positive symptoms accompany hallucinations and
delusions while negative symptoms bring out emotional blunting and decreased
motivation. Although SCZ affects only 1% of the population of the world, it puts
a huge economic and social burden on people that make this psychiatric illness much
more severe (Rosenberg 2012). Moreover, the exact pathogenesis of SCZ is not clear
and thought to be contributed by genetic and environmental factors. Initially the
dopamine hypothesis was considered as a major lead for research of therapeutics in
SCZ. No doubt, this dopamine hypothesis provided a number of therapeutics
including typical and atypical antipsychotics. Growing evidences also suggested
the role of GABA, glutamate, and serotonin in SCZ (Laruelle 2014). The abnormal
activity of circuits of the prefrontal cortex (PFC), medial temporal lobe, and striatal
region also gather attention in recent decades. Post-mortem studies of SCZ patients
8 Pharmacology of GABA and Its Receptors 277

evidenced the alterations in GABA levels to confirm its role in altered activity of
the brain. Decreased activity of GABAergic interneuron observed in PFC along with
reduced quantity of GAT1 and GAD67. The reduction in GABAergic signaling in
PFC may contribute to cognitive, emotional, and enhanced dopaminergic function
like abnormalities of SCZ patients (Nakazawa et al. 2012). The justification behind
this associates with dysfunctioning of mesostriatal dopaminergic neurons in SCZ.
Normally, the dopaminergic projections of mesostrial pathway give input to PFC
GABAergic neurons, and this control becomes mature during younger age with rise
in cortical activity. But any genetic or environmental risk may hinder this control by
causing dysfunction in mesostriatal dopaminergic pathway which reduces GABA in
SCZ (Selten et al. 2016). However, a decrease in dendritic spines and loss of
pyramidal neurons also contribute to a decrease in GABA in SCZ.
Alteration in GABA levels contributes to diverse symptoms of SCZ including
synaptic plasticity, memory, execution, and psychosis. The loss of parvalbumin
GABAergic interneurons in the hippocampus and cortex may contribute to executive
and cognitive symptoms of SCZ (Lewis et al. 2012). One report suggests that
a decrease in GABAergic neurotransmission in the hippocampus contributes
to deficits in working memory in SCZ (Gao and Penzes 2015). Moreover, psychosis
is considered as the positive symptom of schizophrenia that triggers by dysfunction
of the dopaminergic system in the schizophrenic brain (Kesby et al. 2018). The
hyperexcitation of dopamine (D1) in the striatum is reported to crucially control
cortical activity. As if the cortical interneuron gets inhibited, striatum dopamine
neurons get overexcited that may result in schizophrenic symptoms. The debatable
possible mechanism is that it may decrease in cortical GABAergic interneurons
activity that inhibits ventral tegmental area to increase nucleus accumbens dopamine
activity (Nguyen et al. 2014).
Accordingly, a number of drugs targeting dopamine are going to be evaluated for
their beneficial role in SCZ. Direct infusion of GABA agonist muscimol in the hip-
pocampus is shown to mitigate the memory deficits caused by phencyclidine (PCP)
treatment (Riordan et al. 2017). The hormonal drug estradiol rescues memory in
SCZ by upregulating the expression of GABA in the hippocampus. Moreover, a new
compound named as imidazenil modulates GABAergic neurotransmission for bene-
ficial effects in SCZ. It is highly potent partial GABA agonist with anticonvulsant
and anxiolytic property without any side effect of sedation (Guidotti et al. 2005).
Moreover, recent studies are also assessing the therapeutic efficacy of GABA
targeting drugs via their subunit selectivity. The different subunits of GABAA
including α5, α-2/3, and γ2 are ongoing major therapeutic targets in SCZ. The
selective α5 inverse agonists and α-2/3 agonists are under preclinical studies for
their therapeutic benefits over impaired cognitive and executive functions of SCZ
(Charych et al. 2009). The γ2 subunit of GABAA is reported to participate in
physical interaction with D5 receptor of dopamine which may initiate the symptoms
of SCZ (Vinkers et al. 2010) as shown in Table 8.11.
278 S. Kaur et al.

Table 8.11 Drugs Drugs targeting GABAergic system in SCZ

targeting GABA for
MK-077 FG-7142 MRK-536
therapeutic benefit in HD
Tiagabine NS11394 BZDs
Vigabatrin RO4938581 Muscimol
Bretazenil RO4882224 Imidazenil
Abecarnil L-655 ELB-139
TPA023 L-708 Ocinapalon
MRK-409 MRK-016 PWZ-029

8.8.7 Role of GABAergic System in Traumatic Brain Injury

and Enlisting Potential Therapeutic Drug Targets Under
Research

Traumatic brain injury (TBI) is a neurologic disorder resulting from head injury that
leads to temporary or permanent impairment of the structure and function of
the brain. It could occur due to violent blow, injury, or aggressive shakiness to the
head which produces rapid accelerated or decelerated impact on the brain that leads
to structural damage, functional dysfunction, or neurological deficits leading to
the death of patients. The most common TBI-associated cases are prevalent in traffic
accidents, military, sports, violence, construction sites, industrials, etc. (Madikians
and Giza 2006). These cases also occur in infants when sudden shake to babies cause
a violent impact on their heads. The common pathogenic features of TBI involve the
disturbance of the protective endogenous antioxidant system along with altered
mitochondrial function, excitotoxic damage, and cerebral ischemia. Excitotoxicity
is considered as the major pathological event occurring in TBI brain. It is associated
with an imbalance in between the GABA/Glutamate ratio that raises susceptibility of
neurons towards cell death (Yi and Hazell 2006). The glutamate is the major
neurotransmitter present in pyramidal neurons localized in midbrain, hypothalamus,
cortex, and hippocampus, while GABA interneurons are localized in cortical and
thalamocortical regions to regulate motor functions, attention, and memory. The
adequate balance of GABA/Glutamate is necessary for neuron survival; any shift to
it results in cerebral ischemia, TBI, and other neurodegenerative disorders.
During focal TBI, penetrating injury causes local swelling and ischemia that may
irreversibly damage localized tissue through excitotoxicity. One report suggested
that posttraumatic epilepsy is caused by an imbalance in GABA and glutamate. The
imbalance is reported to result from the loss of parvalbumin-positive GABA neurons
(Guerriero et al. 2015). Moreover, brain studies of concussion-affected athletes
reported that there is an increase in levels of GABAB after injury. It is believed to
be proceeded to counteract the excitotoxicity-induced neuronal damage for survival.
Further, synthesis of GABA is occurring from glutamic acid decarboxylase and its
deficiency results in a decrease in GABA levels in the TBI brain. Some evidence has
also contraindicatory reports that speculated that abnormal synaptic activity in
the TBI brain results from a decrease in glutaminergic neurotransmission and
8 Pharmacology of GABA and Its Receptors 279

Table 8.12 Drugs targeting GABA for therapeutic benefit in TBI

Drug Mechanism of action Pharmacological effects in TBI
Levetiracetam Stabilizes imbalance of GABA/ Improves posttraumatic epileptic
glutamate by unknown mechanism symptoms
Diltiazem GABAAR agonist Improves posttraumatic symptoms
Diazepam GABAAR agonist Improves posttraumatic symptoms
MK-801 Stabilizes imbalance of GABA/ Improves posttraumatic symptoms
glutamate
Zolpidem Atypical GABA agonist Insomnia, neurological complications,
improves posttraumatic symptoms
Baclofen GABAB receptor agonist Improves posttraumatic symptoms
Tetrazepam GABAA receptor agonist Improves posttraumatic symptoms
Gabapentin GABA analogue Neuropathic pain
Improves posttraumatic symptoms
Allopragnelone GABA modulator Improves posttraumatic symptoms
Modafinil Decreases GABA and increases Reduces daytime sleepiness
glutamate

overactivation of GABAergic inhibitory impulses (Kochanek et al. 2015). This may

be caused by modulation of GABAA receptor due to Ca2+ ions regulating NMDA
receptors.
On this basis, different GABAergic drugs are evaluated for their neuroprotective
efficacy in TBI including benzodiazepines (BZD) and other modulators of GABA.
Few of these drugs provide successful results including MK-801, Diltiazem, and
diazepam in animal and human studies (Gibson et al. 2010). So, targeting GABA for
therapeutics in TBI could provide more successful results in future studies as shown
in Table 8.12.

8.8.8 Role of GABAergic System in Epilepsy and Enlisting Potential

Therapeutic Drug Targets Under Research

Epilepsy is a CNS disorder characterized by cerebral dysrhythmia. The brief

episodes (seizures) of loss or disturbed consciousness with or without involvement
of body movements are called convulsions. These episodes are unpredictable and
have highly variable intensity depending on the site of origin in the brain. Earlier it
was recognized as the “disease of lightening” (Browne and Holmes 2008).

8.8.8.1 Physiological Role of GABA in Epilepsy

GABA, the principal inhibitory neurotransmitter in the cerebral cortex, maintains the
inhibitory tone and counterbalances neuronal excitation. When this balance is
disturbed, seizures may occur. GABAA (ligand operated channels) receptor binding
influences the early portion of GABA-mediated inhibitory postsynaptic potential,
while GABAB (G-protein-coupled metabotropic receptor) influences the late portion
(Badawy et al. 2009).
280 S. Kaur et al.

Most of the cases of epilepsy are idiopathic while some may be secondary due to
trauma/intracranial tumor/cerebral ischemia. GABA is present relatively in high
concentration in the mammalian brain which is further catalyzed by GABA amino-
transferase into glutamate, i.e., an excitatory neurotransmitter responsible for
the occurrence of epileptic seizures (Khazipov 2016; Rao and Zhang 2011),
Alzheimer’s disease (Solas et al. 2015), Huntington’s disease (Byrne and Wild
2016), Parkinson’s disease (Silverman 2012), tardive dyskinesia, anxiety, schizo-
phrenia, and other behavioral disorders (Gajcy et al. 2010). The explanation for
seizures is the inhibition of glutamate decarboxylase, a synthesizing enzyme of
GABA by lowering brain GABA levels.
Experimental and clinical investigations demonstrate that GABA has a significant
role in the mechanism and treatment of epilepsy: (1) Abnormalities of GABAergic
function have been seen in genetic and acquired animal models of epilepsy;
(2) Reductions of GABA-mediated inhibition, activity of glutamate decarboxylase,
binding to GABAA and benzodiazepine sites, GABA in cerebrospinal liquid and
brain tissue, and GABA detected during microdialysis studies have been accounted
for investigations of human epileptic brain tissue; (3) GABA agonists suppress
seizures, and GABA antagonist produces seizures; (4) Drugs that inhibit GABA
synthesis cause seizures; and (5) Benzodiazepines and barbiturates work by enhanc-
ing GABA-mediated inhibition. Finally, drugs that increase synaptic GABA are
potent anticonvulsants. Two recently developed antiepileptic drugs (AEDs),
vigabatrin (VGB) and tiagabine (TGB), are examples of such agents. However,
their mechanisms of action are quite different (VGB is an irreversible suicide
inhibitor of GABA transaminase, though TGB blocks GABA reuptake into neurons
and glia), which may represent observed differences in drug side effect profile (van
Vliet et al. 2018; Löscher et al. 2013; Treiman 2001).
Hence to increase the level of GABA in the brain in order to prevent the
occurrence of seizure disorders, either one of the following steps should be adopted:

1. Increasing the activity of the enzyme Glutamic Acid Decarboxylase (GAD)

synthesizes GABA.
2. Selective irreversible/reversible inhibition of the GABA-catabolizing enzyme
GABA-transferase.

8.8.8.2 Potentiation in Activity of GAD Activators as a Target

for the Treatment of Epilepsy
Ginkgo biloba L. leaves extract containing various compounds such as flavonoids
and terpenoids has clinical use in the treatment of certain cerebral malfunctioning
and also exerts beneficial effects in the treatment of dementia of AD type and
multi-infarct dementia. Sasaki demonstrated that bilobalide possessed
dose-dependent anticonvulsant activity against many convulsions induced by isoni-
azid and 4-methylpyridoxine (MPN) in mice and causes elevation in hippocampal
GABA levels through potentiation of GAD activity (Abdel-Wahab and Metwally
2011).
8 Pharmacology of GABA and Its Receptors 281

8.8.8.3 GABA Agonist and Antagonist Drugs in Epilepsy

GABA agonist drugs such as muscimol, tetrahydroisooxazolopyridinol (THIP),
cetyl GABA, and progabide (PGB) are potential anticonvulsants in experimental
animals, whereas GABA antagonist such as bicuculline and picrotoxin are
proconvulsants. A number of GABA synthesis inhibitors can cause seizures, includ-
ing 4-deoxypyridoxine, isoniazid, thiosemicarbazide, and L-allylglycine. Drugs that
enhance GABA-mediated inhibition are also anticonvulsants including
benzodiazepines which enhance binding to GABA receptor and increasing the
frequency of chloride channel openings. The two recently developed and marketed
antiepileptic drugs, VGB and TGB, are called “designer drugs” because they were
specifically developed to increase synaptic GABA concentrations and thus inhibit
seizure activity. VGB is an irreversible suicide inhibitor of GABA transaminase and
thus inhibits degradation of GABA while TGB blocks GABA reuptake into neurons
as well as glia. Hence the increased synaptic GABA concentration decreases the
seizure frequency in patients with partial onset seizures (Lasoń et al. 2013; Besag
and Patsalos 2012; Sałat and Kulig 2011).

8.8.8.4 Role of GABA Transporters in Epilepsy

There are 4 cloned isoforms of GABA transporters in rats and humans, termed
GAT1, GAT2, GAT3, BGT-1. GAT1 is localized in presynaptic terminals and
GAT2 in meninges, while BGT-1 in non-neural tissues and in glia. GABA
transporters help in the GABA reuptake by clearing from extracellular space;
blocking GABA transport induces an increase in the level of GABA and hence
decreasing the occurrence of epileptic seizures (Lie et al. 2017).

8.8.8.5 Tonic GABAA Receptors in the Treatment of Temporal Lobe

Epilepsy
Various studies showed that the potentiation of GAD activity causes the elevation in
the level of hippocampal GABA that is contributed to the treatment of seizures and
convulsions. Tonic GABAA receptors are new subpopulation of receptors that lead
to prolonged inhibition and thereby control excitability. GABAA presynaptic
receptors are repetitively activated which results in the increased level of GABA
in the synaptic cleft, then diffuses to extracellular space where it activates postsyn-
aptic receptors (Schipper et al. 2016) as shown in Fig. 8.15.
Secondly, GABAA receptors may be activated by the released GABA from
non-vesicular sources. Extracellular GABA comes from the glial cells or dendrites
by GAT reversal. Thirdly, the opening of GABA can occur spontaneously in the
absence of synaptic activation and even in the absence of GABA. In case of
excessive neuronal firing the GABA may spill into extracellular space.
Neuromodulators can alter tonic GABA signaling. Various therapeutic agents act
by a different facet of action such as GABA facilitatory, GABA mimetic,
antiglutamate, Ca2+ entry reduction by inhibition of t-type Ca2+ current,
prolongation of sodium channel inactivation, facilitation of GABA-mediated chlo-
ride channel opening, etc. Antiepileptic drugs suppress seizures, but do not cure
282 S. Kaur et al.

Tonic GABAA receptor

Glial cell
Phasic GABAB receptor
Non vesicular
release GABA
GABA Transaminase

Spontaneous opening
spill

vesicular Post synaptic

Interneuron release neuron
GAD

GAT-1
Downregulation GABAB
of tonic receptors receptor

Fig. 8.15 Various mechanisms altering the activity of tonic GABAA receptors. Activated by four
mechanisms: (1) spill of synaptically released GABA into extrasynaptic space due to insufficient
clearance by GATs, (2) GAT reversal causes nonvesicular release, (3) spontaneous opening in
absence of extracellular GABA, (4) GABAB receptor activation increases tonic GABAA signaling
via an intracellular mechanism

the disease which may fadeout after years of successful control (Upadhyay et al.
2017) as shown in Table 8.13.

8.8.9 Recent Advances on GABA (Clinical Trials and Investigational

Drugs)

Around 70 years ago, the discovery of neurotransmitter GABA was made and in this
long passage of time, several findings including agonists and antagonists have proven
it to be milestone in the scientific research field. Later, the identification of its
receptors GABAA and GABAB made the elucidation of targets and therapeutic
outcomes more significant. Today, the success of a number of molecules, specifi-
cally the clinical reliability of benzodiazepines and antiepileptic drugs, strongly
attests to the remarked contribution of neurotransmitter GABA in neuro research.
Initially, the benefits of GABA were assessed for only its calming, inhibitory, and
impaired motor-oriented function but now also get diverted towards its role in
neuron survival, memory, and other disease modifying functions. For example,
nowadays the role of GABA receptor is getting explored for altered synapse function
in AD. It is also expected to be a reliable biomarker for earlier diagnosis of disease in
8 Pharmacology of GABA and Its Receptors 283

Table 8.13 Drugs targeting GABA for therapeutic benefit in epilepsy

dementia patients. Moreover, the usual side effects of BZDs also get eliminated as
in-depth structural elucidation of receptors takes place. The knowledge regarding the
different subunits of GABA and its functions raises more possibility with high
accuracy and fewer side effects. Recently, the work is ongoing upon the different
subunits of GABAA receptor for limiting side effects of anxiolytic drugs. Previously,
the target of anxiolytic drug was the interface of α and γ subunits but it shows
sedation rather than calmness. But now the specific agonists and partial agonists of
α2, α3, and α5 subunits are getting evaluated for therapeutic efficacy. Apart from
this, the role of these subunits is also getting explored for other therapeutic actions
including memory, neurotoxicity, and neuron survival in multiple neurodegenerative
disorders. The success of such findings and efforts could be analyzed from a plenty
of molecules undergoing evaluation for GABA modifying properties under recent
clinical trials (Schanzer et al. 2019; Zahn et al. 2019; Nikmaram et al. 2017) as
shown in Table 8.14.
284 S. Kaur et al.

Table 8.14 Recent drug candidates targeting GABA in clinical phases

Clinical
Drug candidate Therapeutic target trial Therapeutic interventions
Allopregnanolone GABAA receptor Phase III Super-refractory status epilepticus
AZD7325 GABAA receptor Phase 2 Autism spectrum disorder
Allopregnanolone GABAA receptor Phase I Increases neurogenesis and
reduces amyloid beta formation in
AD
Ganaxolone GABAA receptor Phase III Refractory partial-onset seizures,
female pediatric epilepsy
Vigabatrin GABA potentiation Completed Complex partial seizures and
infantile spasms
Clobazam GABA potentiation Phase III Lennox–Gastaut syndrome
Muscimol GABAA receptor Phase I Alzheimer’s disease
Clarithromycin Negative allosteric Phase I Parkinson disease
modulator of GABAA
receptors
YKP3089 Enhances GABA Phase III Resistant partial onset seizures
release
SGS742 GABAB receptor Phase II Alzheimer’s disease
antagonist
Retigabine GABA potentiation Phase III Epilepsy
L-830982 GABAA Alpha2/3 Phase II Cognitive disability in
receptor agonist schizophrenia
Zolpidem Positive allosteric Phase III Improves neuropsychiatric
modulator of GABAA symptoms (sleep disorders) in
receptors Alzheimer’s disease
Flumazenil GABAA receptor Phase I Parkinson disease

8.9 Conclusion

GABA is one of the major key players of inhibitory neurotransmission in the central
nervous system and observed to be widely distributed throughout the mammalian
brain. The GABAA receptor influences have been studied for decades and are
beginning to be well understood; research on the GABAB and GABAC receptors
are more recent and currently growing. The crucial regulatory functions of GABAB
have become increasingly evident, whereas GABAC receptor studies are only at the
beginning stage. GABA acts through their receptors and helps to control
the excitability of cortical networks by modulating a network of cortical interneurons
and useful in various CNS-related disorders including Parkinson’s disease, epilepsy,
anxiety, schizophrenia, and dementia. Numerous anxiolytic, sedative, and hypnotics
show their molecular signaling through GABA receptors and dominantly emerge as
powerful drug targets. GABAC receptors show a lower threshold of activation than
GABAA and GABAB receptors, an essential property that is rich in promise for
future clinical applications. Hence, exploring the more molecular mechanisms may
result in a better understanding of their role in CNS disorder.
8 Pharmacology of GABA and Its Receptors 285

References
Abdel-Wahab BA, Metwally ME (2011) Ginkgo biloba enhances the anticonvulsant and
neuroprotective effects of sodium valproate against kainic acid-induced seizures in mice. J
Pharmacol Toxicol 6(8):679–690
Albus H et al (2005) A GABAergic mechanism is necessary for coupling dissociable ventral and
dorsal regional oscillators within the circadian clock. Curr Biol 15(10):886–893. Elsevier
Alexander GE (2004) Biology of Parkinson’s disease: pathogenesis and pathophysiology of a
multisystem neurodegenerative disorder. Dialogues Clin Neurosci 6(3):259. Les Laboratoires
Servier
Allen KL et al (2009) Cannabinoid (CB1), GABAA and GABAB receptor subunit changes in the
globus pallidus in Huntington’s disease. J Chem Neuroanat 37(4):266–281. Elsevier
Anaclet C, Fuller PM (2017) Brainstem regulation of slow-wave-sleep. Curr Opin Neurobiol
44:139–143. Elsevier
Andrews G et al (2010) Generalized worry disorder: a review of DSM-IV generalized anxiety
disorder and options for DSM-V. Depress anxiety 27(2):134–147. Wiley Online Library
Appel K et al (2011) Modulation of the γ-aminobutyric acid (GABA) system by Passiflora incarnata
L. Phytotherapy Res 25(6):838–843. Wiley Online Library
Arnaud C, Gauthier P, Gottesmann C (2001) Study of a GABA C receptor antagonist on sleep-
waking behavior in rats. Psychopharmacology 154(4):415–419. Springer
Aroniadou-Anderjaska V, Qashu F, Braga MFM (2007) Mechanisms regulating GABAergic
inhibitory transmission in the basolateral amygdala: implications for epilepsy and anxiety
disorders. Amino acids 32(3):305–315. Springer
Badawy RAB, Harvey AS, Macdonell RAL (2009) Cortical hyperexcitability and epileptogenesis:
understanding the mechanisms of epilepsy–part 1. J Clin Neurosci 16(3):355–365. Elsevier
Barber RP et al (1978) GABAergic terminals are presynaptic to primary afferent terminals in the
substantia gelatinosa of the rat spinal cord. Brain Res 141(1):35–55. Elsevier
Bateson AN (2004) The benzodiazepine site of the GABAA receptor: an old target with new
potential? Sleep Med 5:S9–S15. Elsevier
Ben-Ari Y (2002) Excitatory actions of gaba during development: the nature of the nurture. Nat Rev
Neurosci 3(9):728. Nature Publishing Group
Bentzen BH, Grunnet M (2011) Central and peripheral GABAA receptor regulation of the heart rate
depends on the conscious state of the animal. Adv Pharmacol Sciences 2011. https://doi.org/10.
1155/2011/578273. Hindawi
Besag FMC, Patsalos PN (2012) New developments in the treatment of partial-onset epilepsy.
Neuropsychiatr Dis Treat 8:455. Dove Press
Bettler B et al (2004) Molecular structure and physiological functions of GABAB receptors,
Physiological reviews. Am Physiol Soc 84(3):835–867
Billinton A et al (2000) GABAB receptor heterodimer-component localisation in human brain. Mol
Brain Res 77(1):111–124. Elsevier
Birnir B, Korpi ER (2007) The impact of sub-cellular location and intracellular neuronal proteins on
properties of GABAA receptors. Curr Pharm Design 13(31):3169–3177. Bentham Science
Publishers
Blackburn T (2010) Gabab receptor pharmacology: a tribute to Norman Bowery. Academic Press,
Cambridge
Błaszczyk JW (2016) Parkinson’s disease and neurodegeneration: GABA-collapse hypothesis.
Front Neurosci 10:269. Frontiers
Bormann J (2000) The ‘ABC’ of GABA receptors. Trends Pharmacol Sci 21(1):16–19. Elsevier
Bowery NG, Smart TG (2006) GABA and glycine as neurotransmitters: a brief history. Br J
Pharmacol 147(S1):S109–S119. Wiley Online Library
Bowery NG, Hill DR, Hudson AL (1982) Guanyl nucleotides decrease the binding affinity of
GABAB but not GABAA receptors in the mammalian CNS. Br J Pharmac 75(Suppl. 86P)
Browne TR, Holmes GL (2008) Handbook of epilepsy. Jones & Bartlett Learning, Burlington, MA
286 S. Kaur et al.

Byrne LM, Wild EJ (2016) Cerebrospinal fluid biomarkers for Huntington’s disease. J Huntington’s
Disease 5(1):1–13. IOS Press
Caldji C et al (2004) Maternal behavior regulates benzodiazepine/GABA A receptor subunit
expression in brain regions associated with fear in BALB/c and C57BL/6 mice.
Neuropsychopharmacology 29(7):1344. Nature Publishing Group
Carver CM, Reddy DS (2013) Neurosteroid interactions with synaptic and extrasynaptic GABA A
receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network
excitability. Psychopharmacology 230(2):151–188. Springer
Castelli MP et al (2012) Characterization of COR627 and COR628, two novel positive allosteric
modulators of the GABAB receptor. J Pharmacol Exp Ther 340(3):529–538. ASPET
Cawley N et al (2015) Reduced gamma-aminobutyric acid concentration is associated with physical
disability in progressive multiple sclerosis. Brain 138(9):2584–2595. Oxford University Press
Chambers CD, Stokes MG, Mattingley JB (2004) Modality-specific control of strategic spatial
attention in parietal cortex. Neuron 44(6):925–930. Elsevier
Chapouthier G, Venault P (2002) GABA-A receptor complex and memory processes. Curr Top
Med Chem 2(8):841–851. Bentham Science Publishers
Charych EI et al (2009) GABAA receptors and their associated proteins: implications in the
etiology and treatment of schizophrenia and related disorders. Neuropharmacology 57
(5–6):481–495. Elsevier
Chebib M, Johnston GAR (2000) GABA-activated ligand gated ion channels: medicinal chemistry
and molecular biology. J Med Chem 43(8):1427–1447. ACS Publications
Chebib M et al (2009) Novel, potent, and selective GABAC antagonists inhibit myopia develop-
ment and facilitate learning and memory. J Pharmacol Exp Ther 328(2):448–457. ASPET
Chen Y et al (2007) Study on olfactory function in GABAC receptor/channel ρ1 subunit knockout
mice. Neurosci Lett 427(1):10–15. Elsevier
Chuhma N et al (2009) Dopamine neuron glutamate cotransmission: frequency-dependent modula-
tion in the mesoventromedial projection. Neuroscie 164(3):1068–1083. Elsevier
Ciranna áL (2006) Serotonin as a modulator of glutamate-and GABA-mediated neurotransmission:
implications in physiological functions and in pathology. Curr Neuropharmacol 4(2):101–114.
Bentham Science Publishers
Collares EF, Vinagre AM (2005) Effect of the GABA B agonist baclofen on dipyrone-induced
delayed gastric emptying in rats. Braz J Med Biol Res 38(1):99–104. SciELO Brasil
Cooper JR, Bloom FE, Roth RH (2003) The biochemical basis of neuropharmacology. Oxford
University Press, USA
Crestani F et al (2000) Mechanism of action of the hypnotic zolpidem in vivo. Br J Pharmacol 131
(7):1251–1254. Wiley Online Library
Cryan JF et al (2004) Behavioral characterization of the novel GABAB receptor-positive modulator
GS39783 (N, N0 -dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4, 6-diamine): anxiolytic-
like activity without side effects associated with baclofen or benzodiazepines. J Pharmacol Exp
Ther 310(3):952–963. ASPET
Cutting GR et al (1992) Identification of a putative γ-aminobutyric acid (GABA) receptor subunit
rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to
human chromosome 6q14–q21 and mouse chromosome 4. Genomics 12(4):801–806. Elsevier
Derwenskus J (2011) Current disease-modifying treatment of multiple sclerosis. Mt Sinai J Med 78
(2):161–175. Wiley Online Library
Dézsi L, Vécsei L (2011) Parkinson’s disease: will therapy move beyond dopaminergic medica-
tion? Clin Invest 1(3):381–398
Drasbek KR, Christensen J, Jensen K (2006) Gamma-hydroxybutyrate–a drug of abuse. Acta
Neurol Scand 114(3):145–156. Wiley Online Library
Du J-L, Yang X-L (2000) Subcellular localization and complements of GABAA and GABAC
receptors on bullfrog retinal bipolar cells. J Neurophysiol 84(2):666–676. American Physiolog-
ical Society Bethesda, MD
8 Pharmacology of GABA and Its Receptors 287

Du Z et al (2017) Differential alteration in expression of striatal GABAAR subunits in mouse

models of Huntington’s disease. Front Mol Neurosci 10:198. Frontiers
Emson PC (2007) GABAB receptors: structure and function. Prog Brain Res 160:43–57. Elsevier
Farrant M, Nusser Z (2005) Variations on an inhibitory theme: phasic and tonic activation of GABA
A receptors. Nat Rev Neurosci 6(3):215. Nature Publishing Group
Filip M, Frankowska M (2008) -GABAB receptors in drug addiction. Pharmacol Rep 60(6):755
Florey E, McLennan H (1959) The effects of factor I and of gamma-aminobutyric acid on smooth
muscle preparations. J physiol 145(1):66. Wiley-Blackwell
Froestl W (2010) Chemistry and pharmacology of GABAB receptor ligands. In: Advances in
pharmacology. Elsevier, Amsterdam, pp 19–62
Froestl W et al (1995) Phosphinic acid analogs of GABA. 2. Selective, orally active GABAB
antagonists. J Med Chem 38(17):3313–3331. ACS Publications
Gajcy K, Lochynski S, Librowski T (2010) A role of GABA analogues in the treatment of
neurological diseases. Curr Med Chem 17(22):2338–2347. Bentham Science Publishers
Gao R, Penzes P (2015) Common mechanisms of excitatory and inhibitory imbalance in schizo-
phrenia and autism spectrum disorders. Curr Mol Mede 15(2):146–167. Bentham Science
Publishers
Garret M et al (2018) Alteration of GABA ergic neurotransmission in Huntington’s disease. CNS
Neurosci Ther 24(4):292–300. Wiley Online Library
Gassmann M, Bettler B (2012) Regulation of neuronal GABA B receptor functions by subunit
composition. Nat Rev Neurosci 13(6):380. Nature Publishing Group
Gibson CJ, Meyer RC, Hamm RJ (2010) Traumatic brain injury and the effects of diazepam,
diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus. J Biomed
Sci 17(1):38. BioMed Central
Griffin CE et al (2013) Benzodiazepine pharmacology and central nervous system–mediated
effects. Ochsner J 13(2):214–223. Ochsner Journal
Guerriero RM, Giza CC, Rotenberg A (2015) Glutamate and GABA imbalance following traumatic
brain injury. Curr Neurol Neurosci Rep 15(5):27. Springer
Guetg N et al (2009) The GABAB1a isoform mediates heterosynaptic depression at hippocampal
mossy fiber synapses. J Neurosci 29(5):1414–1423. Soc Neuroscience
Guidotti A et al (2005) GABAergic dysfunction in schizophrenia: new treatment strategies on the
horizon. Psychopharmacology 180(2):191–205. Springer
Hackett CS et al (2014) Expression quantitative trait loci and receptor pharmacology implicate Arg1
and the GABA-A receptor as therapeutic targets in neuroblastoma. Cell Rep 9(3):1034–1046.
Elsevier
Haefely W et al (1975) Possible involvement of GABA in the central actions of benzodiazepines.
Adv Biochem Psychopharmacol 14:131–151
Hall ZW, Kravitz EA (1967) The metabolism of γ-aminobutyric acid (GABA) in the lobster
nervous system—i: GABA–Glutamate transaminase. J Neurochem 14(1):45–54. Wiley Online
Library
Hasler G et al (2010) Effect of acute psychological stress on prefrontal GABA concentration
determined by proton magnetic resonance spectroscopy. Am J Psychiatry 167
(10):1226–1231. Am Psychiatric Assoc
Jay TM (2003) Dopamine: a potential substrate for synaptic plasticity and memory mechanisms.
Prog Neurobiol 69(6):375–390. Elsevier
Jijón-Lorenzo R et al (2018) Presynaptic dopamine D2 receptors modulate [3H] GABA release at
StriatoPallidal terminals via activation of PLC! IP3! calcineurin and inhibition of AC!
cAMP! PKA signaling cascades. Neuroscience 372:74–86. Elsevier
Johnston GAR et al (2003) GABAC receptors as drug targets. Curr Drug Targets CNS Neurol
Disord 2(4):260–268. Bentham Science Publishers
Jones KA et al (1998) GABA B receptors function as a heteromeric assembly of the subunits GABA
B R1 and GABA B R2. Nature 396(6712):674–679. Nature Publishing Group
288 S. Kaur et al.

Kantamneni S et al (2008) GISP binding to TSG101 increases GABAB receptor stability by down-
regulating ESCRT-mediated lysosomal degradation. J Neurochem 107(1):86–95. Wiley Online
Library
Kaupmann K et al (1997) Expression cloning of GABA B receptors uncovers similarity to
metabotropic glutamate receptors. Nature 386(6622):239–246. Nature Publishing Group
Keberle H, Faigle JW, Wilhelm M (1968) Procedure for the preparation of new amino acids, Swiss
Patent 449046, Publication Date: 11 April 1968, Priority: 9 July 1963, In Chem Abstr
Kerr DIB, Ong J (1996) GABAB receptors: targets for drug development. Drug Discov Today 1
(9):371–380. Elsevier
Kesby JP et al (2018) Dopamine, psychosis and schizophrenia: the widening gap between basic and
clinical neuroscience. Transl Psychiatry 8(1):30. Nature Publishing Group
Khazipov R (2016) GABAergic synchronization in epilepsy. Cold Spring Harb Perspect Med 6(2):
a022764. Cold Spring Harbor Laboratory Press
Kilb W (2012) Development of the GABAergic system from birth to adolescence. Neuroscientist 18
(6):613–630. SAGE Publications Sage CA: Los Angeles, CA
Kim DH et al (2012) GABA a receptor blockade enhances memory consolidation by increasing
hippocampal BDNF levels. Neuropsychopharmacology 37(2):422. Nature Publishing Group
Kochanek PM et al (2015) Emerging therapies in traumatic brain injury. In: Seminars in neurology.
Thieme Medical Publishers, New York, NY, pp 83–100
Korol SV et al (2015) GLP-1 and exendin-4 transiently enhance GABAA receptor–mediated
synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. Diabetes 64
(1):79–89. Am Diabetes Assoc
Kuffler SW (1954) Mechanisms of activation and motor control of stretch receptors in lobster and
crayfish. J Neurophysiol 17(6):558–574
Kumar A, Singh A (2015) A review on Alzheimer’s disease pathophysiology and its management:
an update. Pharmacol Rep 67(2):195–203. Elsevier
Laruelle M (2014) Schizophrenia: from dopaminergic to glutamatergic interventions. Curr Opin
Pharmacol 14:97–102. Elsevier
Lasoń W, Chlebicka M, Rejdak K (2013) Research advances in basic mechanisms of seizures and
antiepileptic drug action. Pharmacol Rep 65(4):787–801. Elsevier
Lazzari C et al (2015) Aβ42 oligomers selectively disrupt neuronal calcium release. Neurobiol
Aging 36(2):877–885. Elsevier
Lee S, Kim K, Zhou ZJ (2010) Role of ACh-GABA cotransmission in detecting image motion and
motion direction. Neuron 68(6):1159–1172. Elsevier
Lewis DA et al (2012) Cortical parvalbumin interneurons and cognitive dysfunction in schizophre-
nia. Trends Neurosci 35(1):57–67. Elsevier
Li J-T et al (2016) Repeated blockade of NMDA receptors during adolescence impairs reversal
learning and disrupts GABAergic interneurons in rat medial prefrontal cortex. Front Mol
Neurosci 9:17. Frontiers
Lie MEK et al (2017) Glial GABA transporters as modulators of inhibitory signalling in epilepsy
and stroke. In: Glial amino acid transporters. Springer, New York, NY, pp 137–167
Lin E et al (2014) Optical mapping of the electrical activity of isolated adult zebrafish hearts: acute
effects of temperature. Am J Physiol Regul Integr Comp Physiol 306(11):R823–R836. Ameri-
can Physiological Society Bethesda, MD
Loma I, Heyman R (2011) Multiple sclerosis: pathogenesis and treatment. Curr Neuropharmacol 9
(3):409–416. Bentham Science Publishers
López-Chávez A, Miledi R, Martínez-Torres A (2005) Cloning and functional expression of the
bovine GABAC ρ2 subunit: molecular evidence of a widespread distribution in the CNS.
Neurosci Res 53(4):421–427. Elsevier
Löscher W et al (2013) New avenues for anti-epileptic drug discovery and development. Nat Rev
Drug Discov 12(10):757–776. Nature Publishing Group
Lukasiewicz PD et al (2004) GABAC receptor-mediated inhibition in the retina. Vision Res 44
(28):3289–3296. Elsevier
8 Pharmacology of GABA and Its Receptors 289

Ma P et al (2015) Effect of GABA on blood pressure and blood dynamics of anesthetic rats. Int J
Clin Exp Med 8(8):14296. e-Century Publishing Corporation
Ma C et al (2019) Sleep regulation by neurotensinergic neurons in a thalamo-amygdala circuit.
Neuron. https://doi.org/10.1016/j.neuron.2019.05.015. Elsevier
Macey PM et al (2016) Obstructive sleep apnea is associated with low GABA and high glutamate in
the insular cortex. J Sleep Res 25(4):390–394. Wiley Online Library
Madikians A, Giza CC (2006) A clinician’s guide to the pathophysiology of traumatic brain injury.
Indian J Neurotrauma 3(01):9–17. Thieme Medical and Scientific Publishers Private Ltd.
Maffucci JA, Gore AC (2009) Hypothalamic neural systems controlling the female reproductive life
cycle: gonadotropin-releasing hormone, glutamate, and GABA. Int Rev Cell Mol Biol
274:69–127. Elsevier
Maiti P, Gregg LC, McDonald MP (2016) MPTP-induced executive dysfunction is associated with
altered prefrontal serotonergic function. Behav Brain Res 298:192–201. Elsevier
Makkar SR, Zhang SQ, Cranney J (2012) Behavioral and neural analysis of GABA in the
acquisition, consolidation, reconsolidation, and extinction of fear memory: Corrigendum.
Neuropsychopharmacology 35(8):1625–1652. Nature Publishing Group
Mandolesi G et al (2015) Synaptopathy connects inflammation and neurodegeneration in multiple
sclerosis. Nat Rev Neurol 11(12):711. Nature Publishing Group
Martin DL, Olsen RW (2000) GABA in the nervous system: the view at fifty years. Lippincott
Williams and Wilkins
Martin SC, Russek SJ, Farb DH (2001) Human GABABR genomic structure: evidence for splice
variants in GABABR1 but not GABABR2. Gene 278(1–2):63–79. Elsevier
Masiulis S et al (2019) GABA A receptor signalling mechanisms revealed by structural pharma-
cology. Nature 565(7740):454–459. Nature Publishing Group
Mason SL, Barker RA (2009) Emerging drug therapies in Huntington’s disease. Expert Opin Emerg
Drugs 14(2):273–297. Taylor & Francis
McDonnell MN, Orekhov Y, Ziemann U (2007) Suppression of LTP-like plasticity in human motor
cortex by the GABA B receptor agonist baclofen. Exp Brain Res 180(1):181–186. Springer
Mingote S et al (2017) Dopamine neuron dependent behaviors mediated by glutamate
cotransmission. Elife 6:e27566. eLife Sciences Publications Limited
Möhler H (2012) The GABA system in anxiety and depression and its therapeutic potential.
Neuropharmacology 62(1):42–53. Elsevier
Morales B, Choi S-Y, Kirkwood A (2002) Dark rearing alters the development of GABAergic
transmission in visual cortex. J Neurosci 22(18):8084–8090. Soc Neuroscience
Naffaa MM et al (2017) GABA-ρ receptors: distinctive functions and molecular pharmacology. Br J
Pharmacol 174(13):1881–1894. Wiley Online Library
Nakazawa K et al (2012) GABAergic interneuron origin of schizophrenia pathophysiology.
Neuropharmacology 62(3):1574–1583. Elsevier
Nguyen R et al (2014) Parvalbumin and GAD65 interneuron inhibition in the ventral hippocampus
induces distinct behavioral deficits relevant to schizophrenia. J Neurosci 34(45):14948–14960.
Soc Neuroscience
Nikmaram N et al (2017) Recent advances in γ-aminobutyric acid (GABA) properties in pulses: an
overview. J Sci Food Agric 97(9):2681–2689. Wiley Online Library
Nowak G et al (2006) Antidepressant-like activity of CGP 36742 and CGP 51176, selective
GABAB receptor antagonists, in rodents. Br J Pharmacol 149(5):581–590. Wiley Online
Library
Nuss P (2015) Anxiety disorders and GABA neurotransmission: a disturbance of modulation.
Neuropsychiatr Dis Treat 11:165. Dove Press
Olsen RW, Sieghart W (2009) GABAA receptors: subtypes provide diversity of function and
pharmacology. Neuropharmacology 56(1):141–148. Elsevier
Orts-Del’Immagine A, Pugh JR (2018) Activity-dependent plasticity of presynaptic GABAB
receptors at parallel fiber synapses. Synapse 72(5):e22027. Wiley Online Library
290 S. Kaur et al.

Pin J-P, Prézeau L (2007) Allosteric modulators of GABAB receptors: mechanism of action and
therapeutic perspective. Curr Neuropharmacol 5(3):195–201. Bentham Science Publishers
Pinard A, Seddik R, Bettler B (2010) GABAB receptors: physiological functions and mechanisms
of diversity. In: Advances in pharmacology. Elsevier, Amsterdam, pp 231–255
Plante DT, Jensen JE Winkelman JW (2012) The role of GABA in primary insomnia. Sleep 35
(6):741–742. Oxford University Press
Polenzani L, Woodward RM, Miledi R (1991) Expression of mammalian gamma-aminobutyric
acid receptors with distinct pharmacology in Xenopus oocytes. Proc Natl Acad Sci 88
(10):4318–4322. Natl Acad Sci
Prud’homme G, Glinka Y, Wang Q (2013) GABA exerts anti-inflammatory and immunosuppres-
sive effects (P5175). Am Assoc Immnol
Rana N et al (2010) Caffeine-induced effects on heart rate in zebrafish embryos and possible
mechanisms of action: an effective system for experiments in chemical biology. Zebrafish 7
(1):69–81. Mary Ann Liebert, Inc. 140 Huguenot Street, 3rd Floor New Rochelle, NY 10801
USA
Rao T, Zhang C (2011) 3, 4-methylenedioxyphenyl inhibitors of gaba aminotransferase and/or gaba
reuptake transporter inhibitor. Google Patents
Riordan AJ et al (2017) Estradiol and luteinizing hormone reverse memory loss in phencyclidine
model of schizophrenia: evidence for hippocampal GABA action. bioRxiv, p 207159. Cold
Spring Harbor Laboratory
Roberts E, Frankel S (1950) γ-aminobutyric acid in brain: its formation from glutamic acid. J Biol
Chem 187:55–63
Rosenberg M (2012) Diagnosis, treatment options, and costs of schizophrenia. J Manage Care Med
3:10–15
Roth FC, Draguhn A (2012) GABA metabolism and transport: effects on synaptic efficacy. Neural
Plast 2012:805830. Hindawi
Rowley NM et al (2012) Glutamate and GABA synthesis, release, transport and metabolism as
targets for seizure control. Neurochem Int 61(4):546–558. Elsevier
Rubinsztein DC (2006) The roles of intracellular protein-degradation pathways in
neurodegeneration. Nature 443(7113):780. Nature Publishing Group
Rudolph U, Möhler H (2014) GABAA receptor subtypes: therapeutic potential in Down syndrome,
affective disorders, schizophrenia, and autism. Annu Rev Pharmacol Toxicol 54:483–507.
Annual Reviews
Rudolph U et al (2000) erratum: Benzodiazepine actions mediated by specific γ-aminobutyric acid
A receptor subtypes. Nature 404(6778):629. Nature Publishing Group
Saito S et al (2010) Interaction between hippocampal γ-aminobutyric acidA and N-methyl-D-
aspartate receptors in the retention of spatial working memory in rats. Biol Pharm Bull 33
(3):439–443. The Pharmaceutical Society of Japan
Sałat K, Kulig K (2011) GABA transporters as targets for new drugs. Future Med Chem 3
(2):211–222. Future Science
Schanzer B et al (2019) Novel investigational therapeutics for generalized anxiety disorder (GAD).
Expert Opin Investig Drugs. https://doi.org/10.1080/13543784.2019.1680638. Taylor &
Francis
Schipper S et al (2016) Tonic GABA A receptors as potential target for the treatment of temporal
lobe epilepsy. Mol Neurobiol 53(8):5252–5265. Springer
Schofield PR et al (1987) Sequence and functional expression of the GABA A receptor shows a
ligand-gated receptor super-family. Nature 328(6127):221–227. Nature Publishing Group
Scotter EL, Abood ME, Glass M (2010) The endocannabinoid system as a target for the treatment of
neurodegenerative disease. Br J Pharmacol 160(3):480–498. Wiley Online Library
Selten MM et al (2016) Increased GABA B receptor signaling in a rat model for schizophrenia. Sci
Rep 6:34240. Nature Publishing Group
8 Pharmacology of GABA and Its Receptors 291

Semyanov AV (2002) GABA-ergic inhibition in the CNS: types of GABA receptors and
mechanisms of tonic GABA-mediated inhibitory action. Neurophysiology 34(1):71–80.
Springer
Shaban H et al (2006) Generalization of amygdala LTP and conditioned fear in the absence of
presynaptic inhibition. Nat Neurosci 9(8):1028. Nature Publishing Group
Sharpley CF (2009) Malfunction in GABA and glutamate as pathways to depression: a review of
the evidence. Clin Med 1, p. CMT-S3481. Therapeutics. SAGE Publications Sage UK: London,
England
Shin LM, Liberzon I (2010) The neurocircuitry of fear, stress, and anxiety disorders.
Neuropsychopharmacology 35(1):169. Nature Publishing Group
Shingai R et al (1997) Functional expression of GABA rho 3 receptors in Xenopus oocytes.
Neurosci Res 26:387–390
Sieghart W et al (2012) A novel GABAA receptor pharmacology: drugs interacting with the α+
β-interface. Br J Pharmacol 166(2):476–485. Wiley Online Library
Sigel E, Steinmann ME (2012) Structure, function, and modulation of GABAA receptors. J Biol
Chem 287(48):40224–40231. ASBMB
Silverman RB (2008) From basic science to blockbuster drug: the discovery of Lyrica. Angew
Chem Int Ed 47(19):3500–3504. Wiley Online Library
Silverman RB (2012) The 2011 EB Hershberg Award for important discoveries in medicinally
active substances:(1 S, 3 S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115), a
GABA aminotransferase inactivator and new treatment for drug addiction and infantile spasms.
J Med Chem 55(2):567–575. ACS Publications
Solas M, Puerta E, J Ramirez M (2015) Treatment options in Alzheimer s disease: the GABA story.
Curr Pharm Design 21(34):4960–4971. Bentham Science Publishers
Steenbergen L et al (2015) γ-Aminobutyric acid (GABA) administration improves action selection
processes: a randomised controlled trial. Sci Rep 5(1):1–7. Nature Publishing Group
Strandwitz P et al (2019) GABA-modulating bacteria of the human gut microbiota. Nat Microbiol
4(3):396–403. Nature Publishing Group
Tadavarty R et al (2015) Are presynaptic GABA-Cρ2 receptors involved in anti-nociception?
Neurosci Lett 606:145–150. Elsevier
Tallman JF, Smith CC, Henneberry RC (1977) Induction of functional beta-adrenergic receptors in
HeLa cells. Proc Natl Acad Sci 74(3):873–877. Natl Acad Sci
Treiman DM (2001) GABAergic mechanisms in epilepsy. Epilepsia 42:8–12. Wiley Online Library
Upadhyay J, Upadhyay G, Rana AJ (2017) A prospective study on prevalence of epilepsy disorders
and drug utilization pattern. Asian J Pharm Clin Res 10(3):136–139
Uusi-Oukari M, Korpi ER (2010) Regulation of GABAA receptor subunit expression by pharma-
cological agents. Pharmacol Rev 62(1):97–135. ASPET
van Vliet EA et al (2018) Neuroinflammatory pathways as treatment targets and biomarker
candidates in epilepsy: emerging evidence from preclinical and clinical studies. Neuropathol
Appl Neurobiol 44(1):91–111. Wiley Online Library
Vinkers CH et al (2010) The inhibitory GABA system as a therapeutic target for cognitive
symptoms in schizophrenia: investigational agents in the pipeline. Expert Opin Investig Drugs
19(10):1217–1233. Taylor & Francis
Waldvogel HJ, Faull RLM (2015) The diversity of GABAA receptor subunit distribution in the
normal and Huntington’s disease human Brain1. In: Advances in pharmacology. Elsevier,
Amsterdam, pp 223–264
Wu C, Sun D (2015) GABA receptors in brain development, function, and injury. Metabolic brain
disease 30(2):367–379. Springer
Wu Z et al (2014) Tonic inhibition in dentate gyrus impairs long-term potentiation and memory in
an Alzheimer’s disease model. Nat Commun 5(1):1–13. Nature Publishing Group
Yadav RK, Khanday MA, Mallick BN (2019) Interplay of dopamine and GABA in substantia nigra
for the regulation of rapid eye movement sleep in rats. Behav Brain Res 376:112169. Springer
292 S. Kaur et al.

Yi J-H, Hazell AS (2006) Excitotoxic mechanisms and the role of astrocytic glutamate transporters
in traumatic brain injury. Neurochem Int 48(5):394–403. Springer
Zahn NM et al (2019) MIDD 0301–A first-in-class anti-inflammatory asthma drug targets GABAA
receptors without causing systemic immune suppression. Basic Clin Pharmacol Toxicol. https://
doi.org/10.1111/bcpt.13206. Wiley Online Library
Zhang D et al (2001) Structure and function of GABAC receptors: a comparison of native versus
recombinant receptors. Trends Pharmacol Sci 22(3):121–132. Elsevier
Zhang C, Liu X, Yuan TF (2017) Somatostatin-positive GABAergic interneuron: new targets for
depression. Nature Publishing Group
Pharmacology of Melatonin and Its
Receptors 9
Shamsher Singh, Arti Rana, Sunpreet Kaur, Jasdeep Singh,
Vikrant Rahi, Hira Choudhury, and Puneet Kumar

Abstract

N-acetyl-5-methoxytryptamine or melatonin is a chronobiotic material, which

proportionally acts as a rhythmic stabilizer of the body. This versatile moiety is
secreted from a highly specialized gland called the pineal gland, located behind
the third ventricle in the center of the brain. Not only the brain but some other
parts are also responsible for the production of melatonin such as the gastrointes-
tinal tract (GIT), skin, and lymphocytes. It is also called the hormone of darkness
because of its versatile release pattern. Its production and release is controlled by
light-dark cycle; the light inhibits the production whereas darkness stimulates
it. Multiple evidence suggested the involvement of melatonin in biologic regula-
tion of circadian rhythms, sleep, mood, and perhaps reproduction, tumor growth,
and aging. For all these processes, receptors are the key element contributing to
elicit all such responses. Melatonin receptors are generally classified into two
subclasses, MT1 and MT2, expressed in different regions of the brain and MT3
receptors located in the liver and kidney. In this chapter, we discuss in detail the
various aspects of melatonin with its key role in the body and how this versatile
hormone and its receptors work in a synchronized way to alter the

S. Singh (*) · A. Rana · S. Kaur

Neuroscience Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab,
India
J. Singh · V. Rahi
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
H. Choudhury
School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia
P. Kumar
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India

# Springer Nature Singapore Pte Ltd. 2020 293

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_9
294 S. Singh et al.

pathophysiological processes of the body according to the biological clock and

circadian rhythm.

Keywords

Melatonin · Antioxidant · Circadian rhythm · Biological clock · Neurological

disorders · Cancer · Cardiovascular diseases · Depression · Parkinson’s disease ·
Alzheimer’s disease

Abbreviation

4P-ADOT 4-Phenyl-2-acetamidotetralin
4P-PDOT 4-Phenyl-2-acetamidotetralin
5-HT 5-Hydroxytryptamine
5-MCA-NAT 5-Methoxycarbonylamino-N-acetyltryptamine
6-SMT 6-Sulphatoxymelatonin
Ab Amyloid β
AD Alzheimer’s disease
AFMK N-acetyl-N-formyl-5-methoxy kynurenine
AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
ATP Adenosine triphosphate
Bcl-2 β-cell lymphoma 2
BDNF Brain-derived neurotrophic factor
BKCa Blocking of calcium-activated potassium channels
cAMP Cyclic adenosine monophosphate
CCK-2 Cholecystokinin-2
cGMP Cyclic guanosine monophosphate
CK1 Casein kinase 1
CK2 Casein kinase
CNS Central nervous system
Cox-2 Cyclooxygenase-2
CREB cAMP response element-binding protein
CVS Melatonin actions on the cardiovascular system
DNA Deoxyribonucleic acid
ERK Extracellular signal-regulated kinase
ETC Electron transport chain
FSH Follicle-stimulating hormone
GABA Gamma-aminobutyric acid
GDNF Glial cell-derived neurotrophic factor
GDNF Glial cell line-derived neurotrophic factor
GIT Gastrointestinal tract
GPCR G-protein-coupled receptor
GPx Glutathione peroxidase
GRd Glutathione reductase
9 Pharmacology of Melatonin and Its Receptors 295

GSK-3 Glycogen synthase kinase 3

HD Huntington’s disease
HIOMT Hydroxyindole-O-methyl transferase
HIV Human immunodeficiency virus
IL Interleukin
IRI Ischemic reperfusion injury
JNK c-Jun N-terminal kinase
LH Luteinizing hormone
MCF-7 Michigan Cancer Foundation
MI Myocardial infarction
MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
mRNA Messenger ribonucleic acid
MTNR1B Melatonin receptor 1B
NADPH Nicotinamide adenine dinucleotide phosphate
NAS N-acetylserotonin
NAT N-acetyl transferase
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
NGF Nerve growth factor
NMDA N-methyl-D-aspartate receptor
NO Nitrogen oxide
NREM Non-rapid eye movement
OH Hydroxyl radical
PD Parkinson’s disease
PKA Protein kinase A
PKC Protein kinase C
PTX Pertussis toxin
QR2 Quinoreductase 2
REM Rapid eye movement
RNS Reactive nitrogen species
RORα Related orphan receptors
ROS Reactive oxygen species
SCN Suprachiasmatic nucleus
SNc Substantia nigra pars compacta
SNRIs Serotonin norepinephrine reuptake inhibitors
SSRIs Selective serotonin reuptake inhibitors
T2D Type 2 diabetes
TLE Temporal lobe epilepsy
TM Transmembrane
TNF Tumor necrosis factor
296 S. Singh et al.

9.1 Introduction

Melatonin is a highly specific moiety, which serves in a versatile manner and

expresses itself throughout the organs as well as tissues in the mammalian system
through several mechanisms of action, whereas the sympathetic nervous system is
directly involved in the control and coordination of melatonin synthesis as well as its
production is in the pineal gland. Circadian rhythm originated from suprachiasmatic
nucleus (SCN) in the hypothalamus and is also responsible for the control and
coordination of melatonin. Not only the pineal gland but other organs like the retina,
GIT, salivary gland, platelets, epithelial hair follicles, lymphocytes, and developing
brain are also responsible for the production of melatonin (Singh and Jadhav 2014;
Baltatu et al. 2017).
Chemically melatonin is N-acetyl-5-methoxytryptamine that was discovered by
Aaron Lerner in 1958. Melatonin is a natural product of body and thus acts naturally
in all living creatures at various stages of the daily cycle from algae to humans. It
performs o’clock calendar functions in body, along with antioxidant action. Biologi-
cally melatonin makes modulations in sexual behavior, sleep, and circadian sleep; a
low level of melatonin has been linked to PD, insomnia, AD, ischemic injury,
epilepsy, and other neurodegenerative disorder (Singh and Jadhav 2014). In most
organisms, these findings suggest its exclusive production and release during
the night (Fig. 9.1) and it arbitrates information about the duration of darkness as
well as the temporal position and therefore considered as a visceral indicator of
darkness (Rocha et al. 2015).
In this chapter, we discuss the significance of melatonin as a hormone along with
its synthesis, storage, and release and how it is involved in the control, coordination,
and regulation of important biological features of mammalian systems such as
circadian rhythm, adolescent growth, and seasonal variation. Furthermore, it is
interesting to know about its essential antioxidant action; in association with

Fig. 9.1 Melatonin stimulation and inhibition

9 Pharmacology of Melatonin and Its Receptors 297

membrane receptors melatonin elicits various biological responses via involving

integral proteins and MT1 and MT2 receptors (Dawoodi et al. 2012). It consists
of MT1 and MT2 melatonin receptors which belong to G-protein-coupled
receptors (GPCRs) family present in different regions of the CNS such as ganglion
cells, retinal horizontal cells as well as distant body parts like ovary, prostate, and
gastrointestinal tract. Quantitative changes in melatonin receptors occur day by day.
The correlation between receptor alteration and alteration in endogenous melatonin
creation is clearly proved in day-to-day rhythm sleep syndromes. Recent evidence
supports the statement of its active role in animal ovary and to furthermore improve
its abilities; it is important to research and investigate the clinical features, effects,
efficacy and safety of melatonin along with its ligand involved in various diseases.

9.1.1 History

Before the discovery of melatonin, researchers used the injection of pineal gland
extract to find out the role of pineal gland in different biological processes of the
body. In some studies the researchers used pineal gland extract in tadpoles, frogs,
toads, and fish to reveal the skin lightening effect of pineal gland biochemicals
(Whitlock et al. 1954). In 1958, Aaron B. Lerner revealed the role of a neurochemi-
cal which reverses the darkening effect of the melanocyte stimulating hormone and
named it “Melatonin” (Lerner et al. 1958) (Table 9.1).

9.2 Synthesis, Storage, Release, and Metabolism

The synthesis of melatonin is a multistep process, which includes the transformation

of circulatory tryptophan to serotonin that further gets converted to melatonin. These
conversational processes depend upon the action of enzymes, and serotonin-N-
acetyl transferase (NAT) and hydroxyindole-O-methyl transferase (HIOMT) are the
enzymes involved in the conversion of serotonin to melatonin as shown in Fig. 9.2
(Ganguly et al. 2002). Acute tryptophan reduces in melatonin synthesis; therefore
melatonin synthesis is dependent on tryptophan availability. Other nutritional factors
that influence melatonin synthesis are folate status and vitamin B6, a coenzyme in
tryptophan decarboxylation that possesses the ability to stimulate melatonin produc-
tion in prepubertal children but not in adults.
Once synthesized, it expeditiously moves in blood and becomes a part of circula-
tion. Therefore, the plasma concentration delivers the actual picture of pineal
secretion. In order to measure melatonin concentration, body fluids like saliva and
urine can be measured. It is estimated that 10–80 μg of nocturnal melatonin
produced endogenously at night and in comparison to the day time, production is
significantly less (Peuhkuri et al. 2012).
Environmental light and endogenous circadian clock are the major factors respon-
sible for controlling the release of melatonin. Light is considered as the major
environmental stimulatory factor of melatonin release (Fig.9.3). Elevation in the
298 S. Singh et al.

Table 9.1 Historical development in the discovery of melatonin and its receptors
Name Year Discoveries Reference
Herophilos 325–280 First time discovering the pineal Kappers
BC organ in man (1979)
Aaron Lerner 1958 Discovered the chemical structure Lerner et al.
of melatonin (1958)
Bubenik 1993 Found melatonin in human Filadelfi and
intestine and the retina of many Castrucci
nonmammalian vertebrates (1996)
Miles, Stankov and Reiter, 1989; The highly specific ligand 2 [‘25I]- Filadelfi and
Stankov Kennaway and 1990; iodomelatonin allowed the Castrucci
Hugel, Almeida 1991; expansion of receptor investigation (1996)
1992; 1951 to peripheral tissues, such as retina,
spleen, gastrointestinal tract,
uterus, ovary, liver, and cultured
normal and tumor cells
Dubocovich 1998, 1981 Melatonin receptors were first Dubocovich
1988, 1995 classified according to classic et al. (2010)
1990,1994 pharmacological criteria
Nosjean 2000 Identification and characterization Nosjean
of a third membrane-bound et al. (2000)
melatonin binding site MT3
receptor
Ebisawa 1994 The MT1 receptor (Mel1c) was Ebisawa
first cloned in frogs et al. (1994)
Reppert 1995 The MT2 receptor was cloned from Reppert
the brain, retina, and human et al. (1995)
pituitary gland
Ayoub 2004 Both MT1 and MT2 can form Ayoub et al.
homo and heterodimers (2004)
Takeda Pharmaceutical 2005 Development of melatonin Neubauer
Company receptor agonist TAK-375, now (2008)
known as ramelteon
Christian de Bodinat 2010 Agomelatine, the first melatonergic De Bodinat
antidepressant et al. (2010)
Daniel P. Cardinali 2013 Role of melatonin and its analogs Cardinali
in insomnia and depression et al. (2012)
Gabriella Gobbi 2019 Differential function of melatonin Gobbi and
MT1 and MT2 receptors in REM Comai
and NREM sleep (2019)

level of pineal melatonin is being observed late in the evening which reaches
maximum up to 4:00 a.m. (2:00 and 4:00 a.m. is a maximum observed range) and
then gets back down to the normal daytime levels. It is very difficult to detect the
daytime level of melatonin. In contrast to sunlight, artificial lights are sufficient to
block the nocturnal release of melatonin (Peuhkuri et al. 2012).
Melatonin possesses rapid metabolism with a half-life that varies between 10 and
60 min in humans following exogenous administration. It is catabolized mostly by
the liver and gets excreted in the urine. Metabolite like 6-sulphatoxymelatonin
9 Pharmacology of Melatonin and Its Receptors 299

L-Tryptophan

Tryptophan
5-hydroxylase

5-hydroxytryptophan

Aromatic L-amino
acid decarboxylase

Serotonin
Serotonin N-
acetyltransferase

N- acetyl serotonin
Hydroxyindole-O-methyl
transferase (HIOMT)

Melatonin

Fig. 9.2 Synthesis of melatonin

Fig. 9.3 Release of melatonin and suppression with light

(6-SMT) in urine indicates that melatonin status can be evaluated melatonin in the
overnight urine sample (Peuhkuri et al. 2012).

9.3 Melatonin Receptors

Two distinct classes of melatonin receptors are found in different regions of the body,
which are expressed in humans and have been reported so far, MT1 and MT2,
formerly designated as Mel1a and Mel1b, respectively (Dubocovich et al. 2003).
The majority of the melatonin receptors are found in the following regions of the
300 S. Singh et al.

body such as cardiovascular system, coronary or cerebral arteries, cardiac ventricular

wall, brain, liver and gallbladder, retina, exocrine pancreas, skin, parotid gland,
colon, duodenal enterocytes, platelets, white and brown adipocytes, kidney, cells of
the immune system, ovary/granulosa cells, placenta, myometrium, and fetal kidney.
In other systems like GIT, these receptors are abundant in colonic mucosa and
jejunal. Melatonin is involved in modulating functions of FSH and LH via affecting
the expression of their receptors (Table 9.2).
Adenylyl cyclase activity gets inhibited due to the coupling of MT1 and MT2
melatonin receptors with higher affinity to pertussis toxin-sensitive G proteins. They
are unique molecules possessing individual chromosomal localization with 60%
amino acid identity. These moieties possess a long chain of amino acids (about
350 and 362) having calculated molecular weight about 39–40 kDa. In N-terminus
MT1 and MT2 receptors contain one or two potential glycosylation sites and protein
kinase A (PKA), protein kinase C (PKC), casein kinase 2 (CK2), and casein kinase
1 (CK1) which promote and regulate the receptor functioning. In the superfamily of
GPCR, melatonin receptors are present as distinct groups due to the presence of
NRY motif (letter amino acid code) which is a DRY (or ERY) variant present G
protein-coupled receptors intracellularly in loop II. This is the particular region
having higher involvement in the process of G protein signal transduction. The
essential melatonin biding sites are Ser 8 (TM III), His 7 (TM IV), Gly 20 (TM VI),
Ser 12 (TM III), Val 4 (TM IV), and Gly 20 (TM VI) in MT1 receptor, whereas two
residues Cys 113 (in EL1) and Cys 190 (in EL2) are present in MT2 melatonin
receptor which are conserved in most GPCRs and estimated to make disulfide bond
which is important for melatonin binding with good affinity (Cajochen et al. 2002;
Pala et al. 2013). Leucine zippers are involved in protein-protein interactions;
melatonin receptors contain 6 leucines in the MT2 and 7 leucines in the MT1 in
TM IV and possess protein-protein interactions.
MT1 and MT2 receptors generally show their effect after adhesion to the cell
surface by G-protein. Forskolin inhibits the protein kinase A (PKA) and cAMP
formation, followed by the activation of MT1 receptor. Similarly, cAMP formation
is inhibited in MT2 receptors stimulated by forskolin; they also inhibit cGMP
formation. In the central nervous system, membrane receptors are present in the brain
as well as in the periphery RZR/RORα is located. Membrane receptors with their
agonists are associated with circadian rhythm, whereas RZR/RORα is responsible
for cellular growth, differentiation of bone, and immunomodulation in the periphery.
To form melatonin effectively, protein kinase C-α activation is a crucial step.

9.3.1 MT1 Receptors

MT1 receptors are high-affinity receptors that fall into the GPCR superfamily and
the binding of melatonin to these receptors inhibits the activity of adenylate cyclase
activity in target cells (Pandi-Perumal et al. 2008). The two subgroups of the MT1
receptors are MT1a receptors and MT1b receptors. Cardiac vessels constriction and
circadian rhythms are modulated by MT1 melatonin receptor generally found in
9 Pharmacology of Melatonin and Its Receptors 301

Table 9.2 Functions of melatonin receptors based on location in the different regions of the body
Receptor
Location type Function Reference
CNS
Hippocampus MT1, MT2 Inhibition of neuronal activity and Savaskan et al. (2005,
excitatory responses in memory 2002)
Modification in Alzheimer disease
Enhancement in seizure threshold via
suppression in GABAA-receptor
function
Cerebellum MT1, MT2 Involvement and interaction with Al-Ghoul et al. (1998)
glutamatergic synapse
Various MT1, MT2 Dopamine release evoked by Reppert et al. (1995)
retinal cell stimulation gets inhibited
Adaptation to low light intensity
Modification in photoreceptor
functions rod phototransduction
pathways
SCN MT1 Initiation and improvisation in sleep
Modification of circadian rhythm
(blind people, phase shift worker, jet
lag)
Central MT1 Modulation of dopamine synthesis and Uz et al. (2005)
dopaminergic release
system Increased sensitivity and activation of
dopamine receptors
CVS
Cerebral MT1 Unknown effects Savaskan et al. (2001)
arteries
Aorta MT1, MT2 Vasodilation may occur Ekmekcioglu et al.
(2003)
Cardiac MT1, MT2 Modulation of beta-adrenergic Ekmekcioglu et al.
ventricular receptor-mediated cAMP signaling (2003, 2001)
wall processes may be initiated
Increased stimulation of voltage-
activated calcium current
Negative inotropic effects may occur
Coronary MT1, MT2 MT2 receptors-mediated vasodilation Ekmekcioglu (2006);
arteries MT1 receptors-mediated Ekmekcioglu et al.
vasoconstriction (2003, 2001)
GIT
Duodenal MT2 Stimulation of HCO3–secretion via Sjöblom et al. (2001);
enterocytes neural stimulation Sjöblom and
Flemström (2003)
Pancreatic MT1 Acid/base homeostasis regulation Ekmekcioglu (2006)
cancer cell (stimulation of HCO3–secretion)
lines
Gallbladder MT1 Gallbladder contraction may occur Aust et al. (2004)
epithelia
302 S. Singh et al.

SCN and cardiac vessels. Not only in these regions but these sets of receptors are
also found in peripheral tissues as well as other parts of the brain.

9.3.1.1 Receptor Signaling Mechanism of Melatonin MT1 Receptors

Pertussis toxin (PTX) sensitive (Gi2 and Gi3) and PTX-insensitive (Gq/11) G
proteins mediate multiple cellular responses through activation of MT1 receptors;
they generate signal transduction as well as physiological response of melatonin.
According to the recent studies, the activation of MT1 receptors is exposed to differ-
ent signal transduction pathways in mammalian cell line. According to these trans-
duction pathways, MT1 receptor activation is responsible for inhibition of
the activity of PKA, cAMP formation followed by forskolin and phosphorylation
of that is cAMP-responsive element binding protein (CREB) (Fig. 9.4).
Further, the activated MT1 receptor also increases the phosphorylation of
mitogen-activated protein kinase or extracellular signal-regulated kinase, kinase
1 and 2 (MEK1 and MEK2), and extracellular signal-regulated kinases 1 and
2-(ERK1/2). Thus, these signaling pathways possibly mediate the induction of
filamentous structures in non-neuronal cells. Potentiation of prostaglandin F2α
(PGF2α) and adenosine triphosphate (ATP)-induced phosphoinositide proceeds
through the activation of βγ subunit of PTX-sensitive G proteins.
Ion channel regulation is responsible for the numerous functional responses
of melatonin such as activation of PTX-insensitive G proteins via an increase in

Fig. 9.4 MT1 receptor-mediated signaling mechanism

9 Pharmacology of Melatonin and Its Receptors 303

intracellular calcium by melatonin in ovine pars tuberalis cells expressing MT1

receptors, but obstructs the influx of calcium at AtT20 cells by PTX-sensitive G
proteins and in pituitary cells of neonatal rats (Dubocovich et al. 2003). Due to MT1
receptors activation, the potentiation of adrenergic vasoconstriction is mediated by
melatonin. In smooth muscles, due to this activation, vasoconstriction is potentiated
by blocking of calcium-activated potassium channels (BKCa), and this blockage
may be due to BKCa channel phosphorylation by PKA and cAMP reduction.
Through inhibition of BKCa channel, melatonin vasoconstricts cerebral artery.
Multiple mechanisms lead to the regulation of the inward rectifier potassium channel
by melatonin in the SCN region. At T20 cells or Xenopus oocytes contain MT1
receptor that activate G protein along with activated inward rectifier potassium
channel, (GIRK) Kir3 through a PTX-sensitive mechanism in which βγ subunits
of the Gi protein are involved. In SCN neuronal firing is inhibited with the elevation
in potassium conductance mediated by melatonin as a result of activation of Kir3
channel. This phenomenon (inhibited neuronal firing) was absent in knockout mice
with MT1 melatonin receptor, which further makes conformation of MT1 receptor
involvement for effect. The MT1 receptor may mediate membrane potential hyper-
polarization in neonatal pituitary cells. There are various tissue-dependent signaling
responses elicited by MT1 receptor activation mediated by PTX-insensitive G or
PTX-sensitive proteins.

9.3.2 MT2 Receptors

The MT2 receptors are low-affinity receptors that are coupled to phosphoinositol
hydrolysis. MT2 receptors are involved in retinal physiology, modulating circadian
rhythms, dilating cardiac vessels, and affecting inflammatory responses in the
microcirculation (Pandi-Perumal et al. 2008). Unlike the MT1, the restriction
is higher with regard to their localization; the involving regions are SCN of the
hypothalamus, cerebellum, cardiac vessels, kidney, ovary, retina, and other multiple
cell lines. The protein reflecting similar binding affinities to MT2 receptor is now
denoted as MT3 and is affinity purified from the Syrian hamster kidney (Nosjean
et al. 2000). It is found that an enzyme involved in detoxification named quinone
reductase 2 shares 95% homology to this enzyme. Lowering of intraocular pressure
and leukocyte adhesion induced by leukotriene B4 is inhibited by MT3 receptors
activation.
There are only one nuclear receptor and three membrane receptors.

9.3.2.1 Type 1a Receptors: Mel 1a, ML1a, ML1, MT1, MTNR1A

It consists of 351 amino acid chain encoded in chromosome number 4 of humans.
Adenylate cyclase is inhibited when MT1 receptors bind to multiple G-proteins.
MT1 receptors are mainly found in the human skin (Emet et al. 2016). Findings
suggest age-dependent decrease in the expression of the MT1 receptor, especially in
cortex SCN and Alzheimer’s disease (AD). The decline in prolactin secretion and
neuronal discharge in SCN is recorded due to MT1 receptor.
304 S. Singh et al.

9.3.2.2 Type 1b Receptors: Mel 1b, ML1b, MT2, MTNR1B

It consist of 363 amino acid chain encoded in chromosome number 11 of humans.
Adenylate cyclase is inhibited when MT1 receptors bind to multiple G-proteins
(Emet et al. 2016). It leads to the inhibition of soluble guanylyl cyclase pathway.
Through activation of melatonin receptor, adenylate cyclase inhibition occurs and
reduces the production of cyclic AMP (cAMP).
Eccrine sweat glands and malign melanocytes are the specific parts of the skin
whereas MT2 receptors are present. In hippocampus region of rats, functions
associated with GABA-A are inhibited. It is found that these receptors are responsi-
ble for antidepressant activity and the expression of MT2 receptors is reduced in
(AD). In the pharmacology and pathophysiology of disorders like AD, anxiety, sleep
disorders, depression, and pain, MT2 receptors play a vital role. For the development
of hypnotic agents MT2 receptors may rise as a new target. For the anxiolytic effects
of melatonin, these types of receptors are responsible.

9.3.2.3 Receptor Signaling Mechanism of Melatonin MT2 Receptors

Physiological response as well as signal transduction mechanism of melatonin
by MT1 activation can be modulated by the levels of both guanosine 30 -50
monophosphate (cGMP) and cAMP. Similarly, melatonin inhibits the forskolin-
stimulated cAMP formation and stimulates JNK through the MT2 receptor activa-
tion (Hardeland 2009). Further, cGMP formation is inhibited by the activation of
MT2 receptor. In SCN, the activity of PKC is increased by MT2 receptor activation
and that response was blocked by the selective MT2 receptor antagonist 4P-PDOT.
It is also reported that the diacylglycerol (DAG) and phospholipase C pathway can
be stimulated by MT2 receptors (Fig. 9.5).
During the activation of the MT2, several physiological responses to melatonin
were identified. These include:

1. Change in the fashion of release of dopamine from the retina of rabbit.

2. Activation of PKC due to phase advance of circadian rhythms in the
isolated SCN.
3. Increase in humoral and cell-mediated immunity.
4. In microvasculature inhibition of rolling leukocyte.
5. Delay of the G1 to S cell cycle transition leads to the inhibition of proliferation of
human choriocarcinoma JAr cells.
6. Activation of MT2 receptors includes the following events: decline expression of
Glut4 (glucose transporter), decrease glucose uptake in human brown adipocytes,
mediates vasodilatation in arterial beds, and modulates neuronal activity in the
hippocampus.
9 Pharmacology of Melatonin and Its Receptors 305

Fig. 9.5 MT2 receptor-mediated signaling mechanism

9.4 Physiological Role of Melatonin

9.4.1 Direct Antioxidant Actions of Melatonin

Melatonin uses multiple mechanisms to decrease the oxidative stress. According to

previous experimental results, it directly acts as a free radical scavenger and indi-
rectly act as antioxidant through stimulating glutathione synthesis (antioxidant),
stimulating antioxidant enzymes, its ability to prevent antioxidative enzymes from
oxidative damage, its potency to elevate the efficiency of, i.e., electron transport
chain (ETC), and hence reducing the generation of free radicle by decreasing leakage
of electrons. As discussed melatonin proved as effective agents in preventing
molecular damage associated with elevated stress conditions. All these above-
mentioned factors which possess the ability to restrain the resulting molecular
mutilation have an unknown mechanism which is to be explored.

9.4.1.1 Direct Scavenger Action of Melatonin at Oxygen Based Free

Radicals and Related Specie\s

Scavenging Action of Melatonin on Hydroxyl Radical (OH)

• 2 OH radicals are scavenger by each melatonin molecule.
• A free radical scavenging action shown by cyclic 3-hydroxy melatonin acts as a
detectable marker which appears in urine.
306 S. Singh et al.

Scavenging Action of Melatonin on Superoxide (O2)

• Xanthine/hypoxanthine O2 producing system is also called the pure chemical
system in which O2 is scavenged by melatonin.

Melatonin Scavenger of H2O2

• Melatonin scavenger H2O2 in a pure chemical system.
• A mechanism of the oxidation of melatonin by H2O2 was suggested based on two
major resulting metabolite, i.e., N-acetyl-N-Formyl-5-Methoxy kynurenine (AFMK).
• Cyclic 3-hydroxyl melatonin and AFMK function as a scavenger of toxic
reactants as like as parent molecule, i.e., melatonin.

9.4.1.2 Scavenger Action of Melatonin on Nitrogen-Based Free Radicals

and Related Species
• Only in the presence of molecule oxygen interaction between NO and melatonin
takes place.
• The chief product of melatonin/NO reaction is N-nitromelatonin.

9.4.2 Indirect Antioxidant Actions of Melatonin

Melatonin can scavenge nitrogen and oxygen-based reactants directly and along
with reducing the oxidative stress by multiple pathways. The relative importance of
the direct and indirect antioxidative processes of melatonin in vivo remains
unknown.

9.4.2.1 Melatonin Stimulation of Glutathione Synthesis

GSH is a well-known antioxidant and free radical scavenger that possess high
abundance intracellularly. According to a single reported result, an increase in
GSH concentrations is observed intracellularly due to glutamylcysteine synthase, a
rate-limiting enzyme stimulated by melatonin. Unlike other functions of melatonin,
there are specific receptors that mediate direct free radical scavenging function of the
indoleamine. Melatonin-mediated GSH synthesis is the major antioxidative action of
melatonin.

9.4.2.2 Synergistic Actions of Melatonin with Classic Antioxidants

The important actions of melatonin at the level of mitochondria are as follows:

• It is an efficient action of ROS/RNS which are abundant in mitochondria than any

other portions of cell and melatonin concentration is higher than serum.
• GPx and GRd are the efficient enzymes stimulated by melatonin useful for GSH
cycling, because mitochondria is not capable of synthesizing GSH by itself.
• Antiapoptotic effects of melatonin have been reported along with mitochondria
originated apoptotic signals.
• Higher concentrations of melatonin may be present in the mitochondria than any
other portions of cell and melatonin concentration is higher than serum.
9 Pharmacology of Melatonin and Its Receptors 307

• Moreover, melatonin was shown to inhibit NADPH-dependent lipid peroxidation

in human placental mitochondria to protect the fetal rat brain against oxidant-
mediated mitochondrial damage and stimulate mitochondrial respiration in the
brain.

9.4.2.3 Action of Melatonin in Immune System

• When melatonin acts on receptor with higher affinity, i.e., MT1 and MT2 it
elicit immunological effects.
• Melatonin may enhance the cytokines production (IL-6, IL-1β, and TNF-α) and
the main function of humoral response is to destroy extracellular microorganisms
and preventing the spread of action.
• In order to fight against infectious diseases such as bacterial infections, HIV,
cancer, etc. useful role of melatonin should be found.

Two GPCRs mediate the physiological actions of melatonin, MT1 and MT2. In
sleep regulation, the role of MT1 and MT2 with high affinity in SCN is observed.
The neuronal firing rate is suppressed by MT1 receptor activation in the SCN, and on
the other hand circadian rhythm phase shifts are induced by MT2 receptors. In
peripheral organs both MT1 and MT2 receptors are present and make their contri-
bution to manage physiological functions. MT1 receptors activation pursue the
following events which include vasoconstriction of cerebral and peripheral arteries,
regulating the expression of Per1 gene in the anterior pituitary, and inhibiting the
secretion of prolactin from the pars tuberalis (Doghramji 2007). Various studies
support the involvement of MT2 receptors in the inhibition of dopamine release in
the retina, vasodilation, in retinal physiology, and also increasing splenocyte prolif-
eration. MT3 binding sites are widely distributed in the brain, liver, heart, kidneys,
and lungs. Several studies performed in vivo suggest its possible role in the regula-
tion of intraocular pressure and inflammatory responses inside the microvasculature.

9.5 Pharmacology

In order to identify and characterize the melatonin receptor in their heterozygous or

native system, a good knowledge of the pharmacological property of radioligands is
required in a receptor system. To characterize the potential therapeutic system, this
knowledge plays an important role in defining ligand selectivity and specificity,
affinity, efficacy, and potency which are further used as an example to demonstrate
the characterization and use in the discovery of functional receptor in native tissue.

9.5.1 General Compounds Acting on Melatoninergic System

Several compounds have revealed the binding efficacy towards melatonin receptors
for significant therapeutic actions. Any compound having 100-fold or more binding
potential for specific receptor is called a ligand. Both the endogenous and exogenous
308 S. Singh et al.

melatonin proved to be potent agonists for MT1 and MT2 receptors providing
a variety of physiological responses (Dawoodi et al. 2012). Another compound
named as N-butanoyl-2-(2-methoxy-6H-isoindolo [2, 1-a] indol-11-yl)-ethanamine
(IIK7) that emerged as a selective MT2 melatonin receptor agonist proved to have
higher affinity towards MT2 in comparison to MT1. On the contrary, luzindole acts
as a selective antagonist for MT2 receptor as it shows a higher affinity towards MT2
in comparison to MT1 receptor. It provides significant results in circadian rhythm
disorders and depression-associated studies. Besides this, other compounds such as
4-phenyl-2-propionamidotetralin (4P-PDOT) and 4-phenyl-2- acetamidotetralin
(4P-ADOT) have also revealed significant antagonist properties for MT2 receptor
(Cecon et al. 2018). Research studies have shown that the success of selective
ligands for MT1 is less as compared to MT2 receptors as their efficacy and binding
affinity are low. To tackle this, a dimer of agomelatine is formed as ligand S26131
which shows 200-fold higher affinities for MT1 receptor in cell line studies (Liu
et al. 2016). Identical to these compounds, a number of different ligands have been
recognized for their specificity towards each melatonin receptor which is enlisted in
Table 9.3.
Moreover, the formation of MT1 or MT2 receptors with 5-HT2C provides suc-
cessful result in higher efficacy in various disorders. Such drugs include agomelatine
and TIK-301 which provide reliable results in depression studies (Liu et al. 2016).
Further on the basis of research data available, several melatonin targeting drugs are
successfully running for their therapeutic efficacy in different clinical conditions
(Dawoodi et al. 2012). Such compounds include ramelteon, agomelatine, TIK-301,
and tasimelteon that have gained clinical reliability for their efficacy in circadian
rhythms-associated disorders (Table 9.4).

Table 9.3 Ligands for melatonin receptors

Receptor MT1 MT2 MT3
Agonists Melatonin, Melatonin, 2-Iodomelatonin,
2-Iodomelatonin, 2-Iodomelatonin, 6-
N-propionyl melatonin, N-propionyl melatonin, Chloromelatonin,
N-butanoyl melatonin, N-butanoyl melatonin, Melatonin
6-Chloromelatonin, 6-Chloromelatonin, (M5250),
2-Methyl-6,7- 2-Methyl-6,7- N-Acetylserotonin,
dichloromelatonin, S20098, dichloromelatonin, 5-MCA-NAT
GR 196429, 8M-PDOT, S20098, GR 196429, 8 M-
S26131 PDOT, IIK7
Partial 5-Methoxyluzindole, 5-Methoxyluzindole, NA
agonists N-acetyltryptamine N-acetyltryptamine
Antagonists Luzindole, S20928 Luzindole, S20928, Luzindole,
4P-PDOT, 4P-ADOT, Prazosin,
K185 Prazosin
9 Pharmacology of Melatonin and Its Receptors 309

Table 9.4 Clinically approved compounds

Clinically approved Mechanism of action—
compounds drug target Therapeutic uses
Melatonin controlled release MT1 and MT2 receptor Treatment of insomnia in
tablets (prolong release agonism amyotrophic lateral sclerosis and
melatonin) other disorders
Ramelteon MT1 and MT2 receptor Treatment of insomnia
agonism
Agomelatine MT1 and MT2 receptor Anxiolytic action
agonism Antidepressive action
Serotonin antagonist Increase daytime alertness
(exhibit little affinity for
5HT2C)
TIK-301 Melatoninergic agonist Antidepressive action
Serotonin antagonist
(exhibit higher affinity
for 5HT2C)
Tasimelteon MT1 and MT2 receptor Sleep-promoting action
agonism Antidepressive action

9.6 Physiological Responses Mediated by Activation

of Specific Melatonin Receptors (MT1, MT2, and MT3)

Melatonin plays a very important role in ensuring that the organism is adjusted to
seasonal and environmental changes. Endogenous and exogenous melatonin play a
very important role in the control and coordination process. The release of endoge-
nous melatonin takes place in seasonal and circadian fashion. On the other hand,
behavioral and physiological responses are regulated by exogenous melatonin. In
this segment multiple mechanisms through which melatonin controls and
coordinates functions are to be discussed such as cardiovascular responses, endo-
crine functions, circadian rhythms, and immune system.

9.6.1 Pharmacology and Function of M1 Receptor (Recent Update

on Involvement of M1 in Normal and Pathophysiological
States)

9.6.1.1 Sleep, Addiction, and Behavioral Associated Disorders

The unique characteristic features of MT1 receptor make it a reliable target in
various disorders like insomnia, circadian sleep disorders, major depression, and
cancer. Regulation of sleep is a major therapeutic action which MT1 receptor
contributes (Liu et al. 2016). It makes melatonin efficacious in insomnia and other
sleep-associated disorders. Here, treatment with melatonin activates the MT1 recep-
tor in the SCN and other limbic regions to decrease neuronal firing for sleep
induction. The region basis localized distribution of MT1 receptor in the brain has
310 S. Singh et al.

been explored for its in-depth analysis. The thalamic reticular nucleus is the major
region to coordinate sleep. Here, the localized MT1 receptor serves to modulate the
REM sleep whereas the localized MT2 receptor takes part in NREM sleep (Ng et al.
2017). The presence and activation of MT1 receptor in the retina regulates the
neurotransmitter release. Once melatonin get secreted from photoreceptors, it imme-
diately binds to melatonin receptors on amacrine cells and affects the subsequent
release of dopamine and GABA. Melatonin and dopamine act as a mutual
antagonists of one another. So, if melatonin binds on dopaminergic amacrine cells
then the subsequent release of dopamine decreases but if itself dopamine interacts
with D2 receptor then it leads to inhibition of melatonin release (Ng et al. 2017).
Normally, the binding of melatonin to MT1 receptor directly inhibits dopamine
release to facilitate dark adaptation.

9.6.1.2 Circadian Rhythm and Melatonin

Melatonin in relation to circadian rhythm has direct effects on sleep-inducing
thermoregulatory mechanisms via loss of body temperature and sleep-inducing
tendency. The disturbed circadian rhythms and abnormal sleep are major symptoms
of depression and other mood-related disorders which make melatonin therapeuti-
cally relevant among them (Kasper and Hamon 2009). Sleep and circadian
rhythms are most commonly affected by melatonin. At pharmacological and physi-
ological dose, melatonin decreases alertness and core body temperature at different
times of the day. Melatonin stimulates both phases, delays and phase advances of
the circadian system, in humans when timed correctly. A decrease in alertness and
temperature is observed in time zone travelers and shift workers, when timed to
advance (Arendt and Deacon 1997).

9.6.1.3 Manipulating Rhythms with Melatonin

Shift work: Sleep disturbance, fatigue, and gastrointestinal problems are primary
health compliance among shift workers. Poor adaptation of the circadian system is
considered as a major cause of all these problems. Circadian state clearly depends
upon core body temperature, and temperature and melatonin rhythms are closely
coupled. The potency of the temperature rhythm is inversely correlated with the
quality of sleep (Arendt and Deacon 1997). Night shift workers tend to sleep in the
morning for short period when there is a rising phase of core temperature rhythms
started, and hence, task performance abilities decrease along with declining core
temperature and imbalanced melatonin. Melatonin improvises the shift worker/jet-
lag conditions. It improvise sleep, performance, alertness, and disturbed homeostasis
(body temperature). Melatonin can be used in conditions where night shift workers
are exposed to morning bright light on the way to their home and it induce natural
sleep.
Jet Lag: The condition of jet lag and shift workers is almost similar. The only
differences between these situations are that time zone travelers can predict the time
to avoid themselves to natural light with the help of natural zeitgebers they can adapt
to situations. They are almost similar to shift workers, subject to other factors such as
meals at inappropriate times, flight times of arrival, stress, difficulty in sleeping on
9 Pharmacology of Melatonin and Its Receptors 311

the aircraft, dehydration, etc. (Arendt and Deacon 1997). Therefore, melatonin is
again considered as a good approach to treat time zone travelers; it improvises sleep
pattern without hindering daily performances and tasks.
Other effects: Other actions like the antidepressant action of melatonin is
confirmed after the discovery of agomelatine which acts synergistically via the
melatonin receptors and 5HT-2C receptors (Kasper and Hamon 2009). The drug
agomelatine has been proven to be better and well tolerated than SSRIs and SNRIs in
clinical studies. This drug not only mitigates stress, depression, neurochemical
abnormalities, and neuronal atrophy but also normalizes distorted sleep and circa-
dian rhythm. The presence of the MT1 receptor in the dorsal raphe nucleus suggests
their involvement in the pathogenesis of depression (Ng et al. 2017). The knockout
model of MT1 receptor resembles the behavioral changes of depression in experi-
mental animal studies.
The role of melatonin was also evaluated for drug addiction, abuse, and reward. It
is observed via locomotor sensitization, a phenomenon involving repetitive
injections of psychostimulants to induce locomotion in rats during drug free period.
Genetic deletion of MT1 receptor enhances locomotor sensitization in rodents
against methamphetamine (Hutchinson et al. 2012). Hence, the MT1 receptor
could be a novel target to treat the addiction behavior induced by psychostimulants.

9.6.1.4 Neurological Disorders

Various studies confirmed that chronic administration of melatonin not only
provides antidepressant action but also enhances hippocampal neurogenesis, neuro-
nal differentiation, and neuronal survival via the activation of melatonin receptors:
both M1 and M2 (Ramírez-Rodríguez et al. 2009). The expression of MT1 receptor
is predominant in the hippocampus to regulate inherent circadian time keeping
capacity, which remains essential to perform basic functions like memory, learning,
neurogenesis, and long-term potentiation. This physiological participation makes
melatonin an important target in hippocampus-dependent disorders such as epilepsy,
dementia, and addiction (Tchekalarova et al. 2015).
The therapeutic efficacy of melatonin is also under evaluation for its role in
excitotoxicity-dependent neuronal cell death. Excitotoxicity is the pathophysiologi-
cal event which involves a variety of neurological disorders. It depends either upon
ionotropic receptors (NMDA or AMPA) or metabotropic receptor (G-protein-cou-
pled receptor). Among these receptors, the NMDA receptor excitotoxicity gets major
concern due to its diverse role in synaptic plasticity, neuroendocrine regulation, and
neuronal injury. Several studies have evaluated the role of melatonin against
excitotoxicity-associated damage to neuronal cells (Escames et al. 2004). It has
been confirmed that melatonin administration significantly ameliorates
excitotoxicity via its anti-inflammatory effect through the MT1 receptor. Further,
few studies have evaluated the specific role of melatonin receptor for the mechanism
behind this action. For example, Das et al. have demonstrated that MT1 and MT2
receptors nonselectively participate in the neuroprotection against glutamate-
associated excitotoxicity (Das et al. 2013). The study also reports that overexpression
of melatonin receptor increases the expression of calcium-binding proteins
312 S. Singh et al.

Fig. 9.6 Role of melatonin receptors in neuroinflammation and neurodegeneration

(Calbindin D28K and parvalbumin) which in turn decreases the calcium-dependent

mediators of cell death including protease, calpains, and pro-apoptotic (Fig. 9.6).
Besides, the mediators of proinflammatory cytokines are expected to decrease as
the expression of their upstream regulators (NF-κβ and COX-2) gets diminished on
melatonin treatment. This anti-inflammatory effect probably gets maintained via the
MT1 receptor. Consequently, neuroinflammation and intracellular calcium-mediated
cell death associated neurodegeneration decrease via the upregulation of melatonin
receptors. The efficacy of MT1 receptor is also proved in striatal disorders like
Huntington’s disease (HD) and Parkinson’s disease (PD). Mutant striatal cell model
evaluates the efficacy of melatonin against mitochondrial dysfunction in HD (Wang
et al. 2011). The efficacy of melatonin in this model is confirmed to be mediated by
the activation of MT1 receptor as mitochondria get reversed by overexpression of
similar receptor. On the other hand in PD, the role of melatonin is under research for
its contribution to motor symptoms and non-motor symptoms like depression,
anxiety, and insomnia. The maintenance of dopaminergic neurons survival is a
primary strategy to treat PD-associated neurodegeneration and symptoms, and the
glial cell-derived neurotrophic factor (GDNF) remains an essential component to
maintain the survival of dopaminergic neurons (Mack et al. 2016). The intrastriatal
melatonin treatment upregulates the GDNF expression in striatum against MPTP-
induced PD. Further, the neural stem cell studies prove it to be upregulated by the
activation of MT1 receptor.
Melatonin has a neuroprotective effect in many other disorders, such as AD,
epilepsy, PD, ischemic, amyotrophic lateral sclerosis, injury, and head injury. Such
disorders concerned with degradation, loss of neurons, and mitochondrial dysfunc-
tion initiating glutamate and free radical overactivity may lead to disease
progression.

Epilepsy
Various studies on the role of epilepsy have revealed that epileptic seizures are
improvised with the help of melatonin but the effect of melatonin as a single
9 Pharmacology of Melatonin and Its Receptors 313

therapeutic moiety is still to be investigated. Due to its ability to cross the blood–
brain barrier it may be used in the treatment of seizures. For example, temporal lobe
epilepsy (TLE) involves progressive development of complex partial seizures that
originate from the temporal lobe (hippocampus) and its various symptoms including
seizures, cognition, and behavioral abnormalities are caused by localized regional
damage in the hippocampus. It includes a gradual decrease of neurogenesis, loss of
GABAergic interneurons, synaptic plasticity, and chronic inflammation which occur
in a bidirectional manner with irregular circadian rhythms. This interaction has been
validated via observing the decreased expression of MT1 receptor in the hippocam-
pus which usually provides an inhibitory effect on the CNS. In this way, agonists of
melatonin receptors could provide an anticonvulsant effect in epilepsy. Both mela-
tonin and agomelatine have been reported to have anticonvulsant action in acute and
chronic preclinical epilepsy. Even the protein and mRNA expression of the binding
site for melatonin named RORα gets decreased in pilocarpine model of epilepsy
(de Alencar Rocha et al. 2017). It has been estimated that RORα may contribute to
temporal epileptic seizures identical to its participation in circadian rhythm, anti-
inflammatory action, and antioxidant properties of melatonin.

Insomnia
High levels of melatonin is observed in adolescence, which declines slowly after the
age of 20. Thus, it affects the age group of 20 more efficiently. Soporific and
hypothermic effects are observed after administration of melatonin during the day
in the young age group. Improvisation in sleep patterns is observed with increased
nocturnal melatonin level via oral administration. Therefore, melatonin therapy is
considered good to treat insomniac conditions.

Depression
It is the property of ideal antidepressant to reduce sleep onset difficulties, without
hindering freshness and daytime alertness. Melatonin does not possess any abuse
potential and adverse effects like “hangover”: it has the ability to improvise patterns
of sleep in patients with insomnia associated with depression.

Alzheimer’s Disease
Accumulation of amyloid β (Aβ) protein and neurofibrillary tangles in the brain is
the main cause of AD. Degeneration takes place as a result of hyper-phosphorylation
of nerve fibers due to age-dependent decline in melatonin. Melatonin plays an
important role in glycogen synthase kinase 3 (GSK-3) modulation; it helps to
prevent neurodegeneration by the influence of AD by the interaction of GSK-3
with presenilin-1, a cofactor for G secretase. It is found that due to its antioxidant
potential, melatonin has direct action in the inhibition of Aβ accumulation and
improving sleep disturbances caused by AD.

Parkinson’s Disease
Oxidative stress is the major factor responsible for the progression of PD; dopamine
metabolism and mitochondrial impairment are the leading causes to generate
314 S. Singh et al.

oxidative stress; these are the major factors responsible for the progression of
PD. Positive therapeutic results are observed when melatonin is used against
rotenone-induced dopamine loss. Due to excessive stimulation of glutamate
receptors, neuronal damage may occur and melatonin has been shown to have a
neuroprotective action against glutamate-induced excitotoxicity. Oxidative stress
and mitochondrial dysfunction in the brains of patients with PD showed elevated
oxidative damage to DNA, decreased levels of glutathione, and increased mono-
amine oxidase activity. Reduced antioxidant defense mechanisms in PD brains are
observed due to a reduction in catalase activity and reduced glutathione (GSH)
(Singh and Jadhav 2014).

9.6.1.5 Cancer Studies

Several cancer studies revealed the remarkable success of melatonin treatment in
lung, gastrointestinal, neck, breast, and head cancer. On one side, melatonin treat-
ment counteracts side effects associated with chemotherapy such as anxiety, depres-
sion, and toxicity but on the other side, it provides some significant results in both
in vitro and in vivo cancer studies. The role of MT1 receptor is specifically
confirmed in breast cancer cell line studies where molecular estimation is performed
on mRNA level of receptor in human specimens. It has been estimated that melato-
nin receptors (MT1) are expressed over the MCF-7 and MDA-MB-231 (human
breast cancer cell lines) (Hill et al. 2015). The exogenous administration of melato-
nin and estradiol in MCF-7 cells reduces the expression of MT-1 receptor as they
control one another receptor binding. In this way, interaction takes place in between
MT1 receptors and estrogen receptors results in antiproliferative effects of melato-
nin. Moreover, melatonin treatment enhances the caspase-independent apoptotic
response via upregulation of Bcl-2/BAX ratio. Overall, melatonin therapy in cancer
cells documented to regulate estrogen receptor binding stimulates the immune
system and mediates apoptosis for anticancer action. It continuously varies in
different cancer cell lines and modulates by melatonin treatment. Overexpression
of MT1 receptor antagonizes the proliferation of MCF-7 cells which confirm its
active participation in antiproliferative action against rapidly growing cancer cells. It
enhances the reliability of selective agonist of MT1 receptor for therapeutic efficacy
in research studies.

9.6.1.6 Cardiac Disorders

Peripheral as well as central interventions are regulated by melatonin; hence cardio-
vascular system is considered as the most important site of action for melatonin. The
presence of melatonin receptors over the ventricular walls, aorta, peripheral and
coronary arteries makes it highly recommended in cardiac disorders. It has been
reported to possess antihypertensive, anti-atherosclerotic, and myocardium protec-
tive properties. The cardioprotective effect of melatonin is thought to be contributed
by its antioxidant and anti-inflammatory potential. In recent years, our changed
lifestyle has encountered late night shifts, indoor work life, and multiple jet lags
which disrupt circadian rhythm leading to melatonin dysfunctional disorders.
9 Pharmacology of Melatonin and Its Receptors 315

A number of epidemiological studies have recognized the circadian rhythm as

a contributory factor to enhance the incidence rate of diabetes, premature aging,
and cardiac disorders (Zhong and Liu 2018). Additionally, the decreased level of
melatonin in blood circulation has been reported in such disorders. For example,
melatonin has been proven to be a potent antiadrenergic molecule in myocardial
infarction. This cardioprotective effect is contributed by the activation of MT1 and
MT2 receptors in coronary arteries as it gets inhibited via luzindole which is a
nonselective blocker of melatonin (Favero et al. 2017). There is one explanation
behind the therapeutic action of melatonin which strikes on the role of adhesion
molecules in the pathophysiology of MI. It has been observed that patients with
impaired circadian rhythm have upregulated endogenous vascular cell adhesion
molecule-1, which is a marker of MI as it rarely occurred in the normal person.
Adhesion molecules become upregulated along with endothelial and platelet activa-
tion after myocardial ischemia. Therefore, the melatonin treatment could effectively
reduce these adhesion molecules to reduce the migration and edema of endothelium
for prevention of heart attacks.
Ischemic perfusion injury remains the most serious complication of ischemic
heart disease. It occurs after hypoxia and plaque removal, when blood starts to
reflow. It frequently damages the cardiac cells through excessive myocardium
contracture, low ventricular pressure, and ventricular fibrillation to enhance the
irreversible damage over the heart. One study report suggests that damage caused
by ischemic reperfusion injury gets enhanced by pinealectomy and significantly
reduced by administration of exogenous melatonin (Nduhirabandi et al. 2011).
Another preclinical study has implicated in the ligation of the coronary artery in
rats which later increases the melatonin receptor expression (MT1 and MT2) to
protect the heart (Lochner et al. 2013). It ensures the cardioprotective role of
melatonin in ischemic reperfusion injury via both endogenous and exogenous
manner.

9.6.1.7 Melatonin and Blood Pressure (BP)

Melatonin actions on the cardiovascular system (CVS) are well known. Since the
classical work of Zanoboni group and Holmes and Sugden, hypertension is observed
in rats after removing circulatory melatonin which gets reversed upon melatonin
therapy is given. BP is regulated by melatonin, either by increasing the parasympa-
thetic or reducing the sympathetic tone, acting centrally in the posttrauma area or
peripherally, acting in the heart, kidney, and directly in the blood vessels, mediating
vasoconstriction and vasodilation. Reduction in diastolic and systolic blood pressure
during the night is the active part of the daily rhythm of blood pressure, and in
humans these rhythms are directly controlled by melatonin. Moreover, as an impor-
tant genetic factor, melatonin participates in programming of adult blood pressure in
neonatal and fetal regulation (Baltatu et al. 2017).
316 S. Singh et al.

9.6.2 Pharmacology and Function of M2 Receptor (Recent Update

on Involvement of M2 in Normal and Pathophysiological
States)

As already discussed, MT2 receptors remain low-affinity receptors which participate

in the regulation of circadian rhythms, inflammation processes, and retinal and
cardiac pharmacology.

9.6.2.1 Neurological Disorders

Recent data implicates the role of MT2 receptor in memory deterioration,
neuroinflammation, and some other neurodegenerative disorders. Hippocampal
expression of MT2 receptor is proved to decrease in AD while treatment drug
ameliorates the memory dysfunction in CA1, CA2, CA3, and dentate gyrus region
by upregulating the MT2 expression (Bahna et al. 2014). Hence, the melatonin
system plays a significant role in the pathophysiology of AD. Besides the hippo-
campus, expression of MT2 receptor is reported to decrease in the pineal gland
and occipital cortex of AD patients. AD involves mitochondrial dysfunction,
neuroinflammation, and cerebral ischemia like abnormalities which accelerate
oxidative stress to damage the neuronal function. Meanwhile, melatonin is well
known for its antioxidant potential which serves via the activation of MT2 receptor.
No doubt, the MT1 receptor is also present in the hippocampus but the compara-
tively high expression of MT2 receptor makes it more significant in AD pathology.
Other major functions of MT2 receptor in the hippocampus are neurogenesis,
neuronal differentiation, and neuronal survival (Ramírez-Rodríguez et al. 2009),
and melatonin treatment proves to be neuroprotective over hippocampal neurons via
upregulating the expression of MT2 receptor.
Several studies have assessed the neuroprotective potential of melatonin in
Parkinson’s disease (PD). Various models of PD such as MPTP, rotenone, and
maneb have induced the PD through a similar mechanism of mitochondrial dysfunc-
tion, which is triggered by oxidative stress and apoptotic pathways. The antioxidant
property of melatonin reported for therapeutic efficacy in PD-associated
neurodegeneration. Even the neuroinflammation that occurs in PD gets significantly
attenuated by melatonin treatment. These neuroprotective effects of melatonin
contribute by the modulation of redox state in neuronal cells during disease
conditions. Besides this, neurotrophic factors including nerve growth factor
(NGF), glial cell line-derived neurotrophic factor (GDNF), and brain-derived
neurotrophic factor (BDNF) are vital key components for neuronal survival in PD
(Mack et al. 2016). Different PD studies have demonstrated that expression of
BDNF gets reduced in SNc of PD patients whereas melatonin treatment using
MT1/MT2 agonist agomelatine enhances the BDNF expression for neuroprotection
in PD.

9.6.2.2 Metabolic Disorders

The crucial role of melatonin in metabolic disorders such as diabetes and obesity has
also gathered specific attention of the researchers. Few studies have shown the
9 Pharmacology of Melatonin and Its Receptors 317

stimulatory action of melatonin on insulin secretion while some indicate it to

inhibitory over the insulin release. An epidemiological study has suggested that
the disruption of circadian rhythm in late night workers raises the chances of type-
2 diabetes (T2D) in their life. Moreover, the presence of melatonin receptors in
adipose tissues, liver, and muscles confirms their participation in glucose homeosta-
sis. The gene encoding for MT2 receptor named MTNR1B is revealed to have a
specific polymorphism that associates with T2D (Karamitri et al. 2018). The exact
mechanism behind this is unclear but it may associate with the dysregulation of
GPCR-dependent downstream signaling of MT2 receptor. Another study has also
confirmed the protective role of melatonin against smoking-induced distortion of
glucose homeostasis via activation of MT2 receptors (Filadelfi and Castrucci 1996;
Cecon et al. 2018). The protective effect is also reported to potentiate via anti-
inflammatory action which is receptor independent action.
In the case of obesity, the disease models of hibernating animals raise queries
regarding the role of melatonin in weight gain. Before going to hibernation, these
animals have more body weight as they take more food as energy reserve while the
hibernation period involves a reduction in weight as food intake decreases with
the reduction of energy consumption. Few animals of such models are reported to
have variation in melatonin release with weight gain due to abnormal circadian
rhythm. The evaluation of the model reveals the modulation of the sympathetic
nervous system through melatonin receptors present on adipose tissue. Further
genetic studies evaluate the role of melatonin on the regulation of body weight,
lipid metabolism, and obesity (Karamitri and Jockers 2018). The polymorphism in
the gene encoding for MT2 receptor in MTNR1B may associate with an increase in
the body mass index and obesity.

9.6.2.3 Sleep, Addiction, and Behavioral Associated Disorders

The role of melatonin in the regulation of sleep disorders is not controversial today.
Several drugs including melatonin and its agonist have gained reliability to treat
insomnia and other sleep-related disorders. Novel compounds including melatonin,
tasimelteon (nonselective MT1/MT2 agonist), and ramelteon (nonselective
MT1/MT2 agonist) have raised the reliability of melatonin in clinical trials. Further
in-depth analysis allows researchers to investigate the role of selective receptor
compounds in circadian rhythm regulation. The presence of MT2 receptor upon
GABAergic neurons enhances its consideration in research studies. As GABA,
produce inhibitory and calming effect and the localized distribution of MT2 receptor
on GABA neurons expected to increase the latency and maintenance of sleep (Comai
and Gobbi 2014). The partial receptor agonist N-{2-[(3-methoxyphenyl)
phenylamino] ethyl} acetamide (UCM765) has provided significant result in the
preclinical model of NREM sleep. These positive results are achieved on reticular
thalamic GABAergic neurons via the preclinical studies (Fig. 9.7).
Moreover, research studies also evaluate the role of melatonin in depression,
anxiety, and pain. Firstly, the administration of melatonin gives antidepressant
effects in depression-associated behavioral studies. A similar study has suggested
that the significant result obtained by melatonin may mediate via the activation of
318 S. Singh et al.

Reduced Postganglionic
Melatonin Pineal Sympathetic
Secretion Gland
NE

Dorsal
Cervical
Ganglion
(-)
Retino-
hypothalamic Preganglionic
pathway
SCN

Light Brain
Stem Spinal Cord
Inferior accessory
optic tract
Retina

Fig. 9.7 Retinal pathway

MT2 receptor. Secondly, the identical receptor has been shown to have analgesic
properties in hot water tail flick test (Yu et al. 2000). The positive result obtained
from the administration of melatonin gets blocked by luzindole and 4P-PDOT which
demonstrate the nociceptive action of MT2 receptor. Besides this, oral administra-
tion of melatonin decreases the flinching behavior associated with tactile allodynia in
diabetic rats (Ambriz-Tututi and Granados-Soto 2007), and this analgesic effect is
reported to get reversed by MT2 receptor antagonist (K-185).

9.6.2.4 Cardiovascular Diseases

The major source of melatonin is the pineal gland, but the presence of numerate
extrapineal sources (mast cell, platelets, leukocytes, and endothelial cells) makes it
possible that melatonin play important role in cardiac disorders. The functions like
maintenance of blood vessel diameter regulate arterial blood pressure and blood flow
in tissues and arteries. Experimental studies proposed the melatonin receptor MT1
and MT2 for their specific role in vasculature such as vasoconstriction and vasodila-
tion respectively. A research study performed in porcine coronary arteries has
accessed the inhibitory role of MT2 receptor in nitric oxide-induced relaxation
(Tunstall et al. 2011). The positive significant effect of melatonin gets decreased
via the MT2 receptor antagonist (4P-PDOT), which confirms it to be a novel
candidate for coronary artery disorders.
Disorders such as ischemic reperfusion injury (IRI), myocardial infarction, and
coronary heart disorders are usually mediated by cardiomyocyte damage that
enhances the infarct size via microvascular dysfunction. The administration of
melatonin has provided significant results in such disorders via receptor dependent
or independent pathway. For example, the administration of luzindole like
9 Pharmacology of Melatonin and Its Receptors 319

nonselective antagonist of melatonin confirms it to be the receptor-dependent action

of melatonin against IRI (Singhanat et al. 2018).

9.6.2.5 Cancer Studies

Cancer studies are associated with uncontrollable proliferation and activation of
specific cell including gastric cell, skin cell, brain cell, etc. Numerous research
studies have evaluated the efficacy of melatonin against a variety of cancer
types including pancreatic cancer, skin cancer, liver cancer, breast cancer, and oral
cancer via its protective action on cell function. The efficacy of melatonin has been
assessed against skin cancer as MT1 and MT2 receptors are present on skin cells.
MT1 receptor is specifically present on keratinocytes and epidermis layer while MT2
receptor is found to be localized on malignant and normal melanocytes (Pourhanifeh
et al. 2019). Melatonin has been evaluated to have anticancer activities against
UV rays and X-ray while the inhibition of cancerous growth in infected cells is
reported to be mediated via the MT2 receptor. Moreover, the administration of
melatonin also provides relief against glucocorticoid-induced suppression caused
by neoplastic infections (Singh et al. 2017). Here, the immunomodulatory action
in the spleen is attained via the activation of the immune response through
overexpression of the MT2 receptor.
Besides this, melatonin acts as a physiological antagonist of serotonin in the gut
via the activation of CCK-2, 5-HT3, and MT2 receptors which make it implement in
gastric cancer (Asghari et al. 2017). The secretion of melatonin increases in the intes-
tine during fasting conditions. It also initiates the secretion of mucosal bicarbonate
by releasing calcium in enterochromaffin cell. Moreover, the activation of MT2
receptor causes the pancreatic secretion of amylase and cholecystokinin along with
free radical scavenging action to heal the ulcerative conditions of the gastrointestinal
tract.

9.6.3 Pharmacology and Function of M3 Receptor (Recent Update

on Involvement of M1 in Normal and Pathophysiological
States)

MT3 receptors are abundantly distributed in the peripheral tissue as well as the brain
of hamster. It is believed that phosphoinositide hydrolysis is stimulated by these
receptors. Both melatonin and its precursor (N-acetylserotonin) possess the ability to
activate MT3 receptor, and they also possess pharmacological profile arranged
according to affinities: the sequence includes 2-iodomelatonin > N-acetyl-seroto-
nin > melatonin and the sequence is slightly similar to the human MT1/MT2
receptors (2-iodomelatonin > melatonin > N-acetyl-serotonin). Specific ligands
for the MT3 melatonin receptor are N-acetyltryptamine and prazosin (Oxenkrug
2005). According to suggested hypothesis, mammalian MT3 site radioligand
2-[125I]-MCA-NAT binds to an enzyme quinone reductase 2 in the kidney mem-
brane of hamster. The cloning of this enzyme is done followed by its purification
from hamster kidney membranes. Decreasing intraocular pressure and inhibiting
320 S. Singh et al.

leukocyte adhesion to vascular endothelial cells were reported when 5-MCA-NAT

activates MT3 receptor (Doghramji 2007). Further investigation is needed to under-
stand the binding affinities of MT3 and furthermore evaluating that MT3 melatonin
binding proteins to represent a binding site for quinone reductase 2 or receptor to G-
protein-coupled receptor. Structurally, this receptor is different from MT1 and MT2
receptors and revealed to be a classic low affinity melatonin membrane receptor (Tan
et al. 2007). The temperature and binding rate based kinetics studies indicate it to be
an enzyme rather than that of a receptor. And, mass spectroscopy and enzymatic
studies confirmed it to be identical to quinoreductase 2 (QR2) which is a detoxifying
agent. This property of MT3 receptor makes it relevantly applicable for therapeutic
applications in different diseases. Currently, several studies are evaluating the
therapeutic potential of MT3 receptor whether the signaling mechanism of MT3
receptor has not been discovered yet.

9.6.3.1 Cancer Studies

The MT3 receptor is reported to present in the liver, kidney, oocytes, brain, and
ovaries. The MT3 receptor proves to be cytotoxic and proapoptotic in cancer studies
which are performed on HT-29 cells (Nair et al. 2018). The anticancer potential of
this receptor gets justified on the basis of its antioxidant efficacy. The MT3 receptor
carries QR2 as binding site in its cleft, which makes it act as a detoxifying and
antioxidant enzyme. An activation of QR2 converts the quinone reductase into more
highly reactive species which exacerbates the cellular damage while the knockdown
model of QR2 in K562 enhances the expression of antioxidant and detoxification
enzymes to reduce proliferation rates in cancer studies. Hence, it is the inhibition of
QR2 which regulates the antioxidant profile of the MT3 receptor.

9.6.3.2 Depression
Melatonin and its immediate precursor N-acetylserotonin (NAS) have been reported
to possess antidepressant action in preclinical and clinical studies. Further different
studies have evaluated the role of selective agonist and antagonist of MT3 receptor in
depression studies which include 5-methoxycarbonylamino-N-acetyltryptamine
(5MCA-NAT) and prazosin, respectively (Oxenkrug et al. 2010). The antidepressant
activity of 5-MCA-NAT has been reported to provide significant results in tail
suspension through QR2/MT3 receptor binding site which reverses via the adminis-
tration MT3 receptor antagonist, prazosin. Another drug like resveratrol is also
proven to be potent antagonists of QR2 binding site of MT3 receptor which may
potentiate via the inhibition of indoleamine 2, 3-dioxygenase, a rate limiting step in
tryptophan metabolism and kynurenine pathway. In other compounds like melatonin
and 5-MCA-NAT, this effect may be achieved through competitive inhibition of
tryptophan 2, 3-dioxygenase. In this way, the protective effect of melatonin via MT3
receptor may provide more reliable results in future preclinical and clinical studies.
9 Pharmacology of Melatonin and Its Receptors 321

9.7 Conclusion

Melatonin is a highly significant moiety, which is not only responsible for inducing
sleep but also contributes in a versatile manner to control and coordinate the different
biological processes of the body which includes maintenance of homeostasis,
secretion of essential hormones, bowel movement, biological clock, circadian
rhythm, and many more. MT1 and MT2 are the two important receptor types, and
its subtypes are located at different regions of the body such as skin, parotid gland,
colon, duodenal enterocytes, platelets, white and brown adipocytes, kidney, cells of
the immune system, ovary/granulosa cells, placenta, and myometrium. These
receptors are generally GPCR type, playing a major role at ground level to achieve
various biological goals. Therefore, this luminary molecule is a crucial component
which directs the smooth biological functioning of the human body.

References
Al-Ghoul WM, Herman MD, Dubocovich ML (1998) Melatonin receptor subtype expression in
human cerebellum. Neuroreport 9(18):4063–4068. LWW
Ambriz-Tututi M, Granados-Soto V (2007) Oral and spinal melatonin reduces tactile allodynia in
rats via activation of MT2 and opioid receptors. Pain 132(3):273–280. Elsevier
Arendt J, Deacon S (1997) Treatment of circadian rhythm disorders–melatonin. Chronobiol Int
14(2):185–204. Taylor & Francis
Asghari MH et al (2017) Melatonin as a multifunctional anti-cancer molecule: implications in
gastric cancer. Life 185:38–45. Elsevier
Aust S et al (2004) The melatonin receptor subtype MT1 is expressed in human gallbladder
epithelia. J Pineal Res 36(1):43–48. Wiley Online Library
Ayoub MA et al (2004) Preferential formation of MT1/MT2 melatonin receptor heterodimers with
distinct ligand interaction properties compared with MT2 homodimers. Mol Pharmacol 66
(2):312–321. ASPET
Bahna SG et al (2014) Regional upregulation of hippocampal melatonin MT2 receptors by valproic
acid: therapeutic implications for Alzheimer’s disease. Neurosci Lett 576:84–87. Elsevier
Baltatu OC et al (2017) Melatonin, mitochondria and hypertension. Cell Mol Life Sci
74(21):3955–3964. Springer
Cajochen C et al (2002) Role of melatonin in the regulation of human circadian rhythms and sleep.
9th Triennial Meeting of the European-Pineal-and-Biological-Rhythm-Society, 15, pp 432–437
Cardinali DP et al (2012) Melatonin and its analogs in insomnia and depression. J Pineal Res 52
(4):365–375. Wiley Online Library
Cecon E, Oishi A, Jockers R (2018) Melatonin receptors: molecular pharmacology and signalling in
the context of system bias. Br J Pharmacol 175(16):3263–3280. Wiley Online Library
Comai S, Gobbi G (2014) Unveiling the role of melatonin MT2 receptors in sleep, anxiety and other
neuropsychiatric diseases: a novel target in psychopharmacology. J Psychiatry Neurosci 39
(1):6–21
Das A et al (2013) Overexpression of melatonin membrane receptors increases calcium-binding
proteins and protects VSC4. 1 motoneurons from glutamate toxicity through multiple
mechanisms. J Pineal Res 54(1):58–68. Wiley Online Library
Dawoodi Z, Shah N, De Sousa A (2012) Melatonin agonists: A brief clinical review. Delhi
Psychiatry J 15:268–273. Citeseer
de Alencar Rocha AKA et al (2017) Altered MT1 and MT2 melatonin receptors expression in the
hippocampus of pilocarpine-induced epileptic rats. Epilepsy Behav 71:23–34. Elsevier
322 S. Singh et al.

De Bodinat C et al (2010) Agomelatine, the first melatonergic antidepressant: discovery, character-

ization and development. Nat Rev Drug Discov 9(8):628. Nature Publishing Group
Doghramji K (2007) Melatonin and its receptors: a new class of sleep-promoting agents. J Clin
Sleep Med 3(5 Suppl):S17. American Academy of Sleep Medicine
Dubocovich ML et al (2003) Molecular pharmacology, regulation and function of mammalian
melatonin receptors. Front Biosci 8(10):1093–1108
Dubocovich ML et al (2010) International Union of Basic and Clinical Pharmacology. LXXV.
Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors.
Pharmacol Rev 62(3):343–380. ASPET
Ebisawa T et al (1994) Expression cloning of a high-affinity melatonin receptor from Xenopus
dermal melanophores. Proc Natl Acad Sci 91(13):6133–6137. National Acad Sciences
Ekmekcioglu C (2006) Melatonin receptors in humans: biological role and clinical relevance.
Biomed Pharmacother 60(3):97–108. Elsevier
Ekmekcioglu C et al (2001) 24h variation in the expression of the mt1 melatonin receptor subtype in
coronary arteries derived from patients with coronary heart disease. Chronobiol Int
18(6):973–985. Taylor & Francis
Ekmekcioglu C et al (2003) The melatonin receptor subtype MT2 is present in the human
cardiovascular system. J Pineal Res 35(1):40–44. Wiley Online Library
Emet M et al (2016) A review of melatonin, its receptors and drugs. Eurasian J Med 48(2):135.
Ataturk University School of Medicine
Escames G et al (2004) Mechanisms of N-methyl-d-aspartate receptor inhibition by melatonin in the
rat striatum. J Neuroendocrinol 16(11):929–935. Wiley Online Library
Favero G et al (2017) Melatonin: protection against age-related cardiac pathology. Ageing Res Rev
35:336–349. Elsevier
Filadelfi AMC, Castrucci AM (1996) Comparative aspects of the pineal/melatonin system of
poikilothermic vertebrates. J Pineal Res 20(4):175–186. Wiley Online Library
Ganguly S, Coon SL, Klein DC (2002) Control of melatonin synthesis in the mammalian pineal
gland: the critical role of serotonin acetylation. Cell Tissue Res 309(1):127–137. Springer
Gobbi G, Comai S (2019) Differential function of melatonin MT1 and MT2 receptors in REM and
NREM sleep. Front Endocrinol 10. https://doi.org/10.3389/fendo.2019.00087. Frontiers Media
SA
Hardeland R (2009) Melatonin: signaling mechanisms of a pleiotropic agent. Biofactors 35
(2):183–192. Wiley Online Library
Hill SM et al (2015) Melatonin: an inhibitor of breast cancer. Endocrine Relat Cancer 22(3):R183–
R204. Bioscientifica Ltd
Hutchinson AJ, Hudson RL, Dubocovich ML (2012) Genetic deletion of MT1 and MT2 melatonin
receptors differentially abrogates the development and expression of methamphetamine-
induced locomotor sensitization during the day and the night in C3H/HeN mice. J Pineal Res
53(4):399–409. Wiley Online Library
Kappers JA (1979) Short history of pineal discovery and research. Prog Brain Res 52:3–22
Karamitri A, Jockers R (2018) Melatonin in type 2 diabetes mellitus and obesity. Nat Rev
Endocrinol:1. https://doi.org/10.1038/s41574-018-0130-1. Nature Publishing Group
Karamitri A, Plouffe B et al (2018) Type 2 diabetes–associated variants of the MT2 melatonin
receptor affect distinct modes of signaling. Sci Signal 11(525):eaan6622. American Association
for the Advancement of Science
Kasper S, Hamon M (2009) Beyond the monoaminergic hypothesis: agomelatine, a new antide-
pressant with an innovative mechanism of action. World J Biol Psychiatry 10(2):117–126.
Taylor & Francis
Lerner A et al (1958) Isolation of melatonin, the pineal gland hormone factor the lightens
melanocytes. Commun Editor 934(2):2907
Liu J et al (2016) MT1 and MT2 melatonin receptors: a therapeutic perspective. Annu Rev
Pharmacol Toxicol 56:361–383. Annual Reviews
9 Pharmacology of Melatonin and Its Receptors 323

Lochner A, Huisamen B, Nduhirabandi F (2013) Cardioprotective effect of melatonin against

ischaemia/reperfusion damage. Front Biosci (Elite Ed) 5:305–315
Mack JM et al (2016) Melatoninergic system in Parkinson’s disease: from neuroprotection to the
management of motor and nonmotor symptoms. Oxid Med Cell Longev 2016:3472032.
Hindawi
Nair S et al (2018) A review on melatonin action as therapeutic agent in cancer. Front Biol 13
(3):180–189. Springer
Nduhirabandi F et al (2011) Chronic melatonin consumption prevents obesity-related metabolic
abnormalities and protects the heart against myocardial ischemia and reperfusion injury in a
prediabetic model of diet-induced obesity. J Pineal Res 50(2):171–182. Wiley Online Library
Neubauer DN (2008) A review of ramelteon in the treatment of sleep disorders. Neuropsychiatr Dis
Treat 4(1):69. Dove Press
Ng KY et al (2017) Melatonin receptors: distribution in mammalian brain and their respective
putative functions. Brain Struct Funct 222(7):2921–2939. Springer
Nosjean O et al (2000) Identification of the melatonin-binding SiteMT 3 as the Quinone reductase
2. J Biol Chem 275(40):31311–31317. ASBMB
Oxenkrug G (2005) ‘Antioxidant effects of N-acetylserotonin. Ann N Y Acad Sci 1053
(1):334–347. Wiley Online Library
Oxenkrug GF et al (2010) Quinone reductase 2 and antidepressant effect of melatonin derivatives.
Ann N Y Acad Sci 1199(1):121–124. Wiley Online Library
Pala D et al (2013) Homology models of melatonin receptors: challenges and recent advances. Int J
Mol Sci 14(4):8093–8121. Multidisciplinary Digital Publishing Institute
Pandi-Perumal SR et al (2008) Physiological effects of melatonin: role of melatonin receptors and
signal transduction pathways. Prog Neurobiol 85(3):335–353. Elsevier
Peuhkuri K, Sihvola N, Korpela R (2012) Dietary factors and fluctuating levels of melatonin. Food
Nutr Res. 56(1):17252. Taylor & Francis
Pourhanifeh MH et al (2019) Potential use of melatonin in skin cancer treatment: a review of current
biological evidence. J Cell Physiol. https://doi.org/10.1002/jcp.28129. Wiley Online Library
Ramírez-Rodríguez G et al (2009) Melatonin modulates cell survival of new neurons in the
hippocampus of adult mice. Neuropsychopharmacology 34(9):2180. Nature Publishing Group
Reppert SM et al (1995) Molecular characterization of a second melatonin receptor expressed in
human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci 92(19):8734–8738.
National Acad Sciences
Rocha CS et al (2015) Melatonin and male reproductive health: relevance of darkness and antioxi-
dant properties. Curr Mol Med 15(4):299–311. Bentham Science Publishers
Savaskan E et al (2001) Cerebrovascular melatonin MT1-receptor alterations in patients with
Alzheimer’s disease. Neurosci Lett 308(1):9–12. Elsevier
Savaskan E et al (2002) Increased melatonin 1a-receptor immunoreactivity in the hippocampus of
Alzheimer’s disease patients. J Pineal Res 32(1):59–62. Wiley Online Library
Savaskan E et al (2005) Reduced hippocampal MT2 melatonin receptor expression in Alzheimer’s
disease. J Pineal Res 38(1):10–16. Wiley Online Library
Singh M, Jadhav HR (2014) Melatonin: functions and ligands. Drug Discov Today
19(9):1410–1418. Elsevier
Singh SS, Deb A, Sutradhar S (2017) Dexamethasone modulates melatonin MT2 receptor expres-
sion in splenic tissue and humoral immune response in mice. Biol Rhythm Res 48(3):425–435.
Taylor & Francis
Singhanat K et al (2018) Roles of melatonin and its receptors in cardiac ischemia–reperfusion
injury. Cell Mol Life Sci 75(22):4125–4149. Springer
Sjöblom M, Flemström G (2003) Melatonin in the duodenal lumen is a potent stimulant of mucosal
bicarbonate secretion. J Pineal Res 34(4):288–293. Wiley Online Library
Sjöblom M, Jedstedt G, Flemström G (2001) Peripheral melatonin mediates neural stimulation of
duodenal mucosal bicarbonate secretion. J Clin Investig 108(4):625–633. Am Soc Clin Investig
324 S. Singh et al.

Tan D et al (2007) Melatonin as a naturally occurring co-substrate of quinone reductase-2, the

putative MT3 melatonin membrane receptor: hypothesis and significance. J Pineal Res 43
(4):317–320. Wiley Online Library
Tchekalarova J et al (2015) The role of the melatoninergic system in epilepsy and comorbid
psychiatric disorders. Brain Res Bull 119:80–92. Elsevier
Tunstall RR et al (2011) MT2 receptors mediate the inhibitory effects of melatonin on nitric oxide-
induced relaxation of porcine isolated coronary arteries. J Pharmacol Exp Ther 336(1):127–133.
ASPET
Uz T et al (2005) The regional and cellular expression profile of the melatonin receptor MT1 in the
central dopaminergic system. Mol Brain Res 136:45–53. https://doi.org/10.1016/j.molbrainres.
2005.01.002
Wang X et al (2011) The melatonin MT1 receptor axis modulates mutant huntingtin-mediated
toxicity. J Neurosci 31(41):14496–14507. Soc Neuroscience
Whitlock FA, Cantab MD, Lond MRCP (1954) Acth and melanin pigmentation. Br J Dermatol 66
(11):388–401. https://doi.org/10.1111/j.1365-2133.1954.tb12567.x
Yu C-X et al (2000) Selective MT2 melatonin receptor antagonist blocks melatonin-induced
antinociception in rats. Neurosci Lett 282(3):161–164. Elsevier
Zhong J, Liu Y (2018) Melatonin and age-related cardiovascular diseases. Aging Med
1(2):197–203. Wiley Online Library
Pharmacology of Adenosine Receptors
10
Pran Kishore Deb, Sarah Falah Kokaz, Sara Nidal Abed,
Balakumar Chandrasekaran, Wafa Hourani,
Abdulmuttaleb Yousef Jaber, Raghu Prasad Mailavaram,
Puneet Kumar, and Katharigatta N. Venugopala

Abstract

Adenosine is an endogenous nucleoside molecule, regulating a myriad of physi-

ological and pathological effects in almost all the organs systems including
central nervous system (CNS), cardiovascular system (CVS), respiratory system,
renal system, and immune system. Biological functions of adenosine are
mediated by its interactions with four subtypes of G-protein-coupled receptors
(GPCRs), namely A1, A2A, A2B, and A3 adenosine receptors (ARs) which are
ubiquitously present throughout the body. However, ubiquitous distribution of
ARs in both healthy and diseased tissues imposed a great challenge to the
researchers in the discovery and development of ligands targeting a particular
AR subtype in a specific tissue, devoid of undesirable side effects. This chapter

P. K. Deb (*)
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
e-mail: [email protected]
S. F. Kokaz · S. N. Abed · B. Chandrasekaran · W. Hourani · A. Y. Jaber
Faculty of Pharmacy, Philadelphia University, Amman, Jordan
R. P. Mailavaram
Department of Pharmaceutical Chemistry, Shri Vishnu College of Pharmacy, Vishnupur (Affiliated
to Andhra University), Bhimavaram, W.G. Dist., AP, India
P. Kumar
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India
K. N. Venugopala
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-
Ahsa, Kingdom of Saudi Arabia
Department of Biotechnology and Food Technology, Durban University of Technology, Durban,
South Africa

# Springer Nature Singapore Pte Ltd. 2020 325

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_10
326 P. K. Deb et al.

provides an overview of the synthesis, metabolism, and cellular transport of

adenosine, with particular emphasis on the distribution and signaling mechanisms
of ARs, including specific examples of agonists/partial agonists, antagonists, and
allosteric modulators of ARs as potential therapeutic agents.

Keywords

Adenosine · A1, A2A, A2B and A3 adenosine receptors · G-protein-coupled

receptors (GPCRs) · Adenosine receptors signaling

Abbreviations

AC Adenylyl cyclase
ADA Adenosine deaminase
AK Adenosine kinase
AMP Adenosine monophosphate
AR Adenosine receptor
ARNO ADP ribosylation factor nucleotide site opener
ATP Adenosine triphosphate
BBB Blood-brain barrier
CADD Computer-aided drug design
cAMP Cyclic adenosine monophosphate
CNT Concentrative nucleoside transporter
COPD Chronic obstructive pulmonary disease
CREB c-AMP-responsive element binding protein
DAG Diacylglycerol
ENT Equilibrative nucleoside transporter
ERK Extracellular signal-regulated kinase
GPCR G-protein-coupled receptor
GSK-3β Glycogen synthase kinase-3β
HFpEF Heart failure with preserved ejection fraction
iNKT cells Invariant natural killer T cells
iNOS Inducible nitric oxide synthase
IP3 Inositol 1,4,5-triphosphate
IR Ischemia-reperfusion
JNK c-Jun N-terminal kinase
LBDD Ligand-based drug design
MAPK Mitogen-activated protein kinase
MPI Myocardial perfusion imaging
OHT Orthotopic heart transplantation
PAM Positive allosteric modulator
PD Parkinson’s disease
PDEs Phosphodiesterases
PKA Protein kinase A
PKC Protein kinase C
10 Pharmacology of Adenosine Receptors 327

PLC Phospholipase C
PLD Phospholipase D
SAHH S-adenosyl-homocysteine hydrolase
SAMe S-adenosylmethionine
SBDD Structure-based drug design
SPECT Single photon emission computed tomography
TNFα Tumor necrosis factor-alpha
TRAX Translin-associated protein X
US FDA United States Food and Drug Administration
USP4 Ubiquitin-specific protease

10.1 Introduction

Adenosine is an endogenous nucleoside molecule, regulating various physiopatho-

logical functions by interacting with four subtypes of G-protein-coupled receptors
(GPCRs): A1, A2A, A2B, and A3 adenosine receptors (ARs). The primary mechanism
of signal transduction of A1 and A3 ARs involves the inhibition of adenylyl cyclase
(AC), thereby reducing the cyclic adenosine monophosphate (cAMP), whereas the
activation of A2A and A2B ARs results in the stimulation of AC and consequent
increase in cAMP levels (Fredholm et al. 2001, 2011). However, adenosine shows
varying affinity for ARs. In particular, A1, A2A, and A3 ARs show moderate to high
affinities towards adenosine, requiring only 10 nM to 1 μM concentration for their
activation, whereas A2B AR is comparatively a low affinity receptor which requires a
higher concentration of adenosine (10 μM) for its activation (Borea et al. 2018a, b;
Fredholm 2014). Table 10.1 provides the molecular characteristics and mechanism
of action of adenosine receptors. All the ARs are ubiquitously present throughout the
body, influencing various physiological and pathological processes of almost all the

Table 10.1 Molecular characteristics and mechanism of action of adenosine receptors

A1 AR A2A AR A2B AR A3 AR
Amino acid residues 326 410 328 318
Amino acid sequence similarity 38.3 44.0 46.5
(%) vs hA1AR
Amino acid sequence similarity 46.6 31
(%) vs hA2AAR
Amino acid sequence similarity 35.7
(%) vs hA2BAR
Affinity for adenosine (nM) 1–10 30 1000 100
G-protein coupling Gi/o Gs GsGq/11 GsGq/11
Signaling system #AC, "PLC #AC, #AC, "PLC, #AC, "PLC,
"PI3 kinase "MAPK "MAPK "PI3 kinase,
"MAPK, "MAPK
"K+, Ca2+
328 P. K. Deb et al.

organ systems including central nervous system (CNS), cardiovascular system

(CVS), respiratory system, renal system, and immune system among others. Thus,
ARs represent potential drug targets for various therapeutic interventions (Borea
et al. 2018a, b, 2016). Various agonists/partial agonists, antagonists, and allosteric
modulators of A1, A2A, A2B, and A3 ARs have been discovered, patented, and are
currently being investigated in clinical trials (Al-attraqchi et al. 2019; Borah et al.
2019; Chandrasekaran et al. 2019; Deb 2019a, b; Deb et al. 2019a, b; Mailavaram
et al. 2019). But only few molecules could successfully reach the market either
due to their poor pharmacokinetic profiles or because of the ubiquitous distribution
of the ARs both in normal and diseased tissues imposing nonspecific actions or
undesirable side effects of the drugs (Borea et al. 2018a, b; Chandrasekaran et al.
2019; Shaik et al. 2019). Istradefylline, the selective A2A AR antagonist, was
initially marketed in Japan (2013) for the treatment of Parkinson’s disease (PD),
but recently (2019) it has got approval from the US FDA as an add-on treatment to
levodopa/carbidopa for PD (Hoffman 2019; Voelker 2019). Table 10.2 provides a
list of clinically approved drugs and their therapeutic applications targeting ARs.
Furthermore, growing advancement in the computer-aided drug design (CADD)
software tools and algorithms has been significantly facilitating both the ligand-
based and structure-based drug design (LBDD and SBDD) strategies for the discov-
ery and development of novel drugs targeting ARs (Agrawal et al. 2019; Al-Shar’i
Nizar and Al-Balas 2019; Deb 2019c; Deb et al. 2018a, b; Deb et al. 2019a, b;
Kishore et al. 2011; N et al. 2019; Samanta et al. 2019). In particular, the recent
discovery of the 3D crystal structure of A1 AR (Cheng et al. 2017; Glukhova et al.
2017) along with the previously identified 3D structure of A2A AR (Jaakola et al.
2008) has augmented the understanding of the molecular structures of ARs as well as
physicochemical requirements of ligands for selective binding with ARs. This
chapter highlights the synthesis, metabolism, and cellular transport of adenosine,
with particular emphasis on the body distribution and signaling mechanisms of ARs
in various physiological and pathological conditions. Important examples of
agonists/partial agonists, antagonists, and allosteric modulators of ARs and their
pathophysiological roles are also briefly discussed.

10.2 Synthesis, Metabolism, and Cellular Transport

of Adenosine

Adenosine metabolism plays an important role in regulating various pathophysio-

logical functions of the body. In physiological conditions, adenosine is available in
low concentration (20–300 nM). However, under metabolic stressful conditions
including pain, inflammation, and various disease states, extracellular adenosine
concentration increases up to 30 μM due to ATP catabolism, where adenosine
exhibits a helper/protective role by restoring the imbalance between energy demand
and availability of working cells like neurons and cardiomyocytes by adapting some
of their activities such as reducing heart inotropic effect, increasing oxygen and
nutrition supply through vasodilation, thereby reducing the ATP requirement (Borea
10 Pharmacology of Adenosine Receptors 329

Table 10.2 Therapeutic applications of clinically approved drugs targeting ARs

Mechanism of
Name and structure of drugs actions Therapeutic applications
NH2 A1 AR agonist Paroxysmal supraventricular
N
tachycardia (PSVT)
N
A2A AR agonist Myocardial perfusion imaging
N N
O
HO
Adenosine (1)
HO HO

O A1 AR Treatment of asthma
H antagonist
H 3C N
N

O N N

Theophylline (2) CH3

O A1 AR Treatment of asthma
O antagonist
H3C O
N N

O N N
Doxofylline (3) CH3

OH A1 AR Treatment of asthma
H3C antagonist
N
O
H 3C N
N

O N N
CH3
Bamifylline (4)

NH2 A2A AR agonist Myocardial perfusion imaging

N N

H3C NH N
N N
O N O
HO

Regadenoson (5) HO
HO

O A2A AR Adjuvant therapy of Parkinson’s

N
antagonist disease
N

O N N O

O
Istradefylline (6)

et al. 2016, a, 2018b). Because of these protective roles, adenosine is considered as a

“retaliatory metabolite” rather than a secondary metabolite of cAMP pathway
(Newby 1984). Adenosine facilitates tissue protection from ischemic damage via
preconditioning cell as well as exerting anti-inflammatory response and promoting
angiogenesis (Linden 2005).
330 P. K. Deb et al.

In physiological conditions, adenosine is synthesized intracellularly from AMP

and S-adenosyl-homocysteine (SAH) hydrolysis by endo-50 -nucleotidase and S-
adenosyl-homocysteine hydrolase (SAHH), respectively (Chen et al. 2013). It
should be noted that the SAH hydrolysis leading to the formation of adenosine
and homocysteine is a reversible process. The formation of SAH from adenosine and
homocysteine is mainly favored under thermodynamic equilibrium conditions, con-
sequently inhibiting the S-adenosylmethionine (SAMe) transmethylation due to
increased levels of SAH. Thus, an effective decrease in adenosine levels mainly
by adenosine kinase (AK) triggers the transmethylation process. Therefore, SAHH
can facilitate both the synthesis and removal of adenosine (Bjursell et al. 2011;
Finkelstein 1998; Moffatt et al. 2002). Extracellularly, adenosine is mainly produced
under stressful conditions in high concentrations from the ATP, ADP, and AMP
dephosphorylation with the help of two hydrolyzing enzymes, namely
ectonucleosidase triphosphate diphosphohydrolase (CD39) and ecto-50 -nucleotidase
(CD73), respectively (Zimmermann 2000). Additionally, extracellular conversion of
cAMP to AMP with the help of ecto-phosphodiesterase (ecto-PDE) can further
trigger the formation of adenosine via CD73 (Godinho et al. 2015; Pleli et al.
2018; Sassi et al. 2014).
Adenosine, once generated, travels across the cell membrane with the help of
concentrative nucleoside transporters (CNTs) and equilibrative nucleoside
transporters (ENTs). There are three isoforms of energy-dependent cation-linked
(Na+) CNTs (1–4) facilitating adenosine influx and four energy-independent
isoforms of ENTs (1–3) which can assist in influx or efflux based on the concentra-
tion of adenosine. In general, adenosine influx takes place from extracellular to
intracellular region, whereas the reverse condition is evident in hypoxia (Bading
et al. 1993; Deussen 2000; Deussen et al. 1999).
Biotransformation of adenosine inside the cell takes place by hydrolysis to SAH,
phosphorylation to AMP, and deamination to inosine with the help of SAHH,
adenosine kinase (AK), and adenosine deaminase (ADA), respectively. Under
physiological conditions, AK is mainly responsible for adenosine metabolism,
whereas under pathological conditions, ADA preferentially facilitates adenosine
clearance. Extracellular adenosine clearance occurs through ecto-ADA and influx
through ENTs (Boison 2018; Boison et al. 2013; Gracia et al. 2012; Pacheco et al.
2005). Figure 10.1 represents the synthesis, metabolism, and cellular transportation
of adenosine.

10.3 Molecular Structure of Adenosine Receptors (ARs)

All the four subtypes of ARs present common molecular structure arrangement,
composed of seven transmembrane helices (TMs 1–7) which are connected to each
other through three intracellular loops (ILs 1–3) and three extracellular loops (ELs
1–3) of varying lengths and functions. These three ELs play important roles in
mediating receptor functions, where cysteine residues connect these ELs by forming
disulfide bonds. The N-terminal containing glycosylation site is present on the
10 Pharmacology of Adenosine Receptors 331

cAMP

ecto-PDE
CD39 CD39 CD73 ADA
ATP ADP 5’-AMP Adenosine Inosine
ENT CNT
Extracellular

5’-nucleotidase SAH hydrolase

Intracellular 5’-AMP Adenosine SAH
AK
ADA

Inosine

Fig. 10.1 Synthesis, biotransformation, and cellular transportation of adenosine

extracellular region, while the intracellular C-terminal possesses phosphorylation

and palmitoylation sites that are responsible for desensitization and internalization of
the receptor. The A2A AR possesses longer C-terminal (122 amino acid residues) as
compared to A1, A2B, and A3 ARs (30–40 amino acid residues). Adenosine receptors
present 41–58% amino acid sequence similarity among human species (Table 10.1)
(Fredholm et al. 2001, 2011, 2000). Among all the subtypes, only the crystal
structures of A1 AR (Cheng et al. 2017; Glukhova et al. 2017) and A2A AR
(Jaakola et al. 2008) have been resolved, based on which several homology models
of A2B and A3 ARs have been constructed to gain insight into their binding
interactions with both agonists and antagonists ligands as well as to facilitate the
structure-based drug design (Deb et al. 2018a, 2018b; Gutiérrez-de-Terán et al.
2017). ARs also exist in the form of homomer, heteromer, and oligomers, such as
A1 AR-A2A AR, A1 AR-A3 AR, A2A AR-D2 dopamine receptor. In particular, A2A
AR-D2 dopamine receptor complex that is present in striatum is considered as a
significant therapeutic target for the treatment of Parkinson’s disease (Brugarolas
et al. 2014; Ferre et al. 2010; Navarro et al. 2016).

10.4 Distribution of Adenosine Receptors

Adenosine receptors are distributed throughout the cardiovascular, nervous, gastro-

intestinal, respiratory, urogenital, as well as immune systems. ARs were also
detected in bones, eyes, joints, and skin (Peleli et al. 2017). Each subtype has a
distinctive cell and tissue distribution, signaling transductors, and hence unique
physiological effects (Fredholm et al. 2001).
332 P. K. Deb et al.

10.4.1 Distribution of A1 AR

A1AR has shown a high abundance in the brain as well as other organs and tissues.
This receptor subtype has been demonstrated by radioligand-receptor binding stud-
ies and imaging (Elmenhorst et al. 2012; Hayashi et al. 2017), along with RNA
expression, Western blot, as well as functional characterization. Therefore, the wide
distribution of this receptor has suggested its important physiological roles including
spanning neurotransmitter release, neuronal excitability dampening, sleep/wakeful-
ness control, reduction of pain, along with the sedative, anxiolytic, anticonvulsant, as
well as locomotor depressant effects (Gessi et al. 2011; Sawynok 2016). In the
central nervous system (CNS), A1AR is mainly expressed in the brain cortex,
hippocampus, cerebellum, spinal cord, autonomic nerve terminals, and glial cells
(Ballesteros-yáñez et al. 2018; Chen et al. 2013). In the heart, the expression of
A1AR has been shown to be higher in atria and much less in the ventricular
myocardium (Stenberg et al. 2003; Varani et al. 2017). At the vascular level,
A1ARs are found on the coronary smooth muscle arteries as well as endothelial
cells (Headrick et al. 2013). Moreover, A1ARs have been detected in the endothelial
cells of the lung, in the airway’s smooth muscles, in the alveolar epithelial cells, and
in immune cells such as macrophages, neutrophils, eosinophils, and monocytes
(Boros et al. 2016; Sachdeva and Gupta 2013; Sun et al. 2005), where they
essentially promote some proinflammatory effects (Ponnoth et al. 2010). A1AR is
also found in the kidney, adipose tissue, and pancreas, where it causes induction of
negative chronotropic, inotropic, as well as dromotropic effects, reduction in the
renal blood flow and renin release, and inhibition of lipolysis and insulin secretion,
respectively (Dhalla et al. 2009; Prystowsky et al. 2003; Rabadi and Lee 2015; Sun
et al. 2001; Vallon and Mu 2006; Vincenzi et al. 2012). In the kidney, A1ARs mostly
present in the papilla’s collecting ducts, inner medulla, in addition to the cells of the
juxtaglomerular apparatus. A1ARs have been also detected in the retina, skeletal
muscle, intestine, and vascular cells of skeletal muscle (Soni et al. 2017; Varani et al.
2017).

10.4.2 Distribution of A2A and A2B ARs

The A2A AR is present centrally and peripherally, where it serves a number of

functions that are related to excitotoxicity, the release of spanning neuronal gluta-
mate, glial reactivity, the permeability of the blood-brain barrier (BBB), as well as
the migration of the peripheral immune cells (Koupenova et al. 2012; Merighi et al.
2015; Pedata et al. 2016), and greatly expressed in the striatum, the olfactory
tubercle, as well as the immune system. However, lower levels are present in the
cerebral cortex, heart, hippocampus, lung, and blood vessels. In the peripheral
immune system, A2A AR has been shown to have a great expression particularly
in leukocytes, platelets, as well as the vasculature, in which it mediates numerous
anti-inflammatory, antiaggregatory, as well as vasodilatory effects, respectively
(Ruiz et al. 2014). A2A ARs are found in the bowel, lung, bladder, vas deferens, as
10 Pharmacology of Adenosine Receptors 333

well as in other different cell types such as fibroblasts, smooth muscles, alveolar
epithelial, chromaffin, and taste cells, platelets, myocardial cells, and retinal, intesti-
nal, endothelial and pulmonary epithelial cells (Aherne et al. 2011).
It has been shown in recent development of A2BAR-knockout/lacZ-knocking
mice (Yang et al. 2006) that A2B AR has a wide distribution in numerous tissues
and organs, and this includes the aortic vascular smooth muscle, vasculature, cecum,
brain, large intestine, and urinary bladder (Wang and Huxley 2006; Yaar et al. 2005).
Moreover, A2B AR was found to be highly expressed in various cell types, including
several immune cells such as mast cells (Hua et al. 2007; Yang et al. 2006),
neutrophils (Ryzhov et al. 2008), dendritic cells (Addi et al. 2008), macrophages
(Novitskiy et al. 2008), as well as lymphocytes (Yang et al. 2006), in addition to
other cell types that include the type II alveolar epithelial cells (Eckle et al. 2008),
endothelial cells (Cagnina et al. 2009), chromaffin cells (Yang et al. 2006),
astrocytes (Peakman and Hill 1994), neurons (Christofi et al. 2001), and taste cells
(Stein et al. 2001).

10.4.3 Distribution of A3 AR

The identification of the A3 AR distribution has been made possible after the
generation of cDNA for this receptor (Nishida et al. 2014). The A3 AR subtype
was found to have wide expression in various primary cells, tissues, as well as cell
lines. In the brain, A3AR has been reported in low levels, where it is expressed
particularly in the hypothalamus, thalamus, hippocampus, cortex, as well as retinal
ganglion cells, and motor nerve terminals, in addition to the pial and intercerebral
arteries (Burnett et al. 2010; Janes et al. 2014). Studies have also shown that the
expression of A3 ARs is also reported in microglia and astrocytes; thus inhibiting the
neuro-inflammatory response in these particular cells was shown to be associated
with the analgesic effect they induce (Borea et al. 2016). Despite the cardio-
protective effects that have been related to the A3 AR, as well as the great expression
of this receptor subtype in the coronary and carotid artery, its precise location in the
heart is not yet reported. At the periphery, A3 AR was found to be expressed in
enteric neurons, epithelial cells, lung parenchyma, colonic mucosa, and bronchi.
Moreover, a broad distribution of A3 AR subtype has been reported in inflammatory
cells (Janes et al. 2014) including mast cells, eosinophils, monocytes, neutrophils,
macrophages, dendritic cells, foam cells, lymphocytes, bone marrow cells,
splenocytes, lymph nodes, chondrocytes, synoviocytes, as well as osteoblasts,
where it is responsible for mediating various anti-inflammatory effects (Borea
et al. 2015). It is worth mentioning that A3 AR subtype is overexpressed in some
cancer cells and tissues, which therefore shows the important antitumoral role of this
receptor subtype (Borea et al. 2016). At cellular level, A3 ARs have shown wide
expression in motor nerve terminals, astrocytes, microglia, cortex, as well as retinal
ganglion cells (Borea et al. 2015; Gessi et al. 2013).
334 P. K. Deb et al.

10.5 Signal Transduction Pathways of Adenosine Receptors

Numerous signal transduction pathways are triggered by all the four G-protein-
coupled ARs based on the activation of a particular type of cell (Fredholm et al.
2001, 2011).

10.5.1 Molecular Signaling of A1 AR

The activation of the Gi-protein-coupled A1 AR causes inhibition of adenylyl

cyclase (AC), leading to the reduction of cyclic adenosine monophosphate
(cAMP) production (Fredholm et al. 2000), resulting in the reduction of cAMP-
dependent protein kinase A (PKA) and cAMP-responsive element-binding protein
1 (CREB-1) phosphorylation (Ellis et al. 1995). A1 AR can stimulate the phospholi-
pase C (PLC)-β, increasing diacylglycerol (DAG) and inositol 1,4,5-triphosphate
(IP3) levels, thus enhancing calcium (Ca2+) concentrations inside the cell,
stimulating the activation of Ca2+-dependent protein kinase C (PKC) and/or other
binding proteins (Basheer et al. 2002; Biber et al. 1997; Borea et al. 2018a, b; Nalli
et al. 2014). Activation of A1 AR also results in the opening of potassium (K+)
channels in neurons and cardiac tissue, while inhibiting Q, P, and N-type Ca2+
channels (Kirsch et al. 1990; Kunduri et al. 2013; Schulte and Fredholm 2003,
2000). Additionally, A1 AR activation is also linked to the phosphorylation of
mitogen-activated protein kinases (MAPK) like p38, ERK1/2, and JNK (Schulte
and Fredholm 2003, 2000). The signal transduction pathway of A1 AR is depicted in
Fig. 10.2.

10.5.2 Molecular Signaling of A2A AR

The activation of Gs-protein-coupled A2A AR triggers AC activity and increases the

cAMP levels, thereby stimulating PKA which causes phosphorylation and further
activation of several proteins including receptors, PDEs, CREB, and dopamine- and
c-AMP-regulated phosphoprotein (DARPP-32) (Preti et al. 2015). Additionally, A2A
ARs inside the brain can stimulate neuron-specific Gs-protein called Golf that is also
connected to c-AMP (Kull et al. 2000). Moreover, in the brain, adenosine level
increases following ischemia-reperfusion injury leading to the stimulation of A2A
AR resulting in the potentiation of neuronal damage by increasing ERK and
consequent stimulation of microglial activation, glial TNFα, glutamate, iNOS, and
apoptosis (Mohamed et al. 2016). In the artery of rat tail, it has been observed that
A2A AR can also regulate the release of norepinephrine through the stimulation of
both PKC and PKA (Fresco et al., 2004). A2A AR is also found to bind with the help
of its C-terminus with various other proteins such as dopamine D2 receptor,
α-actinin, ARNO, USP4, and TRAX (Baraldi et al. 2008). Importantly, A2A AR
can also modulate the signaling of MAPK (Baraldi et al. 2008; Chen et al. 2013).
A2A AR activation also plays an important role in cancer cells by stimulating
10 Pharmacology of Adenosine Receptors 335

A1 AR

AC

+ Gi -
PIP2 DAG
ATP cAMP
IP3 PKC

-
+ - P P
PKA
P
P38 JNK

ERK1/2

K+ channel Ca+2 channel

Fig. 10.2 Molecular signal transduction pathways of A1 AR

proliferation PLC, PKC-δ, ERK, JNK, and AKT (Gessi et al. 2017). Signal trans-
duction pathway of A2A AR is depicted in Fig. 10.3.

10.5.3 Molecular Signaling of A2B AR

Similar to the A2A AR subtype, the A2B AR is also coupled to Gs protein, triggering
the AC activity and thereby increasing the cAMP levels, PKA phosphorylation, and
cAMP-dependent recruitment of different effectors like exchange proteins (Epac)
(Fredholm et al. 2011). A2B AR-stimulated activation of Epac was also found to
affect the proliferation of umbilical vascular endothelial cells and induce early gene
expression reducing the proliferation of smooth muscle cells of coronary artery in
humans (Fang and Olah 2007; Mayer et al. 2011). Unlike A2A AR, the A2B AR is
also coupled to Gq protein, stimulating PLC leading to Ca2+ mobilization, while
regulating the ion channels through the recruitment of γ subunits. A2B AR can
regulate various pathophysiological functions in the central and peripheral system
through the activation of MAPK and AKT (Sun and Huang 2016). Additionally, A2B
AR responses can be influenced by its various binding partners like netrin-1,
E3KARP-EZRIN-PKA, SNARE, NF-κB1/P105, and α-actinin-1. In particular, the
neuronal guidance protein netrin-1 can bind and activate A2B AR during hypoxia,
reducing the migration of neutrophils and consequent inflammation (Rosenberger
336 P. K. Deb et al.

A2A AR

AC

Gs Gs +
GTP
GDP ATP cAMP
+
P
+ PKA
P

P38
P
JNK
+ +
ERK1/2 P P

AKT CREB

Fig. 10.3 Molecular signal transduction pathways of A2A AR

et al. 2009). SNARE protein can bind and translocate the A2B AR from the
cytoplasm to the plasma membrane following agonist binding (Wang et al. 2004)
and consequently, a multiprotein complex with E3KARP (NHERF2) and ezrin
enables the fixation/stabilization of the A2B AR at the cell surface (Sitaraman et al.
2002). Interestingly, α-actinin-1 can promote the dimerization of A2A and A2B ARs,
inducing the cell surface expression of the later (Moriyama and Sitkovsky 2010).
Furthermore, interaction of P105 with A2B AR has shown to reduce the inflamma-
tory effects of NF-κB (Sun et al. 2012). Recently, it has been reported that the
stimulation of A2B AR reduces ERK1/2, p38, and NF-κB induced by RANKL,
thereby reducing osteoclastogenesis in bone (Kim et al. 2017). Several reports also
indicate the role of A2B AR signaling in neuroinflammation (Koscsó et al. 2012;
Merighi et al. 2017), inflammatory bowel disease (Chin et al. 2012; Dammen et al.
2013), cardiac ischemic preconditioning (Yang et al. 2011), atherosclerosis devel-
opment (Gessi et al. 2010a), and reduction of cardiac fibrosis (Phosri et al. 2018,
2017). The signal transduction pathway of A2B AR is depicted in Fig. 10.4.

10.5.4 Molecular Signaling of A3 AR

The A3 AR subtype is coupled to Gi protein and inhibits AC with consequent

reduction of the cAMP levels, while at high concentrations of agonist, A3 AR
couples to Gq protein, thereby stimulating PLC and increasing the Ca2+ release
from the intracellular storage (Borea et al. 2018a, b). A decrease in cAMP level
further causes inhibition of PKA leading to increase in glycogen synthase kinase-3β
(GSK-3β); decrease in β-catenin, cyclin D1, and c-Myc; and reduction of NF-kB
10 Pharmacology of Adenosine Receptors 337

AAB AR

AC
Gq Gs +
+ Gs
PIP2 DAG GTP GTP
GDP ATP cAMP
IP3 PKC +
P
+ PKA
P
P
P38 JNK
AKT
ERK1/2

Fig. 10.4 Molecular signal transduction pathways of A2B AR

DNA binding capability (Fishman et al. 2012, 2004, 2002; Stemmer et al. 2008). A3
AR facilitated neuro- and cardio-protection is regulated via different signaling
pathways including G-protein RhoA and phospholipase D (PLD) (Borea et al.
2018a, b). A3 AR-mediated anti-inflammatory effects are regulated through
MAPK, PI3/Akt, and NF-kB transduction pathways (Ochaion et al. 2008). A3 AR
is also found to induce ERK1/2 and proliferation of cells in human fetal astrocytes,
microglia, glioblastoma, and melanoma among others (Hammarberg et al. 2003;
Merighi et al. 2007; Neary et al. 1998; Soares et al. 2014). Interestingly, reduced
ERK activation was also evident in melanoma, prostate cancer, and glioma cells,
decreasing the proliferation of cells and release of TNF-α (Hyun et al. 2012; Martin
et al. 2006). Activation of A3 AR also modulates p38 and JNK in various cell types
including cancer cells like colon carcinoma (Gessi et al. 2010b). The signal trans-
duction pathway of A3 AR is depicted in Fig. 10.5.
Readers are also encouraged to read the valuable chapter written by Merigi et al.,
highlighting various research findings showcasing the involvement of AR signaling
in diverse pathophysiological conditions (Merighi et al. 2018).

10.6 Agonists, Partial Agonists, Antagonists, and Allosteric

Modulators of Adenosine Receptors

10.6.1 Agonists of Adenosine Receptors

Important agonists of adenosine receptors are presented in Fig. 10.6.

338 P. K. Deb et al.

A3 AR

AC

+ Gi -
PIP2 DAG
ATP cAMP
IP3 PKC P
+
P
-
P PKA
P38 JNK
-
-
ERK1/2
NFkb
+ GSK-3b
PKB

+ - -
Cyclin-D1 - b -catenin

C-Myc

Fig. 10.5 Molecular signal transduction pathways of A3 AR

NH2 NH2
NH2
N N N NH
N N
N
N HN CH3 N
N N O N N O
O N Br
O N O O
OH HN OH

OH HO OH
OH OH
OH
Regadenoson (5) NECA (7) MRS 3997 (8)

I I

HN HN
N N N N
N N N N Cl
O OH O
H OH
H N
N
OH OH
O O

Piclidenoson (9) Namodenoson (10)

Fig. 10.6 Important agonists of ARs

10.6.1.1 Regadenoson
Regadenoson (5), a selective A2A adenosine receptor agonist, was approved by the
FDA (Food and Drug Administration) in 2008 in the injection form as a pharmaco-
logic stress agent for patients unable to perform adequate exercise in order to
increase blood flow in coronary arteries for myocardial perfusion imaging (MPI)
10 Pharmacology of Adenosine Receptors 339

test (Thompson 2008). Regadenoson produces coronary arteries vasodilation by

selectively activating A2A AR; however it shows a very weak agonist activity on
A1 receptors and a negligible affinity for A3 and A2B adenosine receptors.
Regadenoson has longer half-life than adenosine (Vij et al. 2018).
Following the approval of the FDA for regadenoson, many diverse clinical trials
have been performed for the diagnosis and treatment of cardiovascular conditions.
For instance, a phase IIIb study (NCT01618669) sponsored by Astellas Pharma Inc.
on 1147 participants was conducted to compare between administration of
regadenoson after inadequate exercise and administration of regadenoson without
exercise for MPI by using single photon emission computed tomography (SPECT).
Results have shown that the administration of regadenoson after 3 min of inadequate
exercise is well tolerated with careful monitoring in patients without signs and
symptoms of ischemia during exercise or after (Thomas et al. 2017). A study on
123 patients to determine the safety of regadenoson stress testing after orthotopic
heart transplantation (OHT) has shown that dyspnea was the most common side
effect with 66.7% of patients. However, there were no serious adverse effects such as
hemodynamic changes and life-threatening arrhythmias which supports its safety
and tolerability in OHT patients (Lazarus et al. 2018). Several studies have shown
that dyspnea (the most common side effect) is not caused by bronchoconstriction,
which makes regadenoson administration safe for patients with mild to moderate
COPD and mild to moderate asthma (Golzar and Doukky 2014; Raines et al. 2019).
Agonists of A2A AR have shown to decrease hypoxia/reoxygenation-induced
tissue inflammation in mice with SCD (sickle cell disease). A2A agonists reduced
invariant natural killer T (iNKT) cells activation, which is higher than normal in
patients with SCD. A phase II randomized trial (NCT01788631) on patients with
SCD was conducted to test whether regadenoson can reduce iNKT cells activation
and vaso-occlusive crises. After 48-h infusion of regadenoson (1.4 mg/kg/h) during
vaso-occlusive crises the patients did not show significant decrease in iNKT cells
activation as compared to placebo patients which indicates that regadenoson infu-
sion in low doses is not sufficient to induce a significant reduction in iNKT cells
activity (Field et al. 2019). The iNKT cells are also activated after lung transplanta-
tion due to activation of NOX2 (NADPH oxidase 2) causing ischemia-reperfusion
(IR) injury following lung transplantation, and the activation of iNKT cells and
NOX2 increases the production of interleukin-17 (IL-17). An in vivo study showed
that A2A receptor agonists attenuate the production of IL-17 and reduce IR injury in
murine and human iNKT cells which indicates that A2A AR agonists offer a
possible therapeutic strategy to prevent IR injury and graft dysfunction (Sharma
et al. 2016).
Regadenoson has also shown to cause BBB disruption in healthy rodents, which
presents a potential solution for the limitations caused by the BBB in preventing
many therapeutic agents including chemotherapy to reach the brain in higher
concentrations. In a study on healthy rodents, regadenoson increased the concentra-
tion of temozolomide (a chemotherapeutic agent used in the treatment of glioblas-
toma) (Jackson et al. 2016). However, a clinical trial (NCT02389738) by Sidney
Kimmel Comprehensive Cancer Center at Johns Hopkins included six patients with
340 P. K. Deb et al.

recurrent glioblastoma that received regadenoson with temozolomide. Results

showed no increase in temozolomide concentration in brain unlike previous studies
on rodents indicating that further studies and trials with different doses are needed
for determining the optimum regadenoson dose to induce the desired BBB disrup-
tion and increase chemotherapeutic agent concentration in CNS. Another phase I
trial (NCT03971734) is estimated to start in March 2020 by the same cancer center
to determine regadenoson dose that can alter BBB integrity in patients with high-
grade gliomas (Jackson et al. 2018).
Approximately 2–8% of patients experienced gastrointestinal side effects includ-
ing abdominal discomfort, diarrhea, and nausea after receiving regadenoson with
higher side effects frequency in patients with advanced renal disease. However, in
2017, there has been 11 cases of partial seizures and seizure-like adverse effects and
55 cases of convolutions reported to the FDA, which resulted in The American
Society of Nuclear Cardiology (ASNC) guidelines to consider seizure disorders a
relative contraindication with regadenoson administration (Andrikopoulou and Hage
2018; Henzlova et al. 2016).

10.6.1.2 NECA
In recent years, adenosine receptors have shown to be possible pharmacological
targets to alter BBB integrity. A study included intravenous administration of NECA
(50 -N-ethylcarboxamide adenosine) (6), a nonselective ARs agonist, has resulted in
increasing brain concentration of dextrans (both low molecular weight and high
molecular weight). However, NECA pharmacological effect was dose-specific,
producing highest effect at 0.08 mg/kg; lower or higher doses showed less effect.
It was interpreted that doses higher than 0.08 mg/kg of NECA showed less effect due
to adenosine receptors desensitization. The fact that adenosine receptor agonists can
be found in the market and are clinically approved makes these findings even more
valuable presenting a possible less invasive method for BBB disruption (Carman
et al. 2011; Cheng et al. 2016; Malpass 2011).
NECA intraperitoneal administration has shown to increase fasting serum glucose
level. Further investigation showed that NECA administration has elevated glucose
6-phosphatase (G6Pase) enzyme mRNA leading to an increase in the liver G6Pase
enzyme and gluconeogenesis, which is thought to be the cause for serum glucose
elevation (Matsuda et al. 2014). NECA has also been studied for reducing intestinal
IR injury in rats. Results showed that NECA reduced leukocyte activation and
caused a significant improvement in capillary perfusion, thus reducing intestinal
IR injury (Zhou et al. 2015).

10.6.1.3 MRS 3997

MRS 3997 (7) is a potent adenosine receptor agonist that activates mainly A2A and
A2B AR and acts as a weak agonist for A1 and A3 ARs (Adachi et al. 2007; Gao et al.
2014).
10 Pharmacology of Adenosine Receptors 341

10.6.1.4 Piclidenoson, CF101

Piclidenoson or CF101 (8) is a highly specific A3 AR agonist that has proven to have
an anti-inflammatory effect in many preclinical studies for conditions such as uveitis,
rheumatoid arthritis, colitis, and osteoarthritis. Piclidenoson mechanism of action is
mainly through the downregulation of NF-κB signaling pathway which causes an
inhibition in TNF-α. Phase II clinical studies of piclidenoson on patients with plaque
psoriasis have shown its efficacy in reducing signs and symptoms (Cohen et al.
2018).
A phase IIb clinical study (NCT01034306) utilizing piclidenoson as a
monotherapy drug was conducted on 79 patients with rheumatoid arthritis sponsored
by Can-Fite BioPharma. After 12 weeks of twice daily administration of 1 mg of
piclidenoson or placebo, the patients treated with piclidenoson showed a significant
improvement compared to placebo and reduction in rheumatoid arthritis symptoms,
supporting previous clinical studies (Fishman and Cohen 2016; Stoilov et al. 2014).
The same company is currently developing piclidenoson in an oral form as a first-
line treatment for patients with moderate to severe plaque psoriasis (Fellner 2016).

10.6.1.5 Namodenoson, CF102

Namodenoson (CF102) (9) is a potent and selective A3 AR agonist that is considered
safe and tolerable after phase I and II (NCT00790218) clinical trials for hepatocel-
lular carcinoma in combination with sorafenib. In those trials namodenoson has
caused an increase in the median overall survival by approximately 7 months
(Stemmer et al. 2013). Namodenoson has been tested in a phase II trial
(NCT02128958) as a second-line treatment of Child-Pugh B (CPB) advanced
hepatocellular carcinoma (HCC). Despite the fact that the primary end point has
not been met in this trial, the median overall survival of CPB patients increased.
Namodenoson was well tolerated by patients and considered safe for further phase
III trials. Adverse effects that were observed in almost >10% of the patients were
nausea, fatigue, anemia, asthenia, peripheral edema, and abdominal pain (Stemmer
et al. 2019).

10.6.2 Partial Agonists of Adenosine Receptors

Important partial agonists of adenosine receptors are presented in Fig. 10.7.

10.6.2.1 CVT 2759

CVT-2759 (10) is a partial A1 AR agonist that has shown to have the ability to
selectively inhibit AV conduction in a moderate rate without causing an AV block
despite application of high concentrations. It means that CVT-2759 has the ability to
cause a predictable moderate inhibition on the AV nodal conduction while avoiding
the risk of AV blockage. It has been observed in these studies that CVT-2759 has a
minimum effect on the sinoatrial rate or on action potential durations (ventricular
and atrial) (Szentmiklósi et al. 2015; Wu et al. 2001). Accordingly, CVT-2759
administration does not induce flutter of atrial fibrillation. Most importantly, A1
342 P. K. Deb et al.

O OH
HN
N O
N
N
N
O
OH N N
C C
O O OH
N Cl
H2N N S
NH
S
CVT-2759 (11) Capadenoson (BAY68-4986) (12)

O
O
N
H
OH O NH2

O O

N N N N
C C C C

N N Cl
N N S Cl N N S

S S
Neladenoson (13) Neladenoson bialanate (BAY 1067197) (14)

Fig. 10.7 Important partial agonists of ARs

AR partial agonists induce desensitization and downregulation of the receptor much

less than full agonists, which makes these compounds a great option in treating
certain cardiac arrhythmias while avoiding nonspecific adverse effects that are seen
with adenosine administration.

10.6.2.2 Capadenoson (BAY68-4986)

Capadenoson (11) is a non-nucleoside A1 AR partial agonist that has reached clinical
trials, two phase II trials, one for patients with atrial fibrillation and the other for
patients with stable angina (Albrecht-küpper et al. 2012; Szentmiklósi et al. 2015).
Capadenoson was also investigated for advanced heart failure in animal models and
has shown to reduce cardiac remodeling. Neladenoson bialanate is a capadenoson
derivative that has entered clinical trials to treat patients with chronic heart failure.
Mainly the therapeutic action of capadenoson is due to the partial activation of A1
adenosine receptors, but it is important to note that capadenoson can also stimulate
A2B adenosine receptors. A study was conducted to investigate the effect of
capadenoson on A2B AR in cardiomyocytes, cardiac fibroblasts (physiologically
relevant cells). Results have shown a significant effect on A2B AR by capadenoson,
suggesting that capadenoson should be reclassified from an A1 AR partial agonist
into a dual A1AR/A2BAR agonist (Baltos et al. 2017). A phase II clinical trial
(NCT00518921) of capadenoson was also conducted to evaluate the efficacy and
safety in patients with stable angina with 1–4 mg doses; however, the trial was later
withdrawn (Jacobson et al. 2019).
10 Pharmacology of Adenosine Receptors 343

10.6.2.3 Neladenoson
Neladenoson, an A1 AR partial agonist (12), currently is being tested clinically on
patients with chronic heart failure in the form of dipeptide prodrug. Neladenoson
shows higher selectivity to A1 AR as compared to capadenoson. Many promising
effects caused by Neladenoson have been observed including improvement in
cardiac function without causing undesired effects on blood pressure, atrioventricu-
lar blocks, or bradycardia. The preference of using a partial agonist instead of full
agonist of A1 AR is due to the fact that partial agonist can activate the receptors
without producing severe adverse effects as compared to full agonists. A multiple
dose phase II study (NCT02040233) of Neladenoson has been also conducted to
investigate tolerability, pharmacokinetics, and safety in patients with chronic heart
failure (ParSiFAL study) (Jacobson et al. 2019; Voors et al. 2017).

10.6.2.4 Neladenoson Bialanate

Neladenoson bialanate (13), also referred to as BAY-1067197, is a prodrug of
Neladenoson, an A1AR partial agonist with high potency and selectivity. The need
to develop a partial A1 AR agonist comes from the fact that a full agonist produces
extra-cardiac adverse effects including neurological (e.g., sedation) and anti-diuretic
effects due to the vasoconstriction of renal afferent arterioles caused by the activation
A1 AR (Dinh et al. 2017; Greene et al. 2016).
Preclinical studies of Neladenoson bialanate have shown promising results
including anti-ischemic cardio-protective properties, improved mitochondrial func-
tion, and preventing ventricular remodeling, which further supported this compound
for phase II clinical trials such as PANACHE (NCT03098979) and PANTHEON
(NCT02992288) trials. PANACHE trial was to evaluate Neladenoson in patients
with chronic heart failure with preserved ejection fraction (HFpEF) while PAN
THEON trial was for evaluating it on patients with chronic heart failure with reduced
ejection fraction (HFrEF). Both trials were conducted to evaluate the safety and
efficacy of the compound and both of these trials were sponsored by Bayer (Voors
et al. 2018). In PANACHE trial, no significant dose to response relationship has been
detected after 20 weeks of neladenoson administration, which indicates the need for
further investigation and development required for Neladenoson to treat conditions
such as HFpEF (Shah et al. 2019).

10.6.3 Antagonists of Adenosine Receptors

Important antagonists of adenosine receptors are presented in Fig. 10.8.

10.6.3.1 Caffeine and Theophylline

Caffeine (14) (3,7-trimethylpurine-2,6-dione) is a nonselective natural methylamine
that acts as an A2A and A1 AR antagonist. Caffeine can be found in common
beverages such as tea, coffee, products containing cocoa, soft drinks, dietary
sources, and some medications. In the United States, the daily intake of a caffeine
consumer is approximately 280 mg. The main purpose of caffeine consumption is to
344 P. K. Deb et al.

O R O
NH2
H3C N
N N N
N N N O
O N N N O O
O N N
CH3 N N
O N
O N
Caffeine (15), R=CH3
Theophylline (2), R=H Istradefylline (6) Preladenant (16)

H3C

NH2 O F
O F
Br H
N F
N N N N
N N O
N N N N
N N
H2 N N N O O N
N N

PBF 509 (17) CPI 444 (18) CVT 6883 (19)

Fig. 10.8 Important antagonists of ARs

increase energy, alertness, and arousal. In normal population, caffeine consumption

has been associated with mood and cognitive performance changes with more
observed enhancement in performance in fatigued individuals compared to well-
rested ones (López-cruz et al. 2018). Caffeine’s antagonist effect on A2A adenosine
receptors has shown its potentials in treating PD. In vitro and in vivo studies have
both shown that caffeine reduces parkinsonian motor symptoms. Also, drug toler-
ance associated with current PD drugs has been found to be reduced when
co-administered with caffeine (Chen et al. 2010; Roshan et al. 2016). Currently,
caffeine is used as an adjuvant treatment for migraine headache. Clinical trials have
shown that caffeine can reduce postdural puncture headache (PDPH). In addition,
caffeine was reported to produce an effective result in the treatment of hypnic
headache; however, further clinical trials are still needed to prove its efficacy as a
first-line treatment choice (Baratloo et al. 2016, 2015).
Theophylline (dimethylxanthine) (2) is a nonselective A1 and A2 AR antagonist.
It has been used for over 80 years to treat airway diseases. Originally it was used as a
bronchodilator; however the doses that were required were relatively high, which
caused frequent occurrence of adverse effects that lead to the decline of its use and it
was more widely used in inhaled form. Recent studies have shown that theophylline
possess an anti-inflammatory effect in chronic obstructive pulmonary disease
(COPD) and asthma at lower concentrations. Currently, theophylline is used in
patients with asthma as an add-on therapy to inhaled corticosteroids. Theophylline
is also given to patients with severe COPD when symptoms cannot be controlled by
bronchodilators. Side effects of theophylline are related to the plasma concentration
of the drug; most common side effects are headaches, vomiting, and nausea that are
caused by phosphodiesterase (PDE) isoenzymes inhibition. At high concentrations
the inhibition of A1 receptors caused by theophylline induces seizures and cardiac
arrhythmias (Barnes 2013).
10 Pharmacology of Adenosine Receptors 345

10.6.3.2 PBF-680
The PBF-680 is an A1 AR potent antagonist (structure not disclosed) that is currently
in clinical trials for the treatment of asthma. An ongoing phase II trial
(NCT02635945) aimed to evaluate the efficacy of PBF-680 in patients with mild
to moderate asthma. In this study, 10 mg of PBF-680 was administered orally for
5 days; the efficacy was evaluated by the amount to attenuation of late asthmatic
responses that occurs due to allergen broncho-provocation. Previous studies have
shown that the activation of adenosine A1 receptors has a pro-inflammatory role in
certain immune cells and also broncho-constrictory effect in pulmonary tissue.
Adenosine on the other hand has shown to provoke bronchoconstriction in asthmatic
patients, while an adenosine receptor antagonist such as theophylline is an effective
drug for asthma treatment. Selective A1 receptor antagonists may offer a promising
therapeutic option for asthmatic patients in the future (Gao and Jacobson 2017).

10.6.3.3 Istradefylline
Istradefylline (15) was the first selective A2A AR antagonist; initially it was available
only in Japan for treating the wearing-off phenomenon in Parkinson’s disease
patients receiving levodopa-containing treatment (Saki et al. 2013).
A recent clinical trial of Istradefylline on 31 patients with Parkinson’s disease has
proven its effect in decreasing gait disorders including slow walking speed, short
steps, forward-bent posture, toe dragging, and reduced arm swing which improved
the quality of life of those patients without a serious adverse effect detected (Iijima
et al. 2019). Istradefylline has also been investigated in clinical trials for improving
mood disorders in PD patients. Doses between 20 and 40 mg of Istradefylline were
administered for 12 weeks. Results have shown an improvement in overall mood
disorders. However, further trials are needed to confirm the effectiveness of
istradefylline due to the fact that this trial recruited only 30 patients with dropout
rate of 17% and it was an open-label trial which indicates the possibility of placebo
effect in patients (Nagayama et al. 2019). Recently, it has got the US FDA approval
(2019) and available in the market as an add-on to levodopa/carbidopa for the
treatment of PD (Hoffman 2019; Voelker 2019).

10.6.3.4 Preladenant
Preladenant (16) is an A2A AR antagonist; mainly it was developed to treat patients
with PD. However, clinical trials have not been successful and got discontinued. The
development of preladenant was discontinued in 2013 after two phase III clinical
trials to test its efficacy in treating fluctuating motor disturbances in patients. Results
indicated that preladenant had no significant effect as compared to placebo (Pinna
et al. 2018).
A preladenant phase I study (NCT03099161) in combination with
pembrolizumab was conducted to treat neoplasm. Solid tumors that do not respond
to conventional therapy were targeted in the trial. The study was to assess the
efficacy and safety of preladenant as a treatment and to set the recommended dose
for further clinical trials. However, the study was terminated because the data did not
support the study end point (Congreve et al. 2018).
346 P. K. Deb et al.

10.6.3.5 PBF-509
PBF-509 (17) is a non-xanthine potent A2A AR antagonist that has been tested for
the treatment of PD on rodent models. Studies have shown its efficacy in reducing
pilocarpine-induced tremulous jaw movements, haloperidol-mediated catalepsy, and
L-DOPA-induced dyskinesia, which indicates that PBF-509 is an anti-dyskinetic
agent along with reversing parkinsonian motor impairments making it a potential
treatment option for PD in the future (Núñez et al. 2018).

10.6.3.6 CPI-444
CPI-444 (18) is a selective and highly potent A2A AR antagonist for oral adminis-
tration. The adenosine A2A receptors expressed on immune cells have a suppressive
effect on antitumor activity. Blockage of this receptor with a compound such as
CPI-444 has shown to restore IL2 and IFNγ production and T-cell signaling in
in vitro studies. Preclinical studies of CPI-444 on mice have proven its efficacy in
producing antitumor response when anti–PD-L1 immunotherapy failed to produce
the required therapeutic response. The mechanism that explains how blocking of
A2A receptors can overcome the resistance of anti–PD-L1 treatment is still under
investigation (Willingham et al. 2018).
A clinical phase I trial (NCT02655822) is currently ongoing (by Corvus
Pharmaceuticals, Inc.) for dose selection, tolerability, and safety of CPI-444 as a
single antitumor agent or in combination with atezolizumab. Adenosine has shown
to suppress antitumor activity in immune cells (T-cells) (Mobasher et al. 2019).

10.6.3.7 CVT 6883 (GS-6201)

CVT-6883 (19) is a selective and potent A2B AR antagonist. Preclinical studies have
shown that CVT-6883 has an inhibitory effect on pulmonary injury and inflamma-
tion in bleomycin-induced fibrosis models and adenosine deaminase-deficient mice.
CVT-6883 has also shown to reduce airway reactivity induced by allergen or NECA
in sensitized mice. However, CVT-6883 was discontinued from phase I clinical trials
(Basu et al. 2016).
CVT-6883 has also shown to significantly reduce lung fibrosis mediators in
multi-walled carbon nanotube (MWCNT) treated mice. CVT-6883 has also
decreased inflammatory and cytotoxicity in animal models, which indicates that a
selective A2B AR antagonist might offer a possible treatment option for MWCNT-
induced lung fibrosis in humans and requires further investigation and development
(Liu et al. 2019).

10.6.4 Allosteric Modulators of ARs

Important allosteric modulators of adenosine receptors are presented in Fig. 10.9.

10.6.4.1 T-62 and LUF 5484

T62 (20) is a positive allosteric modulator (PAM) of A1 AR. T62 preclinical studies
have shown that oral administration caused a reduction in hypersensitivity
10 Pharmacology of Adenosine Receptors 347

O
R O
N HN
HN HN
NH
H3 C
N OCH3 N N
O N NH N NH
N
S NH2 H
N

T-62 (20), R=4-Cl VUF 5455 (22) Cl Cl

Cl Cl
LUF5484 (21), R=3,4-Cl2 LUF6000 (23) LUF6096 (24) DU124183 (25)

Fig. 10.9 Important allosteric modulators of ARs

neuropathic pain and inflammatory models. It was also noticed to induce sedation
after the initial dosing; 5 days after daily administration tolerance has occurred due to
downregulation of the A1 AR. T62 has progressed into clinical trials, a phase II trial
(NCT00809679) to evaluate the safety and efficacy of this compound as an analgesic
for patients with postherpetic neuralgia. However, some patients experienced tran-
sient elevations in liver enzymes (transaminases) which terminated the study
(Romagnoli et al. 2015; Sawynok 2016). LUF 5484 (2-amino-4,5,6,7-
tetrahydrobenzo[b]thiophen-3-yl)(3,4-dichlorophenyl)methanone (21) is an A1
adenosine receptor allosteric modulator (Bueters et al. 2002).

10.6.4.2 VUF5455
VUF5455 (22) is a 3-(2-pyridinyl) isoquinoline derivative, the first selective PAM of
A3 AR. VUF5455 enhances the binding of A3 receptor agonists and increases the
dissociation rate of antagonist (Briddon et al. 2018; Soudijn et al. 2006).

10.6.4.3 LUF6000
LUF6000 (2-Cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-
amine) (23), an A3 AR PAM, increases the activity of orthosteric agonists. The
maximal effect of the native ligand increases by 45% when an allosteric enhancer
binds to the receptor. LUF6000 has been studied on animal models including mice
and rats, and results have shown that LUF6000 induces anti-inflammatory effect by
slightly stimulating neutrophils and normal white blood cells (Cohen et al. 2014).

10.6.4.4 LUF6096
LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-
amide) (24) is a positive A3 AR allosteric modulator; it was developed by the
scission of the imidazole ring of LUF6000. LUF6096 has been through preclinical
studies on animal models and human cell membranes to evaluate its efficacy in
reducing myocardial ischemia/reperfusion injury. Results have shown that LUF6096
is well tolerated and effective in deceasing the myocardial ischemia/reperfusion
injury on dog models (Du et al. 2018, 2012).
348 P. K. Deb et al.

10.6.4.5 DU124183
DU124183 (2-cyclopentyl-4-phenylamino-1H-imidazo[4,5-c]quinoline) (25) is a
selective allosteric modulator that enhances agonist binding and function of A3
AR (Göblyös and Ijzerman 2009). DU124183 causes a decrease in agonist potency
meanwhile enhancing its maximum effect (Emax) (Gao et al. 2008).

10.7 Conclusions

Adenosine and its four receptor subtypes (A1, A2A, A2B, and A3 ARs) are widely
distributed throughout the body, modulating the physiological and pathological
conditions of almost every organs and tissues. The ubiquitous distribution of ARs
not only signifies their potential drug targets but also imposed a great challenge in
the process of discovery and development of drugs selectively targeting a particular
subtype of AR in disease-specific tissues, while culminating in undesirable side
effects. In the last three decades, extensive research efforts from academia and
pharmaceutical industries resulted in the discovery of various potential ligands
targeting ARs, but only few of them could sustain the clinical trials to successfully
reach the market. Istradefylline, an A2A selective antagonist, is the most recently US
FDA approved (2019) drug available in the market as an add-on to levodopa/
carbidopa for the treatment of PD. Moreover, the recent discovery of the 3D crystal
structure of A1 AR and the previously identified 3D structure of A2A AR have not
only enhanced the understanding of the binding site topology of these receptors but
also facilitated the development of improved homology models of other two AR
subtypes as well as computer-aided structure-based strategies to design and discover
novel AR-specific ligands. In this regard, the future discovery of the 3D crystal
structures of remaining A2B and A3 ARs would further provide a clear insight into all
the four subtypes of ARs, thus boost up the rational drug discovery process and
development of novel clinical candidates, selectively targeting a particular AR
subtype relevant to the therapeutic intervention of specific pathological disorders.

References
Adachi H, Palaniappan KK, Ivanov AA, Bergman N, Gao Z, Jacobson KA (2007) Structure-activity
relationships of 2,N6,50 -substituted adenosine derivatives with potent activity at the A2B
adenosine receptor. J Med Chem 50:1810–1827
Addi AB, Lefort A, Hua X, Libert F, Communi D, Ledent C, Macours P, Tilley SL, Boeynaems J,
Robaye B (2008) Modulation of murine dendritic cell function by adenine nucleotides and
adenosine: involvement of the A 2B receptor. Eur J Immunol 38:1610–1620
Agrawal N, Chandrasekaran B, Al-Aboudi A (2019) Recent advances in the in-silico structure-
based and ligand-based approaches for the design and discovery of agonists and antagonists of
A2A adenosine receptor. Curr Pharm Des 25:774–782
Aherne CM, Kewley EM, Eltzschig HK (2011) The resurgence of A2B adenosine receptor
signaling. Biochim Biophys Acta 1808:1329–1339. https://doi.org/10.1016/j.bbamem.2010.
05.016
10 Pharmacology of Adenosine Receptors 349

Al-attraqchi OHA, Attimarad M, Venugopala KN, Nair A, Noor HA (2019) Adenosine A2A
receptor as a potential drug target - current status and future perspectives. Curr Pharm Des
25:2716–2740
Albrecht-küpper BE, Leineweber K, Nell PG (2012) Partial adenosine A1 receptor agonists for
cardiovascular therapies. Purinergic Signal 8:91–99
Al-Shar’i Nizar A, Al-Balas QA (2019) Molecular dynamics simulations of adenosine receptors:
advances, applications and trends. Curr Pharm Des 25:783–816
Andrikopoulou E, Hage FG (2018) Adverse effects associated with regadenoson myocardial
perfusion imaging. J Nucl Cardiol 25:1724–1731. https://doi.org/10.1007/s12350-018-1218-7
Bading B, Kelm M, Schrader J (1993) Formation and salvage of adenosine by macrovascular
endothelial cells. Am J Physiol Circ Physiol 264:H692–H700
Ballesteros-yáñez I, Castillo CA, Merighi S (2018) The role of adenosine receptors in
psychostimulant addiction. Front Pharmacol 8:1–18
Baltos J, Vecchio EA, Harris MA, Xue C, Ritchie RH, Christopoulos A, White PJ, May LT (2017)
Capadenoson, a clinically trialed partial adenosine A1 receptor agonist, can stimulate adenosine
A2B receptor biased agonism. Biochem Pharmacol 135:79–89. https://doi.org/10.1016/j.bcp.
2017.03.014
Baraldi PG, Tabrizi MA, Gessi S, Borea PA (2008) Adenosine receptor antagonists: translating
medicinal chemistry and pharmacology into clinical utility. Chem Rev 108:238–263
Baratloo A, Negida A, El Ashal G, Behnaz N (2015) Intravenous caffeine for the treatment of acute
migraine: a pilot study. J Caffeine Res 5:1–5
Baratloo A, Rouhipour A, Forouzanfar MM, Safari S, Amiri M, Negida A (2016) The role of
caffeine in pain management: a brief literature review. Anesth Pain Med 6:e33193
Barnes PJ (2013) Theophylline. Am J Respir Crit Care Med 188:901–906
Basheer R, Arrigoni E, Thatte HS, Greene RW, Ambudkar IS, McCarley RW (2002) Adenosine
induces inositol 1,4,5-trisphosphate receptor-mediated mobilization of intracellular calcium
stores in basal forebrain cholinergic neurons. J Neurosci 22:7680–7686
Basu S, Barawkar DA, Ramdas V, Waman Y, Patel M, Panmand A, Kumar S, Thorat S, Bonagiri R,
Jadhav D, Mukhopadhyay P, Prasad V, Reddy BS, Goswami A, Chaturvedi S, Menon S,
Quraishi A, Ghosh I, Dusange S, Paliwal S, Kulkarni A, Karande V, Thakre R, Bedse G,
Rouduri S, Gundu J, Palle VP, Chugh A, Mookhtiar KA (2016) A2B adenosine receptor
antagonists: design, synthesis and biological evaluation of novel xanthine derivatives. Eur J
Med Chem 127:986–996. https://doi.org/10.1016/j.ejmech.2016.11.007
Biber K, Klotz K, Berger M, Gebicke-ha PJ, Van Calker D (1997) Adenosine A1 receptor-mediated
activation of phospholipase C in cultured astrocytes depends on the level of receptor expression.
J Neurosci 17:4956–4964
Bjursell MK, Blom HJ, Cayuela JA, Engvall ML, Lesko N, Halldin M, Falkenberg M, Jakobs C,
Balasubramaniam S, Gustafsson CM, Lundeberg J, Smith D, Struys E, Von Do U, Wedell A
(2011) Adenosine kinase deficiency disrupts the methionine cycle and causes
hypermethioninemia, encephalopathy, and abnormal liver function. Am J Hum Genet
89:507–515
Boison D (2018) Regulation of extracellular adenosine. In: Borea P., Varani, K., Gessi, S., Merighi,
S., Vincenzi, F., (Eds.), The adenosine receptors. pp. 13–32
Boison D, Stone R, Neurobiology D (2013) Adenosine kinase: exploitation for therapeutic gain.
Pharmacol Rev 65:906–943
Borah P, Deka S, Mailavaram RP, Deb PK (2019) P1 receptor agonists/antagonists in clinical trials -
potential drug candidates of the future. Curr Pharm Des 25:2792–2807
Borea PA, Varani K, Vincenzi F, Baraldi PG, Tabrizi MA, Merighi S, Gessi S (2015) The A3
adenosine receptor: history and perspectives. Pharmacol Rev 67(1:74–102.
Borea PA, Gessi S, Merighi S, Varani K (2016) Adenosine as a multi-signalling guardian angel in
human diseases: when, where and how does it exert its protective effects? Trends Pharmacol Sci
37:419–434. https://doi.org/10.1016/j.tips.2016.02.006
350 P. K. Deb et al.

Borea P, Varani K, Gessi S, Merighi S, Vincenzi F (2018a) The adenosine receptors, vol 2018.
Springer, New York, NY
Borea PA, Gessi S, Merighi S, Vincenzi F, Varani K (2018b) Pharmacology of adenosine receptors:
the state of the art. Am Physiol Soc 98:1591–1625
Boros D, Thompson J, Larson DF (2016) Adenosine regulation of the immune response initiated by
ischemia reperfusion injury. Perfusion 31:103–110
Briddon SJ, Kilpatrick LE, Hill SJ (2018) Studying GPCR pharmacology in membrane
microdomains: fluorescence correlation spectroscopy comes of Age. Trends Pharmacol Sci
39:158–174. https://doi.org/10.1016/j.tips.2017.11.004
Brugarolas M, Navarro G, Mart E, Angelats E, Casad V, Lanciego L, Franco R (2014) G-protein-
coupled receptor heteromers as key players in the molecular architecture of the central nervous
system. CNS Neurosci Ther 20:703–709
Bueters TJH, Van Helden HPM, Danhof M, Ijzerman AP (2002) Effects of the adenosine A1
receptor allosteric modulators PD 81,723 and LUF 5484 on the striatal acetylcholine release.
Eur J Pharmacol. 454:177–182
Burnett LA, Blais EM, Unadkat JD, Hille B, Tilley SL, Babcock DF (2010) Testicular expression of
Adora3i2 in Adora3 knockout mice reveals a role of mouse A3Ri2 and human A3Ri3 adenosine
receptors in sperm . J Biol Chem 285:33662–33670
Cagnina RE, Ramos SI, Marshall MA, Wang G, Frazier CR, Linden J, Re C, Si R, Ma M, Wang G,
Cr F (2009) Adenosine A2B receptors are highly expressed on murine type II alveolar epithelial
cells. Am J Physiol Lung Cell Mol Physiol 297:467–475
Carman AJ, Mills JH, Krenz A, Kim D, Bynoe MS (2011) Adenosine receptor signaling modulates
permeability of the blood – brain barrier. J Neurosci 31:13272–13280
Chandrasekaran B, Samarneh S, Jaber A, Kassab G, Agrawal N (2019) Therapeutic potentials of
A2B adenosine receptor ligands: current status and perspectives. Curr Pharm Des 25:2741–2771
Chen X, Ghribi O, Geiger JD (2010) Caffeine protects against disruptions of the blood-brain barrier
in animal models of Alzheimer’s and Parkinson’s diseases. J Alzheimers Dis 20:S127–S141
Chen J, Eltzschig HK, Fredholm BB (2013) Adenosine receptors as drug targets — what are the
challenges? Nat Rev Drug Discov 12:265
Cheng C, Yang YL, Liao KH, Lai TW (2016) Adenosine receptor agonist NECA increases cerebral
extravasation of fluorescein and low molecular weight dextran independent of blood-brain
barrier modulation. Sci Rep 6:1–9. https://doi.org/10.1038/srep23882
Cheng RKY, Segala E, Robertson N, Marshall FH, Cooke RM, Errey JC, Marshall FH, Cooke RM
(2017) Structures of human A1 and A2A adenosine receptors with xanthines reveal
determinants of selectivity. Structure 25:1275–1285
Chin A, Svejda B, Gustafsson BI, Granlund AB, Sandvik AK, Timberlake A, Sumpio B,
Pfragner R, Modlin IM, Kidd M, Chin A, Svejda B, Bi G, Ab G, Ak S, Timberlake A,
Sumpio B, Pfragner R, Im M, The KM (2012) The role of mechanical forces and adenosine
in the regulation of intestinal enterochromaffin cell serotonin secretion. Am J Physiol
Gastrointest Liver Physiol 4:397–405
Christofi FL, Zhang H, Yu J, Guzman J (2001) Differential gene expression of adenosine A1, A2a,
A2b, and A3 receptors in the human enteric nervous system. J Comp Neurol 439:46–64
Cohen S, Barer F, Bar-yehuda S, Ijzerman AP, Jacobson KA, Fishman P (2014) A3 adenosine
receptor allosteric modulator induces an anti-inflammatory effect: in vivo studies and molecular
mechanism of action. Mediators Inflamm 2014:1–8
Cohen S, Barer F, Itzhak I, Silverman MH, Fishman P (2018) Inhibition of IL-17 and IL-23 in
human keratinocytes by the A3 adenosine receptor agonist piclidenoson. J Immunol Res
2018:1–9
Congreve M, Brown GA, Borodovsky A, Lamb ML, Congreve M, Brown GA, Borodovsky A,
Lamb ML, Congreve M, Brown GA, Borodovsky A, Lamb ML (2018) Expert opinion on drug
discovery targeting adenosine A2A receptor antagonism for treatment of cancer. Expert Opin
Drug Discov 13:1–7. https://doi.org/10.1080/17460441.2018.1534825
10 Pharmacology of Adenosine Receptors 351

Dammen R, Haugen M, Svejda B, Alaimo D, Brenna O, Pfragner R, Gustafsson BI, Kidd M (2013)
Correction: the stimulatory adenosine receptor ADORA2B regulates serotonin (5-HT) synthesis
and release in oxygen-depleted EC cells in inflammatory bowel disease. PLoS One 8:e62607
Deb PK (2019a) Therapeutic potentials of adenosine receptors: the state of the art. Curr Pharm Des
25:2789–2791
Deb PK (2019b) Progress in the development of agonists, antagonists and allosteric modulators of
adenosine receptors. Curr Pharm Des 25:2695–2696
Deb PK (2019c) Recent updates in the computer aided drug design strategies for the discovery of
agonists and antagonists of adenosine receptors. Curr Pharm Des 25:747–749
Deb PK, Al-attraqchi O, Al-qattan MN, Prasad MR, Tekade RK (2018a) Applications of computers
in pharmaceutical product formulation, dosage form design parameters. Elsevier, Amsterdam.
https://doi.org/10.1016/B978-0-12-814421-3.00019-1
Deb PK, Mailavaram R, Chandrasekaran B, Kaki VR, Kaur R, Kachler S, Klotz K-N, Akkinepally
RR (2018b) Synthesis, adenosine receptor binding and molecular modeling studies of novel
Thieno[2,3-d]pyrimidine derivatives. Chem Biol Drug Des 91:962–969
Deb PK, Chandrasekaran B, Mailavaram R, Tekade RK, Muttaleb A, Jaber Y (2019a) Molecular
modeling approaches for the discovery of adenosine A2B receptor antagonists: current status
and future perspectives. Drug Discov Today 24:1854–1864. https://doi.org/10.1016/j.drudis.
2019.05.011
Deb PK, Deka S, Borah P, Abed SN, Klotz K (2019b) Medicinal chemistry and therapeutic
potential of agonists, antagonists and allosteric modulators of A1 adenosine receptor: current
status and perspectives. Curr Pharm Des 25:2697–2715
Deussen A (2000) Metabolic flux rates of adenosine in the heart. Naunyn Schmiedebergs Arch
Pharmacol 362:351–352
Deussen A, Stappert M, Schafer S, Kelm M (1999) Quantification of extracellular and intracellular
adenosine production understanding the transmembranous concentration gradient. Circulation
99:2041–2047
Dhalla AK, Chisholm JW, Reaven GM (2009) A 1 adenosine receptor: role in diabetes and obesity.
Handb Exp Pharmacol 193:271–295
Dinh W, Albrecht-k B, Gheorghiade M, Voors AA, Van Der Laan M, Sabbah HN (2017) Partial
adenosine A1 agonist in heart failure. In: Bauersachs J, Butler J, Sandner P (eds) Handbook of
experimental pharmacology, vol 243. Springer, New York, NY, pp 177–203
Du L, Gao Z, Nithipatikom K, Ijzerman AP, Van Veldhoven JPD, Jacobson KA, Gross GJ,
Auchampach JA (2012) Protection from myocardial ischemia/reperfusion injury by a positive
allosteric modulator of the A3 adenosine receptor. JPET 340:210–217
Du L, Gao Z, Paoletta S, Wan TC, Gizewski ET, Barbour S, Van Veldhoven JPD, Ijzerman AP
(2018) Species differences and mechanism of action of A3 adenosine receptor allosteric
modulators. Purinergic Signal 14:59–71
Eckle T, Faigle M, Grenz A, Laucher S, Thompson LF, Eltzschig HK (2008) A2B adenosine
receptor dampens hypoxia-induced vascular leak. Blood 111:2024–2036
Ellis JC, Catherine A, Jones C, Flint J (1995) Activating transcription factor-1 is a specific
antagonist of the cyclic adenosine 3’.5’-monophosphate (cAMP) response element-binding
protein-1-mediated response to cAMP. Mol Endocrinol 9:255–265
Elmenhorst D, Meyer PT, Matusch A, Winz OH, Bauer A (2012) Caffeine occupancy of human
cerebral A1 adenosine receptors: in vivo quantification with 18F-CPFPX and PET. J Nucl Med
53:1723–1730
Fang Y, Olah ME (2007) Cyclic AMP-dependent, protein kinase A-independent activation of
extracellular signal-regulated kinase 1/2 following adenosine receptor stimulation in human
umbilical vein endothelial cells: role of exchange protein activated by cAMP 1 (Epac1). J
Pharmacol Exp Ther 322:1189–1200
Fellner C (2016) More biologic therapies expected to treat advanced plaque psoriasis. Pharm Ther
41:388–390
352 P. K. Deb et al.

Ferre S, Navarro G, Casado V, Cortes A, Mallol J, Canela EI, Lluıs C, Franco R (2010) G protein-
coupled receptor heteromers as new targets for drug development I. Prog Mol Biol Transl Sci
91:41–52
Field JJ, Majerus E, Gordeuk VR, Gowhari M, Hoppe C, Heeney MM, Achebe M, George A,
Chu H, Sheehan B, Puligandla M, Neuberg D, Lin G, Linden J, Nathan DG (2019) Randomized
phase 2 trial of regadenoson for treatment of acute vaso-occlusive crises in sickle cell disease.
Blood Adv 1:1645–1649
Finkelstein JD (1998) The metabolism of homocysteine: pathways and regulation. Eur J Pediatr
157 Suppl:40–44
Fishman P, Cohen S (2016) The A3 adenosine receptor (A3AR): therapeutic target and predictive
biological marker in rheumatoid arthritis. Clin Rheumatol 35:2359–2362
Fishman P, Bar-yehuda S, Madi L, Cohn I, Tikva P, Biopharma IC, Tikva P (2002) A3 adenosine
receptor as a target for cancer therapy. Anticancer Drugs 13:437–443
Fishman P, Bar-yehuda S, Ohana G, Barer F, Ochaion A, Erlanger A, Madi L (2004) An agonist to
the A3 adenosine receptor inhibits colon carcinoma growth in mice via modulation of GSK-3b
and NF-jB. Oncogen 23:2465–2471
Fishman P, Bar-yehuda S, Liang BT, Jacobson KA (2012) Pharmacological and therapeutic effects
of A 3 adenosine receptor agonists. Drug Discov Today 17:359–366. https://doi.org/10.1016/j.
drudis.2011.10.007
Fredholm BB (2014) Adenosine—a physiological or pathophysiological agent? J Mol Med
92:201–206
Fredholm BB, Arslan G, Halldner L, Schulte G, Wasserman W (2000) Structure and function of
adenosine receptors and their genes. Naunyn Schmiedebergs Arch Pharmacol 362:364–365
Fredholm B, IJzerman A, Jacobson K, Klotz K-N, Linden J (2001) International Union of
Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev
53:527–552
Fredholm BB, Ijzerman AP, Jacobson KA, Linden J, Mu CE (2011) International Union of Basic
and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors —
an update. Pharmacol Rev 63:1–34
Fresco P, Diniz C, Gonc J (2004) Facilitation of noradrenaline release by activation of adenosine
A2A receptors triggers both phospholipase C and adenylate cyclase pathways in rat tail artery.
Cardiovasc. Res. 63:739–746
Gao Z, Jacobson KA (2017) Purinergic signaling in mast cell degranulation and asthma. Front
Pharmacol 8:947
Gao Z, Ye K, Göblyös A, Ijzerman AP, Jacobson KA (2008) Flexible modulation of agonist
efficacy at the human A3 adenosine receptor by the imidazoquinoline allosteric enhancer
LUF6000. BMC Pharmacol 8:20
Gao Z, Balasubramanian R, Kiselev E, Wei Q (2014) Probing biased/partial agonism at the G
protein-coupled A2B adenosine receptor. Biochem Pharmacol 90:297–306
Gessi S, Fogli E, Sacchetto V, Merighi S, Varani K, Preti D, Leung E, MacLennan S, Borea PA
(2010a) Adenosine Modulates HIF-1α, VEGF, IL-8, and Foam Cell Formation in a Human
Model of Hypoxic Foam Cells. Arteriosclerosis, Thrombosis, and Vascular Biology 30(1):90-
97
Gessi S, Sacchetto V, Fogli E, Merighi S, Varani K, Giovanni P, Aghazadeh M, Leung E,
Maclennan S, Andrea P (2010b) Modulation of metalloproteinase-9 in U87MG glioblastoma
cells by A 3 adenosine receptors. Biochem Pharmacol 79:1483–1495. https://doi.org/10.1016/j.
bcp.2010.01.009
Gessi S, Merighi S, Fazzi D, Stefanelli A, Varani K, Borea PA (2011) Adenosine receptor targeting
in health and disease. Expert Opin Investig Drugs 20:1591–1609
Gessi S, Merighi S, Stefanelli A, Fazzi D, Varani K, Borea PA (2013) A1 and A3 adenosine
receptors inhibit LPS-induced hypoxia-inducible factor-1 accumulation in murine astrocytes.
Pharmacol Res 76:157–170. https://doi.org/10.1016/j.phrs.2013.08.002
10 Pharmacology of Adenosine Receptors 353

Gessi S, Bencivenni S, Battistello E, Vincenzi F, Colotta V, Catarzi D, Varano F, Merighi S, Borea

PA, Varani K (2017) Inhibition of A2A adenosine receptor signaling in cancer cells proliferation
by the novel antagonist TP455. Front Pharmacol 8:1–13
Glukhova A, Thal DM, Nguyen AT, May LT, Sexton PM, Christopoulos A, Scammells PJ (2017)
Structure of the adenosine A 1 receptor reveals the basis for subtype selectivity article. Cell
168:867–877
Göblyös A, Ijzerman AP (2009) Allosteric modulation of adenosine receptors. Purinergic Signal
5:51–61
Godinho RO, Duarte T, Pacini ESA (2015) New perspectives in signaling mediated by receptors
coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular
cAMP-adenosine pathway. Front Pharmacol 6:1–10
Golzar Y, Doukky R (2014) Regadenoson use in patients with chronic obstructive pulmonary
disease: the state of current knowledge. Int J COPD 4:129–137
Gracia E, Farré D, Cortés A, Ferrer-costa C, Orozco M, Mallol J, Lluís C, Canela EI, Mccormick PJ,
Franco R, Fanelli F, Casadó V (2012) The catalytic site structural gate of adenosine deaminase
allosterically modulates ligand binding to adenosine receptors. FASEB J 27:1–14
Greene SJ, Sabbah HN, Butler J, Voors AA, Albrecht-ku BE (2016) Partial adenosine A1 receptor
agonism: a potential new therapeutic strategy for heart failure. Heart Fail Rev 21:95–102
Gutiérrez-de-Terán H, Sallander J, Sotelo E (2017) Structure-based rational design of adenosine
receptor ligands. Curr Top Med Chem 17:40–58
Hammarberg C, Schulte G, Fredholm BB (2003) Evidence for functional adenosine A3 receptors in
microglia cells. J Neurochem 86:1051–1054
Hayashi S, Inaji M, Nariai T, Oda K, Sakata M, Toyohara J, Ishii K, Ishiwata K, Maehara T,
Medical T, Number F, Medical T (2017) Increased binding potential of brain adenosine A1
receptor in chronic stages of patients with diffuse axonal injury measured with [ 1-methyl-11C ]
8- dicyclopropylmethyl -1- methyl-3-propylxanthine PET imaging. J Neurotrauma 35:1–32
Headrick JP, Ashton KJ, Rose RB, Peart JN (2013) Pharmacology & therapeutics cardiovascular
adenosine receptors: expression, actions and interactions. Pharmacol Ther 140:92–111. https://
doi.org/10.1016/j.pharmthera.2013.06.002
Henzlova MJ, Duvall WL, Einstein AJ, Travin MI, Verberne HJ (2016) ASNC imaging guidelines
for SPECT nuclear cardiology procedures: stress, protocols, and tracers blood pressure. J Nucl
Cardiol 23:606–639
Hoffman M (2019) Istradefylline approved for parkinson add-on therapy. NeurologyTimes https://
www.neurologytimes.com/parkinson-disease/istradefylline-approved-parkinson-add-therapy
Hua X, Kovarova M, Chason KD, Nguyen M, Koller BH, Tilley SL (2007) Enhanced mast cell
activation in mice deficient in the A2b adenosine receptor. J Exp Med 204:117–128
Hyun T, Yong K, Kim K, Erk AÁ, Ros AÁ, Glioma Á (2012) The adenosine A3 receptor agonist
Cl-IB-MECA induces cell death through Ca2+/ROS-dependent down regulation of ERK and
Akt in A172 human glioma cells. Neurochem Res 37:2667–2677
Iijima M, Orimo S, Terashi H, Suzuki M, Shimura H, Mitoma H, Kitagawa K (2019) Expert opinion
on pharmacotherapy efficacy of istradefylline for gait disorders with freezing of gait in
Parkinson’s disease: a single- arm, open-label, prospective, multicenter study. Expert Opin
Pharmacother 20:1405–1411. https://doi.org/10.1080/14656566.2019.1614167
Jaakola V-P, Griffith MT, Hanson MA, Cherezov V, Chien EYT, Lane JR, IJzerman AP, Stevens
RC (2008) The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an
antagonist. Science (80-) 322:1211–1217
Jackson S, Anders NM, Mangraviti A, Wanjiku TM, Sankey EW, Liu A, Brem H, Tyler B, Rudek
MA, Grossman SA (2016) The effect of regadenoson-induced transient disruption of the blood –
brain barrier on temozolomide delivery to normal rat brain. J Neurooncol 126:433–439
Jackson S, Weingart J, Nduom EK, Harfi TT, George RT, Mcareavey D, Ye X, Anders NM, Peer C,
Figg WD, Gilbert M, Rudek MA, Grossman SA (2018) The effect of an adenosine - agonist on
intra-tumoral concentrations of temozolomide in patients with recurrent glioblastoma. Fluids
Barriers CNS 15:1–9. https://doi.org/10.1186/s12987-017-0088-8
354 P. K. Deb et al.

Jacobson KA, Tosh DK, Jain S, Gao Z (2019) Historical and current adenosine receptor agonists in
preclinical and clinical development. Front Cell Neurosci 13:1–17
Janes K, Esposito E, Doyle T, Cuzzocrea S, Tosh DK, Jacobson KA, Salvemini D (2014) A3
adenosine receptor agonist prevents the development of paclitaxel-induced neuropathic pain by
modulating spinal glial-restricted redox-dependent signaling pathways. Pain 155:2560–2567.
https://doi.org/10.1016/j.pain.2014.09.016
Kim BH, Oh JH, Lee NK (2017) Molecules and cells the inactivation of ERK1/2, p38 and NF-kB is
involved in the down-regulation of osteoclastogenesis and function by A2B adenosine receptor
stimulation. Mol Cells 40:752–760
Kirsch GE, Codina J, Birnbaumer L, Brown AM (1990) Coupling of ATP-sensitive K+ channels to
A1 receptors by G proteins in rat ventricular myocytes. Am J Physiol 259:H820–H826
Kishore DP, Balakumar C, Rao AR, Pratim P, Roy K (2011) QSAR of adenosine receptor
antagonists: exploring physicochemical derivatives with human adenosine A3 receptor subtype.
Bioorg Med Chem Lett 21:818–823. https://doi.org/10.1016/j.bmcl.2010.11.094
Koscsó B, Csóka B, Selmeczy Z, Himer L, Pacher P, Virág L, Haskó G (2012) Adenosine augments
IL-10 production by microglial cells through an A2B adenosine receptor-mediated process. J
Immunol 188:445–453
Koupenova M, Johnston-cox H, Vezeridis A, Gavras H, Yang D, Zannis V, Ravid K (2012) A2b
adenosine receptor regulates hyperlipidemia and atherosclerosis. Circulation 125:354–363
Kull B, Svenningsson P, Fredholm BB (2000) Adenosine A2A receptors are colocalized with and
activate G olf in rat STRIATUM. Mol Pharmacol 58:771–777
Kunduri SS, Dick GM, Nayeem M, Mustafa SJ (2013) Adenosine A1 receptor signaling inhibits
BK channels through a PKCα-dependent mechanism in mouse aortic smooth muscle. Physiol
Rep 1:1–11. https://doi.org/10.1002/phy2.37
Lazarus JJ, Saleh A, Ghannam M, Aaronson K, Colvin M, Pagani F, Murthy VL, Konerman MC
(2018) Safety of regadenoson positron emission tomography stress testing in orthotopic heart
transplant patients. J Nucl Cardiol:1–6. https://doi.org/10.1007/s12350-018-01466-1
Linden J (2005) Adenosine in tissue protection and tissue regeneration. Mol Pharmacol
67:1385–1387
Liu B, Bing Q, Li S, Han B, Lu J, Baiyun R, Zhang X, Lv Y, Wu H, Zhang Z (2019) Role of - A2B
adenosine receptor-dependent adenosine signaling in multi-walled carbon nanotube-triggered
lung fibrosis in mice. J Nanobiotechnol 17:1–11. https://doi.org/10.1186/s12951-019-0478-y
López-cruz L, Salamone JD, Correa M (2018) Caffeine and selective adenosine receptor
antagonists as new therapeutic tools for the motivational symptoms of depression. Front
Pharmacol 9:1–14
Mahmod Al-Qattan MN, Mordi MN (2019) Molecular basis of modulating adenosine receptors
activities. Curr Pharm Des 25:817–831
Mailavaram R, Al-Attraqchi O, Kar S, Ghosh S (2019) Current status in the design and develop-
ment of agonists and antagonists of adenosine A3 receptor as potential therapeutic agents. Curr
Pharm Des 25:2772–2787
Malpass K (2011) Pharmacology: new methods to permeabilize the blood–brain barrier. Nat Rev
Neurol 7:597. https://doi.org/10.1038/nrneurol.2011.161
Martin L, Pingle SC, Hallam DM, Rybak LP (2006) Activation of the adenosine A3 Receptor in
RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-a release by
reducing calcium-dependent activation of nuclear factor-kB and extracellular signal-regulated
kinase 1/2. J Pharmacol Exp Ther 316:71–78
Matsuda K, Horikawa Y, Sasaki Y, Sakata SF (2014) The adenosine receptor agonist 5’-N-
ethylcarboxamide-adenosine increases glucose 6-phosphatase expression and gluconeogenesis.
Pharmacol Pharm 5:19–23
Mayer P, Hinze AV, Harst A, Von Ku I (2011) A2B receptors mediate the induction of early genes
and inhibition of arterial smooth muscle cell proliferation via Epac. Cardiovasc Res 90:148–156
10 Pharmacology of Adenosine Receptors 355

Merighi S, Benini A, Mirandola P, Gessi S, Varani K, Leung E, Maclennan S, Baraldi PG, Borea
PA (2007) Hypoxia inhibits paclitaxel-induced apoptosis through adenosine-mediated phos-
phorylation of bad in glioblastoma cells. Mol Pharmacol 72:162–172
Merighi S, Borea PA, Stefanelli A, Bencivenni S, Castillo CA, Varani K, Gessi S (2015) A 2A and
A 2B adenosine receptors Affect HIF-1 a signaling in activated primary microglial cells. Glia
63:1933–1952
Merighi S, Bencivenni S, Vincenzi F, Varani K, Borea PA, Gessi S (2017) Original article A2B
adenosine receptors stimulate IL-6 production in primary murine microglia through p38 MAPK
kinase pathway. Pharmacol Res 117:9–19. https://doi.org/10.1016/j.phrs.2016.11.024
Merighi S, Gessi S, Borea PA (2018) Adenosine receptors: structure, distribution, and signal
transduction. In: Borea P, Varani K, Gessi S, Merighi S, Vincenzi F (eds) The adenosine
receptors, The receptors, vol 34, pp 33–57
Mobasher M, Miller RA, Kwei L, Strahs D, Das V, Luciano G, Powderly JD, Merchan JR, Barve
MA, LoRusso P, Tripathi A (2019) A phase I/Ib multicenter study to evaluate the humanized
anti-CD73 antibody, CPI-006, as a single agent, in combination with CPI-444, and in combina-
tion with pembrolizumab in adult patients with advanced cancers. J Clin Oncol 37:TPS2646–
TPS2646
Moffatt BA, Stevens YY, Allen MS, Snider JD, Pereira LA, Todorova MI, Summers PS,
Weretilnyk EA, Martin-mccaffrey L, Wagner C (2002) Adenosine kinase deficiency is
associated with developmental abnormalities and reduced transmethylation. Plant Physiol
128:812–821
Mohamed RA, Agha AM, Nassar NN (2016) Role of adenosine A 2A receptor in cerebral ischemia
reperfusion injury: signaling to phosphorylated extracellular signal-regulated protein kinase
(pERK1/2). Neuroscience 314:145–159. https://doi.org/10.1016/j.neuroscience.2015.11.059
Moriyama K, Sitkovsky MV (2010) Adenosine A2A receptor is involved in cell surface expression
of A2B receptor . J Biol Chem 285:39271–39288
Nagayama H, Kano O, Murakami H, Ono K, Hamada M, Toda T, Sengoku R, Shimo Y, Hattori N
(2019) Effect of istradefylline on mood disorders in Parkinson’s disease. J Neurol Sci
396:78–83. https://doi.org/10.1016/j.jns.2018.11.005
Nalli AD, Kumar DP, Al-Shboul O, Mahavadi S, Kuemmerle JF, Grider JR, Murthy KS (2014)
Regulation of Gbci-dependent PLC-b 3 activity in smooth muscle: inhibitory phosphorylation
of PLC- b3 by PKA and PKG and stimulatory phosphorylation of Gai -GTPase-activating
protein RGS2 by PKG. Cell Biochem Biophys 70:867–880
Navarro G, Cordomí A, Zelman-femiak M, Brugarolas M, Moreno E, Aguinaga D, Perez-benito L,
Cortés A, Casadó V, Mallol J, Canela EI, Lluís C, Pardo L, García-sáez AJ, Mccormick PJ,
Franco R (2016) Quaternary structure of a G-protein- coupled receptor heterotetramer in
complex with G i and G s. BMC Biol 14:1–12. https://doi.org/10.1186/s12915-016-0247-4
Neary JT, Mccarthy M, Kang Y, Zuniga S (1998) Mitogenic signaling from P1 and P2 purinergic
receptors to mitogen-activated protein kinase in human fetal astrocyte cultures. Neurosci Lett
242:159–162
Newby AC (1984) Adenosine and the concept of ’ retaliatory metabolites ’. Trends Biochem Sci
9:42–44
Nishida K, Dohi Y, Yamanaka Y (2014) Expression of adenosine A2b receptor in rat type II and III
taste cells. Histochem Cell Biol 141:499–506
Novitskiy SV, Ryzhov S, Zaynagetdinov R, Goldstein AE, Huang Y, Oleg Y, Blackburn MR,
Biaggioni I, Carbone DP, Feoktistov I, Mikhail M, Dc W, Novitskiy SV, Ryzhov S,
Zaynagetdinov R, Goldstein AE, Huang Y, Tikhomirov OY (2008) Adenosine receptors in
regulation of dendritic cell differentiation and function. Blood 112:1822–1831
Núñez F, Taura J, Camacho J, López-cano M, Fernández-dueñas V, Castro N, Castro J, Ciruela F,
Aubert J, Castro J (2018) PBF509, an adenosine A 2A receptor antagonist with efficacy in
rodent models of movement disorders. Front Pharmacol 9:1200
Ochaion A, Bar-yehuda S, Cohen S, Amital H, Jacobson KA, Joshi BV (2008) The A 3 adenosine
receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-k B signaling pathway in
synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats. Biochem
Pharmacol 76:482–494
356 P. K. Deb et al.

Pacheco R, Lejeune M, Climent N, Oliva H, Gatell JM, Gallart T, Mallol J (2005) CD26, adenosine
deaminase, and adenosine receptors mediate costimulatory signals in the immunological syn-
apse. PNAS 102:9583–9588
Peakman M, Hill SJ (1994) Adenosine A2B-receptor-mediated cyclic AMP accumulation in
primary rat astrocytes. Br J Pharmacol 111:191–198
Pedata F, Dettori I, Coppi E, Melani A, Fusco I, Corradetti R, Maria A (2016) Neuropharmacology
purinergic signalling in brain ischemia. Neuropharmacology 104:105–130. https://doi.org/10.
1016/j.neuropharm.2015.11.007
Peleli M, Fredholm B, Sobrevia L, Carlström M (2017) Pharmacological targeting of adenosine
receptor signaling. Mol Aspects Med 55:4–8. https://doi.org/10.1016/j.mam.2016.12.002
Phosri S, Arieyawong A, Bunrukchai K (2017) Stimulation of adenosine A 2B receptor inhibits
endothelin-1-induced cardiac fibroblast proliferation and α-smooth muscle actin synthesis
through the cAMP/Epac/PI3K/Akt-signaling pathway. Front Pharmacol 8:1–15
Phosri S, Bunrukchai K, Parichatikanond W, Sato VH, Mangmool S (2018) Epac is required for
exogenous and endogenous stimulation of adenosine A 2B receptor for inhibition of angiotensin
II-induced collagen synthesis and myofibroblast differentiation. Purinergic Signal 14:141–156
Pinna A, Serra M, Morelli M, Simola N (2018) Role of adenosine - A2A receptors in motor control:
relevance to Parkinson’s disease and dyskinesia. J Neural Transm 125:1273–1286. https://doi.
org/10.1007/s00702-018-1848-6
Pleli T, Mondorf A, Ferreiros N, Thomas D, Dvorak K, Biondi RM, Heringdorf DMZ, Zeuzem S,
Geisslinger G, Zimmermann H, Waidmann O, Piiper A (2018) Activation of adenylyl cyclase
causes stimulation of adenosine receptors. Cell Physiol Biochem 45:2516–2528
Ponnoth DS, Nadeem A, Tilley S, Mustafa SJ (2010) Involvement of A 1 adenosine receptors in
altered vascular responses and inflammation in an allergic mouse model of asthma. Am J
Physiol Heart Circ Physiol 299:H81–H87
Preti D, Baraldi PG, Moorman AR, Borea PA, Varani K (2015) History and perspectives of A2A
adenosine receptor antagonists as potential therapeutic agents. Med Res Rev 35:790–848
Prystowsky EN, Niazi I, Curtis AB, Wilber DJ, Bahnson T, Ellenbogen K, Dhala A, Bloomfield
DM, Gold M, Kadish A, Fogel RI, Gonzalez MD, Belardinelli L, Shreeniwas R, Wolff AA
(2003) Termination of paroxysmal supraventricular tachycardia by tecadenoson (CVT-510), a
novel A 1-adenosine receptor agonist. J Am Coll Cardiol 42:1098–1102. https://doi.org/10.
1016/S0735-1097(03)00987-2
Rabadi MM, Lee HT (2015) Adenosine receptors and renal ischaemia reperfusion injury. Acta
Physiol 213:222–231
Raines J, Shakowski C, Page R, Quaife R (2019) Regadenoson associated side effect reversal:
safety and efficacy of IV theophylline. J Nucl Med 60:307
Romagnoli R, Baraldi PG, Moorman AR, Borea PA, Varani K (2015) Current status of A1
adenosine receptor allosteric enhancers. Future Med Chem 7:1247–1259
Rosenberger P, Schwab JM, Mirakaj V, Masekowsky E, Mager A, Morote-garcia JC, Unertl K,
Eltzschig HK (2009) Hypoxia-inducible factor–dependent induction of netrin-1 dampens
inflammation caused by hypoxia. Nat Immunol 10:195–202
Roshan MHK, Tambo A, Pace NP (2016) Potential role of caffeine in the treatment of Parkinson’s
disease. Open Neurol J 10:42–58
Ruiz MDL, Lim Y, Zheng J (2014) Adenosine A2A receptor as a drug discovery target. J Med
Chem 57:3623–3650
Ryzhov S, Zaynagetdinov R, Goldstein AE, Novitskiy SV, Dikov MM, Michael R, Biaggioni I,
Feoktistov I, Ryzhov S, Zaynagetdinov R, Goldstein AE, Novitskiy SV (2008) Effect of A2B
adenosine receptor gene ablation on proinflammatory adenosine signaling in mast cells. J
Immunol 180:7212–7220
Sachdeva S, Gupta M (2013) Adenosine and its receptors as therapeutic targets: an overview. Saudi
Pharm J 21:245–253. https://doi.org/10.1016/j.jsps.2012.05.011
Saki M, Yamada K, Koshimura E (2013) In vitro pharmacological profile of the A2A receptor
antagonist istradefylline. Naunyn Schmiedebergs Arch Pharmacol 386:963–972
10 Pharmacology of Adenosine Receptors 357

Samanta PN, Kar S, Leszczynski J (2019) Recent advances of in-silico modeling of potent
antagonists for the adenosine receptors. Curr Pharm Des 25:750–773
Sassi Y, Laggerbauer B, Sassi Y, Ahles A, Truong DJ, Baqi Y, Lee S, Husse B, Hulot J (2014)
Cardiac myocyte–secreted cAMP exerts paracrine action via adenosine receptor activation find
the latest version: cardiac myocyte–secreted cAMP exerts paracrine action via adenosine
receptor activation. J Clin Invest 124:5385–5397
Sawynok J (2016) Adenosine receptor targets for pain. Neuroscience 338:1–18. https://doi.org/10.
1016/j.neuroscience.2015.10.031
Schulte G, Fredholm BB (2000) Human adenosine A1, A2A, A2B, and A3 receptors expressed in
Chinese hamster ovary cells all mediate the phosphorylation of extracellular-regulated kinase
1/2. Mol Pharmacol 58:477–482
Schulte G, Fredholm BB (2003) Signalling from adenosine receptors to mitogen-activated protein
kinases. Cell Signal 15:813–827
Shah SJ, Voors AA, McMurray JJV, Kitzman D, Viethen T, Wirtz AB, Huang E, Pap AF, Solomon
SD (2019) Effect of neladenoson bialanate on exercise capacity among patients with heart
failure with preserved ejection fraction a randomized clinical trial. JAMA 321:2101–2112
Shaik K, Muttaleb A, Jaber Y, Kachler S, Klotz KN (2019) 7-Amino-2-aryl/hetero-aryl-5-oxo-5,8-
dihydro[1,2,4]triazolo[1,5-a] pyridine-6-carbonitriles: synthesis and adenosine receptor binding
studies. Chem Biol Drug Des 94:1568–1573
Sharma AK, Lapar DJ, Stone ML, Zhao Y, Mehta CK, Kron IL, Laubach VE (2016) NOX2
activation of natural killer T cells is blocked by the adenosine A2A receptor to inhibit lung
ischemia – reperfusion injury. Am J Respir Crit Care Med 193:988–999
Sitaraman SV, Wang L, Wong M, Bruewer M, Hobert M, Yun C, Merlin D, Madara JL (2002) The
adenosine 2b receptor is recruited to the plasma membrane and associates with E3KARP and
Ezrin upon agonist stimulation. J Biol Chem 277:33188–33195
Soares AS, Costa VM, Diniz C (2014) The combination of Cl-IB -MECA with paclitaxel: a new
anti-metastatic therapeutic strategy for melanoma. Cancer Chemother Pharmacol 74:847–860
Soni H, Peixoto-neves D, Buddington RK, Adebiyi XA (2017) Adenosine A 1 receptor-operated
calcium entry in renal afferent arterioles is dependent on postnatal maturation of TRPC3
channels. Am J Physiol Renal Physiol 313(313):1216–1222
Soudijn W, van Wijngaarden I, Ijzerman AP (2006) Allosteric modulation of G-protein–coupled
receptors. In: Bowery NG (ed). Allosteric receptor modulation in drug targeting. pp. 179–206
Stein E, Zou Y, Poo M, Tessier-Lavigne M (2001) Binding of DCC by netrin-1 to mediate axon
guidance independent of adenosine A2B receptor activation. Science (80-) 291:1976–1983
Stemmer SM, Madi L, Castel D, Ochaion A, Cohen S (2008) The A3 adenosine receptor agonist
CF102 induces apoptosis of hepatocellular carcinoma via de-regulation of the Wnt and NF- κ B
signal transduction pathways. Int J Oncol 33:287–295
Stemmer SM, Benjaminov O, Medalia G, Ciuraru NB, Silverman MH, Bar-Yehuda S, Fishman S,
Harpaz Z, Farbstein M, Cohen S, Patoka R, Singer B, Kerns WD, Fishman P (2013) CF102 for
the treatment of hepatocellular carcinoma: a phase I/II, open-label, dose-escalation study.
Oncologist 18:25–26
Stemmer SM, Manojlovic NS, Marinca MV, Petrov P, Cherciu N, Ganea D, Ciuleanu T-E, Puscas
IA, Beg MS, Purcell WT, Croitoru A-E, Ilieva RN, Natošević S, Nita AL, Kalev DN, Harpaz Z,
Farbstein M, Silverman MH, Fishman P, Llovet JM (2019) A phase II, randomized, double-
blind, placebo-controlled trial evaluating efficacy and safety of namodenoson (CF102), an A3
adenosine receptor agonist (A3AR), as a second-line treatment in patients with Child-Pugh B
(CPB) advanced hepatocellular carcinoma. J Clin Oncol 37:2503–2503
Stenberg DAG, Litonius E, Halldner L, Johansson RN, Fredholm BB (2003) Sleep and its
homeostatic regulation in mice lacking the adenosine A 1 receptor. J. Sleep Res 12:283–290
Stoilov RM, Licheva RN, Mihaylova MK, Reitblat T, Dimitrov EA, Shimbova KM, Bhatia G,
Pispati A, Balbir AG, Bagaria B, Oparanov BA, Fishman S, Harpaz Z, Farbstein M, Cohen S,
Bristol D, Silverman MH, Fishman P (2014) Therapeutic effect of oral CF101 in patients with
rheumatoid arthritis: a randomized, double-blind, placebo-controlled phase II study. Immunome
Res 11:1–6
358 P. K. Deb et al.

Sun Y, Huang P (2016) Adenosine A2B receptor: from cell biology to human diseases. Front Chem
4:1–11
Sun D, Samuelson LC, Yang T, Huang Y, Paliege A, Saunders T, Briggs J, Schnermann J (2001)
Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine
1 receptors. PNAS 98:9983–9988
Sun C, Schnermann J, Michael R, Sun C, Young HW, Molina JG, Volmer JB (2005) A protective
role for the A 1 adenosine receptor in adenosine-dependent pulmonary injury. J Clin Invest
115:35–43
Sun Y, Duan Y, Eisenstein AS, Hu W, Quintana A, Lam WK, Wang Y, Wu Z, Ravid K, Huang P
(2012) A novel mechanism of control of NF k B activation and inflammation involving A2B
adenosine receptors. J Cell Sci 125:4507–4517
Szentmiklósi AJ, Galajda Z, Cseppent Á, Hegyi B, Nánási PP (2015) The janus face of adenosine:
antiarrhythmic and proarrhythmic actions. Curr Pharm Des 21:965–976
Thomas GS, Cullom SJ, Kitt TM, Feaheny KM, Ananthasubramaniam K, Gropler RJ, Jain D,
Thompson RC (2017) The EXERRT trial: ‘“EXErcise to Regadenoson in Recovery Trial”’: a
phase 3b, open-label, parallel group, randomized, multicenter study to assess regadenoson
administration following an inadequate exercise stress test as compared to regadenoson with.
J Nucl Cardiol 24:788–802
Thompson C (2008) FDA approves pharmacologic stress agent. Am J Heal Pharm 65:890
Vallon V, Mu B (2006) Adenosine and kidney function. Physiol Rev 86:901–940
Varani K, Vincenzi F, Merighi S, Gessi S, Borea PA (2017) Biochemical and pharmacological role
of A 1 adenosine receptors and their modulation as novel therapeutic strategy. Adv Exp Med
Biol 1051:193–232
Vij A, Golzar Y, Doukky R (2018) Regadenoson use in chronic kidney disease and end-stage renal
disease: a focused review. J Nucl Cardiol 25:137–149
Vincenzi F, Targa M, Corciulo C, Gessi S, Merighi S, Setti S, Cadossi R, Borea PA, Varani K
(2012) The anti-tumor effect of A 3 adenosine receptors is potentiated by pulsed electromag-
netic fields in cultured neural cancer cells. PLoS One 7:e39317
Voelker R (2019) Add-on drug approved for “off” episodes of Parkinson disease. JAMA
322:1246–1246
Voors AA, Düngen H, Senni M, Agostoni P, Ponikowski P, Bax JJ, Voors PAA (2017) Safety and
tolerability of neladenoson bialanate, a novel oral partial adenosine A1 receptor agonist, in
patients with chronic heart failure. J Clin Pharmacol 57:440–451
Voors AA, Shah SJ, Bax JJ, Butler J, Gheorghiade M, Hernandez AF, Kitzman DW, McMurray
JJV, Wirtz AB, Lanius V, van der Laan M, Solomon SD (2018) Rationale and design of the
phase 2b clinical trials to study the effects of the partial adenosine A1-receptor agonist
neladenoson bialanate in patients with chronic heart failure with reduced (PANTHEON) and
preserved (PANACHE) ejection fraction. Eur J Heart Fail 20:1601–1610
Wang J, Huxley VH (2006) Adenosine A2A receptor modulation of juvenile female rat skeletal
muscle microvessel permeability. Am J Physiol Heart Circ Physiol 291:H3094–H3105
Wang L, Kolachala V, Walia B, Balasubramanian S, Hall RA, Merlin D, Sitaraman SV,
Kolachala V, Walia B, Bala S, Hall RA, Merlin D, Shanthi V (2004) Agonist-induced polarized
trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role
of SNARE proteins. Am J Physiol Gastrointest Liver Physiol 287:1100–1107
Willingham SB, Ho PY, Hotson A, Hill C, Piccione EC, Hsieh J, Liu L, Buggy JJ, McCaffery I,
Miller RA (2018) A2AR antagonism with CPI-444 Induces antitumor responses and augments
efficacy to anti–PD-L1 and anti–CTLA-4 in preclinical models. Cancer Immunol Res 6:1–34
Wu LIN, Belardinelli L, Zablocki JA, Palle V, Shryock JC, Belardinelli L, Zablocki JA, Shryock JC
(2001) A partial agonist of the A1-adenosine receptor selectively slows AV conduction in
guinea pig hearts. Am J Physiol Heart Circ Physiol 280:334–343
Yaar R, Jones MR, Chen J, Ravid K (2005) Animal models for the study of adenosine receptor
function. J Cell Physiol 202:9–20
10 Pharmacology of Adenosine Receptors 359

Yang D, Wagner DD, Ravid K, Yang D, Zhang Y, Nguyen HG, Koupenova M, Chauhan AK,
Makitalo M, Schreiber BM, Gavras H, Wagner DD, Ravid K (2006) The A2B adenosine
receptor protects against inflammation and excessive vascular adhesion. J Clin Invest
116:1913–1923
Yang X, Xin W, Yang X-m, Kuno A, Rich TC, Cohen MV, Downey JM (2011) A2B adenosine
receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes
via a mechanism sensitive to Pertussis toxin. Br J Pharmacol 163:995–1006
Zhou J, Zimmermann K, Krieg T, Soltow M, Pavlovic D, Cerny V, Lehmann C (2015) Adenosine
receptor activation improves microcirculation in experimental intestinal ischemia/reperfusion.
Clin Hemorheol Microcirc 59:257–265
Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn
Schmiedebergs Arch Pharmacol 362:299–300
Pharmacology of Angiotensin and Its
Receptors 11
Satyajeet Biswal, Rajat Ghosh, and Pratap Chandra Acharya

Abstract

Angiotensin is a peptide hormone produced by the proteolytic cascade initiated

by the enzyme renin. The physiological effects of angiotensin are articulated by a
particular receptor subtype, and it allows the cells to respond to extracellular
signals. In earlier days, receptors were used to be identified using in vitro
radioimmuno assay methods similar to the method used to identify receptor-
binding properties of antibodies. However, nowadays the validation of receptors
is done by doing the molecular or gene grafting into an unresponsive cell and then
by observing the changes in chemical messengers. These innovative methods of
identifying receptors have led to the discovery of two major angiotensin
receptors, angiotensin type 1 receptor (AT1 receptor) and type 2 receptor (AT2
receptor), which produce cellular signals. Angiotensin has various physiological
functions in different places such as juxtaglomerular cells, aldosterone, heart and
kidney. The pharmacological intervention of renin–angiotensin system can be
done by using beta blockers which create the inhibitory effect on renin secretion
from juxtaglomerular (JG) cells. There is another method which involves the use
of the renin inhibitory peptide. However, this method is not yet proved to be a
successful approach for controlling the renin–angiotensin system. By far the most
appropriate method of controlling the renin–angiotensin system is by using orally
active angiotensin-converting enzyme (ACE) inhibitors, which interrupt the
whole system. However, due to the associated adverse effects of ACE inhibitors,
angiotensin receptor blockers (ARBs) are chosen over them. This chapter
describes the history and origin of angiotensin, its biosynthesis, its mechanism
of action and its physiological role. Further, the chapter also narrates the role of

S. Biswal · R. Ghosh · P. C. Acharya (*)

Department of Pharmacy, Tripura University (A Central University), Suryamaninagar, Tripura (W),
India
e-mail: [email protected]

# Springer Nature Singapore Pte Ltd. 2020 361

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_11
362 S. Biswal et al.

angiotensin as drug target and the use of ARBs for the pharmacotherapeutic
intervention of hypertension.

Keywords

Angiotensin · Angiotensin receptor blockers · Renin–angiotensin systems · Ang

II · AT1 · AT2 · Sartans · Hypertension

Abbreviations

ACE Angiotensin-converting enzyme

Ang II Angiotensin II
ARBs Angiotensin receptor blockers
AT1 Angiotensin type 1 receptor
AT2 Angiotensin type 2 receptor
AT3 Angiotensin type 3 receptor
AT4 Angiotensin type 4 receptor
CHF Congestive heart failure
CKD Chronic kidney disease
GPCR G-Protein-coupled receptor
JG Cells Juxtaglomerular cells
mRNA Messenger RNA
NC-IUPHAR International Union of Pharmacology Committee on Receptor
Nomenclature and Drug Classification
RAS Renin–angiotensin system
US FDA United States Food and Drug Administration
WHO World Health Organization

11.1 Introduction

11.1.1 History

Angiotensin is a peptide hormone produced by the proteolytic cascade which is

initiated by the enzyme renin. The physiological effects of angiotensin are expressed
by a particular receptor subtype, and it allows the cells to respond to extracellular
signals. Primarily, the response to angiotensin signal is mediated by the peptide
hormone after binding to their specific receptors (Goodfriend 2000). The angiotensin
receptors were earlier identified by in vitro radioimmuno assays similar to the
method used to identify receptor-binding properties of antibodies (Goodfriend
et al. 1968; Goodfriend and Lin 1970). It has been observed that angiotensin targets
are distributed in blood vessels and the adrenal glomerulosa by tracking the radioac-
tive angiotensin infused into rats (Bumpus et al. 1964). However, every hormone-
binding site cannot be considered as receptors. In the 1960s, the metabolic enzymes
11 Pharmacology of Angiotensin and Its Receptors 363

used to be particularly confusing with angiotensin receptors, because they do not

transduce the presence of hormone into a cellular response. Nowadays, the identifi-
cation of receptors is done by doing molecular or gene grafting into an unresponsive
cell and then by observing the changes in chemical messengers (Sasaki et al. 1991;
Mukoyama et al. 1993).

11.1.1.1 Discovery of Angiotensin Receptor Subtypes: Insights from

Angiotensin Receptor Binding
The identification of angiotensin receptors was done by performing simpler binding
assays on tissue homogenates. The binding sites for angiotensin II were unknown
until 1970 and were found in tissue homogenates (Lin and Goodfriend 1970;
Goodfriend and Lin 1970). Angiotensin-binding sites have been found outside the
blood vessels in a large number at unpredicted places with the help of autoradiogra-
phy (Mendelsohn et al. 1984; Saavedra et al. 1986). The angiotensin hormone binds
with the cells of the central nervous system, pituitary–reproductive tract, fetus,
bladder, gut and blood vessels (Goodfriend et al. 1996). It has been suggested that
the renin–angiotensin system affects embryogenesis, reproduction and natural
defences against infection. These effects probably have evolutionary significance
on the cardiovascular functions of angiotensin. All other biological functions may
have therapeutic implications; however, the potential uses or side effects of angio-
tensin receptor blockers (ARBs) are yet to be explored (Goodfriend 2000).
The presence of abundant recepto-like binding sites in the kidney of calf fetus has
been observed (Lin and Goodfriend 1970). However, there are vast differences in the
binding properties of angiotensin between foetal and adult sites. This result
suggested the availability of different types or subtypes of receptors for angiotensin
(Simpson et al. 1980). The presence of receptor subtypes was further established by
using specific nonpeptide antagonists leading to the discovery of two receptor
subtypes labelled as AT1 (angiotensin II receptor type 1) and AT2 (angiotensin II
receptor type 2) (Timmermans et al. 1993; De Gasparo et al. 1995).
One of the major challenges in the discovery of angiotensin receptors has been the
cellular response of angiotensin in surprising locations. Sometimes, ‘receptor-like
proteins’ with unknown ligand known as ‘orphan receptors’ or receptors with known
ligand and location but unknown function, called as ‘bureaucrat receptors’, have
been found. Although the working principle of these receptors is known, it is
difficult to predict their physiological functions (Goodfriend 2000).
Angiotensin has been found to bind bovine heart mitochondria leading to incon-
sistent oxidative phosphorylation. This result suggested that the peptide hormone
affects the energy metabolism in myocardial muscle (Goodfriend et al. 1971). This
was further supported by the positive inotropic effect of angiotensin III on cat
papillary muscle, which was exclusively resistant to hypoxia (Kent et al. 1972).
Angiotensin receptors have been located on human platelets (Moore and
Williams 1982). Moreover, aged platelets have more angiotensin receptors com-
pared with the younger ones (Siebers and Goodfriend 1986). Further, animal
experiments suggest that angiotensin can speed up the clotting sequence through
its receptors (Chabielska et al. 1998).
364 S. Biswal et al.

11.1.1.2 Regulation of the Receptor Binding

Inhibition of hormone-receptor binding is one of the lucrative approaches for drug
development. The same approach is also utilized by the human body for physiologi-
cal regulation. The angiotensin receptors are known to interact with intracellular
G-proteins and can affect the angiotensin binding to their receptors (Glossmann et al.
1974).
The angiotensin receptors have been found in the lipid layer of the cell mem-
brane. It is further known that the dietary lipids also influence the tertiary structure
and binding kinetics of the angiotensin receptors. For example, the diets containing
γ-linolenic acid can reduce the angiotensin binding by adrenal cells. The endogenous
steroids and eicosanoids also have similar yet stronger effects. These modulators
also affect the balance between angiotensin hormone and receptor antagonists.
Because of the difference in the diet-induced microenvironments, some patients
respond more to a new drug. The binding of agonist and antagonist at the same place
on the receptors is also not possible precisely due to diet-induced microenvironment
(Simpson et al. 1980; Campanile and Goodfriend 1981; Carroll et al. 1983; Engler
et al. 1998; Carroll and Goodfriend 1984).

11.1.1.3 Effect of Receptor Density

The intensity of the effects of drug, hormone or autacoid depends on the number of
its receptors, assuming that a fixed proportion of the receptor is involved in the signal
transduction process. However, in the case of angiotensin, this generalization may
not be applicable. For example, response of the angiotensin peptide is unimpressive
and delayed in adrenal glomerulosa cells although they have a large number of
angiotensin receptors, whereas vascular smooth muscle cells contain less number of
receptors but mediate a rapid and dramatic response. The number of receptors at the
cell surface is regulated by nuclear events to synthesize new receptors, whereas
internalization of receptors from cell membrane to cytosol is due to either degrada-
tion or recycling. The internalization process is generally accelerated by the binding
of agonists to the receptor, not by the binding of antagonists (Hunyady et al. 1994).
Angiotensin receptors of vascular smooth muscle stick to this scheme, and hence the
hormone downregulates the receptors in the vasculature. However, on the surface of
adrenal glomerulosa cells, angiotensin upregulates its receptors (Conlin et al. 1993).

11.1.1.4 Clinical Relevance of Angiotensin Receptors

Angiotensin plays a vital role in the dysregulation of blood pressure and extracellular
fluid volume due to its effects on blood vessels and aldosterone secretion. Further
studies have revealed that angiotensin contributes to the pathogenesis of a wide
range of diseases, such as vascular fibrosis, cardiac hypertrophy, atherosclerosis and
diabetic nephropathy (Re 1993; Kabour et al. 1994; Weber et al. 1995a; Gansevoort
et al. 1994b). Receptor abnormality is also induced due to the structural defects or
changes in their surroundings. However, factors like tissue distribution, genetic
sequences regulating receptor number, signal transduction coupling and the mem-
brane components can also affect receptor function (Bonnardeaux et al. 1994;
Kainulainen et al. 1999).
11 Pharmacology of Angiotensin and Its Receptors 365

11.1.2 Discovery

The pressor principle ‘renin’ was extracted from the kidney by Tigerstedt and
Bergman in 1897 and provided the first insight into the regulation of blood pressure.
This revolutionary work further directed the discovery of reno-vascular hypertension
in mammals (Goldblatt et al. 1934). When Goldblatt isolated the vasoconstrictor
substance renin from the renal venous blood of a hypertensive dog, nobody believed
it till 1940 (Braun-Menendez et al. 1940). A similar discovery was made simulta-
neously and independently by Page and Helmer in 1940, which showed that the
so-called renin activator, later proved to be angiotensinogen, can be isolated from the
intact animal after injecting it with renin. In Argentina, the pressor substance was
known to be ‘hypertension’ whereas in the United States of America it was called
angiotonin and was later shown to be an octapeptide (Skeggs Jr. et al. 1956; Bumpus
et al. 1957; Elliott and Peart 1956). There were differences between laboratories
concerning the nomenclature, but it was later established that both hypertensin and
angiotonin were the same octapeptide, and the hybrid term ‘angiotensin’ was
unanimously accepted by Braun-Mene’ndez and Page.
Components of angiotensin II-forming cascade such as angiotensinogen,
angiotensin-converting enzyme (ACE), and angiotensins I, II and III were further
characterized. In 1987, a committee was formed comprising members from the
‘International Society for Hypertension’, ‘The American Heart Association’ and
the ‘World Health Organization’ which proposed to abbreviate angiotensin to Ang
using the decapeptide angiotensin I as the reference for numbering the amino acid
sequence of all angiotensin peptides (Dzau et al. 1987). Ang II regulates vascular
resistance and blood volume and has been found in tissues such as kidney, adrenals,
brain, sympathetic nervous system, pituitary gland and vascular smooth muscle. Ang
II is also suggested to function as a paracrine and autocrine hormone in the regula-
tion of cellular growth, proliferation and extracellular matrix formation (Dzau and
Gibbons 1987; Grady et al. 1991; Weber et al. 1995b; Weber et al. 1995c).
Metabolites of angiotensin such as angiotensin 2–8 (Ang III), angiotensin 1–7 or
angiotensin 3–8 (Ang IV) have also been shown to exhibit biological activities
(Peach 1977; Schiavone et al. 1990; Chappell et al. 1991; Iyer et al. 1998; Wright
and Harding 1995).
At the end of the 1980s, it was demonstrated that at least two receptor types exist
for Ang II in many tissues. The conventional peptide analogue such as saralasin and
nonpeptide antagonist such as losartan were found to have a high affinity for both the
receptors but without any selectivity (Chiu et al. 1989; Whitebread et al. 1989; Speth
and Kim 1990). The initial nomenclature of the receptor subtypes was confusing, for
example the receptor sensitive to losartan was called 1, B or a, whereas the receptor
with no affinity for losartan was termed 2, A or b. The ‘High Blood Pressure
Research Council’ in 1990 and the ‘International Union of Pharmacology Commit-
tee on Receptor Nomenclature and Drug Classification’ (NC-IUPHAR) appointed a
subcommittee to address the problem, and a classification was proposed in 1991 and
updated in 1995 (Bumpus et al. 1991; De Gasparo et al. 1995). The details of the
receptor subtype classification are described in Sect. 11.3.
366 S. Biswal et al.

Liver
Secretes Angiotensinogen

Angiotensinogen
Renin
Converts Angiotensinogen
to Angiotensin I
Kidney
Releases Renin
Angiotensin I
ACE
Converts Angiotensin I
Lungs
to Angiotensin II Releases Angiotensin
Converting Enzyme
Angiotensin II
(ACE)

Angiotensin II Receptors
Located in Adrenal glands, Vascular smooth muscle, the heart and the brain

Angiotensin stimulates Angiotensin stimulates Angiotensin stimulates Angiotensin stimulates Angiotensin stimulates
aldosterone secretion sodium & fluid retention in muscle hypertrophy & sympathetic outflow in the vasoconstriction in blood
in the adrenal gland. the kidney fibrosis in the heart brain vessels

Fig. 11.1 Regulation of renin–angiotensin–aldosterone system

11.2 Angiotensin

11.2.1 Synthesis, Storage, Release and Removal

The enzyme renin and its greater precursor, prorenin, are produced by the zona
glomerulosa cells present in renal afferent arterioles of the kidney (Fig. 11.1). The
regulation of the renin–angiotensin–aldosterone system is dependent on several
enzymes with the end product being Ang II. The physiological action of Ang II is
further mediated by the interaction of the peptide with the specific cell surface
receptors namely AT1 and AT2 (Guthrie Jr. 1995).

11.2.2 Physiological Role and Recent Advancement

During the circulation, Ang II contracts vascular smooth muscle by which elevation
of blood pressure happens due to the resistance developed in the arterioles. The renal
and mesenteric beds are known to be most sensitive to the Ang II action. Arteriolar
smooth muscle hypertrophy and increased peripheral resistance are also contributed
by Ang II by acting as a growth factor for blood vessels. This action is particularly
important in the case of vascular injury (Naftilan 1992).
11 Pharmacology of Angiotensin and Its Receptors 367

Ang II is known to play a major role in the fluid and electrolyte balance as well as
cardiovascular functions. However, the circulating Ang II can activate the
angiotensinergic sympatho-excitatory pathway in the brain leading to the develop-
ment and progression of hypertension. Evidences indicate that central sympathetic
nerve activity can trigger pathogenicity of essential hypertension. The sympatho-
adrenomedullary system is known to have an adaptive response to stress. However,
excessive or sustained stress can contribute to the state of hypertension (Mcdougall
et al. 2005; Esler 2009).
Ang II, the peptide hormone, sustains blood pressure and vascular volume
through several actions such as vasoconstriction, stimulation of aldosterone secre-
tion, increased renal tubular absorption of sodium, activation of the sympathetic
nervous system and increased cardiac contractility. However, these actions are
impaired in the pathophysiologic states of hypertension and congestive heart failure
(CHF). Increased release of renin and Ang II has been found in renal diseases like
renal artery stenosis, renin-secreting tumours or kidney injury leading to hyperten-
sion (Guthrie Jr. 1995).
Therefore, drugs such as ACE inhibitors and ARBs are able to antagonize the
actions of the renin–angiotensin axis leading to effective treatment of hypertension
and heart failure.

11.3 Angiotensin Receptor

11.3.1 Types, Subtypes and Localization

11.3.1.1 The Type 1 (AT1) Angiotensin Receptor

The AT1 receptor is better known for its versatile physiological actions of Ang II like
blood pressure regulation in arteries, balancing of water and electrolyte, dehydration,
hormone secretion and renal function in the target cells present in cardiovascular,
endocrine, neuronal, renal and hepatic tissues. The AT1 receptor belongs to the G-
protein-coupled receptor (GPCR) superfamily, and the cellular responses to AT1
receptor signalling include smooth muscle contraction, neuronal activation, neuro-
secretion, ion transport, adrenal steroidogenesis, aldosterone secretion, cell growth
and proliferation. The AT1 receptor is coupled to the Gq-mediated calcium and
protein kinase C signalling pathways as well as the intracellular signalling cascades
extending to the nucleus. These signalling pathways regulate gene transcription and
protein expressions to control cell proliferation in several Ang II target tissues
(De Gasparo et al. 2000).

11.3.1.2 The Type 2 (AT2) Angiotensin Receptor

The differential stability of dithiothreitol towards Ang II binding site suggested
the presence of more than one form of the receptor (Chang et al. 1982; Gunther
1984; Chiu et al. 1989; Speth et al. 1991; Whitebread et al. 1989; Chang and Lotti
1990). The cloning and expression of the receptor types AT1 and AT2 provided clear
evidence regarding the presence of the second isoform of Ang II receptor (Murphy
368 S. Biswal et al.

et al. 1991; Sasaki et al. 1991; Kambayashi et al. 1993; Mukoyama et al. 1993).
However, the biochemical and physiological functions of the AT2 receptor are still a
matter of intense research.
11.3.1.3 AT3 Receptor
The existence of AT3 receptor subtype has been reported with its characteristic
pharmacology. However, the distinct gene for this receptor in humans has not
been established to date (Chaki and Inagami 1992; Inagami et al. 1993).
11.3.1.4 AT4 Receptor
High-affinity binding sites of the Ang IV peptide were found to be concentrated
predominantly in the brain and to some extent in heart, kidney, adrenals and blood
vessels, which were termed as AT4 receptors in 1995 (Harding et al. 1992). The AT4
receptor does not bind the peptide analogues of Ang II as well as the non-peptide
inhibitors of AT1 and AT2 receptors such as losartan, CGD42112A and PD123177
(Karnik et al. 2015).

11.3.2 Regulatory Pathways and Functions

11.3.2.1 Function of AT1 Receptors in Juxtaglomerular Cells

The overexpression of AT1 receptor on juxtaglomerular (JG) cells may hinder the
release and production of renin through short-loop feedback mechanism (Kakinuma
et al. 1993; Matsusaka et al. 1996). Moreover, losartan shows promising results by
inducing mRNA when applying into in vitro culture of freshly isolated renin-releasing
cells (Tufro-Mcreddie et al. 1994). When Ang II is administered in subpressor doses,
the renal renin mRNA gradually decreases and can be stimulated by the treatment with
enalapril. The mechanism behind this release and production of renin is not well
understood because the regulation is primarily done by macula densa and baroreceptor.
The inhibitory effect of Ang II receptor on renin-producing cells cannot be confirmed
from systemic effects like low blood pressure. Thus the production of renin by
overexpression of AT1 receptor on JG cells remains unclear (Matsusaka et al. 1996).

11.3.2.2 Effect of Angiotensin on Aldosterone

Being the primary regulator of aldosterone synthesis, the overexpression of AT1
receptor in the adrenal gland stimulates aldosterone secretion. The expression of
AT1A and AT1B in the rat adrenal by appropriate hybridization has been explored
(Gasc et al. 1994). The overexpression of AT1B mRNA by zona glomerulosa is
higher when compared with AT1A. The stimulation of aldosterone secretion by Ang
II is done in three different ways: (1) stimulation of the proliferation of adrenocorti-
cal cells, (2) induction of enzymes that are required for aldosterone synthesis
and (3) induction of AT1 receptors.

11.3.2.3 Effect of Angiotensin and Its Production in the Heart

In the year 1979, captopril showed a promising result in the case of myocardial
hypertrophy by inhibiting the ventricular myocardial mass of spontaneous
11 Pharmacology of Angiotensin and Its Receptors 369

hypertensive rats (SHR), which cannot be done by the vasodilator drug hydralazine
(Antonaccio et al. 1979). This advanced research resulted in a number of in vitro
studies showing the positive effect of Ang II on cardiomyocytes and vascular smooth
muscle cells and was identified as a promising cardiovascular growth factor.

11.3.2.4 Stimulatory Effect of Potassium on Aldosterone via AT1

Receptor
Potassium has long been thought to be a key mediator for the JG cells in response to
sodium depletion (Boyd et al. 1971). It is also well known that extracellular
potassium concentration carries a vital role in the modulation of aldosterone synthe-
sis. Yet the presence of Ang II is mandatory for the steroidogenic effect of potassium.
Moreover, when potassium supplement downgrades the plasma renin activity,
simultaneously it upgrades the adrenal renin (Nakamaru et al. 1985).

11.3.2.5 Renal AT1 Receptor Regulation

Recent studies show that not renin but angiotensinogen and ACE are rate-limiting
factors, and the involvement of Ang II receptors in the activity of renin–angiotensin
system (RAS) is not yet clear.

11.4 Role of Angiotensin Receptors as Drug Target

11.4.1 Angiotensin II Receptors

Interaction of Ang II with specific cell-surface receptors triggers the secondary

signalling pathways, and the confirmation of the types and subtypes of angiotensin
receptors has been done by the binding studies (Griendling et al. 1994). With the
development of receptor-specific ligands, the different angiotensin receptors are
confirmed as AT1 and AT2 subtypes. The characteristics of AT1 and AT2 receptors
are summarized in Table 11.1.

11.4.1.1 Pharmacological Inhibition of the Renin–Angiotensin System

There are several ways to interrupt the renin–angiotensin system like the inhibition
of renin secretion from the JG cells by using beta blockers. Another method is by
using the peptide which inhibits renin itself; some of them are orally active, yet none
of them is clinically proven. The most appropriate method for interrupting the
system is by using the orally active ACE inhibitors. These ACE inhibitors show
promising results by inhibiting the Ang II production. There are many ACE
inhibitors available in the market which are safe and effective, but due to some of
the side effects, Ang II receptor antagonists are being used (Guthrie Jr. 1995).
370 S. Biswal et al.

Table 11.1 List of characteristics of AT1 and AT2 receptors

AT1 receptor AT2 receptor
Selective antagonists Losartan CGP 42112A
Valsartan PD 123319
Irbesartan
Telmisartan
TCV-116
Signalling pathways (+) Phospholipase C (
) Guanylate cyclase
(+) Phospholipase D
(
) Adenylate cyclase
Tissue distribution Vascular smooth muscle Brain (sensory nuclei)
Heart Uterus
Liver Ovary
Brain (pressor nuclei) Adrenal medulla
Kidney Neointima
Adrenal cortex
Pituitary
Function All major angiotensin II responses (?) Ovulation
(?) Neural tissue
(?) Growth, wound healing
Key: (+) ¼ Stimulates, (
) ¼ inhibits, (?) ¼ unclear

11.4.2 Advantages of the Newer ‘Sartans’

Single dose a day, negligible adverse effect, patient tolerance to side effect and most
importantly the lower cost make these sartans the first-line choice as antihyperten-
sive drugs. These ARBs have a good tolerance profile as compared to the ACE
inhibitors in both long term and short term due to which the patient compliance is
much higher than other antihypertensive drugs. In the initial phase, hypertension is
asymptomatic, and long-term treatment is necessary to control the blood pressure.
Patient compliance is very much essential, and there should not be any interaction
with food. Sartans come into this category and make oral administration very easy
(Guthrie Jr. 1995).

11.4.2.1 Combination Therapy

Patients having poor control over blood pressure are usually prescribed a combina-
tion therapy of calcium channel blocker and angiotensin receptor blocker. The main
strategy for using combination therapy is to provide both inhibitory action and
treatment to the heart diseases. On the one hand, ARBs give renal protection and
stroke protection without causing any metabolic adverse effects, and on the other
hand calcium channel blockers help to treat angina and cardiac ischaemia. During a
randomized double-blind clinical trial of 8- to 16-week weeks duration, it was
observed that the combination therapy of once daily dose of amlodipine and
valsartan showed promising results in lowering and maintaining the blood pressure
level for approximately 1 year. So it is well known that a combination therapy would
give much more benefit to the patient for reducing the blood pressure instead of
using amlodipine or valsartan monotherapy (Sabbah et al. 2013).
11 Pharmacology of Angiotensin and Its Receptors 371

11.4.2.2 ARBs and Chronic Kidney Disease (CKD)

The overexpression of renin–angiotensin–aldosterone system leads to the develop-
ment of CKD despite the initial nephropathy. To preserve the renal function, it is
important to block the renin–angiotensin system. Therefore, the use of ARBs has
been extensively recommended as preferred antihypertensive agents in patients with
pre-existing CKD (Sabbah et al. 2013).

11.4.2.3 Angiotensin II Receptor Antagonists: Effective

Antihypertensive Agents
A numbers of pre-clinical and clinical studies have been done with all Ang II
antagonists to validate their antihypertensive activity. During placebo-controlled
clinical studies, losartan and valsartan showed to be the most effective drugs for
treating hypertension; however, irbesartan and candesartan also showed promising
hypotensive effect in double-blind placebo-controlled trials. But the effect of
irbesartan and candesartan is clearly dose dependent as compared to losartan and
valsartan. The dose-dependent pattern of both irbesartan and candesartan was 75 and
300 mg and 4 and 16 mg, respectively (Sabbah et al. 2013).

11.4.2.4 Angiotensin II Receptor Antagonists: An Excellent Tolerability

Profile
Due to the long-term treatment regimen of ARBs, it is very important to observe the
adverse event profile and tolerability of new therapeutic agents. Studies have shown
that ARBs have an excellent tolerability similar to placebo. Losartan and valsartan
showed a clear and confirmed result in contrast to ACE inhibitors in that these drugs
don’t induce cough (Benz et al. 1997; Lacourciere et al. 1994). The problem with
ACE inhibitors is their lack of specificity, which causes dry cough. Among all
angiotensin II antagonists, losartan is the only drug which has the ability to lower
the plasma uric level by increasing uric acid excretion. However, this uricosuric
effect of losartan, whether its advantage or disadvantage, remains unclear.

11.4.2.5 Renal Effects of Angiotensin II Antagonists: Comparable

to Those of ACE Inhibitors
Ang II receptor antagonists do not interfere with any of the effects caused by kininase
II inhibition. So, the importance of Ang II antagonists on the dynamics of renal blood
flow and the renoprotective effect remains unclear. However, the laboratory
experiments as well as clinical studies postulate that the effects of ACE inhibitors
on kidney are somehow similar to that of ARBs (Burnier et al. 1993; Burnier et al.
1995; Gansevoort et al. 1994a). In contrast to the above facts, a few studies done on
both the normotensive subjects and hypertensive patients suggest that Ang II
antagonists don’t affect the glomerular filtration and increased renal blood flow;
however, it increases the urinary sodium excretion. According to a previous study, it
has been suggested that natriuresis caused by ACE inhibitor is due to the inhibition
of prostaglandin metabolism. Studies on the effects of indomethacin on renal
haemodynamics showed that it acts as an antinatriuretic in both conditions (Burnier
et al. 1993; Minghelli et al. 1998; Burnier et al. 1995). Hence it is proved that
372 S. Biswal et al.

non-steroidal antiinflammatory drugs (NSAIDS) are not class specific, and clinically
it may interfere with the antihypertensive effect of ARBs.

11.5 Clinical Status of Various Angiotensin-II Antagonists

The clinical status of various angiotensin-II antagonists or ARBs has been compiled
in Table 11.2 and described in the following section.
Azilsartan
Azilsartan (Fig. 11.2) is the latest Ang II receptor blocker, developed by Takeda
Pharmaceuticals in the brand name Edarbi, and has been approved by the United
States Food and Drug Administration (US FDA) on 25 February 2011. Azilsartan is
well accepted in the market because of its sustained blood pressure control, which
lasts for 24 h. It is a potent and highly selective Ang II receptor blocker having a
bioavailability of 60% and an elimination half-life of 11 h. The prodrug azilsartan
medoxomil gets hydrolysed to azilsartan very quickly. The dose range of 40 mg or
80 mg once daily shows promising results in systolic and diastolic blood pressure by
reducing 12–15 mm Hg and 7–8 mm Hg respectively. Till now azilsartan is
prescribed only for hypertension, and there are no sufficient human data supporting
the use of azilsartan for the improvement of cardiovascular outcomes.
Candesartan
In the year 1993, candesartan (Fig. 11.3) was first examined by Japanese
scientists, and they published the effectiveness of the compound as an angiotensin
receptor blocker (Mizuno et al. 1992; Ogihara et al. 1993). Candesartan is available
in the market as its prodrug candesartan cilexetil (cyclohexyl 1-hydroxyethyl car-
bonate), which is an ester and gets completely metabolized to its active molecule
candesartan. Candesartan has much lower bioavailability when compared with other
ARBs. It has an absolute bioavailability of 15–40% and an elimination half-life of
9 h. Candesartan can be given in a dose range of 4–16 mg/day for better therapeutic
effectiveness. Candesartan is proved to be lethal if it is taken by pregnant women
during the second or third trimester. Patients with renal artery stenosis are very prone
to face high risk because of the reduction in renal glomerular filtration rate.
Irbesartan
In the year 1990, irbesartan (Fig. 11.4) was patented for use in the treatment of
high blood pressure and heart failure.
Irbesartan is available in the market under the brand name Avapro. This drug is
chosen as an initial treatment for high blood pressure. The dose range of this drug is
150–300 mg. Bioavailability is 60–80% and has an elimination half-life of 11–15 h.
Losartan
Losartan (Fig. 11.5) is the first discovered Ang II receptor antagonist, which was
patented in the year 1986 and got approved by US FDA in 1995. Because of its
effectiveness and less adverse effect, it got placed on the WHO (World Health
Organization) List of Essential Medicines. The primary function of the drug is to
lower the blood pressure, but it shows promising results in the case of renal disease
reduction in patients with type-2 diabetes, hypertension and microalbuminuria or
 11

Table 11.2 Pharmacological characteristics of the angiotensin II receptor antagonists available on the market
Drug name Trade name Prodrug Bioavailability % Dose recommended (mg/day) Half-life (h) Protein binding %
Azilsartan Edarbi Azilsartan medoxomil 60 40–80 11 99
Candesartan Atacand Candesartan cilexetil 42 4–16 9 >99
Irbesartan Avapro – 60–80 150–300 11–15 90
Losartan Cozaar – 25–35 50–100 1.5–2 99.7
Pharmacology of Angiotensin and Its Receptors

Olmesartan Benicar Olmesartan medoxomil 26 5–40 13 99

Telmisartan Micardis – 42–100 20–80 24 99.5
Valsartan Diovan – 25 80–160 6 95
373
374 S. Biswal et al.

Fig. 11.2 Chemical structure O

of azilsartan OH

O HN
N N
O O

Fig. 11.3 Chemical structure O

of candesartan OH

O N
N NH
N
N

Fig. 11.4 Chemical structure

of irbesartan N
N NH
N
N

N
O

Fig. 11.5 Chemical structure N

HN
of losartan
N
N

O N
OH
11 Pharmacology of Angiotensin and Its Receptors 375

N
N N

NH

O O O

O
N O

HO

Fig. 11.6 Chemical structure of olmesartan

proteinuria. However, calcium channel blockers and thiazide diuretics are prescribed
to most patients, but patients who have intolerance to ACE inhibitors are prescribed
with losartan. The bioavailability of losartan is around 25–35% and the elimination
half-life is 1.5–2 h.
Olmesartan
The drug olmesartan (Fig. 11.6) got approved by the FDA in April 2002 for the
treatment of hypertension. Olmesartan medoxomil is a prodrug and gets completely
metabolized to its active metabolite olmesartan rapidly. The bioavailability of
olmesartan is around 26%, and it reaches its peak plasma concentration in 1 or
2 h. The oral dose of olmesartan is 20–40 mg once in a day. Sometimes a diuretic can
be given with olmesartan to the patient when blood pressure cannot be controlled by
only olmesartan.
Telmisartan
Telmisartan (Fig. 11.7) is a nonpeptide Ang II receptor antagonist, which got
FDA approval in the year 1998 for use in the treatment of hypertension.
After oral administration, the peak plasma level is obtained in 0.5–1 h. The
bioavailability of telmisartan is around 42–100% and protein binding is high at
more than 99.5%. The elimination half-life is 24 h. Due to the biliary secretion of the

O
N N HO

Fig. 11.7 Chemical structure of telmisartan

376 S. Biswal et al.

intact drug, telmisartan is cleared completely from circulation. The recommended

dose of telmisartan is 40–80 mg once daily. Combined administration of telmisartan
and digoxin increases the peak and trough plasma concentrations of digoxin by 49%
and 20% respectively.

11.6 Conclusions

The physiological effect of the peptide hormone angiotensin was unknown till the
discovery of two major angiotensin receptors, AT1 and AT2. Research on the
physiological role of angiotensin has led to the finding that Ang II is the primary
culprit in the development of hypertension and related cardiac diseases. However,
Ang II is also known to maintain the fluid and electrolyte balance in the body.
Therefore, the effective treatment of hypertension and heart failure can be achieved
by blocking the angiotensin receptors using ARBs or angiotensin antagonists. The
ARBs have been proven to be a better therapeutic intervention for the management
of hypertension than the ACE inhibitor, due to its fewer adverse effects, although
they both share the same biochemical pathway for the regulation of blood pressure.
In the recent years, the ARBs, also known as ‘saratans’, have been the corner-
stone in the management of hypertension and heart failure. The sartans are typically
the AT1 receptor blockers without any effect on AT2 receptor. It has been more than
three decades since the identification of the AT2 receptor. However, the biochemical
and physiological functions of the AT2 receptor are yet to be established. It is still a
matter of debate whether the AT2 receptor is involved in ovulation, neural tissue
regeneration and wound healing process. Therefore, at this juncture when the role of
AT1 receptor has been fully understood, the exploration of physiological properties
of AT2 receptor is warranted. This could possibly lead to the development of
selective AT2 receptor modulators for the management of diseases other than of
cardiovascular system.

Acknowledgements The authors gratefully acknowledge Tripura University (A Central Univer-

sity), Suryamaninagar-799022, for providing the library facility to write this manuscript. The
authors express gratitude to UGC, New Delhi [(F.30376/2017(BSR)], CSIR, New Delhi [02
(0329)/17/EMR], and DBT, New Delhi [No. BT/PR24783/NER/95/851/2017], for their research
funding. The authors declare no conflict of interest.

References
Antonaccio MJ, Rubin B, Horovitz ZP, Laffan RJ, Goldberg ME, High JP, Harris DN, Zaidi I
(1979) Effects of chronic treatment with captopril (sq 14, 225), an orally active inhibitor of
angiotensin i-converting enzyme, in spontaneously hypertensive rats. Jpn J Pharmacol
29:285–294
Benz J, Oshrain C, Henry D, Avery C, Chiang YT, Gatlin M (1997) Valsartan, a new angiotensin II
receptor antagonist: a double-blind study comparing the incidence of cough with lisinopril and
hydrochlorothiazide. J Clin Pharmacol 37:101–107
11 Pharmacology of Angiotensin and Its Receptors 377

Bonnardeaux A, Davies E, Jeunemaitre X, Fery I, Charru A, Clauser E, Tiret L, Cambien F,

Corvol P, Soubrier F (1994) Angiotensin II type 1 receptor gene polymorphisms in human
essential hypertension. Hypertension 24:63–69
Boyd JE, Palmore WP, Mulrow PJ (1971) Role of potassium in the control of aldosterone secretion
in the rat. Endocrinology 88:556–565
Braun-Menendez E, Fasciolo JC, Leloir LF, Muñoz JM (1940) The substance causing renal
hypertension. J Physiol 98:283–298
Bumpus FM, Schwarz H, Page IH (1957) Synthesis and pharmacology of the octapeptide
angiotonin. Science 125:886–887
Bumpus FM, Smeby RR, Page IH, Khairallah PA (1964) Distribution and metabolic fate of
angiotensin ii and various derivatives. Can Med Assoc J 90:190–193
Bumpus FM, Catt KJ, Chiu AT, Degasparo M, Goodfriend T, Husain A, Peach MJ, Taylor DG Jr,
Timmermans PB (1991) Nomenclature for angiotensin receptors. A report of the nomenclature
committee of the council for high blood pressure research. Hypertension 17:720–721
Burnier M, Rutschmann B, Nussberger J, Versaggi J, Shahinfar S, Waeber B, Brunner HR (1993)
Salt-dependent renal effects of an angiotensin II antagonist in healthy subjects. Hypertension
22:339–347
Burnier M, Hagman M, Nussberger J, Biollaz J, Armagnac C, Brouard R, Waeber B, Brunner HR
(1995) Short-term and sustained renal effects of angiotensin II receptor blockade in healthy
subjects. Hypertension 25:602–609
Campanile CP, Goodfriend TL (1981) Steroids as potential modulators of angiotensin receptors in
bovine adrenal glomerulosa and kidney. Steroids 37:681–700
Carroll JE, Goodfriend TL (1984) Androgen modulation of adrenal angiotensin receptors. Science
224:1009–1011
Carroll JE, Landry AS, Elliott ME, Yatvin MB, Vorpahl J, Goodfriend TL (1983) Cholesteryl
hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal
glomerulosa cells. Life Sci 32:1573–1581
Chabielska E, Pawlak R, Golatowski J, Buczko W (1998) The antithrombotic effect of captopril and
losartan on experimental arterial thrombosis in rats. J Physiol Pharmacol 49:251–260
Chaki S, Inagami T (1992) Identification and characterization of a new binding site for angiotensin
II in mouse neuroblastoma neuro-2A cells. Biochem Biophys Res Commun 182:388–394
Chang RS, Lotti VJ (1990) Two distinct angiotensin II receptor binding sites in rat adrenal revealed
by new selective nonpeptide ligands. Mol Pharmacol 37:347–351
Chang RSL, Lotti VJ, Keegan ME (1982) Inactivation of angiotensin ii receptors in bovine adrenal
cortex by dithiothreitol further evidence for the essential nature of disulfide bonds. Biochem
Pharmacol 31:1903–1906
Chappell MC, Millsted A, Diz DI, Brosnihan KB, Ferrario CM (1991) Evidence for an intrinsic
angiotensin system in the canine pancreas. J Hypertens 9:751–759
Chiu AT, Herblin WF, Mccall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, Wong PC, Wexler
RR, Johnson AL et al (1989) Identification of angiotensin II receptor subtypes. Biochem
Biophys Res Commun 165:196–203
Conlin PR, Moore TJ, Williams GH, Hollenberg NK (1993) Rapid modulation of renal and adrenal
responsiveness to angiotensin II. Hypertension 22:832–838
De Gasparo M, Husain A, Alexander W, Catt KJ, Chiu AT, Drew M, Goodfriend T, Harding JW,
Inagami T, Timmermans PB (1995) Proposed update of angiotensin receptor nomenclature.
Hypertension 25:924–927
De Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T (2000) International Union of Pharma-
cology. XXIII. The angiotensin II receptors. Pharmacol Rev 52:415–472
Dzau VJ, Gibbons GH (1987) Autocrine-paracrine mechanisms of vascular myocytes in systemic
hypertension. Am J Cardiol 60:99i–103i
Dzau V, Baxter J, Cantin M, De Bold A, Ganten D, Gross K, Husain A, Inagami T, Menard J,
Poole S, Robertson J, Tang J, Yamamoto K (1987) Report of the Joint Nomenclature and
378 S. Biswal et al.

Standardization Committee of the International Society of Hypertension, American Heart

Association and the World Health Organization. Hypertension 5:507–511
Elliott DF, Peart WS (1956) Amino-acid sequence in a hypertensin. Nature 177:527–528
Engler MM, Schambelan M, Engler MB, Ball DL, Goodfriend TL (1998) Effects of dietary gamma-
linolenic acid on blood pressure and adrenal angiotensin receptors in hypertensive rats. Proc Soc
Exp Biol Med 218:234–237
Esler M (2009) Heart and mind: psychogenic cardiovascular disease. J Hypertens 27:692–695
Gansevoort RT, De Zeeuw D, De Jong PE (1994a) Is the antiproteinuric effect of ACE inhibition
mediated by interference in the renin-angiotensin system? Kidney Int 45:861–867
Gansevoort RT, De Zeeuw D, Shahinfar S, Redfield A, De Jong PE (1994b) Effects of the
angiotensin II antagonist losartan in hypertensive patients with renal disease. J Hypertens
Suppl 12:S37–S42
Gasc JM, Shanmugam S, Sibony M, Corvol P (1994) Tissue-specific expression of type 1 angioten-
sin II receptor subtypes. An in situ hybridization study. Hypertension 24:531–537
Glossmann H, Baukal A, Catt KJ (1974) Angiotensin II receptors in bovine adrenal cortex.
Modification of angiotensin II binding by guanyl nucleotides. J Biol Chem 249:664–666
Goldblatt H, Lynch J, Hanzal RF, Summerville WW (1934) Studies on experimental hypertension:
i. The production of persistent elevation of systolic blood pressure by means of renal ischemia. J
Exp Med 59:347–379
Goodfriend TL (2000) Angiotensin receptors: history and mysteries. Am J Hypertens 13:442–449
Goodfriend TL, Lin SY (1970) Receptors for angiotensin I and II. Circ Res 27:163–174
Goodfriend TL, Ball DL, Farley DB (1968) Radioimmunoassay of angiotensin. J Lab Clin Med
72:648–662
Goodfriend T, Knych E, Allmann D, Kent K, Cooper T (1971) Angiotensin binding to receptors-a
guide to physiology and therapy. In: Margoulies M, Greenwood F (eds) Proceedings of the
Second International Symposium, Structure-Activity Relationships of Protein and Polypeptide
Hormones, 1971. Excerpta Medica, Amsterdam, pp 243–249
Goodfriend TL, Elliott ME, Catt KJ (1996) Angiotensin receptors and their antagonists. N Engl J
Med 334:1649–1654
Grady EF, Sechi LA, Griffin CA, Schambelan M, Kalinyak JE (1991) Expression of AT2 receptors
in the developing rat fetus. J Clin Invest 88:921–933
Griendling KK, Lassegue B, Murphy TJ, Alexander RW (1994) Angiotensin II receptor pharma-
cology. Adv Pharmacol 28:269–306
Gunther S (1984) Characterization of angiotensin II receptor subtypes in rat liver. J Biol Chem
259:7622–7629
Guthrie GP Jr (1995) Angiotensin receptors: physiology and pharmacology. Clin Cardiol 18:29–34
Harding JW, Cook VI, Miller-Wing AV, Hanesworth JM, Sardinia MF, Hall KL, Stobb JW,
Swanson GN, Coleman JK, Wright JW et al (1992) Identification of an AII(3-8) [AIV] binding
site in guinea pig hippocampus. Brain Res 583:340–343
Hunyady L, Bor M, Balla T, Catt KJ (1994) Identification of a cytoplasmic Ser-Thr-Leu motif that
determines agonist-induced internalization of the AT1 angiotensin receptor. J Biol Chem
269:31378–31382
Inagami T, Iwai N, Sasaki K, Guo DF, Furuta H, Yamano Y, Bardhan S, Chaki S, Makito N, Badr K
(1993) Angiotensin II receptors: cloning and regulation. Arzneimittelforschung 43:226–228
Iyer SN, Ferrario CM, Chappell MC (1998) Angiotensin-(1-7) contributes to the antihypertensive
effects of blockade of the renin-angiotensin system. Hypertension 31:356–361
Kabour A, Henegar JR, Janicki JS (1994) Angiotensin II (AII)-induced myocyte necrosis: role of
the AII receptor. J Cardiovasc Pharmacol 23:547–553
Kainulainen K, Perola M, Terwilliger J, Kaprio J, Koskenvuo M, Syvänen A-C, Vartiainen E,
Peltonen L, Kontula K (1999) Evidence for involvement of the type 1 angiotensin II receptor
locus in essential hypertension. Hypertension 33:844–849
Kakinuma Y, Fogo A, Inagami T, Ichikawa I (1993) Intrarenal localization of angiotensin II type
1 receptor mRNA in the rat. Kidney Int 43:1229–1235
11 Pharmacology of Angiotensin and Its Receptors 379

Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami T (1993)

Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine
phosphatase inhibition. J Biol Chem 268:24543–24546
Karnik SS, Unal H, Kemp JR, Tirupula KC, Eguchi S, Vanderheyden PM, Thomas WG (2015)
International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin receptors:
interpreters of pathophysiological angiotensinergic stimuli [corrected]. Pharmacol Rev
67:754–819
Kent KM, Goodfriend TL, Mccallum ZT, Dempsey PJ, Cooper T (1972) Inotropic agents in
hypoxic cat myocardium: depression and potentiation. Circ Res 30:196–204
Lacourciere Y, Brunner H, Irwin R, Karlberg BE, Ramsay LE, Snavely DB, Dobbins TW, Faison
EP, Nelson EB (1994) Effects of modulators of the renin-angiotensin-aldosterone system on
cough. Losartan Cough Study Group. J Hypertens 12:1387–1393
Lin SY, Goodfriend TL (1970) Angiotensin receptors. Am J Phys 218:1319–1328
Matsusaka T, Nishimura H, Utsunomiya H, Kakuchi J, Niimura F, Inagami T, Fogo A, Ichikawa I
(1996) Chimeric mice carrying ‘regional’ targeted deletion of the angiotensin type 1A receptor
gene. Evidence against the role for local angiotensin in the in vivo feedback regulation of renin
synthesis in juxtaglomerular cells. J Clin Invest 98:1867–1877
Mcdougall SJ, Widdop RE, Lawrence AJ (2005) Central autonomic integration of psychological
stressors: focus on cardiovascular modulation. Auton Neurosci 123:1–11
Mendelsohn FA, Quirion R, Saavedra JM, Aguilera G, Catt KJ (1984) Autoradiographic localiza-
tion of angiotensin II receptors in rat brain. Proc Natl Acad Sci U S A 81:1575–1579
Minghelli G, Seydoux C, Goy J-J, Burnier M (1998) Uricosuric effect of the angiotensin ii receptor
antagonist losartan in heart transplant recipients1. Transplantation 66:268–271
Mizuno K, Niimura S, Tani M, Saito I, Sanada H, Takahashi M, Okazaki K, Yamaguchi M,
Fukuchi S (1992) Hypotensive activity of TCV-116, a newly developed angiotensin II receptor
antagonist, in spontaneously hypertensive rats. Life Sci 51:PL183–PL187
Moore TJ, Williams GH (1982) Angiotensin II receptors on human platelets. Circ Res 51:314–320
Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ (1993) Expression
cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane
receptors. J Biol Chem 268:24539–24542
Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE (1991) Isolation of a cDNA
encoding the vascular type-1 angiotensin II receptor. Nature 351:233–236
Naftilan AJ (1992) The role of angiotensin II in vascular smooth muscle cell growth. J Cardiovasc
Pharmacol 20(Suppl 1):S37–S40
Nakamaru M, Misono KS, Naruse M, Workman RJ, Inagami T (1985) A role for the adrenal renin-
angiotensin system in the regulation of potassium-stimulated aldosterone production. Endocri-
nology 117:1772–1778
Ogihara T, Higashimori K, Masuo K, Mikami H (1993) Pilot study of a new angiotensin II receptor
antagonist, TCV-116: effects of a single oral dose on blood pressure in patients with essential
hypertension. Clin Ther 15:684–691
Peach MJ (1977) Renin-angiotensin system: biochemistry and mechanisms of action. Physiol Rev
57:313–370
Re RN (1993) Myocardial hypertrophy, angiotensin, and ACE inhibitors. Angiology 44:875–881
Saavedra JM, Israel A, Plunkett LM, Kurihara M, Shigematsu K, Correa FM (1986) Quantitative
distribution of angiotensin II binding sites in rat brain by autoradiography. Peptides 7:679–687
Sabbah ZA, Mansoor A, Kaul U (2013) Angiotensin receptor blockers - advantages of the new
sartans. J Assoc Physicians India 61:464–470
Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y, Inagami T (1991)
Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II
type-1 receptor. Nature 351:230–233
Schiavone MT, Khosla MC, Ferrario CM (1990) Angiotensin-[1-7]: evidence for novel actions in
the brain. J Cardiovasc Pharmacol 16(Suppl 4):S19–S24
380 S. Biswal et al.

Siebers MJ, Goodfriend TL (1986) Platelet angiotensin receptors in young and old humans. J
Gerontol 41:574–578
Simpson RU, Campanile CP, Goodfriend TL (1980) Specific inhibition of receptors for angiotensin
II and angiotensin III in adrenal glomerulosa. Biochem Pharmacol 29:927–933
Skeggs LT Jr, Lentz KE, Kahn JR, Shumway NP, Woods KR (1956) The amino acid sequence of
hypertensin. II. J Exp Med 104:193–197
Speth RC, Kim KH (1990) Discrimination of two angiotensin II receptor subtypes with a selective
agonist analogue of angiotensin II, p-aminophenylalanine6 angiotensin II. Biochem Biophys
Res Commun 169:997–1006
Speth RC, Rowe BP, Grove KL, Carter MR, Saylor D (1991) Sulfhydryl reducing agents distin-
guish two subtypes of angiotensin II receptors in the rat brain. Brain Res 548:1–8
Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR,
Saye JA, Smith RD (1993) Angiotensin II receptors and angiotensin II receptor antagonists.
Pharmacol Rev 45:205–251
Tufro-Mcreddie A, Johns DW, Geary KM, Dagli H, Everett AD, Chevalier RL, Carey RM, Gomez
RA (1994) Angiotensin II type 1 receptor: role in renal growth and gene expression during
normal development. Am J Phys 266:F911–F918
Weber K, Sun Y, Cleutjens J (1995a) Structural remodeling of the myocardium postinfarction:
potential mechanisms and influence of therapy. Cardiol Rev 3:53–65
Weber KT, Sun Y, Campbell SE (1995b) Structural remodelling of the heart by fibrous tissue: role
of circulating hormones and locally produced peptides. Eur Heart J 16 Suppl N:12–18
Weber KT, Sun Y, Katwa LC, Cleutjens JP (1995c) Connective tissue: a metabolic entity? J Mol
Cell Cardiol 27:107–120
Whitebread S, Mele M, Kamber B, De Gasparo M (1989) Preliminary biochemical characterization
of two angiotensin II receptor subtypes. Biochem Biophys Res Commun 163:284–291
Wright JW, Harding JW (1995) Brain angiotensin receptor subtypes AT1, AT2, and AT4 and their
functions. Regul Pept 59:269–295
Pharmacology of Endogenous Opioids,
Opiates and Their Receptors 12
Mohammed Noorladeen Al–Qattan, Nirupam Das,
and Rati Kailash Prasad Tripathi

Abstract

The search for analgesia is the main drive for the discovery of opioid receptors
and their endogenous ligand peptides. Although opioid peptides are very similar
in their N-terminal sequence, they are classified into different types according to
their precursor proteins. The peptides activate opioid receptors by binding
to orthosteric-binding sites to mediate intracellular second messengers. Biased
signaling and allosteric modulation is a new approach to obtain receptor subtype
selectivity and separates the desirable from a myriad of unwanted pharmacologi-
cal effects. This chapter describes endogenous opioids and opiates with an
emphasis on structure, origin and processing, receptors, physiological roles, and
potential involvement in therapeutic interventions. Further, it also provides a brief
discussion on the effect of opioids on various ion channels and recent
developments of established and investigational opioid molecules.

Keywords
Opioids receptors · Endorphin · Enkephalin · Dynorphin · GPCR · Neurological
disorders · Ion channels

Abbreviations

ACTH Adrenocorticotropin
BACE1 Beta-site APP cleaving enzyme 1

M. N. Al–Qattan
Department of Pharmacy, Al–Noor University College, Mosul, Iraq
N. Das (*) · R. K. P. Tripathi
Department of Pharmaceutical Science, Sushruta School of Medical and Paramedical Sciences,
Assam University, Silchar, Assam, India

# Springer Nature Singapore Pte Ltd. 2020 381

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_12
382 M. N. Al–Qattan et al.

CLIP Corticotropin-like intermediate peptide

DOR Delta opioid receptor
GPCR G protein-coupled receptor
KOR Kappa opioid receptor
MOR Mu opioid receptor
MSH Melanotropins or melanocyte-stimulating hormone
N/OFQ Nociceptin/orphanin FQ
OP Opioid peptide
OR Opioid receptor
ORL-1 Opioid receptor-like-1
POMC Proopiomelanocortin

12.1 Introduction

The inscription of the ancient Sumerian clay tablet insinuates the use of the natural
extract of poppy plant (Papaver somniferum) as analgesia and post-surgical pain
(Norn et al. 2005; Brownstein 1993). The active ingredient of the extract is mor-
phine, which necessitates the presence of receptors for binding inside the human
body. The search for endogenous ligands acting on those opioid receptors (ORs)
started in the 1960s and succeeded in a few years by using radiolabeled ligands (Pert
and Snyder 1973). On the search for ligands for those ORs, several peptides were
isolated. The early peptides were enkephalins (Hughes et al. 1975) which showed
higher potency than morphine on opioid receptors (Kosterlitz and Hughes 1975).
Soon the untriakontapeptide (endorphin) was discovered (Li and Chung 1976), then
dynorphins (Cox et al. 1975) and endomorphins (Zadina et al. 1997).
Although opioid peptides (OPs) share almost similar N-terminal amino acid
sequence, their precursor proteins are different. The processing of precursor proteins
differs from one tissue to another as well as variation of the half-life of opioid
peptides. Some endogenous opioid peptides like endomorphins act as a selective
agonist for μ-opioid receptor (MOR) and thus have a good potential for clinical use.
However, the low oral bioavailability of endomorphins restricts its use unless given
as glycosylated derivatives (Varamini et al. 2012). Currently, three types of opioid
receptors are known, namely Mu, Delta, and Kappa opioid receptors (MORs, DORs
and KORs, respectively). The receptors can be activated by orthosteric ligands and
cooperated with allosteric ligands to produce modulation of orthosteric affinity/
efficacy as well as biasing the intracellular signaling.

12.2 Opioid Peptides

12.2.1 Classification, Synthesis, Storage, Release, and Removal

Endogenous opioid peptides are classified into three categories, namely endorphins,
enkephalins, and dynorphins. The peptides are endogenously produced by
peptidases digestion of larger precursor proteins, namely proopiomelanocortin,
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 383

proenkephalin and prodynorphin, respectively (Pleuvry 1991). Endomorphins is

regarded as the fourth category of OPs; however, the precursor for endomorphins
is not precisely known from the human genome (Terskiy et al. 2007) and may
involve an oxidative transformation of guanine nucleotide from suspected gene
leading to G ! T transversion in the produced mRNA (messenger ribonucleic
acid) (Matsushima et al. 2019). The proopiomelanocortin (POMC) precursor is
processed to give rise to adrenocorticotropin (ACTH), melanotropins (MSH) and
β-endorphin (Castro and Morrison 1997). The processing of precursor protein
depends on a particular cell type. At anterior pituitary, POMC is processed in
corticotrophs to generate mainly ACTH and β-lipotropin. When processed in
melanotrophs, it produces MSH primarily (De Wied 1999). Both ACTH and
β-lipotropin (1–91 aa) have no analgesic activity. The further processing of
β-lipotropin gives rise to β-endorphin (61–91 aa), which in turn may yield [Met]-
enkephalin (61–65) (Cox et al. 1976; Takeuchi 2001). In melanotrophs of pars
intermediate of the pituitary, POMC is processed further to produce α-MSH (from
N-terminal of POMC) which is attributed to different levels of specific peptidases in
different regions of the pituitary (Day 2009). In addition to the pituitary, POMC is
also detected in the arcuate nucleus of the hypothalamus, solitary tract of the medulla
and several peripheral tissues (Papadimitriou and Priftis 2009).
Proenkephalin peptide contains six copies of [Met]-enkephalin and one copy of
[Leu]-enkephalin, which are separated from each other by dibasic peptides as
digestive site (Takahashi 2016). Although proenkephalin is produced by both the
brain and adrenal gland, however, it is processed differently in each tissue (Geracioti
et al. 2009). Proenkephalin-producing neurons are more widespread in the brain than
POMC neurons (Geracioti et al. 2009). Prodynorphin is also called proenkephalin-B
since it contains three copies of [Leu]-enkephalins in addition to dynorphin A,
dynorphin B and α-neoendorphin (Fig. 12.1 and Table 12.1).
In neuron soma, precursor neuropeptides are synthesized in the endoplasmic
reticulum, and through Golgi apparatus, the peptides are processed by proteases
and undergo functional group modification and then transported to axons as vesicles.
At the nerve terminal, the vesicles may contain both precursor and opioid peptides
(Hökfelt et al. 2000). The processing of precursor peptide is activated by potassium-
induced depolarization of the neuron (Yakovleva et al. 2006). Moreover, the acidic
pH of secretory vacuoles promotes digestion of precursor peptides such as POMC
(Cawley et al. 2016). The opioid peptides vesicles appeared distinct from amino acid
neurotransmitter vesicles, and the former appear denser under a microscope. The
dense vesicles are distributed over neuronal cells but especially abundant in
dendrites, cell body and axon varicosities and can be released from either site with
machinery different from neurotransmitters (Russo 2017; Gu et al. 2017).
Post-translational modifications may take place on opioid peptides such as
glycosylation, acetylation, methylation and phosphorylation which alter their
biological activities (Froehlich 1997). While glycosylation improves the blood-
brain barrier (BBB) penetration (Egleton et al. 2000), acetylation attenuates the
activity of the peptide. Glycosylation may occur even on precursor proteins and
thus controls the degree of digestion to active opioid peptides and protects against
non-specific digestion (Hughes et al. 1980; Loh and Gainer 1978).
384 M. N. Al–Qattan et al.

POMC
± oligosaccharide

± ±
+
ACTH biosynthec intermediate β-LPH

+ +
± ±

16K fragment ACTH γ LPH β-endorphin

γ-MSH α-MSH CLIP β-MSH

Fig. 12.1 The processing of POMC (Retrieved from Mains and Eipper 1981)

The neuropeptides (including opioids) may diffuse to distal sites (up to

millimeters), unlike other neurotransmitters, which remain within the synaptic
space. They exert their effect by interacting with opioid and other receptors and
may have longer extracellular half-lives (van den Pol 2012; Russo 2017). Long-
distance signaling within the brain through no synapses is called volume transmis-
sion (Agnati et al. 1995; Veening et al. 2012). Volume transmission is common with
G protein-coupled receptors (GPCRs) that are more sensitive to neuropeptides where
the nanomolar concentration of opioid peptides is sufficient to activate the receptors.
This is unlike the ionotropic receptors like γ-aminobutyric acid (GABA), where
micromolar concentration is needed (Ludwig and Leng 2006).
Another difference between opioid peptides and neurotransmitters is that opioid
peptides lack reuptake mechanism by releasing neurons. Therefore, the inactivation
mechanism is limited to digestion by peptidases extracellularly or after cellular
internalization. Many peptidases degrade opioid peptides. The major peptidases
involved in the degradation of opioid peptides are endopeptidases (those acting on
N-terminal of peptides, the pharmacophoric domain). However, other peptidases
also contribute such as an insulin-degrading enzyme, angiotensin-converting
enzyme, neprilysin, serine peptidases and other dipeptidyl peptidases (Asvadi
et al. 2014a; Malfroy et al. 1978; Turner 2004; Hersh and Rodgers 2008). Since
the activity of these peptidases is affected by the pH of the tissue, the half-life of
these peptidases increases in inflamed tissues that have acidic pH; therefore, opioid
peptides are degraded more rapidly and differently (Asvadi et al. 2014b; Herath et al.
2012). The inhibition of the degrading enzymes by specific and non-specific
inhibitors is effective in producing analgesia.
 12

Table 12.1 Opioid peptides and their precursor proteins and receptor selectivity (Luca et al. 2007; Coward et al. 1998)
Endogenous peptide Amino acid sequence Receptor affinity Precursor
β-Endorphin YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE δ¼μ Proopiomelanocortin
[Met]-enkephalin YGGFM (YGGFMRF, YGGFMRGL) δ >> μ Proenkephalin
[Leu]-enkephalin YGGFL
Metorphinamide YGGFMRRV-NH2
Dynorphin A YGGFLRRIRPKLKWDNQ κ >> δ ¼ μ Prodynorphin
Dynorphin A(1-8) YGGFLRRI
Dynorphin B YGGFLRRQFKVVT
α-neoendorphin YGGFLRKYPK
YGGFLRKYP
β-neoendorphin
Nociceptin FGGFTGARKSARKLANQ *ORL Pronociceptin
Endomorphin-1 YPWF-NH2 μ Unknown
Endomorphin-2 YPFF-NH2
ORL means orphan opioid-receptor-like
Pharmacology of Endogenous Opioids, Opiates and Their Receptors
385
386 M. N. Al–Qattan et al.

12.2.2 Physiological Role of Opioid Peptides

12.2.2.1 Sites of Action of Opioid Peptides

Opioids act at two sites on the neurons, viz. the presynaptic nerve terminal and the
postsynaptic neuron. The postsynaptic actions of opioids are usually inhibitory,
while presynaptically they inhibit the release of neurotransmitters. Thus, the central
nervous system (CNS) effect of the opioids is an overall result of their actions at the
multiple presynaptic sites of both inhibitory and excitatory neurons and also includes
their actions at postsynaptic sites. As a consequence, the overall effect of opioids
depends upon the location and density of opioid receptors on the neurons. For
instance, presynaptic inhibition of neurotransmitter release leads to the excitatory
effects in target neuron if the neurotransmitter produces an inhibitory effect. On the
other hand, if the opioid also has a postsynaptic inhibitory effect on the target
neuron, then excitatory effects may not occur (Winters et al. 2017; Fields and
Margolis 2015). Morphine acts on μ-receptors and inhibits the discharge of numer-
ous diverse neurotransmitters including acetylcholine, noradrenaline and the neuro-
peptide substance P (Kerage et al. 2019; Commons 2010; Dickenson 1994).

12.2.2.2 Opioid Peptides and Their Effect on Analgesic/Pain Pathways

Pain is usually considered to be associated with augmented activity in primary
sensory neurons provoked either by strong thermal or mechanical stimuli or by the
chemicals released by inflammation or tissue damage (Yam et al. 2018). The primary
sensory neurons implicated in the pain sensation releases glutamate and substance P
in the dorsal horn of the spinal cord principally (Zieglgänsberger 2018; Lembeck
2008). The nociceptive signal is then communicated via the spinothalamic tracts to
the brain, thereby activating the descending pathways from the periaqueductal gray
area in the midbrain which then exerts inhibitory control over the dorsal horn
(Venkatraman et al. 2017; Mendell 2011).
Opioid receptors are located at various regions of the nervous system that are
involved in pain transmission and control, including primary afferent neurons, spinal
cord, midbrain, and thalamus. Though the physiological function of endogenous
opioids toward the pain transmission is still unclear, under pathological conditions,
the endogenous opioid system gets activated. The opioid drugs, however, produce
analgesia by inhibition of neurotransmitter release from the primary afferent
terminals in spinal cord and activation of descending inhibitory controls in the
midbrain (Allouche et al. 2014; McDonald and Lambert 2005; Waldhoer et al.
2004).
Any modulation in the nociceptive pathways may result in profound alterations in
levels of neurotransmitters in primary afferent neurons, thereby causing changes in
sensitivity to opioid analgesia (Yam et al. 2018). Accordingly, neuropathic pain is
related to reduced sensitivity to opioids; on the contrary, inflammatory pain is linked
with increased opioid sensitivity. Moreover, the alterations that occur in pain
sensitivity during the states of chronic pain have been ascribed to the activation of
glutamate NMDA (N-Methyl-D-aspartate) receptor (Greenwald and Shafritz 2018;
Latremoliere and Woolf 2009; Petrenko et al. 2003; Bennett 2000).
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 387

12.2.2.3 Activity of Opioid Peptides

Opioid peptides are produced by different tissues, including mainly pituitary and
adrenal glands (Przewlocki 2013). Enkephalins, for instance, are also released by
heart, skeletal muscle, kidney, and intestinal cells, thus playing an important role in
behavior, pain, cardiac function, cellular growth, immunity, and ischemic tolerance
(Denning et al. 2008). The expression of prodynorphin is mainly observed in
the cerebral cortex and basal ganglia in addition to reproductive tissues of testis,
uterus, and ovary (Collard et al. 1990; Douglass et al. 1987).
Opioid peptides are neuromodulators rather than neurotransmitters—that is, OPs
can alter the release and neuronal response to neurotransmitter by changing the
hyperpolarizing neuronal cell membrane (North and Williams 1983; Loose et al.
1990; Wu et al. 2007). The activation of ORs by endogenous peptides transmits or
modulates signal transmission for other neurotransmitters. Therefore, the peptides
have a wide range of activities that include central and peripheral antinociception,
endocrine, immune, motor activity, feeding, sexual behavior, regulation of body
temperature, respiration and cardiovascular and gastrointestinal functions
(Przewlocki 2013).
The β-endorphin is regarded as the most important opioid peptide. The peptide is
involved in interneuron communication through synapses as well as extracellular
spaces and cerebrospinal fluid (volume transmission, VT) (Veening and Barendregt
2015). The peptide showed central and peripheral analgesic activities (Sprouse-
Blum et al. 2010). Both ACTH and β-endorphin are stress hormones released during
painful stimulations. The release of ACTH and β-endorphin from a culture of
pituitary tumor cells can be evoked by epinephrine (Mains and Eipper 1981).
Therefore β-endorphin is responsible for stress-induced analgesia (Rubinstein et al.
1996). The N-terminal amino acids of β-endorphin are likely accountable for anal-
gesic activity, while the C-terminal amino acids are more related to potency. The
removal of eight amino acids from N-terminal abolishes the analgesic activity of
β-endorphin (Deakin et al. 1980). Although the analgesic potency of β-endorphin is
higher than [Met]-enkephalin, the potency is reduced by N-α-acetylation (Deakin
et al. 1980).
Enkephalins are another group of opioid peptides that include mainly [Met]-
enkephalin and [Leu]-enkephalin. The affinity of enkephalins for MOR is similar to
that of morphine and is ten times lower then affinity for DOR. Besides analgesia,
enkephalins may be involved in emotional and motivational behavior (Nieto et al.
2005) and sexual activities for male (Rodrı́guez-Manzo et al. 2002) and controls
gastrointestinal motility and secretions (Mitznegg et al. 1977; Holzer 2009).
Dynorphin is an opioid peptide which is released at the level of the spinal cord
and augments the afferent pain signal (Podvin et al. 2016), thus provoking allodynia,
pain sensation for usually non-painful stimuli. Therefore, dynorphin has a significant
role in the mediation of chronic pain (Podvin et al. 2016) as well as tolerance to
antinociceptive opioids (Vanderah et al. 2001). Moreover, dynorphin promotes
anxiety, stress, and dysphoria-driven cravings for another dose in addiction through
its action on KORs (Chavkin and Koob 2016; Knoll and Carlezon 2010).
388 M. N. Al–Qattan et al.

Endomorphins (1 and 2) are reported to be the only discovered opioid peptide that
are selective for MOR (Zadina et al. 1997). The two peptides differ from other opioid
peptides in having NH2-Tyr-Pro-Trp(Phe)-Phe-CO-NH2, that is, having
carboxamide (aminocarbonyl) terminal. Some studies showed that endomorphin-1
produces higher analgesia and without reward effect compared to morphine (Wilson
et al. 2000). Although the reward effect is known to be mediated by binding MOR, it
is limited to a specific conformational set of receptors; thus it is ligand-dependent
property. However, the potential of respiratory depression, urinary retention, toler-
ance, addiction and cardiac side effects and the low intrinsic bioavailability restrict
the clinical use of endomorphins (Gu et al. 2017). Glycosylation through succinamic
acid linker at the N-terminal enhances metabolic stability and membrane permeabil-
ity of endomorphins while maintaining potency and efficacy (Varamini et al. 2012).
Nociceptin is structurally related to dynorphin; however, it binds to opioid
receptor-like (ORL) while having no affinity for other ORs. Similar to dynorphin,
nociceptin antagonizes the analgesic effect of other opioid ligands (Mika et al.
2011). Activation of ORL inhibits adenylyl cyclase and Ca+2 channels
while activating K+ channels like opioid receptors (Calo et al. 2000). However, the
pharmacological behavior observed upon activation by nociceptin produces potent
anti-analgesic action supra-spinally and analgesic action spinally (Mogil and
Pasternak 2001).

12.3 Receptor (Types, Subtypes, Localization, Down Signaling

Pathways, Functions, Agonist, Antagonist)

Endogenous opioids produce effects on the neurons by binding to three types of

receptors located on neuronal cell membranes: namely, mu (μ), delta (δ) and kappa
(κ) receptors. Other receptors such as opioid receptor-like-1 (ORL1) have sequence
similarity to ORs, however not classified as ORs since being unresponsive to
classical opioids ligands (Snyder 2004). Naturally occurring opioids, β-endorphins
interact preferentially with μ-receptors, while the enkephalins and dynorphin interact
respectively with δ-receptors and κ-receptors (Ghelardini et al. 2015; Al-Hasani and
Bruchas 2011; Dhawan et al. 1996). The relative selectivity for endogenous and
exogenous ORs ligands are listed in Table 12.2. The crystal structures for opioid
receptors are available for mu, delta, and recently kappa receptors. The structures are
for inactive conformations that are stabilized by bound antagonists (Manglik et al.
2012; Wu et al. 2012; Granier et al. 2012) and for active conformations that are
stabilized by either bound agonist or conformation-specific nanobody or G-protein
(Huang et al. 2015; Koehl et al. 2018; Che et al. 2018). The further sorting of main
OR classes into subtypes is only approved from the anesthetic perspective in a way
that μ1 is responsible for analgesia and dependence, μ2 is for euphoria, dependence
and respiratory depression, and μ3 is for vasodilation. However, currently, there is
no clear evidence for the existence of subtypes for these receptors (Dietis et al.
2011).
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 389

Table 12.2 Selectivity of μ-receptor δ-receptor κ-receptor

naturally occurring
Opioid peptides (endogenous opioids)
endogenous opioids and
opiates for opioid receptors β-endorphin +++ +++ +++
[Leu]-enkephalin + +++ –
[Met]-enkephalin ++ +++ –
Dynorphin ++ + +++
Opioid drugs (agonists)
Morphine +++ + ++
Codeine + + +
Pethidine ++ + +
Fentanyl +++ + –
Opioid drugs (partial/mixed agonists)
Pentazocine + +

Buprenorphine – –

Opioid drugs (antagonists)
Naloxone +++ ++ ++
Naltrexone +++ ++ ++
+ indicates agonist;  indicates partial agonist; number of + or 
indicates potency

Opioid receptors are classical G protein-coupled receptors (Fig. 12.2), thus

interacting with intracellular G-protein that has GTPase activity to mediate intracel-
lular signaling. The G-protein is a heterotrimeric protein composed of Gα, Gβ, and
Gγ. The activation of ORs leads to the dissociation of G-protein. The Gβγ subunits
may interact directly with the membrane ion channels, thereby causing inhibition of
voltage-gated calcium channels (VGCCs) or rectifying potassium channels (Kir).
The Gβγ subunits also contribute to the activation of inositol triphosphate kinase
(IP3K), phospholipase C—phosphatidyl inositol triphosphate (PLC-IP3), or
mitogen-activated protein kinase (MAPK) (Khan et al. 2013; Smrcka 2008). With
respect to Gα subunit, it is classified into four families: Gαs (activates adenylyl
cyclase), Gαi/o (inhibits adenylyl cyclase), Gαq/11 (activation of phospholipase Cβ)
and Gα12/13 (activation of GTPase activating protein for Ras)(Ras, a small
GTP-binding protein named after Rat sarcoma) (Neves et al. 2002). The inhibition
of adenylyl cyclase by Gαi/o subunit leads to reduced production of cyclic adenosine
monophosphate (cAMP). Decreased concentration of cAMP further modulates
membrane sodium or calcium channels (Law 2011; Catterall 2011; Scheuer 2011).
Additionally, cAMP may also interact with and modulate the inward rectifying
potassium channels (Li et al. 2013; Butt and Kalsi 2006; North 1993). Chronic
consumption of opiates inhibits the production of cAMP; however, this inhibition is
offset in the long run by other cAMP production mechanisms (Ramaswamy and
Langford 2017; Al-Hasani and Bruchas 2011; Kosten and George 2002). When no
opiates are available, this increased cAMP production capacity comes to the force
and results in neural hyperactivity, thus causing a sensation of craving the drug
(Al-Hasani and Bruchas 2011; Bie 2005; Kosten and George 2002).
390 M. N. Al–Qattan et al.

Top view (Extracellular)

Side view (Transmembranal helices)

Bottom view (Intracellular)

Fig. 12.2 Crystal structure of active conformation of Mu opioid receptors (PDB ID: 6DDE) with
missing H8 being added from PDB 5C1M and the position within cell membrane is optimized using
OPM database

Besides interaction with G-protein, ORs interact with other regulatory

proteins and may end up with its sequestration or desensitization. Specific phosphor-
ylation of ORs by GPCR kinases leads to interaction with β-arrestin and subsequent
internalization of the receptor. Probably, Gi/o mediates the antinociceptive effect
produced by activation of MOR (Lamberts et al. 2011; Connor and Christie 1999),
while respiratory depression and tolerance is mediated by activation of β-arrestin.
Specific phosphorylation of ORs by GPCR kinases leads to interaction with
β-arrestin and subsequent internalization of the receptor further causes tolerance to
morphine analgesia (Bohn et al. 1999). Avoiding activation of β-arrestin-2 pathway
reduces internalization of MOR and consequently tolerance; however, it has little
effect on the up-regulation of adenylyl cyclase activity that is correlated to physical
dependence (Bohn et al. 2000).
Opioid receptors may exist as homo- or heteromers to provide a cellular specific
response for the same ligand. The ORL-1 or nociceptin/orphanin FQ (N/OFQ)
receptor undergoes heteromerization to form heteromers with μ-opioid receptor
(MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR) (Evans et al.
2010). Additionally, MOR may also form heteromers with DOR in small dorsal
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 391

root ganglion neurons (DRGn) and in homologous expression systems (Gendron

et al. 2015).
All three ORs produce analgesia when opioid binds to them; however, it is higher
for μ then κ and δ subtypes and is associated with euphoria, dysphoria, and anxio-
lytic effects, respectively (Lutz and Kieffer 2013). Activation of κ-receptors
produces comparatively less physical dependence as activation of μ-receptors
(Machelska and Celik 2018; Feng et al. 2012). Each of these receptors is coupled
to intracellular mechanisms via G-protein and mediates its effect through the second
messengers, thereby influencing the probability of opening of the ion channels,
which in some instances may reduce the excitability of the neurons (Machelska
and Celik 2018, Feng et al. 2012). This reduced excitability contributes as a potential
source of the euphoric effect of opiates and probably appears to be mediated by μ-
and δ-receptors (Machelska and Celik 2018; Reisine and Bell 1993).
Like many GPCRs, ORs have binding sites for orthosteric and allosteric ligands.
While orthosteric ligand can modulate receptor conformation independently, allo-
steric ligand depends on orthosteric ligand for modulating receptor conformation.
The orthosteric active opioid peptides (Livingston and Traynor 2018) share a
common N-terminal sequence of Tyr-Gly-Gly-Phe that starts with an ionized
amine. The peptides are proposed to have two pharmacophores named as message
and address pharmacophores. In other words, the address leads the peptide for opioid
receptor subtype, while the message stabilizes particular receptor conformations to
mediate specific set of intracellular signals. This proposal has been used to design
highly potent and selective non-peptide DOR antagonists (Portoghese et al. 1988).
The extracellular loops of opioid receptors have long been thought to involve in
controlling ligand selectivity to receptor subtypes by interaction with the address
part of the ligand (Metzger and Ferguson 1995). On the other hand, allosteric ligands
(or modulators) are classified as positive (PAM), negative (NAM) and neutral/silent
allosteric (SAM) modulators, which increases, decreases, and has no effect on
orthosteric ligand activity, respectively (Mahmod Al-Qattan and Mordi 2019).
Most of the allosteric ligands require bound orthosteric ligand in order to exert
activity.
The conformational ensembles stabilized by orthosteric  allosteric ligand(s)
modulate the downstream signaling of the receptor (i.e. interaction with intracellular
second messengers). Therefore, any ligand binding to ORs has a 2D fingerprint of
intracellular signaling paradigm as affinities versus efficacies. Accordingly, some
ligands are biased toward activating a particular group of second messengers over
others, which is referred to as biased agonism. Biased signaling (also known as
differential efficacy or functional selectivity) is the way of separating wanted
pharmacological effect from other unwanted effects mediated by the same receptor
(Bologna et al. 2017). The biased activation of ORs was observed among endoge-
nous as well as exogenous ligands. Unlike other endogenous OPs, α-neoendorphin,
[Met]-enkephalin, [Met]-enkephalin-RF, endomorphin-1 and endomorphin-2-
showed biased agonistic activities compared to reference DAMGO (a synthetic
peptide with high μ-opioid receptor specificity and its structure is H-Tyr-D-Ala-Gly-
N-MePhe-Gly-ol) across multiple signaling pathways as shown in Fig. 12.3
(Thompson et al. 2015).
392 M. N. Al–Qattan et al.

Fig. 12.3 Webs of bias of

endogenous opioid peptides
and reference ligands at the
MOP. (a) Ligands with
profiles similar to DAMGO.
(b) Ligands with profiles that
differ from that of DAMGO.
The score of τ/KA (or efficacy/
affinity) values was
normalized to the reference
ligand DAMGO and to the
cAMP assay. Statistically
significant differences
(P  0.05) are denoted by
black circles as determined by
two-tailed t test. For the
purposes of visualization
only, a τ/KA for Met-enk-RF
in the internalization assay
was estimated using the
incomplete concentration
response curve for plasma
membrane marker (Thompson
et al. 2015)

With respect to exogenous ligands, biased activation of MOR toward G-protein

was observed for a fungal peptide, which is opposite to the biased activity produced
by endogenous endomorphin-2 that is toward β-arrestin-2 (Dekan et al. 2019).
Biased agonist at MOR opens a new opportunity of agonists with lower side effects
(Madariaga-MazÓN et al. 2017). Oliceridine is a biased agonist at MOR, which
provides lower β-arrestin binding leading to lower receptor internalization and thus
lower respiratory depression and tolerance compared to unbiased morphine agonist
(Dewire et al. 2013). Similar natural products such as mitragynine and
7-hydroxymitragynine (Kruegel et al. 2016) and salvinorin-analogues (Groer et al.
2007) also showed a biased effect with lower β-arrestin binding. The PZM21, a
molecule obtained by structure-based screening and design, showed selective MOR
agonistic effect with minimal β-arrestin activation (Manglik et al. 2016). Biased
agonist at KOR was developed to produce antinociception and anti-itching activities
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 393

without the concomitant dysphoria and sedation usually associated with this receptor
(Brust et al. 2016). Several biased agonists at KOR were successfully developed, and
some are being used clinically (Mores et al. 2019; Brust et al. 2016). Despite the
previous successes, there are unresolved experimental limitations of measuring
differences in biased factor among various ligands in addition to the implications
of cellular environments (Mores et al. 2019; Ho et al. 2018). Although beneficial
results observed by using biased agonists to stabilize receptor conformational
ensembles that said to activate intracellular Gαi over β-arrestin-2 recruitment
(Ranjan et al. 2017), such simplification of the story might not be precise (Conibear
and Kelly 2019; Bermudez et al. 2019).
Like other GPCRs, the allosteric sites of ORs are therapeutically promising to get
biased signaling (Livingston and Traynor 2018; Livingston et al. 2018). Moreover,
allosteric ligands can enhance efficacy/affinity for endogenous opioid peptides as
well as provide selectivity toward particular OR type, which usually is difficult to
achieve using orthosteric ligands (Livingston et al. 2018). Interestingly, some
endogenous compounds act as PAM for ORs, for example, oxytocin which is a
peptide hormone principally involved in labor and lactation act as PAM for
orthosteric endomorphin-1, β-endorphin, and morphine by enhancing efficacy and
not affinity toward MOR (Meguro et al. 2018). The tuber of the species Aconitum is
traditionally used in Japan to relieve pain, currently shown to have ignavine, that has
selectivity for MOR over KOR and acts as PAM for endomorphin-1 and morphine
(Ohbuchi et al. 2016). Biased signaling can also be produced using allosteric
modulators. The BMS-986187, a synthetic compound discovered by high-
throughput screening (HTS), can function as PAM selectively for DOR (Burford
et al. 2015) and produce biased signaling toward G-protein over β-arrestin-2, which
is elicited by lower receptor internalization (Stanczyk et al. 2019).

12.4 Opioid System as Potential Target for Neurological

Disorders

The ongoing developments, including a plethora of available drugs and various

promising investigational molecules, suggest the potentiality of opioid receptors as
viable drug targets for therapeutic interventions of numerous disorders. Targeting
and modulating these molecular switches mediates the alleviation of Alzheimer’s
disease (AD) (Torres-Berrio and Nava-Mesa 2019), mood disorder (Lutz and Kieffer
2013; Lalanne et al. 2014; Ehrich et al. 2014; Browne and Lucki 2019) and
psychiatric disorders (Tejeda et al. 2011; Carlezon and Krystal 2016; Guerrero
et al. 2019) and treatment of alcohol and opioid use disorder (AUD and OUD)
(Niciu and Arias 2013; Schuckit 2016). Besides, the target-specific treatment inter-
vention related to bowel disorders (Lacy et al. 2016; Corsetti et al. 2019), chronic
pain (Günther et al. 2017; Markman et al. 2019), ischemic stroke (Wang and Subedi
2020) and respiratory disorders (Zebraski et al. 2000) have also been demonstrated
amongst others. This section focuses on the current updates on targeting the
opioidergic system in an effort to treat neurological disorders.
394 M. N. Al–Qattan et al.

12.4.1 Alzheimer’s Disease (AD)

Numerous evidential researches suggest the possible association of the opioidergic

system and pathogenesis of AD. The opioid receptors are innervated exten-
sively in the specific region of the central nervous system that is vital for
cognition and memory and includes hippocampus and cortex. Any abnormality
within the opioid system homeostasis may result in the hyperphosphorylation of
tau proteins and subsequent generation of amyloid-beta (Aβ) proteins. This event
cause neuroinflammation followed by cholinergic neurons’ deterioration and
impairment of cognitive functions (Mathieu-Kia et al. 2001). Alterations in the
level of G-protein coupled opioid receptors (μ, δ, and κ) indicate a significant
association of the endogenous opioid in AD pathology (Nandhu et al. 2010). The
ORs can modulate the imbalance of various neurotransmitters of cholinergic, adren-
ergic, GABAergic, serotonergic, and glutaminergic systems that are involved with
the progression of AD (Cai and Ratka 2012). Studies on morphine (1) and endoge-
nous opioids such as endomorphin-1 (2) and endomorphin-2 (3) showed substantial
protection against intracellular Aβ toxicity in both human and rat brains in vivo.
Morphine mediated its activity by stimulating estradiol release in the hippocampus
and increases the activity of P450 cytochrome aromatase. This chain of events
stimulates the heat shock protein 70 (Hsp70) and confers protection against
neurodegeneration (Cui et al. 2011). Furthermore, morphine attenuates the Aβ
induced neurotoxicity by activation of MOR and upregulates the mammalian target
of rapamycin (mTOR) signaling as evaluated by cell viability and neurite outgrowth
assay (Wang et al. 2014).

HO

H2N NH
O H O O
O H
N
N CH3 N N NH2
H
H
O
HO
1 2

H2N
O O
O H H
O O H3 C O
H O
N
N NH2 CH3
N O
H
O
CH3
O O

3 CH3
4
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 395

Unlike morphine, salvinorin A (4), a novel non-nitrogenous hallucinogenic

neoclerodane diterpene isolated from Salvia divinorum (Roth et al. 2002), exhibited
potent hallucinogenic property by selective agonism at κ-opioid receptor (KOR).
This activity opens up new avenues and insights for the development of selective
KOR antagonists. Targeting and blocking KOR will reverse the hallucinosis and
altered perception as observed in AD and Pick’s and Huntington’s diseases
(Cunningham et al. 2011; Sheffler and Roth 2003). Besides, salvinorin A also
displayed modulatory activity on cholinergic systems and may signify a valuable
molecular entity for alleviating the progression of AD (Motel et al. 2013). Research
on an elevated level of dynorphin A (5) (Yakovleva et al. 2007) and enkephalins,
namely [Met]-enkephalin (6) and [Leu]-enkephalin (7), observed that they induce
neurodegeneration in transgenic mouse models and AD patients (Meilandt et al.
2008).

SCH3

O
H
HOOC N
H H
O HN
H O HOOC N
N O
H2N H H
N
H 6
H
5
HN NH2 HN NH2
OH
NH NH

O O O
H H
N N N
N N N
O NH H H H
O O O NH
O O
HN
NH HN
O
O NH HN NH2
O
7 HN
NH2
H2N
O

OH
OH NH2

The dynorphins were profoundly expressed during the aging process and AD and
have been reported to stimulate the KOR and thought to induce stress-related
memory impairments. Additionally, they also affect glutamate neurotransmission
and perturb their function of synaptic plasticity essential for memory (Ménard et al.
2013).
The rate-determining step in the production of Aβ is the proteolytic breakdown of
Amyloid Precursor Protein (APP) by β-secretase (BACE1). The activation of DOR
396 M. N. Al–Qattan et al.

by an agonist has been shown to increase the secretase activity probably through the
post-translational mechanism and causing an increased formation of Aβ
(Sarajarvi et al. 2015). The cascade of reaction makes DOR and BACE1 as prospec-
tive targets in the design of DOR antagonist (Zhao et al. 2015) and BACE1 inhibitors
(Coimbra et al. 2018) for ameliorating the neurodegeneration underlying AD. The
hypermethylation of opioid receptor δ 1 (OPRD1) promoter was also linked with the
risk of AD (Ji et al. 2017). Subsequent studies suggest that in addition to OPRD1,
elevated methylation of opioid receptor κ1 and opioid receptor μ1 genes are also
involved in the progression of AD. Therefore, the genes of the opioid receptors could
serve as potential biomarkers for AD diagnosis (Xu et al. 2018).

12.4.2 Schizophrenia

Schizophrenia is a multifaceted, diverse mental disorder that affects the behavior and
cognitive function and has genetic or environmental predisposition, or both.
Antipsychotics, along with psychological therapies, are the primary line of manage-
ment available to alleviate the disorder. In recent years, much research to gain insight
into the pathophysiology of schizophrenia has been undertaken (Owen et al. 2016;
Patel and Shulman 2015) and identification of novel targets is under investigation
(Gill et al. 2018; Yang and Tsai 2017). The cardinal features of schizophrenia are
negative symptoms (reduced enthusiasm and withdrawal from society), cognitive
symptoms (disruption of attentiveness and dementia), and positive symptoms
(hallucinations accompanied by delusions). KOR agonists could elicit these specific
symptoms and drugs blocking this receptor might result in a fruitful therapeutic
outcome. Antipsychotics are capable of effectively combating the positive
symptoms; however, presently, efficient therapeutic managements for controlling
the symptoms (negative or cognitive) of schizophrenia are not available. The
potentiality of a pan-opioid antagonist either naloxone (8) or naltrexone (9) could
make KOR a promising target option for overall treatment benefits in schizophrenia
(Clark and Abi-Dargham 2019; Shekhar 2019). Alongside, contemporary research
on the discovery of novel KOR antagonist by Guerrero et al. has shown encouraging
results. The promising drug candidate BTRX-335140/CYM-53093 (10) exhibited
potent (IC50-0.8 nM) and selective KOR antagonistic activity with a favorable
pharmacokinetic profile. Currently, the compound is undergoing phase I clinical
trials for the possible treatment of various psychiatric disorders (Guerrero et al.
2019). A recent double-blind phase II study found that a combination of olanzapine
and fixed dose of MOR antagonist samidorphan (11) demonstrated clinically and
statistically significant reduction of weight gain and adverse metabolic effect of
olanzapine without compromising the antipsychotic efficacy of olanzapine. The
combination was considerably tolerated and comparable to that of olanzapine-
placebo in terms of safety (Chaudhary et al. 2019; Martin et al. 2019).
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 397

HO HO

O OH O OH

N N

O O
8 9
O

O N H2N

N HO

N N O N
OH
F
N O
10 H 11

12.4.3 Post-traumatic Stress Disorder (PTSD), Opioid Use Disorder

(OUD), and Alcohol Use Disorder (AUD)

Post-traumatic stress disorder (PTSD) is a psychiatric disorder that may arise after a
single encounter or exposures to life-threatening chronic events. PTSD deteriorates
physical health and is mostly accompanied by cardiorespiratory, musculoskeletal,
gastrointestinal, immunological, endocrine, and metabolic problems. It is also
associated with psychiatric comorbidity and an increased suicidal tendency (Bisson
et al. 2015; Yehuda et al. 2015). Existing approaches in the treatment of PTSD
involve cognitive behavioral therapy and the use of anxiolytic/antidepressant agents
to ameliorate the symptoms (Shalev et al. 2017). Interestingly, studies suggest that
endogenous opioid peptides exhibited a placebo effect in PTSD, and the mood-
enhancing effects of the peptides may be initiated by exercise and light therapy to
relieve the stress. It is suggested that the interaction between dopaminergic pathways
and the endogenous opioids may be responsible for the placebo effect (Sher 2004),
although it would not be a stand-alone option and may be concomitantly utilized
along with standard drugs. As discussed earlier, the dynorphin mediates its action via
KOR and the dynorphin/KOR interrelationship is associated in several brain
disorders (De Lanerolle et al. 1997; Mathieu-Kia et al. 2001; Mello and Negus
2006). Various works of the literature suggest that in PTSD, there is a substantial
expression of the KOR and mediate the symptoms of anxiety. Therefore, in line of
the evidence, targeting the KOR might be a viable option in the management of
PTSD (Bailey et al. 2013). The opioid analgesics prescribed in PTSD often result in
comorbidity between PTSD and OUD, and they are frequently considered as two
sides of the same coin (Elman and Borsook 2019; Hassan et al. 2017).
Centrally acting competitive MOR antagonist opioid receptor antagonists such as
naloxone is the ideal choice in emergencies related to opioid overdose. On the
contrary, naltrexone, which mediates its action via KOR antagonism, is employed
mainly in OUD and AUD for maintaining abstinence by decreasing the cravings
398 M. N. Al–Qattan et al.

(Theriot et al. 2019). However, both opioid agonist and antagonist are utilized in
substance abuse therapies to combat the withdrawal syndromes and for the inhibition
of return usage. Agonists such as morphine and methadone (12), partial agonist
buprenorphine (13) and opioid antagonist such as extended-release injectable nal-
trexone are recommended for overall treatment and tackling the relapsing of OUD.
The mechanism by which opioid antagonist maintain abstinence in OUD and AUD
is by reducing the mesolimbic dopaminergic neurotransmission (McCarty et al.
2018; Williams et al. 2008).

HO

N
O
O

O
OH
H

12 13

12.4.4 Parkinson’s Disease (PD)

It is a chronic neurodegenerative disease having marked motor (rigidity, tremor,

bradykinesia, firmness, and defective gait) and nonmotor features (hyposmia, sleep
disorders, depression, etc.) (Schapira et al. 2017; Xia and Mao 2012). The disease is
related to formation of Lewy bodies and dopaminergic neuronal damage in the
substantia nigra. The main challenge in the treatment of Parkinson’s disease
(PD) is the failure to make a conclusive diagnosis at the earliest stages and
difficulties in late-stage management of symptoms. Presently, there are no effective
treatments for slowing down the neurodegenerative process and involve significal
physical and mental co-morbidity (Demaagd and Philip 2015; Kalia and Lang 2015).
The currently available pharmacotherapeutic options in PD are dopamine precursor
(first line), dopamine agonist and monoamine oxidase B inhibitors (second line), the
antiviral drug amantadine (third line) and a newer United States Food and Drug
Administration (USFDA)-approved second-generation antipsychotic pimavanserin
(Young and Mendoza 2018). The increasing perspective of the opioid receptors as a
promising target in numerous brain disorders is well established. The treatment with
levodopa is almost always associated with dyskinesia. Recent data suggest that
selective opioid antagonists (MOR and DOR) can efficiently improve the dyskinetic
side effect in animal models (Pan and Cai 2017; Sgroi and Tonini 2018).
Nevertheless, a study on DOR agonist UFP-512 to mitigate motor insufficiencies
in hemilesioned rodents resulted in a mixed response. At a low dose involving
rotarod test, it largely improved the gait, whereas at high dose UFP-512 (14) was
found inefficacious or to exacerbate the symptoms of Parkinsonism. Further, when
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 399

locally microinjected in the globus pallidus (GP), increased akinesia was observed,
and vice versa, when injected in the substantia nigra pars reticulate. Adverse effects
such as convulsions may restrict the use of DOR agonist in Parkinsonism. The
convulsion was avoided by a synergistic combination of a DOR agonist, SNC-80,
(15) and J-113397, an N/OFQ antagonist (Mabrouk et al. 2009; Mabrouk et al.
2014). Conversely, UF-512 showed encouraging activities as anxiolytic/antidepres-
sant and treatment against chronic and neuropathic pain (Polo et al. 2019; Vergura
et al. 2008). Recently, a new mixed DOR agonist/MOR antagonist, DPI-289 (16), in
combination with levodopa elicited improved activity without increasing dyskinesia,
and it was superior when compared to high dose levodopa (Johnston et al. 2018).

HO

H2N
O O

O N N

HN O N

OH
N N
HN

O 15
14

N
H

OH

16
400 M. N. Al–Qattan et al.

12.4.5 Mood Disorder

Mental illness such as bipolar disorder (BD) and major depressive disorder (MDD)
are recalcitrant to treat due to the chronic nature and due to inter-individual variation
(Jeon et al. 2016). Studies found that opioid analgesic demonstrated potent mood-
elevating effect on patients with bipolar disorder and involve positive interaction
between the opioid and the dopaminergic systems (Schaffer et al. 2007). Preclinical
evaluations revealed that the dynorphin system is related to mood, motor, cognitive,
and endocrine functionality and subjects with MDD and BD showed a decreased
level of prodynorphin mRNA expression (Hurd 2002). Besides, a novel KOR
antagonist MCL-144B (17) displayed antidepressant activity in the forced swim
test (Reindl et al. 2008). Berrocoso et al. reported that a combination of a selective
serotonin reuptake inhibitor (SSRI) with a weak MOR agonist, (+)-tramadol (18),
produced better antidepressant activity than SSRI alone (Berrocoso and Mico 2009).
As previously discussed, UF-512, a DOR agonist, showed anxiolytic/antidepressant
properties (Polo et al. 2019). However, a combination of opioid-based samidorphan
(MOR antagonist) and buprenorphine (ALKS 5461) in phase III trials was recently
rejected by USFDA because of inadequate data to prove its effectiveness.

N
N

O H
O
H

O CH2)8 O 2HCl

17

OH

18

12.5 Effect of Opioids on Various Ion Channels

The body of human beings normally generates substances similar to opiates and
utilizes them as neuromodulators. These opiate-like substances comprise of
β-endorphins, dynorphins, and enkephalins, and are frequently conjointly called as
opioid peptides or endogenous opioids (Corder et al. 2018; Li et al. 2012; Pathan and
Williams 2012). Opioid peptides are implicated in the regulation and/or control of
various body functions such as tolerance and drug dependency, stress and pain,
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 401

cognition, immunological, muscle-related, cardiovascular (CVS) and endocrine. Not

only these, endogenous opioids also play a vital role in monitoring various sensory
functions (Bodnar 2018). Opioids are enormously expressed in several parts of the
brain including non-neuronal tissues as well, namely central nervous system (CNS)
and peripheral nervous system (PNS). Within the CNS, opioids exert their actions in
spinal cord; while in PNS, they have been found to act not only in myenteric but also
in submucous plexus situated within the stomach wall and are accountable for
producing vigorous constipation. Besides, opioids have been entailed in reduction
of pain stimuli and inflammation in several peripheral tissues like joints
(Iwaszkiewicz et al. 2013).

12.5.1 Calcium Channels

The entry of Ca2+ ions through the voltage-gated Ca2+ channels (VGCCs) results in
depolarization of nerve terminals that further causes the discharge of
neurotransmitters from the nerve cells. Three types of voltage-gated Ca2+ channels
are reported, namely, T-type channels which are capable of showing small conduc-
tance, N-type channels which are inept to demonstrate intermediate conductance,
and L-type channels which illustrate large conductance. Opioids act through the
inhibition of N-type voltage-gated Ca2+ channels and reduce the passage of Ca2+
ions inside the cell, thereby inhibiting the release of neurotransmitters (Zamponi
et al. 2015; Seseña et al. 2014; Catterall et al. 2013; Zamponi and Currie 2013).
However, this action of opioids exclusively is not accountable for the total cumula-
tive effect of opioids on neurotransmitter release (Chieng and Bekkers 2001).

12.5.2 GABA Channels

The euphoric effect of opioids may be due to another mechanism in which the
GABA inhibitory interneurons of the ventral tegmental area (VTA) are involved
(Listos et al. 2019; Creed et al. 2014; Xi 2002). By attaching to the μ-receptors, the
exogenous opioids like morphine and heroine decrease the amount of GABA
(a neurotransmitter) released. More often than not, GABA reduces the amount of
dopamine released in the nucleus accumbens (NAcc). Hence, by inhibiting GABA,
the opiates eventually increase the concentration of dopamine produced and conse-
quently the amount of pleasure felt (Dubhashi 2018; Nuechterlein 2016; Shirayama
and Chaki 2006). Opiates also have dopamine-independent effects within the NAcc,
which play an important role in opiate reward (Ting-A-Kee and Van Der Kooy 2012;
Tomkins and Sellers 2001; O’malley et al. 1992; Shippenberg and Elmer 1998;
Koob and Bloom 1988).
Besides this, the periaqueductal gray (PAG) in the midbrain region being rich in
endogenous opioids and opioid receptors is a major target of analgesic action in CNS
(Tsagareli et al. 2012; Pathan and Williams 2012; Mansour et al. 1995). The
analgesic action of opioids on PAG is exerted by the suppression of inhibitory
402 M. N. Al–Qattan et al.

influence of neurotransmitter GABA on neurons that form part of a descending

antinociceptive pathway (Tsagareli et al. 2012; Basbaum and Fields 1984). Opioids
inhibit GABA-mediated (GABAergic) synaptic transmission in the PAG and other
brain regions by reducing the probability of presynaptic neurotransmitter release
(Wilson-Poe et al. 2017; Vaughan and Christie 1997), but the mechanisms involved
remain uncertain. Literatures have reported that opioid inhibition of GABAergic
synaptic currents in the PAG is controlled by a presynaptic voltage-dependent
potassium conductance. Opioid receptors of μ-type in GABAergic presynaptic
terminals are specifically coupled to this potassium conductance by a pathway
involving phospholipase A2, arachidonic acid and 12-lipoxygenase. Additionally,
opioid inhibition of GABAergic synaptic transmission is also found to be potentiated
by inhibitors of the enzymes cyclooxygenase and 5-lipoxygenase, presumably
because more arachidonic acid is available for conversion to 12-lipoxygenase
products (Zhang and Pan 2011; Heinke et al. 2011; Finnegan et al. 2006; Ingram
et al. 1998). These mechanisms account for the analgesic action of cyclooxygenase
inhibitors in the PAG and their synergism with opioids (Leith et al. 2007; Vaughan
1998; Vaughan et al. 1997; Tortorici and Vanegas 1995; Maves et al. 1994).

12.5.3 Sodium Channels

Voltage-gated sodium channel plays a critical role in nociception by interacting with

the δ-opioid receptor. The dorsal root ganglia (DRG) neurons being rich in voltage-
gated sodium channels (Wang et al. 2011; Rush et al. 2007; Wang and Wessendorf
2001; Zhang et al. 1997) can be correlated with the emergence of pain-related
behavior characteristic of painful diabetic neuropathy (PDN). Activation of presyn-
aptic δ-opioid receptor by enkephalin prevents the increase in neuronal Na+ in DRG
through inhibition of protein kinase C (PKC) and p38 mitogen-activated protein
kinase. This can be implicating presynaptic receptors of primary sensory afferents in
modulating the amount of voltage-gated sodium channels and can be a useful
therapy for PDN (Chattopadhyay et al. 2008).

12.6 Conclusion

Opioid peptides are endogenous ligands for opioid receptors. Proteolytic processing
of larger precursor proteins generates the peptides. The peptides are stored in dense
vesicles within neurons and released upon activation. The release is not restricted to
synaptic space; thus peptides may signal other neurons by volume transmission.
Opioid peptides inhibit the release of neurotransmitters by the affected neurons, thus
modulating their signal propagations. The action of opioid peptides is mediated by
binding GPCR group of receptors (opioid receptors) by binding to the orthosteric
binding site. However, the affinity/efficacy of orthosteric ligand is affected by
cooperation with ligand at allosteric site. Interestingly, many orthosteric and alloste-
ric ligands show biased activation of intracellular second messengers, which
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 403

provide an opportunity of separating the desired pharmacological properties from

a myriad of unwanted effects. Due to the interaction between opioid receptors and
other types of receptors and ion channels, opioid peptides find a wide range of
applications in managing several types of neurological disorders besides their
primary use as analgesics.

12.7 Remark

The authors declare that theory of biological evolution and its related terms men-
tioned in this chapter and in references are not considered per se by them.

References
Agnati LF, Bjelke B, Fuxe K (1995) Volume versus wiring transmission in the brain: a new
theoretical frame for neuropsychopharmacology. Med Res Rev 15:33–45
Al-Hasani R, Bruchas MR (2011) Molecular mechanisms of opioid receptor-dependent signaling
and behavior. Anesthesiology 115:1363–1381
Allouche SP, Noble F, Marie N (2014) Opioid receptor desensitization: mechanisms and its link to
tolerance. Front Pharmacol 5:280
Asvadi NH, Morgan M, Herath HM, Hewavitharana AK, Shaw PN, Cabot PJ (2014a) Beta-
endorphin 1-31 biotransformation and cAMP modulation in inflammation. PLoS One 9:e90380
Asvadi NH, Morgan M, Hewavitharana AK, Shaw PN, Cabot PJ (2014b) Biotransformation of
beta-endorphin and possible therapeutic implications. Front Pharmacol 5:18–18
Bailey CR, Cordell E, Sobin SM, Neumeister A (2013) Recent progress in understanding the
pathophysiology of post-traumatic stress disorder. CNS Drugs 27:221–232
Basbaum AI, Fields HL (1984) Endogenous pain control systems: brainstem spinal pathways and
endorphin circuitry. Annu Rev Neurosci 7:309–338
Bennett GJ (2000) Update on the neurophysiology of pain transmission and modulation: focus on
the NMDA-receptor. J Pain Symptom Manage 19:2–6
Bermudez M, Nguyen TN, Omieczynski C, Wolber G (2019) Strategies for the discovery of biased
GPCR ligands. Drug Discov Today 24:1031–1037
Berrocoso E, Mico J-A (2009) Cooperative opioid and serotonergic mechanisms generate superior
antidepressant-like effects in a mice model of depression. Int J Neuropsychopharmacol
12:1033–1044
Bie B (2005) cAMP-mediated mechanisms for pain sensitization during opioid withdrawal. J
Neurosci 25:3824–3832
Bisson JI, Cosgrove S, Lewis C, Roberts NP (2015) Post-traumatic stress disorder. BMJ 351:h6161
Bodnar RJ (2018) Endogenous opiates and behavior: 2016. Peptides 101:167–212
Bohn LM, Lefkowitz RJ, Gainetdinov RR, Peppel K, Caron MG, Lin FT (1999) Enhanced
morphine analgesia in mice lacking beta-arrestin 2. Science 286:2495–2498
Bohn LM, Gainetdinov RR, Lin FT, Lefkowitz RJ, Caron MG (2000) Mu-opioid receptor desensi-
tization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature
408:720–723
Bologna Z, Teoh JP, Bayoumi AS, Tang Y, Kim IM (2017) Biased G protein-coupled receptor
signaling: new player in modulating physiology and pathology. Biomol Ther (Seoul) 25:12–25
Browne CA, Lucki I (2019) Targeting opioid dysregulation in depression for the development of
novel therapeutics. Pharmacol Ther. https://doi.org/10.1016/j.pharmthera.2019.04.009
Brownstein MJ (1993) A brief history of opiates, opioid peptides, and opioid receptors. Proc Natl
Acad Sci U S A 90:5391–5393
404 M. N. Al–Qattan et al.

Brust TF, Morgenweck J, Kim SA, Rose JH, Locke JL, Schmid CL, Zhou L, Stahl EL, Cameron
MD, Scarry SM, AubÉ J, Jones SR, Martin TJ, Bohn LM (2016) Biased agonists of the kappa
opioid receptor suppress pain and itch without causing sedation or dysphoria. Sci Signal 9:ra117
Burford NT, Livingston KE, Canals M, Ryan MR, Budenholzer LM, Han Y, Shang Y, Herbst JJ,
O’connell J, Banks M, Zhang L, Filizola M, Bassoni DL, Wehrman TS, Christopoulos A,
Traynor JR, Gerritz SW, Alt A (2015) Discovery, synthesis, and molecular pharmacology of
selective positive allosteric modulators of the delta-opioid receptor. J Med Chem 58:4220–4229
Butt AM, Kalsi A (2006) Inwardly rectifying potassium channels (Kir) in central nervous system
glia: a special role for Kir4.1 in glial functions. J Cell Mol Med 10:33–44
Cai Z, Ratka A (2012) Opioid system and Alzheimer’s disease. Neuromol Med 14:91–111
Calo G, Guerrini R, Rizzi A, Salvadori S, Regoli D (2000) Pharmacology of nociceptin and its
receptor: a novel therapeutic target. Br J Pharmacol 129:1261–1283
Carlezon WA, Krystal AD (2016) Kappa-opioid antagonists for psychiatric disorders: from bench
to clinical trials. Depress Anxiety 33:895–906
Castro MG, Morrison E (1997) Post-translational processing of proopiomelanocortin in the pitui-
tary and in the brain. Crit Rev Neurobiol 11:35–37
Catterall WA (2011) Voltage-gated calcium channels. Cold Spring Harb Perspect Biol 3:a003947–
a003947
Catterall WA, Leal K, Nanou E (2013) Calcium channels and short-term synaptic plasticity. J Biol
Chem 288:10742–10749
Cawley NX, Li Z, Loh YP (2016) 60 years of POMC: biosynthesis, trafficking, and secretion of
pro-opiomelanocortin-derived peptides. J Mol Endocrinol 56:T77–T97
Chattopadhyay M, Mata M, Fink DJ (2008) Continuous δ-opioid receptor activation reduces
neuronal voltage-gated sodium channel (NaV1. 7) levels through activation of protein kinase
C in painful diabetic neuropathy. J Neurosci 28:6652–6658
Chaudhary AMD, Khan MF, Dhillon SS, Naveed S (2019) A review of samidorphan: a novel
opioid antagonist. Cureus. https://doi.org/10.7759/cureus.5139
Chavkin C, Koob GF (2016) Dynorphin, dysphoria, and dependence: the stress of addiction.
Neuropsychopharmacology 41:373–374
Che T, Majumdar S, Zaidi SA, Ondachi P, Mccorvy JD, Wang S, Mosier PD, Uprety R, Vardy E,
Krumm BE, Han GW, Lee MY, Pardon E, Steyaert J, Huang XP, Strachan RT, Tribo AR,
Pasternak GW, Carroll FI, Stevens RC, Cherezov V, Katritch V, Wacker D, Roth BL (2018)
Structure of the nanobody-stabilized active state of the kappa opioid receptor. Cell 172:55–67.
e15
Chieng B, Bekkers JM (2001) Inhibition of calcium channels by opioid-and adenosine-receptor
agonists in neurons of the nucleus accumbens. Br J Pharmacol 133:337–344
Clark SD, Abi-Dargham A (2019) The role of dynorphin and the kappa opioid receptor in the
symptomatology of schizophrenia: a review of the evidence. Biol Psychiatry 86:502–511
Coimbra JRM, Marques DFF, Baptista SJ, Pereira CMF, Moreira PI, Dinis TCP, Santos AE,
Salvador JAR (2018) Highlights in BACE1 inhibitors for Alzheimer’s disease treatment.
Front Chem 6:178
Collard MW, Day R, Akil H, Uhler MD, Douglass JO (1990) Sertoli cells are the primary site of
prodynorphin gene expression in rat testis: regulation of mRNA and secreted peptide levels by
cyclic adenosine 3’,5’-monophosphate analogs in cultured cells. Mol Endocrinol 4:1488–1496
Commons KG (2010) Neuronal pathways linking substance P to drug addiction and stress. Brain
Res 1314:175–182
Conibear AE, Kelly E (2019) A biased view of mu-opioid receptors? Mol Pharmacol 96:542–549
Connor M, Christie MJ (1999) Opioid receptor signalling mechanisms. Clin Exp Pharmacol Physiol
26:493–499
Corder G, Castro DC, Bruchas MR, Scherrer G (2018) Endogenous and exogenous opioids in pain.
Annu Rev Neurosci. https://doi.org/10.1146/annurev-neuro-080317-061522
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 405

Corsetti M, Pannemans J, Whorwell P (2019) Targeting mu opioid receptors to modulate gastroin-

testinal function: what have we learnt so far from the studies in functional bowel disorders?
F1000Res 8:257
Coward P, Wada HG, Falk MS, Chan SDH, Meng F, Akil H, Conklin BR (1998) Controlling
signaling with a specifically designed Gi-coupled receptor. Proc Natl Acad Sci 95:352–357
Cox BM, Opheim KE, Teschemacher H, Goldstein A (1975) Purification and properties. Life Sci
16:1777–1782
Cox BM, Goldstein A, Hi CH (1976) Opioid activity of a peptide, beta-lipotropin-(61-91), derived
from beta-lipotropin. Proc Natl Acad Sci U S A 73:1821–1823
Creed MC, Ntamati NR, Tan KR (2014) VTA GABA neurons modulate specific learning behaviors
through the control of dopamine and cholinergic systems. Front Behav Neurosci 8:8
Cui J, Wang Y, Dong Q, Wu S, Xiao X, Hu J, Chai Z, Zhang Y (2011) Morphine protects against
intracellular amyloid toxicity by inducing estradiol release and upregulation of Hsp70. J
Neurosci 31:16227–16240
Cunningham CW, Rothman RB, Prisinzano TE (2011) Neuropharmacology of the naturally
occurring κ-opioid hallucinogen salvinorin A. Pharmacol Rev 63:316–347
Day R (2009) Proopiomelanocortin. In: Squire LR (ed) Encyclopedia of neuroscience. Academic
Press, Oxford
De Lanerolle NC, Williamson A, Meredith C, Kim JH, Tabuteau H, Spencer DD, Brines ML (1997)
Dynorphin and the kappa 1 ligand [3H]U69,593 binding in the human epileptogenic hippocam-
pus. Epilepsy Res 28:189–205
De Wied D (1999) Behavioral pharmacology of neuropeptides related to melanocortins and the
neurohypophyseal hormones. Eur J Pharmacol 375:1–11
Deakin JF, DostrÖvsky JO, Smyth DG (1980) Influence of N-terminal acetylation and C-terminal
proteolysis on the analgesic activity of beta-endorphin. Biochem J 189:501–506
Dekan Z, Sianati S, Yousuf A, Sutcliffe KJ, Gillis A, Mallet C, Singh P, Jin AH, Wang AM,
Mohammadi SA, Stewart M, Ratnayake R, Fontaine F, Lacey E, Piggott AM, Du YP, Canals M,
Sessions RB, Kelly E, Capon RJ, Alewood PF, Christie MJ (2019) A tetrapeptide class of biased
analgesics from an Australian fungus targets the μ-opioid receptor. Proc Natl Acad Sci U S A
116:22353–22358
Demaagd G, Philip A (2015) Parkinson’s disease and its management: part 1: disease entity, risk
factors, pathophysiology, clinical presentation, and diagnosis. Pharm Ther 40:504
Denning GM, Ackermann LW, Barna TJ, Armstrong JG, Stoll LL, Weintraub NL, Dickson EW
(2008) Proenkephalin expression and enkephalin release are widely observed in non-neuronal
tissues. Peptides 29:83–92
Dewire SM, Yamashita DS, Rominger DH, Liu G, Cowan CL, Graczyk TM, Chen XT, Pitis PM,
Gotchev D, Yuan C, Koblish M, Lark MW, Violin JD (2013) A G protein-biased ligand at the
mu-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunc-
tion compared with morphine. J Pharmacol Exp Ther 344:708–717
Dhawan B, Cesselin F, Raghubir R, Reisine T, Bradley P, Portoghese PS, Hamon M (1996)
International Union of Pharmacology. XII. Classification of opioid receptors. Pharmacol Rev
48:567–592
Dickenson AH (1994). Where and how do opioids act?. In: Gebhart GF, Hammond DL, Jensen TS
(eds) Progress in pain research and management. Vol. 2. Proceedings of the 7th World Congress
on Pain, Seattle: IASP Press, Seattle
Dietis N, Rowbotham DJ, Lambert DG (2011) Opioid receptor subtypes: fact or artifact? Br J
Anaesth 107:8–18
Douglass J, Cox B, Quinn B, Civelli O, Herbert E (1987) Expression of the prodynorphin gene in
male and female mammalian reproductive tissues. Endocrinology 120:707–713
Dubhashi J (2018) Analysis of the genetic and neurological components of opioid addiction, with
public health perspectives of the opioid epidemic in the United States of America. Discovery 4:4
406 M. N. Al–Qattan et al.

Egleton RD, Mitchell SA, Huber JD, Janders J, Stropova D, Polt R, Yamamura HI, Hruby VJ,
Davis TP (2000) Improved bioavailability to the brain of glycosylated Met-enkephalin analogs.
Brain Res 881:37–46
Ehrich E, Turncliff R, Du Y, Leigh-Pemberton R, Fernandez E, Jones R, Fava M (2014) Evaluation
of opioid modulation in major depressive disorder. Neuropsychopharmacology 40:1448–1455
Elman I, Borsook D (2019) The failing cascade: comorbid post traumatic stress- and opioid use
disorders. Neurosci Biobehav Rev 103:374–383
Evans RM, You H, Hameed S, Altier C, Mezghrani A, Bourinet E, Zamponi GW (2010)
Heterodimerization of ORL1 and opioid receptors and its consequences for N-type calcium
channel regulation. J Biol Chem 285:1032–1040
Feng Y, He X, Yang Y, Chao D, Lazarus LH, Xia Y (2012) Current research on opioid receptor
function. Current Drug Targets 13:230–246
Fields HL, Margolis EB (2015) Understanding opioid reward. Trends Neurosci 38:217–225
Finnegan TF, Chen S-R, Pan H-L (2006) μ opioid receptor activation inhibits GABAergic inputs to
basolateral amygdala neurons through Kv1.1/1.2 channels. J Neurophysiol 95:2032–2041
Froehlich JC (1997) Opioid peptides. Alcohol health Res World 21:132–135
Gendron L, Mittal N, Beaudry H, Walwyn W (2015) Recent advances on the δ opioid receptor:
from trafficking to function. Br J Pharmacol 172:403–419
Geracioti TD, Strawn JR, Ekhator NN, Wortman M, Kasckow J (2009) 82 - Neuroregulatory
peptides of central nervous system origin: from laboratory to clinic. In: PFAFF DW, Arnold AP,
Etgen AM, Fahrbach SE, Rubin RT (eds) Hormones, brain and behavior, 2nd edn. Academic
Press, San Diego
Ghelardini C, Mannelli LDC, Bianchi E (2015) The pharmacological basis of opioids. Clin Cases
Miner Bone Metab 12:219
Gill BJA, Khan FA, Mckhann GM (2018) You’re not hallucinating: potential new targets for
schizophrenia treatment. Neurosurgery 84:E146–E147
Granier S, Manglik A, Kruse AC, Kobilka TS, Thian FS, Weis WI, Kobilka BK (2012) Structure of
the delta-opioid receptor bound to naltrindole. Nature 485:400–404
Greenwald JD, Shafritz KM (2018) An integrative neuroscience framework for the treatment of
chronic pain: from cellular alterations to behavior. Front Integr Neurosci 12:18
Groer CE, Tidgewell K, Moyer RA, Harding WW, Rothman RB, Prisinzano TE, Bohn LM (2007)
An opioid agonist that does not induce mu-opioid receptor – arrestin interactions or receptor
internalization. Mol Pharmacol 71:549–557
Gu ZH, Wang B, Kou ZZ, Bai Y, Chen T, Dong YL, Li H, Li YQ (2017) Endomorphins: promising
endogenous opioid peptides for the development of novel analgesics. Neurosignals 25:98–116
Guerrero M, Urbano M, Kim E-K, Gamo AM, Riley S, Abgaryan L, Leaf N, Van Orden LJ, Brown
SJ, Xie JY, Porreca F, Cameron MD, Rosen H, Roberts E (2019) Design and synthesis of a
novel and selective kappa opioid receptor (KOR) antagonist (Btrx-335140). J Med Chem
62:1761–1780
Günther T, Dasgupta P, Mann A, Miess E, Kliewer A, Fritzwanker S, Steinborn R, Schulz S (2017)
Targeting multiple opioid receptors – improved analgesics with reduced side effects? Br J
Pharmacol 175:2857–2868
Hassan AN, Foll BL, Imtiaz S, Rehm J (2017) The effect of post-traumatic stress disorder on the
risk of developing prescription opioid use disorder: results from the National Epidemiologic
Survey on Alcohol and Related Conditions III. Drug Alcohol Depend 179:260–266
Heinke B, Gingl E, SandkÜhler J (2011) Multiple targets of μ-opioid receptor-mediated presynaptic
inhibition at primary afferent Aδ- and C-fibers. J Neurosci 31:1313–1322
Herath HMDR, Cabot PJ, Shaw PN, Hewavitharana AK (2012) Study of beta endorphin metabo-
lism in inflamed tissue, serum and trypsin solution by liquid chromatography–tandem mass
spectrometric analysis. Anal Bioanal Chem 402:2089–2100
Hersh LB, Rodgers DW (2008) Neprilysin and amyloid beta peptide degradation. Curr Alzheimer
Res 5:225–231
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 407

Ho JH, Stahl EL, Schmid CL, Scarry SM, Aube J, Bohn LM (2018) G protein signaling-biased
agonism at the kappa-opioid receptor is maintained in striatal neurons. Sci Signal 11. https://doi.
org/10.1126/scisignal.aar4309
Hökfelt T, Broberger C, Xu Z-QD, Sergeyev V, Ubink R, Diez M (2000) Neuropeptides — an
overview. Neuropharmacology 39:1337–1356
Holzer P (2009) Opioid receptors in the gastrointestinal tract. Regul Pept 155:11–17
Huang W, Manglik A, Venkatakrishnan AJ, Laeremans T, Feinberg EN, Sanborn AL, Kato HE,
Livingston KE, Thorsen TS, Kling RC, Granier S, Gmeiner P, Husbands SM, Traynor JR, Weis
WI, Steyaert J, Dror RO, Kobilka BK (2015) Structural insights into micro-opioid receptor
activation. Nature 524:315–321
Hughes J, Smith T, Morgan B, Fothergill L (1975) Purification and properties of enkephalin — The
possible endogenous ligand for the morphine receptor. Life Sci 16:1753–1758
Hughes J, Beaumont A, Fuentes JA, Malfroy B, Unsworth C (1980) Opioid peptides: aspects of
their origin, release and metabolism. J Exp Biol 89:239–255
Hurd YL (2002) Subjects with major depression or bipolar disorder show reduction of
prodynorphin mRNA expression in discrete nuclei of the amygdaloid complex. Mol Psychiatry
7:75–81
Ingram SL, Vaughan CW, Bagley EE, Connor M, Christie MJ (1998) Enhanced Opioid Efficacy in
Opioid Dependence Is Caused by an Altered Signal Transduction Pathway. J Neurosci
18:10269–10276
Iwaszkiewicz KS, Schneider JJ, Hua S (2013) Targeting peripheral opioid receptors to promote
analgesic and anti-inflammatory actions. Front Pharmacol 4:132
Jeon HJ, Baek JH, Ahn Y-M, Kim SJ, Ha TH, Cha B, Moon E, Kang H-J, Ryu V, Cho C-H, Heo
J-Y, Kim K, Lee H-J (2016) Review of cohort studies for mood disorders. Psychiatry Investig
13:265
Ji H, Wang Y, Liu G, Chang L, Chen Z, Zhou D, Xu X, Cui W, Hong Q, Jiang L, Li J, Zhou X,
Li Y, Guo Z, Zha Q, Niu Y, Weng Q, Duan S, Wang Q (2017) Elevated OPRD1 promoter
methylation in Alzheimer’s disease patients. PLoS One 12:e0172335
Johnston TH, Versi E, Howson PA, Ravenscroft P, Fox SH, Hill MP, Reidenberg BE, Corey R,
Brotchie JM (2018) DPI-289, a novel mixed delta opioid agonist / mu opioid antagonist
(DAMA), has L-DOPA-sparing potential in Parkinson’s disease. Neuropharmacology
131:116–127
Kalia LV, Lang AE (2015) Parkinson’s disease. Lancet 386:896–912
Kerage D, Sloan EK, Mattarollo SR, Mccombe PA (2019) Interaction of neurotransmitters and
neurochemicals with lymphocytes. J Neuroimmunol 332:99–111
Khan SM, Sleno R, Gora S, Zylbergold P, Laverdure J-P, LabbÉ J-C, Miller GJ, HÉbert TE (2013)
The expanding roles of Gβγ subunits in G protein–coupled receptor signaling and drug action.
Pharmacol Rev 65:545–577
Knoll AT, Carlezon WA (2010) Dynorphin, stress, and depression. Brain Res 1314:56–73
Koehl A, Hu H, Maeda S, Zhang Y, Qu Q, Paggi JM, Latorraca NR, Hilger D, Dawson R, Matile H,
Schertler GFX, Granier S, Weis WI, Dror RO, Manglik A, Skiniotis G, Kobilka BK (2018)
Structure of the micro-opioid receptor-Gi protein complex. Nature 558:547–552
Koob GF, Bloom FE (1988) Cellular and molecular mechanisms of drug dependence. Science
242:715–723
Kosten T, George T (2002) The neurobiology of opioid dependence: implications for treatment. Sci
Pract Perspect 1:13–20
Kosterlitz HW, Hughes J (1975) Some thoughts on the significance of enkephalin, the endogenous
ligand. Life Sci 17:91–96
Kruegel AC, Gassaway MM, Kapoor A, VÁradi A, Majumdar S, Filizola M, Javitch JA, Sames D
(2016) Synthetic and receptor signaling explorations of the mitragyna alkaloids: mitragynine as
an atypical molecular framework for opioid receptor modulators. J Am Chem Soc
138:6754–6764
408 M. N. Al–Qattan et al.

Lacy BE, Mearin F, Chang L, Chey WD, Lembo AJ, Simren M, Spiller R (2016) Bowel disorders.
Gastroenterology 150:1393–1407.e5
Lalanne L, Ayranci G, Kieffer BL, Lutz P-E (2014) The kappa opioid receptor: from addiction to
depression, and back. Front Psychiatry 5:170
Lamberts JT, Jutkiewicz EM, Mortensen RM, Traynor JR (2011) mu-Opioid receptor coupling to
Galpha(o) plays an important role in opioid antinociception. Neuropsychopharmacology
36:2041–2053
Latremoliere A, Woolf CJ (2009) Central sensitization: a generator of pain hypersensitivity by
central neural plasticity. J Pain 10:895–926
Law PY (2011) Opioid receptor signal transduction mechanisms. In: Pasternak GW (ed) The opiate
receptors. Humana Press, Totowa, NJ
Leith JL, Wilson AW, Donaldson LF, Lumb BM (2007) Cyclooxygenase-1-derived prostaglandins
in the periaqueductal gray differentially control C- versus A-fiber-evoked spinal nociception. J
Neurosci 27:11296–11305
Lembeck F (2008) The archeology of substance P. Neuropeptides 42:444–453
Li CH, Chung D (1976) Isolation and structure of an untriakontapeptide with opiate activity from
camel pituitary glands. Proc Natl Acad Sci U S A 73:1145–1148
Li Y, Lefever MR, Muthu D, Bidlack JM, Bilsky EJ, Polt R (2012) Opioid glycopeptide analgesics
derived from endogenous enkephalins and endorphins. Future Med Chem 4:205–226
Li J, Blankenship ML, Baccei ML (2013) Inward-rectifying potassium (Kir) channels regulate
pacemaker activity in spinal nociceptive circuits during early life. J Neurosci 33:3352–3362
Listos J, Łupina M, Talarek S, Mazur A, Orzelska-GÓrka J, KotliŃska J (2019) The mechanisms
involved in morphine addiction: an overview. Int J Mol Sci 20:4302
Livingston KE, Traynor JR (2018) Allostery at opioid receptors: modulation with small molecule
ligands. Br J Pharmacol 175:2846–2856
Livingston KE, Stanczyk MA, Burford NT, Alt A, Canals M, Traynor JR (2018) Pharmacologic
evidence for a putative conserved allosteric site on opioid receptors. Mol Pharmacol 93:157–167
Loh YP, Gainer H (1978) The role of glycosylation on the biosynthesis, degradation, and secretion
of the ACTH-beta-lipotropin common precursor and its peptide products. FEBS Lett
96:269–272
Loose M, Ronnekleiv O, Kelly M (1990) Membrane properties and response to opioids of identified
dopamine neurons in the guinea pig hypothalamus. J Neurosci 10:3627–3634
Luca G, Federico S, Roberto A (2007) Re-discussion of the importance of ionic interactions in
stabilizing ligand-opioid receptor complex and in activating signal transduction. Curr Drug
Targets 8:185–196
Ludwig M, Leng G (2006) Dendritic peptide release and peptide-dependent behaviours. Nat Rev
Neurosci 7:126–136
Lutz PE, Kieffer BL (2013) Opioid receptors: distinct roles in mood disorders. Trends Neurosci
36:195–206
Mabrouk OS, Marti M, Salvadori S, Morari M (2009) The novel delta opioid receptor agonist
UFP-512 dually modulates motor activity in hemiparkinsonian rats via control of the nigro-
thalamic pathway. Neuroscience 164:360–369
Mabrouk OS, Viaro R, Volta M, Ledonne A, Mercuri N, Morari M (2014) stimulation of opioid
receptor and blockade of nociceptin/orphanin FQ receptor synergistically attenuate Parkinson-
ism. J Neurosci 34:12953–12962
Machelska H, Celik MÖ (2018) Advances in achieving opioid analgesia without side effects. Front
Pharmacol 9. https://doi.org/10.3389/fphar.2018.01388
Madariaga-MazÓN A, Marmolejo-Valencia AF, Li Y, Toll L, Houghten RA, Martinez-Mayorga K
(2017) Mu-opioid receptor biased ligands: a safer and painless discovery of analgesics? Drug
Discov Today 22:1719–1729
Mahmod Al-Qattan MN, Mordi MN (2019) Molecular basis of modulating adenosine receptors
activities. Curr Pharm Design 25:817–831
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 409

Mains RE, Eipper BA (1981) Coordinate, equimolar secretion of smaller peptide products derived
from pro-ACTH/endorphin by mouse pituitary tumor cells. J Cell Biol 89:21–28
Malfroy B, Swerts JP, Guyon A, Roques BP, Schwartz JC (1978) High-affinity enkephalin-
degrading peptidase in brain is increased after morphine. Nature 276:523–526
Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis WI,
Kobilka BK, Granier S (2012) Crystal structure of the micro-opioid receptor bound to a
morphinan antagonist. Nature 485:321–326
Manglik A, Lin H, Aryal DK, Mccorvy JD, Dengler D, Corder G, Levit A, Kling RC, Bernat V,
Hubner H, Huang XP, Sassano MF, Giguere PM, Lober S, Da D, Scherrer G, Kobilka BK,
Gmeiner P, Roth BL, Shoichet BK (2016) Structure-based discovery of opioid analgesics with
reduced side effects. Nature 537:185–190
Mansour A, Fox CA, Akil H, Watson SJ (1995) Opioid-receptor mRNA expression in the rat CNS:
anatomical and functional implications. Trends Neurosci 18:22–29
Markman J, Gudin J, Rauck R, Argoff C, Rowbotham M, Agaiby E, Gimbel J, Katz N, Doberstein
SK, Tagliaferri M, Lu L, Siddhanti S, Hale M (2019) SUMMIT-07. Pain 160:1374–1382
Martin WF, Correll CU, Weiden PJ, Jiang Y, Pathak S, Dipetrillo L, Silverman BL, Ehrich EW
(2019) Mitigation of olanzapine-induced weight gain with samidorphan, an opioid antagonist: a
randomized double-blind phase 2 study in patients with schizophrenia. Am J Psychiatry
176:457–467
Mathieu-Kia A-M, Fan L-Q, Kreek MJ, Simon EJ, Hiller JM (2001) μ-, δ- and κ-opioid receptor
populations are differentially altered in distinct areas of postmortem brains of Alzheimer’s
disease patients. Brain Res 893:121–134
Matsushima A, Sese J, Koyanagi KO (2019) Biosynthetic short neuropeptides: a rational theory
based on experimental results for the missing pain-relief opioid endomorphin precursor gene.
Chembiochem 20:2054–2058
Maves TJ, Pechman PS, Meller ST, Gebhart G (1994) Ketorolac potentiates morphine
antinociception during visceral nociception in the rat. Anesthesiology 80:1094–1101
McCarty D, Priest KC, Korthuis PT (2018) Treatment and prevention of opioid use disorder:
challenges and opportunities. Annu Rev Public Health 39:525–541
McDonald J, Lambert D (2005) Opioid receptors. Contin Educ Anaesth Crit Care Pain 5:22–25
Meguro Y, Miyano K, Hirayama S, Yoshida Y, Ishibashi N, Ogino T, Fujii Y, Manabe S, Eto M,
Nonaka M, Fujii H, Ueta Y, Narita M, Sata N, Yada T, Uezono Y (2018) Neuropeptide oxytocin
enhances μ opioid receptor signaling as a positive allosteric modulator. J Pharmacol Sci
137:67–75
Meilandt WJ, Yu G-Q, Chin J, Roberson ED, Palop JJ, Wu T, Scearce-Levie K, Mucke L (2008)
Enkephalin elevations contribute to neuronal and behavioral impairments in a transgenic mouse
model of Alzheimer’s disease. J Neurosci 28:5007–5017
Mello NK, Negus SS (2006) Interactions between kappa opioid agonists and cocaine: preclinical
studies. Annal N Y Acad Sci 909:104–132
Ménard C, Herzog H, Schwarzer C, Quirion R (2013) Possible role of dynorphins in Alzheimer’s
disease and age-related cognitive deficits. Neurodegener Dis 13:82–85
Mendell LM (2011) Computational functions of neurons and circuits signaling injury: relationship
to pain behavior. Proc Natl Acad Sci U S A 108:15596–15601
Metzger TG, Ferguson DM (1995) On the role of extracellular loops of opioid receptors in
conferring ligand selectivity. FEBS Lett 375:1–4
Mika J, Obara I, Przewlocka B (2011) The role of nociceptin and dynorphin in chronic pain:
implications of neuro-glial interaction. Neuropeptides 45:247–261
Mitznegg P, Domschke W, Sprugel W, Domschke S, Subramanian N, Wunsch E, Moroder L,
Demling L (1977) Enkephalins inhibit intestinal motility: mode of action. Acta
Hepatogastroenterol (Stuttg) 24:119–120
Mogil JS, Pasternak GW (2001) The molecular and behavioral pharmacology of the orphanin
Fq/nociceptin peptide and receptor family. Pharmacol Rev 53:381–415
410 M. N. Al–Qattan et al.

Mores KL, Cummins BR, Cassell RJ, Van Rijn RM (2019) A review of the therapeutic potential of
recently developed G protein-biased kappa agonists. Front Pharmacol 10
Motel CW, Coop A, Cunningham CW (2013) Cholinergic modulation by opioid receptor ligands:
potential application to Alzheimer’s disease. Mini Rev Med Chem 13:456–466
Nandhu MS, Naijil G, Smijin S, Jayanarayanan S, Paulose CS (2010) Opioid system functional
regulation in neurological disease management. J Neurosci Res 88:3215–3221
Neves SR, Ram PT, Iyengar R (2002) G protein pathways. Science 296:1636–1639
Niciu MJ, Arias AJ (2013) Targeted opioid receptor antagonists in the treatment of alcohol use
disorders. CNS Drugs 27:777–787
Nieto MM, Guen SL, Kieffer BL, Roques BP, Noble F (2005) Physiological control of emotion-
related behaviors by endogenous enkephalins involves essentially the delta opioid receptors.
Neuroscience 135:305–313
Norn S, Kruse PR, Kruse E (2005) History of opium poppy and morphine. Dan Medicinhist Arbog
33:171–184
North R (1993) Opioid actions on membrane ion channels. In: Opioids. Springer, New York, NY
North RA, Williams JT (1983) How do opiates inhibit neurotransmitter release? Trends Neurosci
6:337–339
Nuechterlein EB (2016) Alterations in endogenous opioid neurotransmission associated with acute
and long-term use of drugs of abuse. PhD Thesis, University of Michigan, Ann Arbor, USA.
https://deepblue.lib.umich.edu/bitstream/handle/2027.42/120787/emilybn_1.pdf?sequence¼1
O’malley SS, Jaffe AJ, Chang G, Schottenfeld RS, Meyer RE, Rounsaville B (1992) Naltrexone
and coping skills therapy for alcohol dependence: a controlled study. Arch Gen Psychiatry
49:881–887
Ohbuchi K, Miyagi C, Suzuki Y, Mizuhara Y, Mizuno K, Omiya Y, Yamamoto M, Warabi E,
Sudo Y, Yokoyama A, Miyano K, Hirokawa T, Uezono Y (2016) Ignavine: a novel allosteric
modulator of the μ opioid receptor. Sci Rep 6:31748
Owen MJ, Sawa A, Mortensen PB (2016) Schizophrenia. Lancet 388:86–97
Pan J, Cai H (2017) Opioid system in L-Dopa-induced dyskinesia. Transl Neurodegener. https://
deepblue.lib.umich.edu/bitstream/handle/2027.42/120787/emilybn_1.pdf?sequence¼1
Papadimitriou A, Priftis KN (2009) Regulation of the hypothalamic-pituitary-adrenal axis.
Neuroimmunomodulation 16:265–271
Patel RM, Shulman ST (2015) Kawasaki disease: a comprehensive review of treatment options. J
Clin Pharm Ther 40:620–625
Pathan H, Williams J (2012) Basic opioid pharmacology: an update. Br J Pain 6:11–16
Pert CB, Snyder SH (1973) Properties of opiate-receptor binding in rat brain. Proc Natl Acad Sci U
S A 70:2243–2247
Petrenko AB, Yamakura T, Baba H, Shimoji K (2003) The role of N-methyl-D-aspartate (NMDA)
receptors in pain: a review. Anesth Analg 97:1108–1116
Pleuvry BJ (1991) Opioid receptors and their ligands: natural and unnatural. Br J Anaesth
66:370–380
Podvin S, Yaksh T, Hook V (2016) The emerging role of spinal dynorphin in chronic pain: a
therapeutic perspective. Annu Rev Pharmacol Toxicol 56:511–533
Polo S, DÍaz AF, Gallardo N, LeÁnez S, Balboni G, Pol O (2019) Treatment with the delta opioid
agonist Ufp-512 alleviates chronic inflammatory and neuropathic pain: mechanisms implicated.
Front Pharmacol 10:283
Portoghese PS, Sultana M, Nagase H, Takemori AE (1988) Application of the message-address
concept in the design of highly potent and selective non-peptide .delta. opioid receptor
antagonists. J Med Chem 31:281–282
Przewlocki R (2013) Opioid peptides. In: Pfaff DW (ed) Neuroscience in the 21st century: from
basic to clinical. New York, NY, Springer
Ramaswamy S, Langford R (2017) Antinociceptive and immunosuppressive effect of opioids in an
acute postoperative setting: an evidence-based review. BJA Educ 17:105–110
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 411

Ranjan R, Pandey S, Shukla AK (2017) Biased opioid receptor ligands: gain without pain. Trends
Endocrinol Metab 28:247–249
Reindl JD, Rowan K, Carey AN, Peng X, Neumeyer JL, McLaughlin JP (2008) Antidepressant-like
effects of the novel kappa opioid antagonist MCL-144B in the forced-swim test. Pharmacology
81:229–235
Reisine T, Bell GI (1993) Molecular biology of opioid receptors. Trends Neurosci 16:506–510
Rodrı́guez-Manzo G, Asai M, Fernández-Guasti A (2002) Evidence for changes in brain enkephalin
contents associated to male rat sexual activity. Behav Brain Res 131:47–55
Roth BL, Baner K, Westkaemper R, Siebert D, Rice KC, Steinberg S, Ernsberger P, Rothman RB
(2002) Salvinorin A: a potent naturally occurring nonnitrogenous opioid selective agonist. Proc
Natl Acad Sci 99:11934–11939
Rubinstein M, Mogil JS, JapÓN M, Chan EC, Allen RG, Low MJ (1996) Absence of opioid stress-
induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis. Proc Natl Acad
Sci 93:3995–4000
Rush AM, Cummins TR, Waxman SG (2007) Multiple sodium channels and their roles in
electrogenesis within dorsal root ganglion neurons. J Physiol 579:1–14
Russo AF (2017) Overview of neuropeptides: awakening the senses? Headache 57(Suppl 2):37–46
Sarajarvi T, Marttinen M, Natunen T, Kauppinen T, MÄkinen P, Helisalmi S, Laitinen M,
Rauramaa T, Leinonen V, PetÄJÄ-Repo U, Soininen H, Haapasalo A, Hiltunen M (2015)
Genetic variation in δ-opioid receptor associates with increased β- and γ-secretase activity in the
late stages of Alzheimer’s disease. J Alzheimer’s Dis 48:507–516
Schaffer CB, Nordahl TE, Schaffer LC, Howe J (2007) Mood-elevating effects of opioid analgesics
in patients with bipolar disorder. J Neuropsychiatry 19:449–452
Schapira AHV, Chaudhuri KR, Jenner P (2017) Non-motor features of Parkinson disease. Nat Rev
Neurosci 18:435–450
Scheuer T (2011) Regulation of sodium channel activity by phosphorylation. Semin Cell Dev Biol
22:160–165
Schuckit MA (2016) Treatment of opioid-use disorders. N Engl J Med 375:357–368
Seseña E, Vega R, Soto E (2014) Activation of μ-opioid receptors inhibits calcium-currents in the
vestibular afferent neurons of the rat through a cAMP dependent mechanism. Front Cell
Neurosci 8:90
Sgroi S, Tonini R (2018) Opioidergic modulation of striatal circuits, implications in Parkinson’s
disease and levodopa induced dyskinesia. Front Neurol 9:524
Shalev A, Liberzon I, Marmar C (2017) Post-traumatic stress disorder. N Engl J Med
376:2459–2469
Sheffler DJ, Roth BL (2003) Salvinorin A: the ‘magic mint’ hallucinogen finds a molecular target in
the kappa opioid receptor. Trends Pharmacol Sci 24:107–109
Shekhar A (2019) Role of kappa opioid receptors in symptoms of schizophrenia: what is the
neurobiology? Biol Psychiatry 86:494–496
Sher L (2004) The role of endogenous opioids in the placebo effect in post-traumatic stress disorder.
Complement Med Res 11:354–359
Shippenberg TS, Elmer GI (1998) The neurobiology of opiate reinforcement. Crit Rev Neurobiol
12:267–303
Shirayama Y, Chaki S (2006) Neurochemistry of the nucleus accumbens and its relevance to
depression and antidepressant action in rodents. Curr Neuropharmacol 4:277–291
Smrcka A (2008) G protein βγ subunits: central mediators of G protein-coupled receptor signaling.
Cell Mol Life Sci 65:2191–2214
Snyder SH (2004) Opiate receptors and beyond: 30 years of neural signaling research. Neurophar-
macology 47:274–285
Sprouse-Blum AS, Smith G, Sugai D, Parsa FD (2010) Understanding endorphins and their
importance in pain management. Hawaii Med J 69:70–71
412 M. N. Al–Qattan et al.

Stanczyk MA, Livingston KE, Chang L, Weinberg ZY, Puthenveedu MA, Traynor JR (2019) The
delta-opioid receptor positive allosteric modulator BMS 986187 is a G-protein-biased allosteric
agonist. Br J Pharmacol 176:1649–1663
Takahashi A (2016) Subchapter 7A - enkephalin. In: TAKEI Y, Ando H, Tsutsui K (eds) Handbook
of hormones. Academic Press, San Diego
Takeuchi M (2001) The mammalian pars intermedia—structure and function. Zoolog Sci 18
(133-144):12
Tejeda HA, Shippenberg TS, Henriksson R (2011) The dynorphin/κ-opioid receptor system and its
role in psychiatric disorders. Cell Mol Life Sci 69:857–896
Terskiy A, Wannemacher KM, Yadav PN, Tsai M, Tian B, Howells RD (2007) Search of the
human proteome for endomorphin-1 and endomorphin-2 precursor proteins. Life Sci
81:1593–1601
Theriot J, Azadfard M, Kum B (2019) Opioid antagonists. In: StatPearls. StatPearls Publishing,
Treasure Island (FL)
Thompson GL, Lane JR, Coudrat T, Sexton PM, Christopoulos A, Canals M (2015) Biased
agonism of endogenous opioid peptides at the mu-opioid receptor. Mol Pharmacol 88:335–346
Ting-A-Kee R, Van Der Kooy D (2012) The neurobiology of opiate motivation. Cold Spring Harb
Perspect Med 2:a012096–a012096
Tomkins DM, Sellers EM (2001) Addiction and the brain: the role of neurotransmitters in the cause
and treatment of drug dependence. CMAJ 164:817–821
Torres-Berrio A, Nava-Mesa MO (2019) The opioid system in stress-induced memory disorders:
from basic mechanisms to clinical implications in post-traumatic stress disorder and
Alzheimer’s disease. Prog Neuropsychopharmacol Biol Psychiatry 88:327–338
Tortorici V, Vanegas H (1995) Anti-nociception induced by systemic or PAG-microinjected lysine-
acetylsalicylate in rats. Effects on tail-flick related activity of medullary off-and on-cells. Eur J
Neurosci 7:1857–1865
Tsagareli MG, Tsiklauri N, Nozadze I, Gurtskaia G (2012) Tolerance effects of non-steroidal anti-
inflammatory drugs microinjected into central amygdala, periaqueductal grey, and nucleus
raphe. Neural Regen Res 7:1029–1039
Turner AJ (2004) 108 - Neprilysin. In: Barrett AJ, Rawlings ND, Woessner JF (eds) Handbook of
proteolytic enzymes, 2nd edn. Academic Press, London
van den Pol AN (2012) Neuropeptide transmission in brain circuits. Neuron 76:98–115
Vanderah TW, Ossipov MH, Lai J, Malan PTJ, Porreca F (2001) Mechanisms of opioid-induced
pain and antinociceptive tolerance: descending facilitation and spinal dynorphin. Pain 92:5–9
Varamini P, Mansfeld FM, Blanchfield JT, Wyse BD, Smith MT, Toth I (2012) Synthesis and
biological evaluation of an orally active glycosylated endomorphin-1. J Med Chem
55:5859–5867
Vaughan CW (1998) Enhancement of opioid inhibition of GABAergic synaptic transmission by
cyclo-oxygenase inhibitors in rat periaqueductal grey neurones. Br J Pharmacol 123:1479–1481
Vaughan C, Christie M (1997) Presynaptic inhibitory action of opioids on synaptic transmission in
the rat periaqueductal grey in vitro. J Physiol 498:463–472
Vaughan CW, Ingram SL, Connor MA, Christie MJ (1997) How opioids inhibit GABA-mediated
neurotransmission. Nature 390:611–614
Veening JG, Barendregt HP (2015) The effects of beta-endorphin: state change modification. Fluids
Barriers CNS 12:3–3
Veening JG, Gerrits PO, Barendregt HP (2012) Volume transmission of beta-endorphin via the
cerebrospinal fluid; a review. Fluids Barriers CNS 9:16–16
Venkatraman A, Edlow BL, Immordino-Yang MH (2017) The brainstem in emotion: a review.
Front Neuroanat 11:15
Vergura R, Balboni G, Spagnolo B, Gavioli E, Lambert DG, McDonald J, Trapella C, Lazarus LH,
Regoli D, Guerrini R, Salvadori S, CalÓ G (2008) Anxiolytic- and antidepressant-like activities
of H-Dmt-Tic-Nh-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor
agonist. Peptides 29:93–103
12 Pharmacology of Endogenous Opioids, Opiates and Their Receptors 413

Waldhoer M, Bartlett SE, Whistler JL (2004) Opioid receptors. Annu Rev Biochem 73:953–990
Wang H, Subedi K (2020) δ-Opioid receptor as a potential therapeutic target for ischemic stroke.
Neural Regen Res 15:20
Wang H, Wessendorf MW (2001) Equal proportions of small and large DRG neurons express
opioid receptor mRNAs. J Comp Neurol 429:590–600
Wang W, Gu J, Li Y-Q, Tao Y-X (2011) Are voltage-gated sodium channels on the dorsal root
ganglion involved in the development of neuropathic pain? Mol Pain 7:16
Wang Y, Wang Y-X, Liu T, Law P-Y, Loh HH, Qiu Y, Chen H-Z (2014) μ-opioid receptor
attenuates AβOligomers-induced neurotoxicity through mTOR signaling. CNS Neurosci Ther
21:8–14
Williams WA, Grant JE, Winstanley CA, Potenza MN (2008) Current concepts in the classification,
treatment, and modeling of pathological gambling and other impulse control disorders. In:
McArthur RA, Borsini F (eds) Animal and translational models for CNS drug discovery,
Academic Press, San Diego
Wilson AM, Soignier RD, Zadina JE, Kastin AJ, Nores WL, Olson RD, Olson GA (2000)
Dissociation of analgesic and rewarding effects of endomorphin-1 in rats. Peptides
21:1871–1874
Wilson-Poe AR, Jeong H-J, Vaughan CW (2017) Chronic morphine reduces the readily releasable
pool of GABA, a presynaptic mechanism of opioid tolerance. J Physiol 595:6541–6555
Winters BL, Gregoriou GC, Kissiwaa SA, Wells OA, Medagoda DI, Hermes SM, Burford NT,
Alt A, Aicher SA, Bagley EE (2017) Endogenous opioids regulate moment-to-moment neuronal
communication and excitability. Nat Commun 8:14611
Wu ZQ, Chen J, Chi ZQ, Liu JG (2007) Involvement of dopamine system in regulation of Na+,K+-
ATPase in the striatum upon activation of opioid receptors by morphine. Mol Pharmacol
71:519–530
Wu H, Wacker D, Mileni M, Katritch V, Han GW, Vardy E, Liu W, Thompson AA, Huang XP,
Carroll FI, Mascarella SW, Westkaemper RB, Mosier PD, Roth BL, Cherezov V, Stevens RC
(2012) Structure of the human kappa-opioid receptor in complex with JDTic. Nature
485:327–332
Xi Z-X (2002) Gabaergic mechanisms of opiate reinforcement. Alcohol Alcohol 37:485–494
Xia R, Mao Z-H (2012) Progression of motor symptoms in Parkinson’s disease. Neurosci Bull
28:39–48
Xu C, Liu G, Ji H, Chen W, Dai D, Chen Z, Zhou D, Xu L, Hu H, Cui W, Chang L, Zha Q, Li L,
Duan S, Wang Q (2018) Elevated methylation of OPRM1 and OPRL1 genes in Alzheimer’s
disease. Mol Med Rep. https://doi.org/10.3892/mmr.2018.9424
Yakovleva T, Bazov I, Cebers G, Marinova Z, Hara Y, Ahmed A, Vlaskovska M, Johansson B,
Hochgeschwender U, Singh IN, Bruce-Keller AJ, Hurd YL, Kaneko T, Terenius L, EkstrÖM
TJ, Hauser KF, Pickel VM, Bakalkin G (2006) Prodynorphin storage and processing in axon
terminals and dendrites. FASEB J 20:2124–2126
Yakovleva T, Marinova Z, Kuzmin A, Seidah NG, Haroutunian V, Terenius L, Bakalkin G (2007)
Dysregulation of dynorphins in Alzheimer disease. Neurobiol Aging 28:1700–1708
Yam M, Loh Y, Tan C, Khadijah Adam S, Abdul Manan N, Basir R (2018) General pathways of
pain sensation and the major neurotransmitters involved in pain regulation. Int J Mol Sci
19:2164
Yang A, Tsai S-J (2017) New targets for schizophrenia treatment beyond the dopamine hypothesis.
Int J Mol Sci 18:1689
Yehuda R, Hoge CW, McFarlane AC, Vermetten E, Lanius RA, Nievergelt CM, Hobfoll SE,
Koenen KC, Neylan TC, Hyman SE (2015) Post-traumatic stress disorder. Nat Rev Dis Primers
1:15057. https://doi.org/10.1038/nrdp.2015.57
Young J, Mendoza M (2018) Parkinson’s disease: A treatment guide. J Fam Pract 67:276–286
Zadina JE, Hackler L, Ge LJ, Kastin AJ (1997) A potent and selective endogenous agonist for the
mu-opiate receptor. Nature 386:499–502
414 M. N. Al–Qattan et al.

Zamponi GW, Currie KPM (2013) Regulation of CaV2 calcium channels by G protein coupled
receptors. Biochim Biophys Acta 1828:1629–1643
Zamponi GW, Striessnig J, Koschak A, Dolphin AC (2015) The physiology, pathology, and
pharmacology of voltage-gated calcium channels and their future therapeutic Potential.
Pharmacol Rev 67:821–870
Zebraski SE, Kochenash SM, Raffa RB (2000) Lung opioid receptors: pharmacology and possible
target for nebulized morphine in dyspnea. Life Sci 66:2221–2231
Zhang Z, Pan ZZ (2011) Signaling cascades for δ-opioid receptor-mediated inhibition of GABA
synaptic transmission and behavioral antinociception. Mol Pharmacol 81:375–383
Zhang X, Bao L, Arvidsson U, Elde R, HÖkfelt T (1997) Localization and regulation of the delta-
opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: evidence for
association with the membrane of large dense-core vesicles. Neuroscience 82:1225–1242
Zhao J, Li X, Li B, Chen L, Pei G (2015) A new delta opioid receptor antagonist as a novel drug
against Alzheimer’s disease. Alzheimer’s Dement 11:P371
Zieglgänsberger W (2018) Substance P and pain chronicity. Cell Tissue Res 375:227–241
Pharmacology of Endocannabinoids
and Their Receptors 13
Gaurav Gupta, Wafa Hourani, Pran Kishore Deb, Satyendra Deka,
Pobitra Borah, Juhi Tiwari, Sacchidanand Pathak, and Puneet Kumar

Abstract

The identification of cannabinoid (CB) receptors has contributed to the state-of-the-art

on the endocannabinoid system and its elements. Endocannabinoids (eCBs) are the
endogenous agonists, derived from the conjugation of arachidonic acid with either
ethanolamine (i.e. anandamide) or glycerol (i.e. 2-arachidonoylglycerol) acting as a
lipid signaling mediator via two types of cannabinoid receptors (i.e. CB1 and CB2).
Introduction of selective CB antagonists, inhibitors of eCB transport and metabolism,
cannabinoid receptor-deficient mice and highlights on amidohydrolase have greatly
facilitated the subsequent investigation of the eCB system. Moreover, modulation of
the eCB system holds a promising therapeutic potential in the management of a myriad
of pathophysiological conditions such as anxiety or mood disorders, neuropathic pain,
multiple sclerosis, neurodegenerative diseases, osteoporosis, obesity and cancer

G. Gupta · S. Pathak
School of Pharmacy, Suresh Gyan Vihar University, Jaipur, India
W. Hourani (*)
Faculty of Pharmacy, Philadelphia University, Amman, Jordan
e-mail: [email protected]
P. K. Deb
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
S. Deka · P. Borah
Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam, India
J. Tiwari
Alwar Pharmacy College, Alwar, India
P. Kumar
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India

# Springer Nature Singapore Pte Ltd. 2020 415

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_13
416 G. Gupta et al.

among others. This chapter is comprehensively focused on the signal transduction and
metabolic pathways, physiological roles, pharmacology and therapeutic potential of
the endocannabinoids, with particular emphasis on cannabinoid addiction.

Keywords

Cannabinoids · Cannabinoid receptors · Biosynthesis of endocannabinoids ·

Pharmacology of endocannabinoids · Phytocannabinoids · Therapeutic potentials
of cannabinoid receptors · Addiction of cannabinoids

Abbreviations

Abh4 α/β-Hydrolase 4
2-AG 2-Arachidonoylglycerol
CB Cannabinoid
CB1R Cannabinoid 1 receptor
CB2R Cannabinoid 2 receptor
CBD Cannabidiol
CBN Cannabinol
DAG Diacylglycerol
eCBs Endocannabinoids
FAAH Fatty acid amide hydrolase
GPCRs G protein-coupled receptors
MAGL Monoacylglycerol lipase
MAPK Mitogen-activated protein kinase
NAPE N-arachidonoyl phosphatidylethanolamine
PA2 Phospholipase A2
pCBs Phytocannabinoids
PLA1 PI-explicit phospholipase A1
PLC Phospholipase C
THC Δ9-Tetrahydrocannabinol

13.1 Introduction

The medicinal use of Cannabis sativa or marijuana has a long and rich history dating
back to the sixth century B.C. and it was introduced into western medicine during the
nineteenth century (Gaoni and Mechoulam 1964). Being the oldest source of textile
fibers, cultivation of cannabis originated in Western Asia and Egypt, and later
expanded to Europe and America. Under federal laws in the United States, the
cultivation, use and possession of cannabis is illegal (1982). The legalization of
cannabis for medical and recreational use is supported by many countries, and it has
been legalized in some states of America.
Cannabis sativa is a dioecious species, that is, there is a distinct male and female
flower on separate plants, and it belongs to the Cannabinaceae family (Small et al.
2002). It is generally reported that the psychomimetic property of cannabis is
13 Pharmacology of Endocannabinoids and Their Receptors 417

Fig. 13.1 Cannabis sativa plant

associated with the female plant and the sticky resins are secreted from the glandular
hair located on the female flowers and their adjacent leaves (Fig. 13.1). A number of
constituents are found in the plant possessing their crucial role in the treatment of
various diseases, among which Δ13-tetrahydrocannabinol (THC) is the most active
constituent (Baron 2018). Cannabis sativa also consists of a hempseed fixed oil
which is found in the seed part of the plant.
This plant is therapeutically indicated in the management of nausea, pain, glau-
coma, neuralgia, cardiovascular disorder, epilepsy, inflammation, cancer, neuropsy-
chological disorder, neurodegenerative diseases, addiction, arthritis, depression and
headache (Adler and Deleo 2019; Akram et al. 2019; Alipour et al. 2019). Recent
data also suggests the use of phytocannabinoids in the management of multiple
sclerosis and HIV/AIDS symptoms.
The compounds identified or isolated from the plant have been continuously
increasing over time. Approximately 565 compounds have been identified from
C. sativa, among which approximately 120 are called cannabinoids. Cannabinoids
(CBs) are the compounds possessing the typical C21 terpenophenolic ring or its
derivatives/transformation products. CBs can be classified into two broad categories
on the basis of the location where they are found (Baron 2018). The CBs found in the
plant are known as phytocannabinoids (pCBs), whereas the CBs obtained from
animals are known as endocannabinoids (eCBs). Phytocannabinoids are either
found in the cannabis plant or in agricultural hemp (Gertsch et al. 2010). CBs are
lipophilic in nature due to which previously it was speculated that the drug directly
disrupts the cellular membrane without any specified pathway. But after the discov-
ery of some phytocannabinoids, the presence of certain receptors was observed
showing an affinity toward the CBs. CBs show their pharmacological effect by
binding with a specific cannabinoid receptor (CBR) found in the animal body, which
can be classified into two types: cannabinoid 1 receptor (CB1R) and cannabinoid
2 receptor (CB2R). These CBRs are G protein-coupled receptors (GPCRs).
Endocannabinoids are lipid-based endogenous cannabimimetic neurotransmitters
which bind to the CBR and CBR-proteins found in both the central nervous system
(CNS) and the peripheral nervous system (PNS) (Andrade et al. 2019; Borsoi et al.
2019; Breit et al. 2019). The eCBs form the endocannabinoid system, which
418 G. Gupta et al.

is involved in the maintenance of the homeostasis of the human body (Battista et al.
2012). The development of the endocannabinoid system depends upon the intake of
nutritional and dietary co-factors. The formation of eCBs occurs according to the
necessity and requirement of the body, and can bind with the receptors after being
produced. The endocannabinoids found in the human body are N-arachidonoyl-
ethanolamine (AEA; anandamide) and 2-arachidonoylglycerol (2-AG). They serve as
the endogenous agonists of CBRs and regulate the CNS as well as the PNS, thus
controlling various physiological functions of the body including the immune system.
Therefore, any changes in the endocannabinoid system will result in various disorders,
from neurodegenerative disorder to arthritis. Homeostasis is maintained due to the
constant enzymatic degradation of eCBs. Anandamide shows higher affinity but less
efficacy toward the CB1R, whereas 2-AG exhibits less affinity but high efficacy for
both the receptors (Carr et al. 2019; Chye et al. 2019; Cohen et al. 2019). The eCBs are
hydrophobic in nature and exhibit slower diffusion. The degradation of eCBs inside
the cell occurs either by oxidation or by hydrolysis. Anandamide is hydrolyzed by
fatty acid amide hydrolase into free arachidonic acid and ethanolamine, whereas 2-AG
is hydrolysed into arachidonic acid and glycerol in the presence of monoacylglycerol
lipase (MAGL). Cyclooxygenase-2 and several lipoxygenases are responsible for
hydrolysing the eCBs by the process of oxidation. Apart from maintaining homeosta-
sis, the eCBs are also responsible for recovery and repair of cells. This may confer
various properties including anti-oxidant, anti-inflammatory, anti-anxiety, anti-psy-
chotic, anti-epilepsy, anti-cancer, anti-nausea, anti-bacterial, anti-diabetic, anti-arthri-
tis, pain relief, bone stimulant, immune modulator, neuroprotective and cardio
protective activities. Some endogenous fatty acid derivatives are also found in the
body, which are known as eCB-like compounds. These substances are known to
promote the activity of classic eCBs by the entourage effect (Dale et al. 2019; Diao and
Huestis 2019; Dinis-Oliveira 2019). Apart from the above two eCBs, other reported
eCBs are noladin ether (2-arachidonylglyceryl ether), arachidonoyl dopamine and
virodhamine. The pharmacological profile and biochemical properties of these eCBs
are not known. This chapter discusses the signal transduction and metabolic pathways,
physiological roles, pharmacology and therapeutic potential of the endocannabinoids.

13.2 Cannabinoid Receptors: Signaling Pathway

and Distribution

Cannabinoid receptors are a class of cell membrane receptors that belong to the
family of rhodopsin-like G protein-coupled receptors (GPCR). There are thus two
cannabinoid receptor classes, CB1 and CB2 receptors, which are both coupled to Gi
or Go protein, via negative coupling to adenylyl cyclase (AC) and positive coupling
to the mitogen-activated protein (MAP) kinase family. CB1 receptors are also
coupled to ion channels through the Gi/o proteins. There is precisely positive
coupling to the A-type inward rectifier potassium channels with negative coupling
to N-type, P/Q-type voltage-gated calcium channels and to D-type potassium
channels (Pertwee et al. 2010). Moreover, it is presumed that CB1 also activates
adenylate cyclase types II, IV and VIII via Gsα (Rhee et al. 1998). Both receptors are
13 Pharmacology of Endocannabinoids and Their Receptors 419

Ca+2 K+ Extracellular
CB2

Adenylyl cyclase Intracellular

α Βγ
-/+ +
Gi/Go
-
cAMP ATP

+
-
PKA AKT/PKB mTOR Raf

MEK 1/2
- + +
-/+
Gene expression EKK 1/2

Fig. 13.2 Cannabinoid receptor signaling. CB Cannabinoid receptor, mTOR Mechanistic target of
rapamycin, Akt Protein kinase B, PI3K Phosphatidylinositol-3-kinase, PKA Protein kinase A, ERK
Extracellular signal-regulated kinase

located pre-synaptically and modulate neurotransmitter release. Different impacts

ascribed to the CB receptor seem to include actuation of the PI3K/AKT pathway.
Activation of the anandamide/CB/PI3K pathway was known to be associated with
protection of rodent brain from cocaine-initiated neurotoxicity (Fig. 13.2) (Crowley
et al. 2018; Curran et al. 2019; Cyr et al. 2018; Da Silva et al. 2018; Daris et al.
2019).
However, one of the essential differences between the receptor subtypes is their
distribution, whereby the CB1 is expressed on the presynaptic peripheral and central
nerve terminals as well as in the peripheral organs. It is presumed to be distributed
throughout the brain, with the highest concentration of these receptors located in the
cerebellum (motor control and cognitive function), cerebral cortex (sensation, infor-
mation processing and cognitive function), hippocampus (short- and long-term
memory), hypothalamus (hormone release, metabolic process and sexual orienta-
tion), basal ganglia (voluntary movements control, learning and emotion), amygdala
(memory and emotions), nucleus accumbens (motor and reward system), spinal cord
(transmission of neural signals) and to a lesser extent the cardiopulmonary center of
the brainstem, which seemingly justifies its lack of respiratory depression as opposed
to the opioids (Pertwee 1997). Several pronounced effects of (-)Δ9-THC can be
accounted for by the distribution pattern of CB1 receptors within the CNS. Some of
these effects include the ability of ( )Δ9-THC to decrease motor activity, as
manifested in rodents by catalepsy and hypokinesia, to stimulate food intake, and
420 G. Gupta et al.

to produce analgesia (Pertwee et al. 2010; Matsuda et al. 1990; Herkenham et al.
1991).
CB2 receptors are predominantly associated with expression in the peripheral
cells derived from the immune system. Expression of the CB2 receptor gene
transcripts was detected in the thymus, mast cells, spleen, tonsils and blood cells
(Galiègue et al. 1995; Munro et al. 1993), where they tend to modulate inflammatory
and immunosuppressive activity and control cytokines release.
In contrast, the CB1 was also detected in several peripheral tissues including the
reproductive system, cardiovascular system and gastrointestinal tract. Recently, the
CB2 was reported to be located in the CNS, for example, in the microglial cells
(Svíženská et al. 2008). It has been demonstrated that CB2Rs expression level in the
cerebrum is much lower than CB1Rs in healthy subjects (Onaivi et al. 2008, 2006;
Nunez et al. 2004). In the brain, CB2A is the significant transcript isoform, while
both CB2A and CB2B transcripts are available in larger amounts in the peripheral
tissue such as spleen, skeletal, cardiovascular, thymus and renal systems (Jordan and
Xi 2019; Rossi et al. 2018) and in immune cells, especially cells of macrophage,
lineage thymus, tonsils, T-lymphocytes, monocytes, natural killer cells, and poly-
morphonuclear cells and B-lymphocytes (Howlett et al. 2002; Schatz et al. 1997;
Galiegue et al. 1995). This prevalent distribution of cannabinoid receptors explains
their wide therapeutic applications in almost every system in the human body.
It has been reported that the CB1 receptor conserves its identity across different
species such as mammals, amphibians and fish (Yamaguchi et al. 1996; Soderstrom
et al. 2000). In contrast, cannabinoid CB2 receptors are more varied. Mukherjee and
co-workers (2004) and Bingham et al. (2007) reported that rat CB2, mouse CB2 and
human CB2 receptors show different pharmacological profiles, although all of them
are considered CB2 receptors (Mukherjee et al. 2004; Bingham et al. 2007). There is
an 81% amino acid sequence in rodent and human CB2 receptors, contrasted with
93% amino acid identity among rodent and mouse CB2 receptors (Griffin et al.
2000).
Notwithstanding CB1 and CB2 receptors, pharmacological studies recommend
the presence of non-CB1, non-CB2 receptors interceding the impacts of
CB. Although a few proteins have been examined as contenders for a potential
“CB3” receptor, the reality is questionable and not yet established (Chen 2016).

13.3 Cannabinoid Receptor Agonists and Antagonists

13.3.1 Endocannabinoids

The extensively studied endocannbainoids (eCB) are the arachidonic acid

derivatives anandamide and 2-AG. The first putative endocannabinoid to be
identified is known as Anandamide, where ‘Ananda’ is the Sanskrit word for
‘bliss’ and ‘amide’ describes the ‘amide linkage’ in its chemical structure, which
was detected in porcine brain (Devane et al. 1992).
13 Pharmacology of Endocannabinoids and Their Receptors 421

This ligand behaves as a CB1 and CB2 receptor partial agonist and demonstrates
higher intrinsic activity toward CB1 than CB2 (Mackie et al. 1993). Gonsiorek and
co-workers reported that anandamide and not its metabolite arachidonic acid
antagonizes hCB2 activation by 2-AG in CHO-hCB2 and anandamide can function
as an endogenous antagonist at the peripheral cannabinoid receptor. This strengthens
the hypothesis that the in vivo immunosuppressive effects of 2-AG or other
cannabinoids are dependent on the local concentration of anandamide and 2-AG
(Gonsiorek et al. 2000).
2-AG was the second endogenous cannabinoid receptor ligand to be discovered
following anandamide (Mechoulam et al. 1995) and better illustrates selective
binding toward CB1 than CB2 and functions as a full agonist. Anandamide and
2-AG show similar affinity toward hCB2. Some other substances have been found to
function as endocannabinoids. These substances comprise N-
dihomo-γ-linolenoylethanolamine, N-docosatetraenoylethanolamine,
arachidonoylethanolamine (virodhamine), oleamide, N-arachidonoyl dopamine
(NADA), Arachidonoyl-serine (ARA-S) and Noladin etherand N-oleoyl dopamine
(for a review, see Pertwee et al. 2010). Table 9.1 represents various eCBs and their
type of interaction with CB1R and CB2R, respectively.

13.3.1.1 Biosynthesis of Endocannabinoids

Two classes of eCBs have been recognized and completely contemplated. Ananda-
mide and 2-AG are bioactive lipids, having a place with the subdivisions called
monoacylglycerols and N-acylethanolamines, respectively.
As opposed to the majority of the intercellular signaling systems where
neurotransmitters are synthesized in advance and stored in vesicles for future use,
endocannabinoids are synthesized and used immediately. The endocannabinoids are
synthesized on demand in a stimulus-dependent pathway, activating the cannabinoid
receptor where it is required. The action of the cannabinoids is terminated once they
enter the cells, where they are metabolised by enzymatic hydrolysis (Varma et al.
2001).
Several mechanisms have been postulated for the synthesis of AEA from its
corresponding N-acyl phosphatidyl ethanolamine (NAPE) precursor. The most
widely studied pathway is based on NAPE-phospholipase D (PLD) interaction
(Okamoto et al. 2004; Schmid et al. 1983). However, two additional parallel
pathways have recently been proposed. One pathway involves deacylation of
NAPE by α,β-hydrolase 4 (ABHD4) followed by the cleavage of glycerolphosphate
producing anandamide (Liu et al. 2008). The other pathway involves phospholipase
C-mediated hydrolysis of NAPE-producing phosphoanandamide. The latter is then
dephosphorylated by phosphatases such as tyrosine phosphates PTPN22 and the
inositol 5ˋ phosphatase SHIP1. The functional relevance of these different pathways
has not yet been confirmed; however, it is quite certain that synthesis of AEA
depends on the tissues in which it is synthesized (Liu et al. 2008). Biosynthesis of
2-AG involves a two-step mechanism whereby the first step involves the generation
of 1-acyl-2-arachidonoylglycerol (diacylglycerol, DAG) from phosphatidylinositol
Table 13.1 Nature of endocannabinoids with receptor
422

S. No Name of eCB Chemical Name Chemical Structure CB1R CB2R

1. Anandamide N-arachidonoyl- Partial or full Low efficiency as an agonist and may act
ethanolamine agonist as an antagonist

2. 2- 2- Agonist Agonist
Arachidonoylglycerol arachidonoylglycerol

3. Noladin ether 2- More affinity Less affinity than CB1R

arachidonylglyceryl
ether
G. Gupta et al.
 13

4. Arachidonoyl N-arachidonoyl Antagonist Partial Agonist

dopamine dopamine

5. Virodhamine O-arachidonoyl Antagonist Agonist

ethanolamine
Pharmacology of Endocannabinoids and Their Receptors
423
424 G. Gupta et al.

N-Acylphosphatidylethanolamine Diacylglycerol

NAPE-PLD DAGL Synthesis

N-acylethanolamine Phosphatidylethanolamine Monoacylglycerol Fatty acid

FAAH MGL
FAAH 2 ABHD6 Hydrolysis
NAAA ABHD12

Ethanolamine Glycerol Fatty acid

Fatty acid

Fig. 13.3 Schematic representation of the endocannabinoid synthesis and hydrolysis. DAGL
Diacylglycerol lipase, NAPE-PLD N-acylphosphatidylethanolamine phospholipase D, FAAH
fatty acid amide hydrolase, NAAA N-acylethanolamine-hydrolyzing acid amidase, MGL
Monoacylglycerol lipase, ABHD6 α,β-hydrolase 6, ABHD12 α,β-hydrolase 12

by PLC activity and the second step comprises the hydrolysis of DAG by
diacylglycerol (Stella et al. 1997).

13.3.1.2 Hydrolysis of Endocannabinoids

The chief pathways involving the metabolism of endocannabinoids specifically,
AEA and 2-AG, are the hydrolase and oxygenase pathways (Alexander 2016).
AEA hydrolysis is mainly mediated by fatty acid amide hydrolase (FAAH) through
the hydrolytic cleavage of the amide linkage to form arachidonic acid and ethanol-
amine (Giang and Cravatt 1997). Two other enzymes have also been identified,
namely FAAH2 and N-acylethanolamine-hydrolyzing acid amidase (NAAA). The
hydrolysis of 2-AG is mainly directed by monoacylglycerol lipase (MGL). Other
2-AG hydrolases such as ABHD12 and ABHD6 have been identified (Blankman
et al. 2007). AEA is also converted by cyclooxygenase type-2 (COX-2) to
prostamides (prostaglandin-ethanolamides) while 2-AG is converted by COX-2 to
prostaglandin glycerol esters (Fowler 2007). Biosynthesis and the hydrolysis mech-
anism of eCBs are presented in Fig. 9.3.

13.3.1.3 Pharmacodynamics of Endocannabinoids

Anandamide and 2-AG are understood as two noteworthy endogenous agonists of
CBRs. 2-AG alone is a full agonist for CB1 and CB2 and intercedes retrograde signs
at the neural connection, strongly suggesting that 2-AG is physiologically more
significant than anandamide, which is essentially a partial agonist for the CB1
receptor and CB2 receptors, but has higher affinity for the CB1R (Howlett et al.
2002). Both eCBs produce biological activity through initiation of Gi/o G proteins,
coupled to calcium channel (N-and P/Q-type) hindrance and potassium channels
13 Pharmacology of Endocannabinoids and Their Receptors 425

opening in the cell membrane, which are associated with the regulation of synapse
discharge (either restraint of glutamate or GABA). Through hindrance of AC and a
decrease of cAMP, or through MAP kinase pathways, eCB could likewise prolong
cell action (Miller and Devi 2011; Tsuboi et al. 2018).

13.3.1.4 Physiological Role of Endocannabinoids

The eCB opened up new methodologies in the treatment of pain, obesity and
neurological diseases including multiple sclerosis and other mental disorders such
as drug addiction (Bilbao et al. 2004). Motor work, control of tremor and spasticity,
psychological capacity (e.g. learning and memory), thermogenesis, regulation of
sleep/wake cycle, adult neurogenesis, stress reaction by regulating the
hypothalamic-pituitary-adrenal axis (HPA), conceptive capacity through
hypothalamic-pituitary-gonadal pivot (HPG) and sex conduct, retinal neurotrans-
mission from the retina to the essential visual cortex (Chianese and Meccariello
2016) may improve by endocannabinoids. The well-recorded inhibitory impacts of
the CB1 receptor agonists on the arrival of GABA, glutamate, acetylcholine and
noradrenaline were investigated in different preclinical settings solely as clinical
examinations (Schlicker and Kathmann 2001; Piomelli 2003).

13.3.1.5 Pharmacology of Endocannabinoids

More than 4000 years ago, the eCB system was first reported in India, as well as the
remedial and psychotropic activity of the plant Cannabis sativa. The eCB system
involves the CB receptors and endogenous lipid ligands. Restorative uses of
antagonists of CB receptor for obesity treatment as well as control of eating conduct,
intake of food and vitality metabolism have been examined in several studies. The
excessive medicinal, religious and recreational utilization of marijuana in all ages
was clearly not adequate to start cautious and broad research up to a few decades of
20th era on CB. The endocannabinoid system is associated with a wide range of
physiological activities, a considerable number of which are identified with stress
recuperation systems and the support of homeostatic equalization. Among other
capacities, the endocannabinoid system is associated with neuroprotection, adjust-
ment of nociception, regulating motor-related activities and the control of specific
points of the memory process. Likewise, the endocannabinoid system is associated
with balancing the resistant and provocative reactions. It additionally impacts the
cardiovascular and respiratory system by controlling pulse, pressure and bronchial
capacities. Endocannabinoids are known to exhibit significant antiproliferative
activities in tumor cells (Elliott et al. 2019; Friedman et al. 2019; Goncalves et al.
2019; He et al. 2019; Henschke 2019).
In 1992, the first endogenous CB, AEA, additionally known as anandamide, was
distinguished. Later, a second endocannabinoid, 2-arachidonoyl glycerol (2-AG),
was found (Devane et al. 1992). Both these compounds are arachidonic acid
derivatives and bind to CB1 and CB2 receptors with contrast in affinities and
initiation efficacies. Neuropsychopharmacology has portrayed the eCB system
activity alongside the distinguishing proof of CB1R and CB2R and their endogenous
ligands. Nerve center, amygdaloid complex, hippocampus, mesencephalic
426 G. Gupta et al.

structures, substantia nigra, periaqueductal gray, superior colliculus and inferior

colliculus comprise a complex neural circuit that handles the dread and resistance
responses. For the control of anxiety and delirium states, substantia nigra pars
reticulata act as a key structure in the modulator circuit. Recently, eCB system in
the brain has developed as a significant subject of experiments focusing on stress-
related reactions. Endocannabinoids have a wide scope of neuronal reactions such as
perception, suffering, nervousness and anxiety-related symptoms. N-
arachidonoylethanolamide and 2-arachidonyl glycerol are the two most examined
eCBs that have been found to bind with CBRs. Both CB1R and CB2R, being the
GPCR, share similar signaling nature (Pertwee 1997). CB2R manages the
constrained restriction in the brain and furthermore controls the processes that are
not quite the same as CB1R. CB1R is associated with both glutamatergic excitatory
pathways and GABAergic inhibitory neurons. CB1R is otherwise called ‘cerebrum
type’ CBR as it is primarily located in the cerebrum, whereas CB2R is widely
distributed in immune and blood cells. Several studies conducted on striatonigral
neurons and the nigrostriatal pathway have demonstrated the presence of CB
receptors in the presynaptic terminals of striatonigral neurons (Ho et al. 2019;
Huestis et al. 2019; Khuja et al. 2019; Krebs et al. 2019).
In highly evolved creatures, the capacity of the ECS is to control a wide
assortment of physiological procedures at both central and peripheral level. How-
ever, the potential job of the endocannabinoid system in skeletal muscle tissue
remains obscure. Recently, it has been demonstrated that the muscle dimensions of
2-AG are diminished amid both myotube arrangement in vitro from C2C12
myoblasts and mouse muscle advancement in vivo. It has been additionally revealed
that in primary human myoblasts the activation of CB1 by endogenous 2-AG or
synthetic agonists, for example, arachidonoyl-2-chloroethylamide (ACEA),
invigorates myoblast expansion while checking myoblast separation. Inverse
impacts were seen with rimonabant (SR141716) or AM251, two CB1 antagonists/
reverse agonists. Several experiments showed that the activation of CB1 actuates a
commonplace GPCR-intervened signaling component, that is, the hydrolysis of
4,5-bisphosphate, along these lines causing the hindrance of myogenesis-advancing
voltage-gated potassium Kv7.4 channels (Kumar et al. 2019; Lattanzi et al. 2019).
The development of diacylglycerols and inositol trisphosphate is suggested as
occurring as an outcome of protein Gq-interceded PIP2 hydrolysis. CB ligands
likewise bind to GPR55, a vagrant GPCR, suggesting that this receptor may show
a novel focus of CB activity. CB1R is the standout among the most plentiful GPCR
in the human brain. ECBs are lipophilic and therefore cannot be stored in vesicles
such as different neurotransmitters (Lossignol 2019; Lowin et al. 2019).
Thus, the endocannabinoid signaling regulation is firmly constrained by its
synthesis, release, uptake and degradation. A few other stimuli, including layer
depolarization and expanded intracellular Ca2+ and receptor activation, can initiate
complex enzymatic machineries, which lead to the cleavage of membrane
phospholipids and finally to eCB synthesis. Significantly, various proteins are
associated with the synthesis of particular eCB, showing an autonomous inclusion
of eCB in various conditions. After synthesis, eCB can activate CB receptors, either
13 Pharmacology of Endocannabinoids and Their Receptors 427

after the past discharge into the extracellular space or straightforwardly moving
inside the cell membrane. Endocannabinoid flagging is restricted by extremely
proficient degradation processes, including encouraging the uptake from the extra-
cellular space into the cell and enzymatic catabolism interceded by explicit intracel-
lular compounds. The molecular nature of the transporter protein(s) associated with
endocannabinoid uptake has not yet been illustrated. Nonetheless, the compounds’
capacity to degrade eCB is quite well characterized (Bara et al. 2018; Baron 2018;
Bonini et al. 2018). They are FAAH for anandamide and related eCBs, and
monoglycerollipase for 2-AG, albeit different chemicals may be in part engaged
with the degradation of this last compound. An intriguing part of endocannabinoid
function is the quick induction of their synthesis, receptor activation and degrada-
tion. The endocannabinoid framework has in this manner been proposed to act on
interest, with a firmly controlled spatial and temporal selectivity. The framework
applies its modulatory activities only when and where it is required. It represents a
significant refinement among physiological elements of the endocannabinoid frame-
work and the biological activities of exogenous CB receptor agonists, which need
such selectivity. With regard to regulation of endocrine, it is intriguing to note here
that hormonal incitement with glucocorticoids can prompt eCB synthesis in the
nerve center by quick nongenomic systems (Bonn-Miller et al. 2018; Bramness et al.
2018; Cardenia et al. 2018). It was likewise demonstrated later that phospholipase C
communicates to an intracellular event finder of membrane depolarization and
receptor incitement, prompting the combination and, potentially, the arrival of
eCB in the hippocampus. This information uncovers a novel component for activa-
tion of the endocannabinoid framework, which could be engaged with the regulation
of endocrine frameworks. This also concerns eCB degradation, which communicates
to a significant regulatory part of the movement of the endocannabinoid framework
(Citti et al. 2018; Colizzi et al. 2018).

13.3.2 Phytocannabinoids

More than 500 compounds have been identified from Cannabis sativa, which are
phytocannabinoids in nature as well as non-phytocannabinoids. A total of
104 phytocannabinoids have been isolated to date, classified into 11 chemical classes
as follows: (–)-delta-9-trans-tetrahydrocannabinol (Δ9-THC), (–)-delta-8-trans-tetra-
hydrocannabinol (Δ8-THC), cannabidiol (CBD), cannabinodiol (CBND),
cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabicyclol
(CBL), cannabielsoin (CBE), cannabitriol (CBT) and miscellaneous-type
cannabinoids (Bilbao et al. 2004). Miscellaneous cannabinoids represent compounds
that are composed of different chemical structures and these compounds tend to
differ in their psychoactive properties. In particular, the psychoactive properties of
cannabis have been attributed to the most abundant constituent, Δ9-THC, which was
initially discovered in 1964 by Ganoi and Mechoulam (Gaoni and Mechoulam
1964), while CBD is the major non-psychotropic cannabinoid found in cannabis.
The other noncannabinoid constituents that have been isolated from cannabis since
2005 belong to 8 major chemical classes, steroids, flavonoids, fatty acids,
428 G. Gupta et al.

phenanthrenes, spiroindans, xanthones, biphenyls and nitrogenous compounds

(Baron 2018).

13.3.3 Examples of Cannabinoids

13.3.3.1 Δ9-Tetrahydrocannabinol (THC)

Cannabis sativa and its subsidiary, that is, marijuana, are among the best-known
substances utilized by humans for centuries and are still among the most mishandled
substances around the world. Δ9-THC is a partial agonist at both the CB1 and CB2
receptors and the major psychoactive component of cannabis. Δ9-THC was isolated
and the structure was characterized by Gaoni and Mechoulam in 1964. Δ9-THC
induces reactions such as excess craving, decrease in the severity of sickness,
reduction of intraocular stress, euphoria and dysphoria (Volkow et al. 2014; Wang
et al. 2008; Moreira and Crippa 2009). Increasingly serious antagonistic impacts
include respiratory problems, hypertension, tachycardia, chest pain, muscle jerks,
intense renal disappointment, nervousness, disturbance, psychosis, self-destructive
ideation and intellectual impedance (Cohen and Weinstein 2018). A purified active
compound of Δ9-THC is (-)trans-Δ9-tetrahydrocannabinol (dronabinol), as of late
affirmed for the treatment of chronic pain and chemotherapy-induced nausea and
vomiting (Kowal et al. 2016; May and Glode 2016). Nabilone, a more established
commercial Δ9-THC, is used in the management of chemotherapy-induced nausea
and vomiting (Ware et al. 2008).

13.3.3.2 Cannabidiol (CBD)

Recent studies suggest that cannabidiol (CBD) might be helpful for the treatment of
various neuropsychiatric disorders. Cannabidiol does not show tranquilizing abuse
potential in experiments on mice. The researchers demonstrated that CBD, the
second most significant component of cannabis, has reduced misuse potential
(Viudez-Martinez et al. 2019).

13.3.3.3 GW405833
CB receptor, especially cerebrum CB2R, expression, shows dynamic and inducible
profiles under different neurotic conditions. GW405833 sub-atomic compound is
under preclinical investigation showing a particular CB2R agonistic activity. A
recent report revealed that CB2R agonist GW405833 secures liver cells and shows
therapeutic impact by lessening serum aminotransferase levels, and diminishes
hepatocyte apoptosis against intense concanavalin A-initiated poisonous quality
through the CB2 receptors communicated in liver resistant cells (Huang et al. 2019).

13.3.3.4 URB597 ([3-(3-carbamoylphenyl)

phenyl]-N-cyclohexylcarbamate)
The compound URB597 is an FAAH inhibitor which prompts delay in the degrada-
tion of anandamide, leading to increased centralization of anandamide accessible for
its natural movement by means of CB1 receptor. In addition, URB597 has been
13 Pharmacology of Endocannabinoids and Their Receptors 429

exhibited to be dynamic in discouragement (Gobbi et al. 2005), inflammation (Holt

et al. 2005), neuropathy (Russo et al. 2007), and acute pain and anxiety (Kathuria
et al. 2002).

13.3.4 CB Receptor Blockers

Medications that modify the endogenous eCB levels by blocking CB receptors

intervene in downregulatory signaling and may give another medication focus to
treating different neuropsychiatric and obesity issues. Receptor blockers that have
been broadly explored include Rimonabant, Taranabent, AM4113SR141716A and
SR144528.

13.3.4.1 Rimonabant and Taranabent

Rimonabant (Sanofi-Aventis) and Taranabant (Merk pharmaceutical) can block
agonist-induced activation of cannabinoid CB1R2 stuck an aggressive way and
imbroglio with fundamentally more noteworthy partiality to cannabinoid CB1 than
cannabinoid CB2 receptors (Pertwee et al. 2010).
In spite of the fact that these mixes are lacking in their capacity to activate CB1
receptors when regulated alone, there is proof that in some CB1 receptor-containing
tissues, they can initiate inverse reactions from those evoked by a CB1 receptor
agonistic; they are CB1 receptor inverse agonists (Pertwee 2005; Fong et al. 2007).
They are suggested for the treatment of obesity by creating activity on CB1 receptors
in CNS to control hunger. Other proposed components are activities on receptors in
the GIT that may tweak satiety as a peripheral means of controlling nourishment
intake or on those communicated in the fat tissue that may improve metabolic
confusions regularly found in those who are overweight thus diminishing insulin
opposition, coronary artery disease and dyslipidemia (Kaur et al. 2013). A recent
investigation revealed knowledge on the anti-tumor adequacy of Rimonabant, firmly
recommending that it could be a novel lead compound for colorectal cancer treat-
ment (Fiore et al. 2018). Alongside the clinical preliminaries in obesity that produced
the information submitted to regulatory authorities, Rimonabant was additionally
examined in clinical preliminaries as a potential treatment for different conditions,
including diabetes, atherosclerosis and smoking cessation (Hollander et al. 2010;
Dol-Gleizes et al. 2009; Steinberg and Foulds 2007). Psychiatric adverse events such
as depression and nervousness were observed to be increasingly regular with
Rimonabant (20 mg/day), which was tested by the US Food and Drug Administra-
tion through distributed and non-distributed preliminaries (Dol-Gleizes et al. 2009).
In addition, two deaths from suicide were accounted for in patients who consumed
Rimonabant. Moreover, 10% of individuals experienced sickness and upper respira-
tory tract infections and around 1–10% of individuals suffered from unfavorable
impacts such as gastroenteritis, tension, sleeping disorder, hot flashes, loose bowels,
heavy, dry or irritated skin, tendonitis, muscle aches, fatigue and flu-like symptoms.
430 G. Gupta et al.

Rimonabant was never endorsed in the United States for the treatment of obesity.
The marketing endorsement for the drug was canceled by the European Regulatory
Authorities in 2009 (Sam et al. 2011; Moreira and Crippa 2009).

13.3.5 Therapeutic Potential of Targeting Cannabinoid Receptors

The undesired psychotropic effects of cannabinoid agonists represent a drawback in

the therapeutic development of compounds for direct activation of CB1 receptors.
On the other hand, activation of CB2 receptors has no psychotropic side effects such
as hypolocomotion or catalepsy (Volkow et al. 2014). Currently, there is an
approach to developing medications that activate CB2 receptors at doses that have
little or no CB1 receptor activation. This approach appears tempting because there is
much evidence that the undesired effects induced by CB1/CB2 receptor agonists are
attributed to CB1 rather than CB2 receptor activation. This indicates that there are
important potential therapeutic applications for CB2 selective agonists. For instance,
therapies based on agonists targeting CB2 receptors have been proposed for the
treatment of a wide range of conditions. Table 13.2 represents therapeutic potentials
of cannabinoids or their derivatives.

13.3.5.1 Pain
Therapies based on agonists targeting CB2 receptors have been proposed for the
management of an array of painful conditions. Such conditions include acute pain,
nociceptive, neuropathic pain, and chronic inflammatory pain (Whiteside et al.
2007). CB2 agonists exerted analgesic effects in animal models of neuropathic
pain such as partial sciatic nerve ligation model, spinal nerve ligation model and
chemotherapy-induced neuropathy. In addition, cannabinoids also showed analgesic
effects in diverse persistent inflammatory pain models such as carrageenan, capsai-
cin, complete Freund’s adjuvant, formalin and arachidonic acid (Guindon and
Hohmann 2008). The mechanisms through which cannabinoids alleviate pain
involve decreasing the sensitivity of transient receptor potential channel vanilloid
1 (TRPV1) to noxious stimuli (Jeske et al. 2006), inhibiting NF-κB activity and
microglial production of IL-1β, IL-6 and TNFα (Klegeris et al. 2003).
In clinical trials, there were controversial findings regarding the effect of
cannabinoids in pain management, since some qualitative systematic reviews
reported that cannabinoids are no more effective than codeine in pain management
and the risks associated with their use outweigh their benefit because of their
depressant effects (Campbell et al., 2001).

13.3.5.2 Metabolic Disorders

A hypothesis proposed that when mice are exposed to cannabinoids, CB1 receptors
inhibit hypothalamic pro-opiomelanocortin (POMC) neurons which participate in
feelings of satiety resulting in the release of beta-endorphins (Koch et al. 2015). The
first human study assessing the effect of cannabinoids on appetite was reported in
13 Pharmacology of Endocannabinoids and Their Receptors 431

Table 13.2 Summary of certain diseases that could be targeted by cannabinoids or their
derivatives
Biological Disease Targets Therapeutic Potentials
Pain • Decrease sensitivity of TRPV1 to Acute pain, nociceptive,
noxious stimuli neuropathic and chronic
• Inhibit NF-κB activity and inflammatory pain
microglial production of IL-1β,
IL-6 and TNFα
Metabolic • CB2R blockade Insulin resistance associated with
disorders obesity and obesity-associated
fatty liver
Asthma • Inhibitory effects on mast cells Immunosuppressive, anti-
and eosinophils in lung tissue inflammatory and bronchodilator
• Reduce levels of cytokines effects
involved in the immune response to
an allergen
Glaucoma • Neuroprotective and vasorelaxant Decrease intraocular pressure, and
properties via CB2R activation, hence decrease optic nerve damage
increased aqueous humor outflow due to inadequate blood supply
via enhancing the p42/44 MAP
kinase
Autoimmune • Enhance levels of anti- • Maintain normoglycemia
diseases inflammatory mediators while • Decrease inflammation
decreasing the levels of associated with rheumatoid
pro-inflammatory cytokines arthritis
• Decrease neurodegeneration in
multiple sclerosis
• Improve the symptoms of
Crohn’s, multiple sclerosis
Bone diseases • Stimulation of the CB2R • Prevent osteoclast formation
• Decrease arthritis progression
• Enhance fracture healing
Cardiovascular • CB2R activation • Diminish infract size
disorders
Gastrointestinal • Targeting the CB1 and CB2 • GastricB58 ulcers
disorders receptors • Gastroesophageal reflux
• Irritable bowel syndrome,
• Secretory diarrhea,
• Crohn’s disease,
• Paralytic ileus
• Hepatitis C
Mood and anxiety • CB2R stimulation • Bipolar disorders
disorders • Drug abuse
• Post-traumatic stress disorder
Neurodegenerative • Selective targeting of the CB2R • Multiple sclerosis
diseases • Amyotrophic lateral sclerosis
• Parkinson’s disease
• Huntington’s disease
(continued)
432 G. Gupta et al.

Table 13.2 (continued)

Biological Disease Targets Therapeutic Potentials
Cancer • Down-regulated Id-1 gene • Breast and prostate cancer
expression and increase in the • Glioblastoma, glioma
generation of reactive oxygen • Pancreatic and oral cancer
species leading to induction of • Liver and colorectal cancer
apoptosis and autophagy • Thyroid, ovarian and cervical
• Induce apoptosis via pro-caspase- cancer
3 cleavage to caspase-3 • Skin and gastric cancer
• Inhibition of angiogenesis by the
reduction of pro-angiogenic factors
VEGF, inhibiting forskolin-
induced cAMP formation and
activation of RAF1 translocation
and MAPK activity

1971 in which there was a reported increase in food intake following use of
Cannabis (Hollister 1971). Another study also presented that oral Δ9-THC doses
of up to 15 mg/day stimulated appetite and produced significant weight gain in
advanced cancer patients (Regelson et al. 1976). Later on, a more comprehensive
study demonstrated clearly that smoking Cannabis leads to a substantial increase in
food intake (Foltin et al. 1986). The expression of CB1 receptor, MAGL and FAAH
in the human pancreas was reported (Kim et al. 2011) and it was recognized that CB1
suppresses β-cell proliferation by hindering insulin secretion. As a result, CB1
receptor blockade leads to elevated β-cell mass in diabetic mice and enhanced insulin
sensitivity. Additionally, the contribution of cannabinoids to the pathogenesis of
diabetic neuropathy, retinopathy and nephropathy has been explored (Horváth et al.
2012).
CB2 receptor stimulation enhanced insulin resistance associated with obesity and
obesity-associated fatty liver and was improved in CB2 knock-out mice, suggesting
that CB2 blockade might be beneficial for the treatment of insulin resistance and fatty
liver (Deveaux et al. 2009).

13.3.5.3 Asthma
Several studies postulated that targeting cannabinoid receptors might be promising
for treating patients with asthma. Cannabinoids demonstrated immunosuppressive,
anti-inflammatory and bronchodilatory effects. In vivo models showed that
cannabinoids exerted inhibitory effects on mast cells and eosinophils in lung tissue
(Giannini et al. 2008) and reduced the levels of cytokines involved in the immune
response to an allergen (Vuolo et al. 2015). Moreover, cannabinoids demonstrated
antibacterial effects against Staphylococci and Streptococci in broth (Van Klingeren
and Ten Ham 1976). CB2 receptors also regulate the function of natural killer cells
by inhibiting cytokine production in a murine model of asthma (Ferrini et al. 2017).
In human studies, early studies found that Cannabis smoke, unlike cigarette
smoke, caused bronchodilatation rather than bronchoconstriction and, unlike
13 Pharmacology of Endocannabinoids and Their Receptors 433

opiates, did not cause central respiratory depression (Vachon et al. 1973). Another
study also demonstrated that doses of THC provided by aerosol caused
bronchodilatation as measured by the enhancement of lung function (Hartley et al.
1978).

13.3.5.4 Glaucoma
Recent studies have demonstrated that the neuroprotective and vasorelaxant
properties of cannabinoids might be effective in reducing intraocular pressure
(Tomida et al. 2004). CB2 receptor activation increased aqueous humor outflow by
enhancing the p42/44 MAP kinase activity in cultured porcine trabecular meshwork
cells (Zhong et al. 2005). According to the American Glaucoma Society,
cannabinoids can decrease intraocular pressure briefly, and reduce blood pressure
and hence can decrease optic nerve damage due to inadequate blood supply (Jampel
2009).

13.3.5.5 Autoimmune Diseases

CB2 is primarily expressed in peripheral tissues of the immune system (leukocytes,
spleen, tonsils, thymus, bone marrow), and it has been established that cannabinoids
display immunosuppressant effects and hence can be beneficial in treating autoim-
mune diseases. They can amend immune balance by enhancing levels of anti-
inflammatory mediators while decreasing the levels of pro-inflammatory cytokines
(Nagarkatti et al. 2009). Recently, the immunomodulatory effect of THC has been
linked to its ability to affect epigenetic regulation by modifying histones (Yang et al.
2014).
In animal studies, cannabinoids produced a decreased risk of hypoglycemia along
with a significant reduction in beta-cell damage in autoimmune diabetes (Li et al.
2001). This, in turn, helps in maintaining normoglycemia (Weiss et al. 2008).
Moreover, cannabinoids were found to contribute to decreasing inflammation
associated with rheumatoid arthritis (Costa et al. 2004), as well as arresting the
neurodegeneration in multiple sclerosis (Pryce et al. 2003). In clinical studies, there
has been evidence that cannabinoids can improve the symptoms of Crohn’s disease
(Naftali et al. 2014), fibromyalgia (Schley et al. 2006) and multiple sclerosis (Collin
et al. 2007). A correlation has been established between the negative regulation of
the cAMP signaling pathway, leading to less cAMP response element-binding
protein affecting gene expression and immunomodulatory effects mediated by
cannabinoids (Kaminski 1998), suggesting that selective targeting of CB2 receptors
can cause immunosuppressive effects without eliciting psychotropic effects
(Turcotte et al. 2016).

13.3.5.6 Bone Diseases

Multiple studies have recognized the involvement of cannabinoids in osteoporosis as
CB receptors are substantially expressed in osteoblasts, osteoclasts and osteocytes. A
correlation between CB2 receptor expression and bone density, as well as polymor-
phism of the gene responsible for coding CB2 receptor and post-menopausal osteo-
porosis in humans, has been reported, and thus it has been found that stimulation of
434 G. Gupta et al.

CB2 prevents osteoclast formation (Ofek et al. 2006; Karsak et al. 2005). Other
studies involving in vivo models demonstrated that cannabinoids might improve
fracture healing (Kogan et al. 2015) and prevent the progression of arthritis (Malfait
et al. 2000).

13.3.5.7 Cardiovascular Diseases

In animal and human studies, cannabidiol has been found to exert anti-arrhythmic,
vasodilator, antioxidant and anti-inflammatory effects (Stanley et al. 2013), while
acute administration of low doses of THC before ischemia also produced
cardioprotective effects by diminishing myocardial damage (Waldman et al. 2013).
CB2 receptor stimulation has been demonstrated to diminish the infarct size of
myocardium in ischemia/reperfusion (I/R) injury by inhibiting apoptosis and
enhancing Akt phosphorylation (Li et al. 2013).

13.3.5.8 Gastrointestinal Disorders

The most commonly used indication of cannabinoids is nausea and vomiting since
former studies have revealed that THC and its analogues were effective in managing
chemotherapy-induced nausea and vomiting (Shiling et al. 1981). Paradoxically,
cannabis can be problematic if used chronically and has been reported in very few
cases to lead to cannabinoid hyperemesis syndrome (Lu and Agito 2015). Stimula-
tion of CB1 receptors has been shown to suppress gastrointestinal motility, gastric
acid secretion and intestinal secretion.
CB2 receptors are also involved in modulating the inflammatory response in the
gastrointestinal system by reducing the secretion of proinflammatory cytokines
(Wright et al. 2008). Thus, targeting cannabinoid receptors can be effective in
multiple conditions including, gastric ulcers, gastroesophageal reflux disease, irrita-
ble bowel syndrome, secretory diarrhea, Crohn’s disease, paralytic ileus and hepati-
tis C (Izzo and Camilleri 2008).

13.3.5.9 Mood and Anxiety Disorders

Numerous investigators suggested that CB2 agonists can be beneficial in the man-
agement of bipolar disorders, personality disorders and drug abuse disorders
(Navarrete et al. 2012). Moreover, recent evidence demonstrated that cannabinoids
can alleviate the symptoms of post-traumatic stress disorder (Korem and Akirav
2014). CB2 receptors have also been reported to modulate the midbrain, ventral
tegmental area, and dopamine activity responsible for reward and addiction (Zhang
et al. 2014).

13.3.5.10 Neurodegenerative Diseases

The undesired psychotropic side effects of CB1 receptor agonists represent the
downside in drug discovery. Activation of CB2 receptors, however, has no psycho-
tropic side effects such as hypolocomotion and catalepsy (Volkow et al. 2014).
Moreover, CB2 receptors are present in limited amounts in the brain, and this
indicates that selective targeting of CB2 receptors could be useful in treating diseases
with neuroinflammatory or neurodegenerative components. Such diseases include
multiple sclerosis, amyotrophic lateral sclerosis, Parkinson’s disease, Huntington’s
13 Pharmacology of Endocannabinoids and Their Receptors 435

disease and inflammatory peripheral disorders (Pertwee 2010). Recent evidence

acknowledged the involvement of the endocannabinoid system in neuroprotection
induced by minocycline against brain edema, microglial stimulation and neurologi-
cal damage in traumatic brain injury in mice (Lopez-Rodriguez et al. 2013).
The down-regulation of CB2 receptors has been described in patients with
Parkinson’s disease. Hence, the activation of CB2 receptors has been postulated to
delay the progression of the disease (Javed et al. 2016).

13.3.5.11 Attention-deficit Hyperactivity Disorder (ADHD)

THC has been reported to reduce hyper impulsivity associated with ADHD by
binding to CB1 receptors and enhancing retrograde signaling inhibition in the
neuronal synapses, thus decreasing hyperactivity (Adriani et al. 2003). A clinical
case reported in Germany showed that THC improved cognitive performance in
individuals suffering from ADHD similar to stimulant medications and with limited
side effects when combined with CBD (Strohbeck-Kuehner et al. 2008). Over-
activation of mTOR signaling has been implicated in autism spectrum disorders by
inhibiting autophagy that results in autism-like behaviors (Tang et al. 2014),
suggesting that targeting mTOR signaling might provide a new therapeutics for
autism spectrum disorders.

13.3.5.12 Cancers
In addition to palliative effects, preclinical studies demonstrated that cannabinoids
exert antitumor, antiproliferative, antiangiogenic or proapoptotic effects both in vitro
and in vivo against several types of cancer. Such cancers include glioblastoma
(Guzman et al. 2006), glioma (Massi et al. 2004), pancreatic cancer (Carracedo
et al. 2006), oral cancer (Whyte et al. 2010), breast cancer (Caffarel et al. 2010),
prostate cancer (De Petrocellis et al. 2013), lung cancer (Preet et al. 2011), blood
cancer (Gustafsson et al. 2006), liver cancer (Knowles et al. 1980), colorectal cancer
(Kogan et al. 2007), thyroid cancer (Portella et al. 2003), ovarian cancer (Afaq et al.
2006), cervical cancer (Lukhele and Motadi 2016), gastric cancer (Park et al. 2011)
and skin cancer (Blázquez et al. 2006).
In breast cancer cells, cannabinoids have shown the ability to down-regulate Id-1
gene expression and increase the generation of reactive oxygen species leading to
induction of apoptosis and autophagy (Śledziński et al. 2018). In addition, the
antitumor effects of cannabinoids, phytocannabinoids, and synthetic and endoge-
nous ligands have been reported in various types of breast cancer including
hormone-dependent and hormone-independent breast cancer cell lines. These
antitumor effects include induction of apoptosis via pro-caspase-3 cleavage to
caspase-3 and cell cycle arrest, inhibition of angiogenesis by the reduction of
pro-angiogenic factors VEGF, inhibiting forskolin-induced cAMP formation, and
activation of RAF1 translocation and MAPK activity (Kisková et al. 2019).
One of the primary advantages of the potential use of cannabinoids in cancer
management is selectivity and lack of cytotoxicity to normal cells (Guzman 2003).
These effects were observed in cultured cells originating from human, mouse and
436 G. Gupta et al.

rodent tumor models. Clinical trials are needed to demonstrate the effectiveness and
safety of cannabinoids in cancer patients.

13.3.5.13 Cannabinoids and their Potential for Addiction

According to the National Institute on Drug Abuse, marijuana (cannabis) is the most
commonly used illicit substance. One of the key issues associated with the use of
cannabis is the manifestation of psychological effects. Former clinical studies
investigating the effects of marijuana were conducted by Moreau and it was
concluded that consuming large doses causes personality changes and hallucinations
(Wilkinson et al. 2014).
It has been found that the subjective effects of marijuana consumption are
partially mediated by CB1R. The effects of cannabis differ broadly depending on
many factors including the product, its route of administration, dosage form, dura-
tion of use, drug–cannabis interaction, pharmacokinetics and pharmacogenetics
properties. The acute effects of cannabis tend to be transient and dose dependent
and can be both physiological and psychological. At low doses, cannabis use can
cause difficulties in concentration and thinking, lack of coordination, temporal
distortion, restlessness, memory impairment, tachycardia and increase in blood
pressure. On the other hand, higher doses can lead to anxiety, visual hallucinations,
panic attacks and sensory distortion (Chopra and Smith 1974). These effects are
mainly attributed to the psychoactive component of cannabis, THC. CBD, on the
other hand, the nonpsychoactive constituent of cannabis, produces the opposite
effects, such as anti-anxiety, anti-psychotic, neuroprotective and bradycardia.
Due to the highly lipophilic and protein-bound nature of cannabis, it has a
prolonged half-life and the retention of THC can persist from several hours to
days depending on the amount consumed and its route of administration. Therefore,
abrupt cessation of cannabis is generally tolerated. Marijuana Abstinence Syndrome
usually commences within 48 h of cessation and can last up to 2 weeks and manifests
in the form of craving, anger or aggression, fatigue, anxiety, shakiness, sweating,
insomnia, vivid dreams, decreased appetite, irritability and depression. Substantial
clinical data is still required to confirm the safety and efficacy of cannabis consump-
tion (Abood and Martin 1992).

13.3.6 Conclusion

The underlying disclosure and ensuing thorough research of the endocannabinoid

system over the last three decades have uncovered presumably the most notable
retrograde neurotransmission system. As the fundamental mediator of the psychoac-
tive impact of THC, CB1R has gained remarkable attention over these years. Its
widespread expression and versatile functions not only increase its promising
potential as a medication focus for different illnesses, but additionally make the
undesired reactions almost inescapable. This hindrance drives specialists to give
more consideration to the science that quite a while ago disregarded CB2R and other
endo-/phyto-cannabinoids. In addition, as a neuromodulator, the crosstalk among the
13 Pharmacology of Endocannabinoids and Their Receptors 437

endocannabinoid and other synapse systems, by means of nearby neural circuits, or

receptor heteromerization, or downstream pathway, has been accentuated. Produc-
tive investigations have been carried out, unraveling the intricacy of the entire
endocannabinoid system. It is essential to remember that the investigation of the
endocannabinoid system should be region- and condition-explicit, alongside the
consideration of other neurotransmission systems.
Even though cannabis has existed for more than 5000 years, there is anecdotal
evidence that cannabis is therapeutically beneficial for a variety of human diseases.
Nevertheless, we still have insufficient data to accept or reject the use of
cannabinoids in many clinical conditions. Few randomized controlled trials exist
and the majority of the data is based on case reports and small retrospective reviews.
The availability of a varied range of cannabinoids products, doses and routes of
administration renders it difficult to compare studies and derive appropriate
conclusions.

References
Abood M, Martin B (1992) Neurobiology of marijuana abuse. Trends Pharmacol Sci 13:201–206
Adler BL, Deleo VA (2019) Allergenic ingredients in commercial topical cannabinoid preparations.
J Am Acad Dermatol. https://doi.org/10.1016/j.jaad.2019.03.015
Adriani W, Caprioli A, Granstrem O, Carli M, Laviola G (2003) The spontaneously hypertensive-
rat as an animal model of ADHD: evidence for impulsive and non-impulsive subpopulations.
Neurosci Biobehav Rev 27:639–651
Afaq F, Sarfaraz S, Syed DN, Khan N, Malik A, Bailey HH, Mukhtar H (2006) Cannabinoid
receptors as a target for therapy of ovarian cancer. AACR Meeting, Washington, DC
Akram H, Mokrysz C, Curran HV (2019) What are the psychological effects of using synthetic
cannabinoids? A systematic review. J Psychopharmacol 33:271–283
Alexander SP (2016) Therapeutic potential of cannabis-related drugs. Prog Neuropsychopharmacol
Biol Psychiatry 64:157–166
Alipour A, Patel PB, Shabbir Z, Gabrielson S (2019) Review of the many faces of synthetic
cannabinoid toxicities. Ment Health Clin 9:93–99
Andrade AK, Renda B, Murray JE (2019) Cannabinoids, interoception, and anxiety. Pharmacol
Biochem Behav 180:60–73
Bara A, Manduca A, Bernabeu A, Borsoi M, Serviado M, Lassalle O, Murphy M, Wager-Miller J,
Mackie K, Pelissier-Alicot AL, Trezza V, Manzoni OJ (2018) Sex-dependent effects of in utero
cannabinoid exposure on cortical function. Elife 7
Baron EP (2018) Medicinal properties of cannabinoids, terpenes, and flavonoids in cannabis, and
benefits in migraine, headache, and pain: an update on current evidence and cannabis science.
Headache 58:1139–1186
Battista N, Di Tommaso M, Bari M, Maccarrone M (2012) The endocannabinoid system: an
overview. Front Behav Neurosci 6:9
Bilbao A, Cippitelli A, Bermudez-Silva FJ, Del Arco I, Navarro M, Rodríguez De Fonseca F (2004)
The endocannabinoid system: physiology and pharmacology. Alcohol Alcohol 40:2–14
Bingham B, Jones P, Uveges A, Kotnis S, Lu P, Smith V, Sun SC, Resnick L, Chlenov M, He Y
(2007) Species-specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2)
selective ligand AM1241 and its resolved enantiomers. Br J Pharmacol 151:1061–1070
Blankman JL, Simon GM, Cravatt BF (2007) A comprehensive profile of brain enzymes that
hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem Biol 14:1347–1356
438 G. Gupta et al.

Blázquez C, Carracedo A, Barrado L, Real PJ, Fernández-Luna JL, Velasco G, Malumbres M,

GuzmáN M (2006) Cannabinoid receptors as novel targets for the treatment of melanoma.
FASEB J 20:2633–2635
Bonini SA, Premoli M, Tambaro S, Kumar A, Maccarinelli G, Memo M, Mastinu A (2018)
Cannabis sativa: a comprehensive ethnopharmacological review of a medicinal plant with a
long history. J Ethnopharmacol 227:300–315
Bonn-Miller MO, Elsohly MA, Loflin MJE, Chandra S, Vandrey R (2018) Cannabis and cannabi-
noid drug development: evaluating botanical versus single molecule approaches. Int Rev
Psychiatry 30:277–284
Borsoi M, Manduca A, Bara A, Lassalle O, Pelissier-Alicot AL, Manzoni OJ (2019) Sex differences
in the behavioral and synaptic consequences of a single in vivo exposure to the synthetic
cannabimimetic WIN55,212-2 at puberty and adulthood. Front Behav Neurosci 13:23
Bramness JG, Dom G, Gual A, Mann K, Wurst FM (2018) A survey on the medical use of cannabis
in Europe: a position paper. Eur Addict Res 24:201–205
Breit KR, Zamudio B, Thomas JD (2019) Altered motor development following late gestational
alcohol and cannabinoid exposure in rats. Neurotoxicol Teratol 73:31–41
Caffarel MM, Andradas C, Mira E, Pérez-Gómez E, Cerutti C, Moreno-Bueno G, Flores JM,
García-Real I, Palacios J, Mañes S (2010) Cannabinoids reduce ErbB2-driven breast cancer
progression through Akt inhibition. Mol Cancer 9:196
Cardenia V, Gallina Toschi T, Scappini S, Rubino RC, Rodriguez-Estrada MT (2018) Development
and validation of a Fast gas chromatography/mass spectrometry method for the determination of
cannabinoids in Cannabis sativa L. J Food Drug Anal 26:1283–1292
Carr C, Vertelney H, Fronk J, Trieu S (2019) Dronabinol for the treatment of paraneoplastic night
sweats in cancer patients: a report of five cases. J Palliat Med. https://doi.org/10.1089/jpm.2018.
0551
Carracedo A, Gironella M, Lorente M, Garcia S, GuzmáN M, Velasco G, Iovanna JL (2006)
Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress–
related genes. Cancer Res 66:6748–6755
Chen C (2016) Endocannabinoid metabolism in neurodegenerative diseases. Neuroimmunol
Neuroinflammation 3:268–270
Rosanna Chianese and Rosaria Meccariello (2016) The endocannabinoid system in human physi-
ology. In: Meccariello R (ed.) Cannabinoids in health and disease. IntechOpen. https://doi.org/
10.5772/63818
Chopra GS, Smith JW (1974) Psychotic reactions following cannabis use in East Indians. Arch Gen
Psychiatry 30:24–27
Chye Y, Christensen E, Solowij N, Yucel M (2019) The endocannabinoid system and cannabidiol’s
promise for the treatment of substance use disorder. Front Psychiatry 10:63
Citti C, Palazzoli F, Licata M, Vilella A, Leo G, Zoli M, Vandelli MA, Forni F, Pacchetti B,
Cannazza G (2018) Untargeted rat brain metabolomics after oral administration of a single high
dose of cannabidiol. J Pharm Biomed Anal 161:1–11
Cohen K, Weinstein AM (2018) Synthetic and non-synthetic cannabinoid drugs and their adverse
effects-a review from public health prospective. Front Public Health 6:162–162
Cohen K, Abraham W, Aviv W (2019) Modulatory effects of cannabinoids on brain neurotrans-
mission. Eur J Neurosci. https://doi.org/10.1111/ejn.14407
Colizzi M, McGuire P, Giampietro V, Williams S, Brammer M, Bhattacharyya S (2018) Previous
cannabis exposure modulates the acute effects of delta-9-tetrahydrocannabinol on attentional
salience and fear processing. Exp Clin Psychopharmacol 26:582–598
Collin C, Davies P, Mutiboko I, Ratcliffe S (2007) Randomized controlled trial of cannabis-based
medicine in spasticity caused by multiple sclerosis. Eur J Neurol 14:290–296
Costa B, Colleoni M, Conti S, Parolaro D, Franke C, Trovato AE, Giagnoni G (2004) Oral anti-
inflammatory activity of cannabidiol, a non-psychoactive constituent of cannabis, in acute
carrageenan-induced inflammation in the rat paw. Naunyn-Schmiedeberg’s Arch Pharmacol
369:294–299
13 Pharmacology of Endocannabinoids and Their Receptors 439

Crowley K, De Vries ST, Moreno-Sanz G (2018) Self-reported effectiveness and safety of Trokie
((R)) lozenges: a standardized formulation for the buccal delivery of cannabis extracts. Front
Neurosci 12:564
Curran HV, Hindocha C, Morgan CJA, Shaban N, Das RK, Freeman TP (2019) Which biological
and self-report measures of cannabis use predict cannabis dependency and acute psychotic-like
effects? Psychol Med 49:1574–1580
Cyr C, Arboleda MF, Aggarwal SK, Balneaves LG, Daeninck P, Neron A, Prosk E, Vigano A
(2018) Cannabis in palliative care: current challenges and practical recommendations. Ann
Palliat Med 7:463–477
Da Silva VK, De Freitas BS, Garcia RCL, Monteiro RT, Hallak JE, Zuardi AW, Crippa JAS,
Schroder N (2018) Antiapoptotic effects of cannabidiol in an experimental model of cognitive
decline induced by brain iron overload. Transl Psychiatry 8:176
Dale T, Downs J, Olson H, Bergin AM, Smith S, Leonard H (2019) Cannabis for refractory epilepsy
in children: a review focusing on CDKL5 Deficiency Disorder. Epilepsy Res 151:31–39
Daris B, Tancer Verboten M, Knez Z, Ferk P (2019) Cannabinoids in cancer treatment: therapeutic
potential and legislation. Bosn J Basic Med Sci 19:14–23
De Petrocellis L, Ligresti A, Schiano Moriello A, Iappelli M, Verde R, Stott CG, Cristino L,
Orlando P, Di Marzo V (2013) Non-THC cannabinoids inhibit prostate carcinoma growth
in vitro and in vivo: pro-apoptotic effects and underlying mechanisms. Br J Pharmacol
168:79–102
Devane W, Hanus L, Breuer A, Pertwee R, Stevenson L, Griffin G, Gibson D, Mandelbaum A,
Etinger A, Mechoulam R (1992) Isolation and structure of a brain constituent that binds to the
cannabinoid receptor. Science 258:1946–1949
Deveaux V, Cadoudal T, Ichigotani Y, Teixeira-Clerc F, Louvet A, Manin S, Tran-Van Nhieu J,
Belot MP, Zimmer A, Even P (2009) Cannabinoid CB2 receptor potentiates obesity-associated
inflammation, insulin resistance and hepatic steatosis. PLoS One 4:e5844
Diao X, Huestis MA (2019) New synthetic cannabinoids metabolism and strategies to best identify
optimal marker metabolites. Front Chem 7:109
Dinis-Oliveira RJ (2019) [The clinical toxicology perspective on the therapeutic use of cannabis and
cannabinoids]. Acta Med Port 32: 87–90
Dol-Gleizes F, Paumelle R, Visentin V, Mares AM, Desitter P, Hennuyer N, Gilde A, Staels B,
Schaeffer P, Bono F (2009) Rimonabant, a selective cannabinoid CB1 receptor antagonist,
inhibits atherosclerosis in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol
29:12–18
Elliott J, McCoy B, Clifford T, Potter BK, Skidmore B, Wells GA, Coyle D (2019) Cost-
effectiveness of cannabinoids for pediatric drug-resistant epilepsy: protocol for a systematic
review of economic evaluations. Syst Rev 8:75
Ferrini M, Hong S, Stierle A, Stierle D, Stella N, Roberts K, Jaffar Z (2017) CB 2 receptors regulate
natural killer cells that limit allergic airway inflammation in a murine model of asthma. Allergy
72:937–947
Fiore D, Ramesh P, Proto MC, Piscopo C, Franceschelli S, Anzelmo S, Medema JP, Bifulco M,
Gazzerro P (2018) Rimonabant kills colon cancer stem cells without inducing toxicity in normal
colon organoids. Front Pharmacol 8:949
Foltin RW, Brady JV, Fischman MW (1986) Behavioral analysis of marijuana effects on food
intake in humans. Pharmacol Biochem Behav 25:577–582
Fong TM, Guan X-M, Marsh DJ, Shen C-P, Stribling DS, Rosko KM, Lao J, Yu H, Feng Y, Xiao
JC, Van Der Ploeg LHT, Goulet MT, Hagmann WK, Lin LS, Lanza TJ, Jewell JP, Liu P, Shah
SK, Qi H, Tong X, Wang J, Xu SS, Francis B, Strack AM, Macintyre DE, Shearman LP (2007)
Antiobesity efficacy of a novel cannabinoid-1 receptor inverse agonist, N-[(1S,2S)-3-
(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-
2-yl]oxy]propanamide (MK-0364), in rodents. J Pharmacol Exp Ther 321:1013–1022
Fowler C (2007) The contribution of cyclooxygenase-2 to endocannabinoid metabolism and action.
Br J Pharmacol 152:594–601
440 G. Gupta et al.

Friedman D, French JA, Maccarrone M (2019) Safety, efficacy, and mechanisms of action of
cannabinoids in neurological disorders. Lancet Neurol 18:504–512
Galiègue S, Mary S, Marchand J, Dussossoy D, CarriÈre D, Carayon P, Bouaboula M, Shire D,
Fur G, Casellas P (1995) Expression of central and peripheral cannabinoid receptors in human
immune tissues and leukocyte subpopulations. Eur J Biochem 232:54–61
Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, Bouaboula M, Shire D, Le
Fur G, Casellas P (1995) Expression of central and peripheral cannabinoid receptors in human
immune tissues and leukocyte subpopulations. Eur J Biochem 232:54–61
Gaoni Y, Mechoulam R (1964) Isolation, structure, and partial synthesis of an active constituent of
hashish. J Am Chem Soc 86:1646–1647
Gertsch J, Pertwee RG, Di Marzo V (2010) Phytocannabinoids beyond the Cannabis plant - do they
exist? Br J Pharmacol 160:523–529
Giang DK, Cravatt BF (1997) Molecular characterization of human and mouse fatty acid amide
hydrolases. Proc Natl Acad Sci U S A 94:2238–2242
Giannini L, Nistri S, Mastroianni R, Cinci L, Vannacci A, Mariottini C, Passani M, Mannaioni P,
Bani D, Masini E (2008) Activation of cannabinoid receptors prevents antigen-induced asthma-
like reaction in guinea pigs. J Cell Mol Med 12:2381–2394
Gobbi G, Bambico FR, Mangieri R, Bortolato M, Campolongo P, Solinas M, Cassano T, Morgese
MG, Debonnel G, Duranti A, Tontini A, Tarzia G, Mor M, Trezza V, Goldberg SR, Cuomo V,
Piomelli D (2005) Antidepressant-like activity and modulation of brain monoaminergic trans-
mission by blockade of anandamide hydrolysis. Proc Natl Acad Sci U S A 102:18620–18625
Goncalves J, Rosado T, Soares S, Simao AY, Caramelo D, Luis A, Fernandez N, Barroso M,
Gallardo E, Duarte AP (2019) Cannabis and its secondary metabolites: their use as therapeutic
drugs, toxicological aspects, and analytical determination. Medicines (Basel) 6. https://doi.org/
10.3390/medicines6010031
Gonsiorek W, Lunn C, Fan X, Narula S, Lundell D, Hipkin RW (2000) Endocannabinoid
2-arachidonyl glycerol is a full agonist through human type 2 cannabinoid receptor: antagonism
by anandamide. Mol Pharmacol 57:1045–1050
Griffin G, Tao Q, Abood ME (2000) Cloning and pharmacological characterization of the rat CB
(2) cannabinoid receptor. J Pharmacol Exp Ther 292:886–894
Guindon J, Hohmann A (2008) Cannabinoid CB2 receptors: a therapeutic target for the treatment of
inflammatory and neuropathic pain. Br J Pharmacol 153:319–334
Gustafsson K, Christensson B, Sander B, Flygare J (2006) Cannabinoid receptor-mediated apopto-
sis induced by R (+)-methanandamide and Win55, 212-2 is associated with ceramide accumu-
lation and p38 activation in mantle cell lymphoma. Mol Pharmacol 70:1612–1620
Guzman M (2003) Cannabinoids: potential anticancer agents. Nature reviews cancer 3:745
Guzman M, Duarte M, Blazquez C, Ravina J, Rosa M, Galve-Roperh I, Sanchez C, Velasco G,
Gonzalez-Feria L (2006) A pilot clinical study of Δ9-tetrahydrocannabinol in patients with
recurrent glioblastoma multiforme. Br J Cancer 95:197–203
Hartley J, Nogrady S, Seaton A (1978) Bronchodilator effect of delta1-tetrahydrocannabinol. Br J
Clin Pharmacol 5:523–525
He Y, De Witte LD, Schubart CD, Van Gastel WA, Koeleman BPC, De Jong S, Ophoff RA, Hol
EM, Boks MP (2019) Liprin alfa 2 gene expression is increased by cannabis use and associated
with neuropsychological function. Eur Neuropsychopharmacol 29:643–652
Henschke P (2019) Cannabis: an ancient friend or foe? What works and doesn’t work. Semin Fetal
Neonatal Med 24:149–154
Herkenham M, Lynn AB, Johnson MR, Melvin LS, De Costa BR, Rice KC (1991) Characterization
and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic
study. J Neurosci 11:563–583
Ho C, Martinusen D, Lo C (2019) A review of cannabis in chronic kidney disease symptom
management. Can J Kidney Health Dis 6:2054358119828391
Hollander PA, Amod A, Litwak LE, Chaudhari U (2010) Effect of rimonabant on glycemic control
in insulin-treated type 2 diabetes: the ARPEGGIO trial. Diabetes Care 33:605–607
13 Pharmacology of Endocannabinoids and Their Receptors 441

Hollister LE (1971) Hunger and appetite after single doses of marihuana, alcohol, and dextroam-
phetamine. Clin Pharmacol Ther 12:44–49
Holt S, Comelli F, Costa B, Fowler CJ (2005) Inhibitors of fatty acid amide hydrolase reduce
carrageenan-induced hind paw inflammation in pentobarbital-treated mice: comparison with
indomethacin and possible involvement of cannabinoid receptors. Br J Pharmacol 146:467–476
Horváth B, Mukhopadhyay P, Haskó G, Pacher P (2012) The endocannabinoid system and plant-
derived cannabinoids in diabetes and diabetic complications. Am J Pathol 180:432–442
Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, Felder CC, Herkenham M,
Mackie K, Martin BR, Mechoulam R, Pertwee RG (2002) International Union of Pharmacol-
ogy. Xxvii. Classification of cannabinoid receptors. Pharmacol Rev 54:161–202
Huang Z-B, Zheng Y-X, Li N, Cai S-L, Huang Y, Wang J, Hu X-W, Wang Y, Wu J, Fan X-G
(2019) Protective effects of specific cannabinoid receptor 2 agonist GW405833 on concanavalin
A-induced acute liver injury in mice. Acta Pharmacol Sinica
Huestis MA, Blount BC, Milan DF, Newmeyer MN, Schroeder J, Smith ML (2019) Correlation of
creatinine- and specific gravity-normalized free and glucuronidated urine cannabinoid
concentrations following smoked, vaporized, and oral cannabis in frequent and occasional
cannabis users. Drug Test Anal. https://doi.org/10.1002/dta.2576
Izzo AA, Camilleri M (2008) Emerging role of cannabinoids in gastrointestinal and liver diseases:
basic and clinical aspects. Gut 57:1140–1155
Jampel H (2009) Position statement on marijuana and the treatment of glaucoma. American
Glaucoma Society, San Francisco
Javed H, Azimullah S, Haque ME, Ojha SK (2016) Cannabinoid type 2 (CB2) receptors activation
protects against oxidative stress and neuroinflammation associated dopaminergic
neurodegeneration in rotenone model of Parkinson’s Disease. Front Neurosci 10:321
Jeske NA, Patwardhan AM, Gamper N, Price TJ, Akopian AN, Hargreaves KM (2006) Cannabi-
noid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons. J Biol Chem
281:32879–32890
Jordan CJ, Xi ZX (2019) Progress in brain cannabinoid CB2 receptor research: from genes to
behavior. Neurosci Biobehav Rev 98:208–220
Kaminski NE (1998) Inhibition of the cAMP signaling cascade via cannabinoid receptors: a
putative mechanism of immune modulation by cannabinoid compounds. Toxicol Lett
102:59–63
Karsak M, Cohen-Solal M, Freudenberg J, Ostertag A, Morieux C, Kornak U, Essig J, Erxlebe E,
Bab I, Kubisch C (2005) Cannabinoid receptor type 2 gene is associated with human osteopo-
rosis. Human Mol Genet 14:3389–3396
Kathuria S, Gaetani S, Fegley D, ValiñO F, Duranti A, Tontini A, Mor M, Tarzia G, Rana GL,
Calignano A, Giustino A, Tattoli M, Palmery M, Cuomo V, Piomelli D (2002) Modulation of
anxiety through blockade of anandamide hydrolysis. Nat Med 9:76
Kaur R, Singh P, Kaur A, Mahajan D, Kaur H (2013) Effect of rimonabant on the components of
metabolic syndrome: a randomized, controlled study done on Punjabi population. J Pharm
Negat Results 4:46–53
Khuja I, Yekhtin Z, Or R, Almogi-Hazan O (2019) Cannabinoids reduce inflammation but inhibit
lymphocyte recovery in murine models of bone marrow transplantation. Int J Mol Sci 20. https://
doi.org/10.3390/ijms20030668
Kim W, Doyle ME, Liu Z, Lao Q, Shin Y-K, Carlson OD, Kim HS, Thomas S, Napora JK, Lee EK
(2011) Cannabinoids inhibit insulin receptor signaling in pancreatic β-cells. Diabetes
60:1198–1209
Kisková T, Mungenast F, Suváková M, Jäger W, Thalhammer T (2019) Future aspects for
cannabinoids in breast cancer therapy. Int J Mol Sci 20:1673
Klegeris A, Bissonnette CJ, Mcgeer PL (2003) Reduction of human monocytic cell neurotoxicity
and cytokine secretion by ligands of the cannabinoid-type CB2 receptor. Br J Pharmacol
139:775–786
Knowles BB, Howe CC, Aden DP (1980) Human hepatocellular carcinoma cell lines secrete the
major plasma proteins and hepatitis B surface antigen. Science 209:497–499
442 G. Gupta et al.

Koch M, Varela L, Kim JG, Kim JD, Hernández-NuñO F, Simonds SE, Castorena CM, Vianna CR,
Elmquist JK, Morozov YM (2015) Hypothalamic POMC neurons promote cannabinoid-
induced feeding. Nature 519:45–50
Kogan NM, Schlesinger M, Peters M, Marincheva G, Beeri R, Mechoulam R (2007) A cannabinoid
anticancer quinone, HU-331, is more potent and less cardiotoxic than doxorubicin: a compara-
tive in vivo study. J Pharmacol Exp Ther 322:646–653
Kogan NM, Melamed E, Wasserman E, Raphael B, Breuer A, Stok KS, Sondergaard R, Escudero
AV, Baraghithy S, Attar-Namdar M (2015) Cannabidiol, a major non-psychotropic cannabis
constituent enhances fracture healing and stimulates lysyl hydroxylase activity in osteoblasts. J
Bone Miner Res 30:1905–1913
Korem N, Akirav I (2014) Cannabinoids prevent the effects of a footshock followed by situational
reminders on emotional processing. Neuropsychopharmacology 39:2709–2722
Kowal M, Hazekamp A, Grotenhermen F (2016) Review on clinical studies with cannabis and
cannabinoids 2010-2014
Krebs MO, Kebir O, Jay TM (2019) Exposure to cannabinoids can lead to persistent cognitive and
psychiatric disorders. Eur J Pain. https://doi.org/10.1002/ejp.1377
Kumar A, Premoli M, Aria F, Bonini SA, Maccarinelli G, Gianoncelli A, Memo M, Mastinu A
(2019) Cannabimimetic plants: are they new cannabinoidergic modulators? Planta
249:1681–1694
Lattanzi S, Trinka E, Russo E, Striano P, Citraro R, Silvestrini M, Brigo F (2019) Cannabidiol as
adjunctive treatment of seizures associated with Lennox-Gastaut syndrome and Dravet syn-
drome. Drugs Today (Barc) 55:177–196
Li X, Kaminski NE, Fischer LJ (2001) Examination of the immunosuppressive effect of Δ
9-tetrahydrocannabinol in streptozotocin-induced autoimmune diabetes. Int Immunopharmacol
1:699–712
Li Q, Wang F, Zhang Y-M, Zhou J-J, Zhang Y (2013) Activation of cannabinoid type 2 receptor by
JWH133 protects heart against ischemia/reperfusion-induced apoptosis. Cell Physiol Biochem
31:693–702
Liu J, Wang L, Harvey-White J, Huang BX, Kim H-Y, Luquet S, Palmiter RD, Krystal G, Rai R,
Mahadevan A (2008) Multiple pathways involved in the biosynthesis of anandamide. Neuro-
pharmacology 54:1–7
Lopez-Rodriguez AB, Siopi E, Finn DP, Marchand-Leroux C, Garcia-Segura LM, Jafarian-
Tehrani M, Viveros M-P (2013) CB1 and CB2 cannabinoid receptor antagonists prevent
minocycline-induced neuroprotection following traumatic brain injury in mice. Cereb Cortex
25:35–45
Lossignol D (2019) Cannabinoids: a new approach for pain control? Curr Opin Oncol. https://doi.
org/10.1097/CCO.0000000000000523
Lowin T, Schneider M, Pongratz G (2019) Joints for joints: cannabinoids in the treatment of
rheumatoid arthritis. Curr Opin Rheumatol 31:271–278
Lu M, Agito MD (2015) Cannabinoid hyperemesis syndrome: marijuana is both antiemetic and
proemetic. Cleve Clin J Med 82:429–434
Lukhele ST, Motadi LR (2016) Cannabidiol rather than Cannabis sativa extracts inhibit cell growth
and induce apoptosis in cervical cancer cells. BMC Complement Altern Med 16:335
Mackie K, Devane WA, Hille B (1993) Anandamide, an endogenous cannabinoid, inhibits calcium
currents as a partial agonist in N18 neuroblastoma cells. Mol Pharmacol 44:498–503
Malfait A, Gallily R, Sumariwalla P, Malik A, Andreakos E, Mechoulam R, Feldmann M (2000)
The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in
murine collagen-induced arthritis. Proc Natl Acad Sci U S A 97:9561–9566
Massi P, Vaccani A, Ceruti S, Colombo A, Abbracchio MP, Parolaro D (2004) Antitumor effects of
cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines. J Pharmacol Exp Ther
308:838–845
Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bronner TI (1990) Structure of a cannabinoid
receptor and functional expression of the cloned cDNA. Nature 346:561
13 Pharmacology of Endocannabinoids and Their Receptors 443

May MB, Glode AE (2016) Dronabinol for chemotherapy-induced nausea and vomiting unrespon-
sive to antiemetics. Cancer Manag Res 8:49–55
Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR, Gopher A,
Almog S, Martin BR, Compton DR (1995) Identification of an endogenous 2-monoglyceride,
present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 50:83–90
Miller LK, Devi LA (2011) The highs and lows of cannabinoid receptor expression in disease:
mechanisms and their therapeutic implications. Pharmacol Rev 63:461–470
Moreira FA, Crippa JAS (2009) The psychiatric side-effects of rimonabant. Braz J Psychiatry
31:145–153
Mukherjee S, Adams M, Whiteaker K, Daza A, Kage K, Cassar S, Meyer M, Yao BB (2004)
Species comparison and pharmacological characterization of rat and human CB 2 cannabinoid
receptors. Eur J Pharmacol 505:1–9
Munro S, Thomas KL, Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for
cannabinoids. Nature 365:61
Naftali T, Mechulam R, Lev LB, Konikoff FM (2014) Cannabis for inflammatory bowel disease.
Dig Dis 32:468–474
Nagarkatti P, Pandey R, Rieder SA, Hegde VL, Nagarkatti M (2009) Cannabinoids as novel anti-
inflammatory drugs. Future Med Chem 1:1333–1349
National Research Council (US) Committee on Substance Abuse and Habitual Behavior 1982) An
analysis of Marijuana Policy [Online]. Accessed https://www.ncbi.nlm.nih.gov/books/
NBK217602/
Navarrete F, Pérez-Ortiz JM, Manzanares J (2012) Cannabinoid CB2 receptor-mediated regulation
of impulsive-like behaviour in DBA/2 mice. Br J Pharmacol 165:260–273
Nunez E, Benito C, Pazos MR, Barbachano A, Fajardo O, Gonzalez S, Tolon RM, Romero J (2004)
Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain:
an immunohistochemical study. Synapse 53:208–213
Ofek O, Karsak M, Leclerc N, Fogel M, Frenkel B, Wright K, Tam J, Attar-Namdar M, Kram V,
Shohami E (2006) Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc Natl Acad
Sci U S A:103, 696–701
Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N (2004) Molecular characterization of a
phospholipase D generating anandamide and its congeners. J Biol Chem 279:5298–5305
Onaivi ES, Ishiguro H, Gong JP, Patel S, Perchuk A, Meozzi PA, Myers L, Mora Z, Tagliaferro P,
Gardner E, Brusco A, Akinshola BE, Liu QR, Hope B, Iwasaki S, Arinami T, Teasenfitz L, Uhl
GR (2006) Discovery of the presence and functional expression of cannabinoid CB2 receptors
in brain. Ann N Y Acad Sci 1074:514–536
Onaivi ES, Ishiguro H, Gong JP, Patel S, Meozzi PA, Myers L, Perchuk A, Mora Z, Tagliaferro PA,
Gardner E, Brusco A, Akinshola BE, Hope B, Lujilde J, Inada T, Iwasaki S, Macharia D,
Teasenfitz L, Arinami T, Uhl GR (2008) Brain neuronal CB2 cannabinoid receptors in drug
abuse and depression: from mice to human subjects. PLoS One 3:e1640
Park JM, Xian XS, Choi MG, Park H, Cho YK, Lee IS, Kim SW, Chung IS (2011) Antiproliferative
mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells. J Cell
Biochem 112:1192–1205
Pertwee R (1997) Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther
74:129–180
Pertwee RG (2005) The therapeutic potential of drugs that target cannabinoid receptors or modulate
the tissue levels or actions of endocannabinoids. AAPS J 7
Pertwee R (2010) Receptors and channels targeted by synthetic cannabinoid receptor agonists and
antagonists. Curr Med Chem 17:1360
Pertwee R, Howlett A, Abood ME, Alexander S, Di Marzo V, Elphick M, Greasley P, Hansen HS,
Kunos G, Mackie K (2010) International Union of Basic and Clinical Pharmacology. LXXIX.
Cannabinoid receptors and their ligands: beyond CB1 and CB2. Pharmacol Rev 62:588–631
Piomelli D (2003) The molecular logic of endocannabinoid signalling. Nat Rev Neurosci
4:873–884
444 G. Gupta et al.

Portella G, Laezza C, Laccetti P, De Petrocellis L, Di Marzo V, Bifulco M (2003) Inhibitory effects

of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on
signals involved in angiogenesis and metastasis. FASEB J 17:1771–1773
Preet A, Qamri Z, Nasser MW, Prasad A, Shilo K, Zou X, Groopman JE, Ganju RK (2011)
Cannabinoid receptors, CB1 and CB2, as novel targets for inhibition of non–small cell lung
cancer growth and metastasis. Cancer Prev Res 4:65–75
Pryce G, Ahmed Z, Hankey DJ, Jackson SJ, Croxford JL, Pocock JM, Ledent C, Petzold A,
Thompson AJ, Giovannoni G (2003) Cannabinoids inhibit neurodegeneration in models of
multiple sclerosis. Brain 126:2191–2202
Regelson W, Butler J, Schulz J, Kirk T, Peek L, Green M, Zalis M (1976) Delta-9-THC as an
effective antidepressant and appetite-stimulating agent in advanced cancer patients. In: Braude
MC, Szara S (eds). The pharmacology of marihuana. Raven press, New york, pp. 763-776.
Rhee MH, Bayewitch M, Avidor-Reiss T, Levy R, Vogel Z (1998) Cannabinoid receptor activation
differentially regulates the various adenylyl cyclase isozymes. J Neurochem 71:1525–1534
Rossi F, Punzo F, Umano GR, Argenziano M, Miraglia Del Giudice E (2018) Role of cannabinoids
in obesity. Int J Mol Sci 19
Russo R, Loverme J, La Rana G, Compton TR, Parrott J, Duranti A, Tontini A, Mor M, Tarzia G,
Calignano A, Piomelli D (2007) The fatty acid amide hydrolase inhibitor URB597
(cyclohexylcarbamic acid 3’-carbamoylbiphenyl-3-yl ester) reduces neuropathic pain after
oral administration in mice. J Pharmacol Exp Ther 322:236–242
Sam AH, Salem V, Ghatei MA (2011) Rimonabant: from RIO to Ban. J Obes 2011:432607–432607
Schatz AR, Lee M, Condie RB, Pulaski JT, Kaminski NE (1997) Cannabinoid receptors CB1 and
CB2: a characterization of expression and adenylate cyclase modulation within the immune
system. Toxicol Appl Pharmacol 142:278–287
Schley M, Legler A, Skopp G, Schmelz M, Konrad C, Rukwied R (2006) Delta-9-THC based
monotherapy in fibromyalgia patients on experimentally induced pain, axon reflex flare, and
pain relief. Curr Med Res Opin 22:1269–1276
Schlicker E, Kathmann M (2001) Modulation of transmitter release via presynaptic cannabinoid
receptors. Trends Pharmacol Sci 22:565–572
Schmid P, Reddy P, Natarajan V, Schmid H (1983) Metabolism of N-acylethanolamine
phospholipids by a mammalian phosphodiesterase of the phospholipase D type. J Biol Chem
258:9302–9306
Shiling DJ, Stillman RC, Chang AE, Goldberg NH, Seipp CA, Barofsky I, Rosenberg SA (1981) A
prospective evaluation of delta-9-tetrahydrocannabinol as an antiemetic in patients receiving
adriamycin and cytoxan chemotherapy. Cancer 47:1746–1751
Śledziński P, Zeyland J, SŁomski R, Nowak A (2018) The current state and future perspectives of
cannabinoids in cancer biology. Cancer Med 7:765–775
Small E, Pocock T, Cavers PB (2002) The biology of Canadian weeds. 119. Cannabis sativa L. Can
J Plant Sci 83:217–237
Soderstrom K, Leid M, Moore FL, Murray TF (2000) Behavioral, pharmacological, and molecular
characterization of an amphibian cannabinoid receptor. J Neurochem 75:413–423
Stanley CP, Hind WH, O’sullivan SE (2013) Is the cardiovascular system a therapeutic target for
cannabidiol? Br J Clin Pharmacol 75:313–322
Steinberg MB, Foulds J (2007) Rimonabant for treating tobacco dependence. Vasc Health Risk
Manag 3:307–311
Stella N, Schweitzer P, Piomelli D (1997) A second endogenous cannabinoid that modulates long-
term potentiation. Nature 388:773–778
Strohbeck-Kuehner P, Skopp G, Mattern R (2008) Cannabis improves symptoms of ADHD.
Cannabinoids 3:1–3
Svíženská I, Dubový P, Šulcová A (2008) Cannabinoid receptors 1 and 2 (CB1 and CB2), their
distribution, ligands and functional involvement in nervous system structures—a short review.
Pharmacol Biochem Behav 90:501–511
Tang G, Gudsnuk K, Kuo S-H, Cotrina ML, Rosoklija G, Sosunov A, Sonders MS, Kanter E,
Castagna C, Yamamoto A (2014) Loss of mTOR-dependent macroautophagy causes autistic-
like synaptic pruning deficits. Neuron 83:1131–1143
13 Pharmacology of Endocannabinoids and Their Receptors 445

Tomida I, Pertwee R, Azuara-Blanco A (2004) Cannabinoids and glaucoma. Br J Ophthalmol

88:708–713
Tsuboi K, Uyama T, Okamoto Y, Ueda N (2018) Endocannabinoids and related
N-acylethanolamines: biological activities and metabolism. Inflamm Regen 38:28
Turcotte C, Blanchet M-R, Laviolette M, Flamand N (2016) The CB 2 receptor and its role as a
regulator of inflammation. Cell Mol Life Sci 73:4449–4470
Vachon L, Fitzgerald MX, Solliday NH, Gould IA, Gaensler EA (1973) Single-dose effect of
marihuana smoke: bronchial dynamics and respiratory-center sensitivity in normal subjects. N
Engl J Med 288:985–989
Van Klingeren B, Ten Ham M (1976) Antibacterial activity of Δ 9-tetrahydrocannabinol and
cannabidiol. Antonie Van Leeuwenhoek 42:9–12
Varma N, Carlson GC, Ledent C, Alger BE (2001) Metabotropic glutamate receptors drive the
endocannabinoid system in hippocampus. J Neurosci 21:188
Viudez-Martinez A, Garcia-Gutierrez MS, Medrano-Relinque J, Navarron CM, Navarrete F,
Manzanares J (2019) Cannabidiol does not display drug abuse potential in mice behavior.
Acta Pharmacol Sin 40:358–364
Volkow ND, Baler RD, Compton WM, Weiss SR (2014) Adverse health effects of marijuana use. N
Engl J Med 370:2219–2227
Vuolo F, Petronilho F, Sonai B, Ritter C, Hallak JE, Zuardi AW, Crippa JA, Dal-Pizzol F (2015)
Evaluation of serum cytokines levels and the role of cannabidiol treatment in animal model of
asthma. Mediators Inflamm 2015
Waldman M, Hochhauser E, Fishbein M, Aravot D, Shainberg A, Sarne Y (2013) An ultra-low dose
of tetrahydrocannabinol provides cardioprotection. Biochem Pharmacol 85:1626–1633
Wang T, Collet J-P, Shapiro S, Ware MA (2008) Adverse effects of medical cannabinoids: a
systematic review. CMAJ:178, 1669–1678
Ware MA, Daeninck P, Maida V (2008) A review of nabilone in the treatment of chemotherapy-
induced nausea and vomiting. Ther Clin Risk Manag 4:99–107
Weiss L, Zeira M, Reich S, Slavin S, Raz I, Mechoulam R, Gallily R (2008) Cannabidiol arrests
onset of autoimmune diabetes in NOD mice. Neuropharmacology 54:244–249
Whiteside GT, Lee G, Valenzano KJ (2007) The role of the cannabinoid CB2 receptor in pain
transmission and therapeutic potential of small molecule CB2 receptor agonists. Curr Med
Chem 14:917–936
Whyte DA, Al-Hammadi S, Balhaj G, Brown OM, Penefsky HS, Souid A-K (2010) Cannabinoids
inhibit cellular respiration of human oral cancer cells. Pharmacology 85:328–335
Wilkinson ST, Radhakrishnan R, D’souza DC (2014) Impact of cannabis use on the development of
psychotic disorders. Curr Addict Rep 1:115–128
Wright K, Duncan M, Sharkey K (2008) Cannabinoid CB2 receptors in the gastrointestinal tract: a
regulatory system in states of inflammation. Br J Pharmacol 153:263–270
Yamaguchi F, Macrae AD, Brenner S (1996) molecular cloning of two cannabinoid type 1-like
receptor genes from the puffer fishfugu rubripes. Genomics 35:603–605
Yang X, Hegde VL, Rao R, Zhang J, Nagarkatti PS, Nagarkatti M (2014) Histone modifications are
associated with Δ9-tetrahydrocannabinol-mediated alterations in antigen-specific T cell
responses. J Biol Chem 289:18707–18718
Zhang H-Y, Gao M, Liu Q-R, Bi G-H, Li X, Yang H-J, Gardner EL, Wu J, Xi Z-X (2014)
Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-
related behavior in mice. Proc Natl Acad Sci U S A 111:E5007–E5015
Zhong L, Geng L, Njie Y, Feng W, Song Z-H (2005) CB2 cannabinoid receptors in trabecular
meshwork cells mediate JWH015-induced enhancement of aqueous humor outflow facility.
Invest Ophthalmol Vis Sci 46:1988–1992
Hormones and Steroids
as Neurotransmitters 14
Sarapynbiang Marwein, Satyajeet Biswal,
and Pratap Chandra Acharya

Abstract

Neurotransmitters are chemical messengers synthesized by neurons, which enable

interconnection of nerve fibers within their vicinity. Neurotransmitters tradition-
ally consist of amino acids and their derivatives, chains of amino acids, peptides or
proteins. However, several studies report that steroids and hormones also exert an
acute effect on the physiology of neuronal activity and the expression of behavior
that can happen within minutes. Those steroids that can bind to the neurotransmit-
ter receptors and modulate the neurotransmission signal are included together
within the term neurosteroids or neuroactive steroids. The examples of neuroactive
steroids include progesterone, estradiol, testosterone, DHEA, glucocorticoid,
allotetrahydrodeoxycorticosterone (THDOC), androstanediol (AD), ganaloxone,
androsterone, pregnenolone, allopregnanolone and their sulfate esters. Addition-
ally, several synthetic steroids such as alphaxalone and 3ɑ-hydroxy-5β-pregnan-
20-one hemisuccinate possess similar characteristics of modulating neuronal
activities to the endogenous steroids. These hormonal steroids exert their neuronal
excitability functions through various receptors and ion channels such as the
estrogen receptor, progesterone receptor, androgen receptor, GABAA, AMPA and
NMDA receptors. These neuroactive steroids are also involved in the pathology
and physiology of various neurological disorders such as epilepsy, schizophrenia
and traumatic brain injury. Additionally, these neuroactive steroids have agonistic
or antagonistic effects toward the neurotransmission action of various other
neurotransmitters some of which have undergone clinical trials for the treatment
of various neurological disorders. Thus, these steroids and hormones can act as
neurotransmitters, exert either agonistic or antagonistic effects on receptors and
have potential benefits in the treatment of neurological disorders.

S. Marwein · S. Biswal · P. C. Acharya (*)

Department of Pharmacy, Tripura University (A Central University), Suryamaninagar, Tripura (W),
India
e-mail: [email protected]

# Springer Nature Singapore Pte Ltd. 2020 447

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_14
448 S. Marwein et al.

Keywords

Neurotransmitter · Neuroactive steroids · DHEA · Estradiol · Ganaloxone ·

Neurological disorders

Abbreviations

AD Androstanediol
AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ANT Adenine nucleotide transporter protein
ARH Arcuate nucleus of the hypothalamus
BLSA Baltimore Longitudinal Study of Aging
CB1 Type-1 cannabinoid
CNS Central nervous system
CREB cAMP response element binding protein
D1 Dopamine 1
D2 Dopamine 2
DHEA Dehydroepiandrosterone
DHP Dihydroprogesterone
ERɑ Estrogen receptor ɑ
ERs Estrogen receptors
ERβ Estrogen receptor β
ET Essential tremor
GABA Gamma-aminobutyric acid
GAMSA GABAA receptor modulating steroid antagonists
GPER G protein-coupled estrogen receptor
GPER1 G protein-coupled estrogen receptor 1
GSK3b Glycogen synthase kinase 3b
HPA Hypothalamic-pituitary-adrenal
IP3 Inositol 1,4,5-trisphoshate
LXRs Liver X receptors
MAPK Mitogen-activated protein kinase
MDD Major depressive disorder
mERɑ Membrane-associated estrogen receptor ɑ
mERs Membrane-associated estrogen receptors
mERβ Membrane-associated estrogen receptor β
mGluR Metabotropic glutamate receptor
MPN Medial preoptic nucleus
mPRs Membrane-bound progesterone receptors
MPTP 1-Methyl 4-phenyl-1,2,3,6 tetrahydropyridine
NADPH Nicotinamide adenine dinucleotide phosphate
NMDA N-methyl-D-aspartate
P4507ɑ Cytochrome P450 7ɑ-hydroxylase
14 Hormones and Steroids as Neurotransmitters 449

P450C11β Cytochrome P45011β-hydroxylase

P450C17 Cytochrome P450 17ɑ-hydroxylase
P450C21 Cytochrome P450 21-hydroxylase
PGRMC1 Progesterone receptor membrane component 1
PI3K Phosphoinositide-3 kinase
PNS Peripheral nervous system
PPD Postpartum depression
PPT Propyl-pyrazoletriol
PREs Progesterone response elements
PTZ Pentylenetetrazol
PXR Pregnane X receptor
SERM Selective estrogen receptor modulator
Src Src kinase
SRSE Super refractory status epilepticus
StAR Steroidogenic acute regulatory protein
THDOC Tetrahydrodeoxycorticosterone
THP Tetrahydroprogesterone
TSPO Translocator protein of 18kDa
VDAC Voltage-dependent anion channel protein
17PA (3ɑ5ɑ)-17-Phenylandrost-16-en-3-ol
17β-HSD 17β-Hydroxysteroid dehydrogenase
3ɑ-HSD 3ɑ-Hydroxysteroid dehydrogenase
3α-diol 5α-Androstane-3α,17β-diol
3α-HSOR 3ɑ-Hydroxysteroid oxidoreductase
3β-diol 5α-Androstane-3β,17β-diol
3β-HSD 3β-Hydroxysteroid dehydrogenase
3β-HSOR 3β-Hydroxysteroid oxidoreductase
6-OHDA 6-Hydroxydopamine

14.1 Introduction

Neurons in the brain signal to their neighbors via the aid of endogenous chemicals
called neurotransmitters. Neurotransmitters are chemical messengers synthesized by
neurons, which enable interconnection of nerve fibers within their vicinity. In a
broader sense, neurotransmitters are chemical messengers which communicate
signals between chemical synapses including neuromuscular junctions, from one
neuron to another "target" neuron, muscle cells or gland cells (Badgaiyan 2011). Till
the very recent past, neurotransmitters belonging to the categories of amino acid and
their derivatives, chains of amino acids, peptides or proteins was a very stated fact
(Rudolph et al. 2016). Molecules such as steroids, cholesterol derivatives and
hormones were never believed to be involved in the neurotransmission system
since they easily pass through cell membranes and distribute themselves in tissue.
It was found that steroids exert an acute effect on physiology (Szego and Davis
1967), neuron activity (Kelly et al. 1976) and the expression of behavior (Hayden-
450 S. Marwein et al.

Hixson and Ferris 1991) that can happen within minutes. Their ability to modulate
the neuronal function within minutes and to rapidly stimulate cognitive functions
and behaviors suggest their neurotransmitter-like action. The understanding of the
role of neuroactive steroids was conceived from the study by Etienne-Emile Baulieu
on the levels of dehydroepiandrosterone (DHEA) sulfate in the brain of adult male
rats (Corpechot et al. 1981; Baulieu 1998). In another study, Corpéchot and his
group attained a significant finding which describes the endogenous production of
DHEA sulfate within the brain, without considering the steroids secreted from the
adrenals and the gonads (Corpechot et al. 1981). The steroids produced within the
nervous system were referred to as neurosteroids (Corpechot et al. 1981). The
documentation of other classes of steroid receptor such as the pregnane X receptor
(PXR) that can be stimulated by steroids like pregnenolone and progesterone
(Kliewer et al. 1998; Moore et al. 2000) additionally augments to the element of
brain action modulating activity of steroids. From the 1970s onward, the potential
effects of steroids such as estradiol, a cholesterol derivative, in the nervous system
were appreciated owing to their ability to cause rapid alteration of the electrical
impulse fired by specific neurons within the brain. This rapid change and the
response time are not achievable by the binding of estrogen to the nuclear steroid
receptor within the neurons, which is a slow process that takes hours to be initiated
(Majewska et al. 1986; Paul and Purdy 1992). This was the initial evidence of the
possible mechanism that certain steroids and hormones can modulate excitability of
neurons by interacting with specific cell surface neurotransmitter receptors (Lambert
et al. 1995; Rupprecht 1997; Rupprecht and Holsboer 1999). The modulatory effects
of steroids in between the neurons occurs rapidly, ranging from milliseconds to
seconds, which is distinguishable from the steroid’s action at the genome, since it
involves an event ranging from minutes to hours depending on the proportion of
protein biosynthesis (McEwen 1991). Therefore, the activity of steroids within the
brain that can be either their genomic or non-genomic effects establishes the
understanding at the molecular level of a comprehensive effect of steroids on
neuronal excitability and plasticity. Several studies demonstrate that steroids can
and do function in ways that are “neurotransmitter-like,” as they are produced locally
in a specific region within neural circuits whereby they can exert their modulating
activity locally within minutes to stimulate neuronal functions such as cognition and
behavior-related activity (Balthazart et al. 2006; Dewing et al. 2007; Saldanha et al.
2011; Remage-Healey 2014).
Thus, steroids which have the capability of binding to the neurotransmitter
receptors and modulate the neurotransmission signal are included together within
the term neurosteroids or neuroactive steroids. However, neurosteroids are actually
metabolic products of cholesterol which are produced continuously inside the brain
from the readily available metabolic precursors and enzymes and they initiate instant
effects on neuronal excitability (Carver and Reddy 2013). Neuroactive steroids are
steroids synthesized in the endocrine gland that circulates through the bloodstream
into the brain to exert their effects on brain function. Therefore, neuroactive steroids
include neurosteroids and other steroids that are produced within the adrenal, testis
14 Hormones and Steroids as Neurotransmitters 451

Exogenous steroid (e.g. Alphaxalone, etc.)

Estradiol
Neuroactive steroid Progesterone

Hormonal steroid Testosterone

Glucocorticoid

Endogenous steroid Dehydroepiandrosterone

Pregnenolone

Neurosteroid Pregnenolone sulfate

Progesterone

Allopregnenolone

Dehydroepiandrosterone

Dehydroepiandrosterone
sulfate

Fig. 14.1 Broad classification of neuroactive steroids

and ovary, and are transported into the brain through the blood-brain barrier to
stimulate brain action in a similar way to the neurosteroids (Girdler et al. 2012).
Neuroactive steroids have the ability to regulate neuronal excitability function
and are of different categories depending on their source and site of production
(Zheng 2009). Neuroactive steroids are divided into two categories on the basis of
the source and production site, that is, the exogenous steroids (these include the
synthetic steroids) and endogenous steroids (Fig. 14.1). Further, based on the site of
production, endogenous steroids are subdivided into hormonal steroid (steroids
synthesized within the endocrine glands) and neurosteroids (steroids synthesized
within the brain) (Melcangi et al. 2008). The examples of hormonal steroids mostly
consist of steroids synthesized in the ovary such as progesterone and estradiol, in the
testis such as testosterone, and in the adrenal gland including DHEA and glucocor-
ticoid (Rhodes et al. 2004; Scharfman and MacLusky 2006), whereas the
neurosteroids include steroids produced by the neuronal and glial cells, with
DHEA, pregnenolone, allopregnanolone, progesterone, and their sulfate esters as
the well-known examples (Baulieu 1998). Additionally, several synthetic steroids
such as alphaxalone and steroid-3ɑ-hydroxy-5β-pregnan-20-one hemisuccinate pos-
sess similar characteristics of modulating neuronal activities to the endogenous
steroids (Melcangi et al. 2008). Neuroactive steroids also include
allotetrahydrodeoxycorticosterone (THDOC), and androstanediol (AD),
ganaloxone, androsterone, etiocholanolone (Akk et al. 2005, 2007), and 17-α and
17-β estradiol (Nguyen et al. 2017).
452 S. Marwein et al.

14.2 Synthesis, Storage, Release of Hormonal and Steroidal

Neurotransmitters

The synthesis of neuroactive steroids occurs in the brain, the gonads, the adrenal
glands and even in the fetoplacental unit within the body (Midzak et al. 2011). In
peripheral tissues, the gonads and adrenal gland are the main site of neuroactive
steroid production. Further, the metabolization process into active steroid
metabolites occurs in tissues including the endocrine tissues and liver (Akk et al.
2009). The synthesis of neuroactive steroids within the body is catalyzed with the aid
of various enzymes. For instance, the steroid 5α-reductase, an NADPH-dependent
enzyme, catalyzes the reduction of testosterone to the neuroactive steroid, dihydro-
testosterone (Reddy and Rogawski 2010). These neuroactive steroids are then
circulated into the bloodstream, crossing through the blood-brain barrier into the
brain (Haage and Johansson 1999; Baulieu et al. 2001). 5α-Reductase enzyme also
catalyzes the reduction of steroids such as deoxycorticosterone, progesterone and
various 3-keto-pregnane steroids and is available in tissues such as the brain, liver,
prostate, genitalia and testis. The enzyme 5α-reductase exerts its activity in brain
regions such as the neurons, glial cells, neocortex, subcortical white matter and the
hippocampus (Appelgren 1967). Other enzymes necessary for the biosynthesis of
neuroactive steroids include aromatase, sulfotransferase sulfatase, cytochrome 7ɑ-
hydroxylase (P4507ɑ), 11β-hydroxylase (P450C11β), cytochrome P450 17ɑ-
hydroxylase (P450C17), cytochrome P450 21-hydroxylase (P450C21), 3ɑ-
hydroxysteroid dehydrogenase (3ɑ-HSD), 3β-hydroxysteroid dehydrogenase
(3β-HSD), 5ɑ-reductase and 17β-hydroxysteroid dehydrogenase (17β-HSD)
(Do Rego et al. 2009).
Within the nervous system neuroactive steroids are produced locally, mainly
from cholesterol (Ellsworth et al. 1998), in specific brain tissues such as the pineal
gland which is the main neurosteroidogenic organ, glutamatergic neurons, cortex
and the hippocampal region (Appelgren 1967). These sites are steroidogenic sites
and produce steroidogenic enzymes such as 5α-reductase, aromatase,
sulfotransferase sulfatase, cytochrome 7ɑ-hydroxylase, 11β-hydroxylase and many
other enzymes necessary for steroid production. Neuroactive steroids are
synthesized directly from cholesterol via a progressive A-ring reduction process
(Melcangi et al. 2008). The primary stage in steroid synthesis is the metabolization
of cholesterol into pregnenolone. This step involves the transportation of cholesterol
from the outer to the inner mitochondrial membrane with the aid of molecular
complex comprising proteins such as the translocator protein of 18 kDa (TSPO),
the adenine nucleotide transporter protein (ANT), the steroidogenic acute regulatory
protein (StAR), and the voltage-dependent anion channel protein (VDAC)
(Morohaku et al. 2014; Papadopoulos et al. 2018; Selvaraj and Stocco 2015; Selvaraj
et al. 2015). It is located inside the inner mitochondrial membrane, where cholesterol
is metabolized into pregnenolone in the presence of the steroidogenic enzyme
P450scc (Midzak et al. 2011). Pregnenolone then undergoes transformation to
other steroids via sequent stages inside the endoplasmic reticulum. For instance,
pregnenolone when catalyzed by 3β-HSD enzyme or cytochrome P450C17 is
14 Hormones and Steroids as Neurotransmitters 453

changed into progesterone or dehydroepiandrosterone respectively. Consequently,

DHEA acts as a precursor for the synthesis of other neuroactive steroids like
testosterone. DHEA and progesterone can be converted further into other neuroac-
tive steroids with the help of the enzyme 5α-reductase. Specifically, testosterone is
metabolized into dihydrotestosterone and progesterone into dihydroprogesterone
(DHP), which can be further metabolized into other neuroactive metabolites in the
presence of 3α-HSOR or 3β-HSOR enzymes. For example, DHP is converted into
tetrahydroprogesterone (THP) or isopregnanolone, while testosterone is converted
into 5α-androstane-3α,17β-diol (3α-diol) or into 5α-androstane-3β,17β-diol
(3β-diol). Testosterone can also be converted in to 17β-estradiol with the help of
aromatase enzyme (Giatti et al. 2019). The reduction of the A-ring in steroid
hormones such as progesterone, testosterone and deoxycorticosterone results in the
production of other neuroactive steroids (Reddy and Rogawski 2010). The synthesis
of sulfate-conjugated steroids such as DHEA sulfate and pregnenolone sulfate from
their respective neuroactive steroids is catalyzed by two enzymes, that is,
sulfotransferase and sulfatase (Do Rego et al. 2009). Moreover, the steroidogenic
enzymes such as 3α-HSOR and 5α-reductase can convert the steroid precursors
present in peripheral tissues such as liver to produce androstanediol,
allopregnanolone and tetrahydrodeoxycorticosteone (THDOC), which can be
circulated systematically into the brain as they are lipophilic in nature and have an
effect on neuronal brain function (Do Rego et al. 2009; Kushida and Tamura 2009;
Yagishita et al. 2012). Figures 14.2 and 14.3 depict the biosynthetic pathway of
different neuroactive steroids.

14.3 Receptors and Channels Involved in Neurotransmitter

Action of Hormones and Steroids

Primarily, steroids and hormones were conceived to act solely through the traditional
genomic pathway by binding to the known steroid receptors (Evans 1988; Paul and
Purdy 1992). However, the documentation of new binding sites for neuroactive
steroids such as progesterone, testosterone (Ramirez and Zheng 1996),
glucocorticoids (Orchinik et al. 1991), estradiol (Pappas et al. 1995) or aldosterone
(Wehling 1997) in the membrane of cells and tissues with different signal transduc-
tion pathways that differ from the conventional transduction pathway involved in
steroid action (Wehling 1997) has shed new light on the primarily known concept.
The neuroactive steroids exert several physiological activities within the brain
facilitated by their interaction with the nuclear/membrane steroid receptors (estrogen
and progesterone receptors), androgen receptors and glucocorticoid receptors and
through their modulating activity toward the ion channels, which include the
GABAA (gamma-aminobutyric acid), NMDA (N-methyl-D-aspartate), AMPA
(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and kainate receptors
(Ogden and Traynelis 2011). The neuroactive steroids also exert their neuronal
modulation function through various voltage-dependent ion channels which include
the T-type Ca2+ channel, Na+ channel, Ca2+ activated K+ channel and anion
 454

17,20-lyase 17 -HSD 3 -HSD

Dehydroepiandroste Androstenediol
OMM Testosterone
17 -hdroxypregnenolone rone
TSPO
P450
StAR aromatase
Cholesterol 17 -
VDAC Estradiol
hydroxylase

IMM ANT Cholesterol

5 -reductase
Pregnenolone
P450scc
3 -HSD
Pregnenolone Dihydrotestosterone
3 ,5 -tetrahydroprogesterone
Progesterone

3 -HSD
5 -reductase
5 -dihydroprogesterone
Endoplasmic
reticulum

Fig. 14.2 Synthesis of neuroactive steroids within the brain

S. Marwein et al.
14 Hormones and Steroids as Neurotransmitters 455

O
O HO
HO
HO

HO
O H
HST
Corticosterone Allotetradeoxycorticosterone
O STS
HO OH
HO S O
HO 7-alpha hydroxy pregnenolone
O
Cholesterol sulfate Cholesterol

O P450scc
P450
O O
HO HO
O O
HST
O
HO S O STS
O O
Pregnenolone sulfate O
HO O H
Pregnenolone 11-Deoxycorticosterone
Progesterone Dihydrodeoxycorticosterone

O
O O
O O
OH
OH

HO
O H
HO H
HO OH O
Dihydroprogesterone Tetrahydroprogesterone
7-alpha hydroxy DHEA 17-hydroxy pregnenolone 17-Hydroxy progesterone

O
O
O

O
HO S O
O
O HO
DHEA sulfate Androstenedione
Dehydroepiandrosterone
(DHEA)

O OH

HO O
Testosterone
Estrone OH
OH
OH

HO
O
H
HO
Androstanediol
Estradiol Dihydrotestosterone

Fig. 14.3 Biochemical pathways for the synthesis of neuroactive steroids in the brain. AROM,
aromatase; HST, sulfotransferase; STS, sulfatase; P450scc, cytochrome P450 side-chain cleavage;
P4507ɑ, cytochrome 7ɑ-hydrolase; P450c11β, 11β-hydrolase; P450C17, cytochrome P450 17ɑ-
-hydrolase/C17,20-lyase; P450C21, 21-hydrolase; 3ɑ-HSD, 3ɑ-hydroxysteroid dehydrogenase;
3β-HSD, 3β-hydroxysteroid dehydrogenase; 5ɑ-R, 5ɑ-reductase; 17β-HSD, 17β-hydroxysteroid
dehydrogenase

channels, and the high-voltage activated Ca2+ channel (Carrer et al. 2003; Todorovic
et al. 2004; Pathirathna et al. 2005; Joksovic et al. 2007; Cheng et al. 2008). It is
understood that the physiological effects of neuroactive steroids result from their
interaction with definite receptors. Therefore, it is necessary to study the mechanism
by which these steroids activate these receptors and affect brain physiology and the
impact of the neurotransmitter receptors on neuroactive steroid actions (Rossetti
et al. 2016).
456 S. Marwein et al.

14.3.1 Estrogen Receptors

The discovery of estradiol binding sites in different rat tissues by Elwood V. Jensen
in the early 1950s led to the proposition of the occurrence of an explicit receptor for
estradiol (Jensen 1962). Thereafter, Toft and Gorski (1966) isolated an estrogen
receptor from rat uterus, which was later identified as estrogen receptor α (ERα)
shortly after the discovery of the additional estrogen receptor known as estrogen
receptor β (ERβ). These two functionally similar estrogen receptors (ERs) originated
from different genes (Kuiper et al. 1996). Both the ERɑ and ERβ receptors are also
expressed on the cell membrane (Coleman and Smith 2001; Pedram et al. 2007;
Boonyaratanakornkit 2011; Meitzen et al. 2013), but there is still a lack of under-
standing of their cell membrane association. However, it is suggested to be involved
in post-translational lipid modification and communication with membrane/cyto-
plasmic scaffolding proteins (Boonyaratanakornkit 2011; Meitzen et al. 2013;
Pedram et al. 2007). In addition to the membrane-associated mERɑ and mERβ,
one more membrane-associated estrogen receptor is also known to be involved in the
neurotransmission function of the neuroactive steroids, G protein-coupled estrogen
receptor 1 (GPER1) (Carmeci et al. 1997). All these estrogen receptors were found to
be commonly expressed in the neuronal and glial cells within the central nervous
system (Cardona-Gomez et al. 2000; Chaban et al. 2004; Quesada et al. 2007). Brain
regions such as astrocytes were also found to express ERα, ERβ, GPER and
Gq-mER (Kuo et al. 2010; Almey et al. 2012), and all these receptors are activated
by neuroactive steroids such as estradiol and contribute to the regulation of the
astrocytic functions (Pawlak et al. 2005; Kuo et al. 2010; Karki et al. 2014). For
instance, triggering membrane-associated estrogen receptor by neuroactive steroid
can alter membrane permeability (Fu and Simoncini 2008), initiate second messen-
ger cascades (Coleman and Smith 2001) and hyperpolarize neurons in the preoptic
area (Bologa et al. 2006; Prossnitz et al. 2008). Additionally, cumulative data from
different studies indicate that the dihydrotestosterone metabolite, 3β-diol, can inter-
act with and trigger the ERβ, thereby exerting its oestrogenic effects on various
pathophysiological brain functions, including anxiety, depression, affection
disorders and cognition (Fugger et al. 2000; Krȩżel et al. 2001; Österlund et al.
2005; Kudwa et al. 2006; Handa et al. 2008).

14.3.2 Progesterone Receptors

The progesterone receptors are important for the neuronal function of steroids such
as progesterone, metabolites of progesterone and sulfated progesterone. Comparable
to the estrogen receptors, progesterone receptors are of two isoforms, that is PR-A
and PR-B, and unlike ERɑ and ERβ originated from an identical gene (Pratt and Toft
2003). Bound progesterone receptors dissociate from the chaperone proteins, and
undertake conformational alteration leading to their dimerization, thus resulting in
the direct interaction with progesterone response elements (PREs) in promoter
regions of targeted genes through their binding with the steroid receptor coactivators
14 Hormones and Steroids as Neurotransmitters 457

(Leonhardt et al. 2003). Furthermore, several membrane-bound progesterone

receptors (mPRs) have also been identified (Lösel et al. 2005; Tang et al. 2005;
Thomas et al. 2007). Additionally, progesterone also binds to membrane progester-
one receptors and to the membrane-associated protein PGRMC1 (Progesterone
receptor membrane component 1), termed 25-Dx (Schumacher et al. 2007; Brinton
et al. 2008; Guennoun et al. 2008). Progesterone receptor PR-B has been reported to
have rapid neurotransmitter-like actions by inducing sexual receptivity when pro-
gesterone binds to this receptor (Mani et al. 1994; Serey et al. 2014). Rapid actions of
PR-B signaling are involved in a subcircuit regulating lordosis, which originates in
the arcuate nucleus of the hypothalamus (ARH) and projects to the medial preoptic
nucleus (MPN) (Sinchak and Wagner 2012; Sinchak et al. 2015). This phenomenon
was verified by a study which described that instillation of progesterone into the
ARH of estradiol-primed ovariectomized rats enables receptivity within 30 min by
inhibiting ARH β-endorphin neurons that project to the MPN, which induces sexual
receptivity (Huss et al. 2011). Although most evidence of neurotransmitter-like
actions of steroids has focused on estrogens, progesterone and reproduction, other
steroids have also been shown to act via non-classical mechanisms.

14.3.3 Glucocorticoid Receptors

Neuroactive glucocorticoids such as corticosterone and cortisol are also known for
their stimulating activity in various brain-related functions and exhibit their effect
mainly through the glucocorticoid receptors. Glucocorticoid receptors are nuclear
receptors located in the cytoplasm that can be included in the group of receptors by
which the neuroactive steroids (glucocorticoids) facilitate their neuronal signaling
activity (Aranda and Pascual 2001). Classical glucocorticoid receptor signaling
requires ligand binding to initiate glucocorticoid receptor dimerization and translo-
cation to the nucleus (Oakley and Cidlowski 2013) where they bind directly with
DNA and other transcription factors to regulate gene transcription. Glucocorticoids
can activate these receptors at the membrane level to alter the physiology functioning
more like neurotransmitters. The enzymes necessary for corticosterone production
are widely distributed throughout the brain (MacKenzie et al. 2000), and de novo
corticosterone synthesis from pregnenolone has been observed in the hippocampus
of adrenalectomized rats (Higo et al. 2011). This suggests the synthesis of
glucocorticoids, the expression of their receptors within the nervous system and
their responsibility in modulating brain function.

14.3.4 Other Neurotransmitter Receptors

The neuroactive steroids usually bind with the steroid receptors to exert their
physiological actions. However, they are also known to modulate various other
ion channels including the voltage-dependent ion channels, especially GABAA,
NMDA and AMPA receptors. There is ample evidence indicating how neuroactive
458 S. Marwein et al.

Presynaptic
terminal

GAT Neurosteroid site

GAT

1 β2 4 β2 GABA site
Neurosteroid
site γ2 δ 4
GABA site β2 1 β2

Synaptic receptor Extrasynaptic receptor

Cl- Phasic inhibition Cl-
Tonic inhibition

Fig. 14.4 Schematic illustration of neurosteroid-sensitive synaptic and extrasynaptic GABAA

receptors in the brain

steroids modulate neuronal excitability by their direct interaction with GABAA

receptors. Due to their high affinity and inherent modulating activity toward the
GABAA receptor, numerous steroids are often mentioned as endogenous modulators
of GABAA receptors (Majewska et al. 1986; Reddy 2003). These receptors also act
as the primary neurotransmitter receptor in the brain for several other neuroactive
steroids (Mellon and Griffin 2002). GABAA receptors are ligand-gated chloride
channels which when activated hyperpolarize the neurons through the influx of
chloride ions. GABAA receptors can be grouped into two classes: the synaptic and
extrasynaptic GABAA receptors (Fig. 14.4). However, the synaptic and
extrasynaptic GABAA receptors possess different degrees of affinity and sensitivity
toward neuroactive steroid neurotransmitters (Brown et al. 2002; Bianchi and
Macdonald 2002; Mortensen et al. 2012), desensitization rates and agonist efficacy
(Bianchi and Macdonald 2002, 2003). For instance, the measure by which various
progesterone derivatives modulate the GABAA receptors depicts the importance of
these receptors for the neuroactive steroids to mediate their neuronal excitability
actions. Studies show that fluctuations in the level of neuroactive steroids within the
brain and fluctuations in GABAergic signaling correlate with the manifestation of
pathophysiological brain conditions, such as depression, anxiety, seizure, stress and
epilepsy (Charalampopoulos et al. 2008; MacKenzie and Maguire 2013; Murphy
et al. 1998; Pluchino et al. 2015), suggesting that GABAA receptors play a
14 Hormones and Steroids as Neurotransmitters 459

Cl-
Ca2+ Neurosteroids
Neurosteroids GABA Glutamate Na+
Glycine
NR2 NR1
γ
extracellular extracellular NR2 NR1
β β

intracellular intracellular
K+

Fig. 14.5 Schematic illustration of neuroactive steroid actions mediated by GABAA receptor and
NMDA receptor

significant role in mediating the neuronal excitability function of these neuroactive

compounds.
The neuroactive steroids also have an affinity with the NMDA receptors in
addition to the GABAA receptors (Fig. 14.5). These receptors are tetrameric ion
channels consisting of two GluN1 and GluN2 subunits (formerly designated as NR1
and NR2). The GluN2 subunits of the NMDA receptors are the main contributor to
the multiple functionality of the NMDA receptors and they are specifically expressed
only during development in the brain regions (Ogden and Traynelis 2011). Some
neuroactive steroids such as estradiol act as negative modulators, whereas DHEA,
pregnenolone and their sulfate esters act as positive allosteric NMDA receptor
modulators (Charalampopoulos et al. 2008; Mellon and Griffin 2002). Although
GABAA and NMDA receptors seem to be the main ion channel receptors that the
neuroactive steroids interact with and bind to, other kinds of receptors such as
AMPA receptors have also been documented (Mellon and Griffin 2002; Niitsu
et al. 2012). Neuroactive steroids such as pregnenolone sulfate, DHEA sulfate and
progesterone have been known to interact allosterically with the AMPA, and their
involvement in the cental nervous system functions and pathologies has
been established.
In addition to the GABAA, NMDA and AMPA receptors, studies also demon-
strate that neuroactive steroids have an affinity with the kainate, serotonin and
X-receptors of the liver, sigma-1, nicotinic receptors (Rupprecht et al. 2001;
Dubrovsky 2006; Strous et al. 2006) and various voltage-dependent ion channels
such as T-type Ca2+ channel, high-voltage activated Ca2+ channel, Na+ channel and
anion channels (Kelly et al. 2002; Carrer et al. 2003; Darbandi-Tonkabon et al. 2004;
Todorovic et al. 2004; Pathirathna et al. 2005; Joksovic et al. 2007; Cheng et al.
2008). Neuroactive steroid metabolites can also trigger non-classical steroid
receptors such as the dopamine 1 (D1) receptor, the α1 adrenergic receptor and
L-type Ca2+ channels (Giatti et al. 2015; Vega-Vela et al. 2017). Studies have
demonstrated the neuroactive steroids’ ability to modulate the neuronal excitability
460 S. Marwein et al.

which results in brain function stimulating activity such as regulation of mood and
learning via their indirect involvement with the T-type voltage-gated Ca2+ channels
(Smith and Woolley 2004; Benarroch 2007; Grassi et al. 2007; Fatehi and Fatehi-
Hassanabad 2008). They have also been demonstrated to modulate mitochondrial
function and synaptic plasticity and to play a vital role in neuroprotective activity
due to their interaction with the voltage-dependent anion channel receptors (Tuem &
Atey 2017). Furthermore, neuroactive steroids are described to have modulating
effects on every synaptic transmission, namely GABAergic, glutamatergic, dopami-
nergic, cholinergic, serotonergic and noradrenergic synaptic transmission, through
their ability to alter the receptivity of postsynaptic receptors or to alter presynaptic
neurotransmitter release (Belelli et al. 2006; Gibbs et al. 2006; Mitsushima et al.
2007; Pérez-Neri et al. 2008; Laconi et al. 2007). Though with less efficacy, the
neuroactive steroids tetrahydroprogesterone and 3α-diol (Carver and Reddy 2013)
indeed stimulate neurotransmission via the GABAA receptor, thereby mediating
their effects on affection and behavior (Belelli and Lambert 2005). Similarly,
sulfate-conjugated neuroactive steroids such as pregnenolone and DHEA also
exert a weak antagonistic effect on the NMDA receptor at the micromolar level,
thereby producing a stimulating neuronal excitatory action (Rupprecht 2003). Neu-
roactive steroids including progesterone can also bind with the membrane receptors
such as the membrane-associated protein PGRMC1, also known as 25-Dx, and the
membrane progesterone receptors (mPRs) (Singh et al. 2013; Giatti et al. 2016). The
recently identified nuclear pregnane-X receptor (PXR) presents an additional site for
neuroactive steroids to bind and exert their actions (Frye 2011).
It is clear that neuroactive steroids exert various types of physiological activity in
the brain (Lapchak and Araujo 2001; Belelli et al. 2006; Strous et al. 2006) either
through interaction with nuclear steroid receptors such as the estrogen receptors,
androgen receptors and progesterone receptors or through interaction with mem-
brane receptors such as membrane progesterone receptors (Fig. 14.6). In particular,
neuroactive steroids interact with various ion channels (Fig. 14.6) and act as
allosteric modulators of the GABAA receptors (Covey et al. 2001; Belelli and
Lambert 2005), NMDA receptors (Mameli et al. 2005; Monnet and Maurice
2006), kainate receptors (Costa et al. 2000), AMPA receptors (Dubrovsky 2005),
sigma receptors (Maurice et al. 2006), serotonin receptors (Shannon et al. 2005a, b),
nicotinic receptors (Paradiso et al. 2000) and muscarinic receptors (Horishita et al.
2005). Additionally, it has been reported that neuroactive steroids may directly
trigger G protein-coupled membrane receptors (Meyer et al. 2002; Schiess and
Partridge 2005) or indirectly control the binding of neuropeptides to their receptors
(Torres and Ortega 2003).
14 Hormones and Steroids as Neurotransmitters 461

Sigma 1
AMPA

mPR
PGRMC1

GABAA
ER receptor
PXR

Nucleus
PR
Alpha 1 AR
adrenergic
L-type
Ca2+
NMDA
D1

Nicotine
receptor

Fig. 14.6 Schematic illustration of different receptors involved in the neuronal function of the
neuroactive steroids

14.4 Mechanism of Action and Signaling Pathways

for Neuroactive Steroids

Neuroactive steroids usually exhibit their mechanism of action through:

1. Classical intracellular binding to steroid receptors that facilitates interaction with

specific nucleotide sequences stimulating gene expression (Cato et al. 2002).
2. Effect on membrane receptors and ion channels (i.e., GABAA, NMDA, AMPA,
sigma 1, kainate receptors) described as a rapid non-genomic effect of neuroac-
tive steroids (Rupprecht et al. 1993; Le Mellédo and Baker 2002) or
3. Their metabolic interconversion to other neuroactive steroids in the brain or
through their interaction with certain neurotransmitter receptors (Rupprecht
et al. 1993; Le Mellédo and Baker 2002).

The general down signaling pathway for most of the neuroactive steroids occurs
through a series of processes as depicted in Fig. 14.7. First, neuroactive steroids
might bind to ion channels (i.e., GABAA, NMDA, AMPA, sigma 1, kainite
receptors) associated with neurotransmitter receptors (1). Second, they can bind
and activate the known steroid receptors widely distributed in the plasma membrane
(2), or they may bind to the nuclear plasma membrane-associated steroid receptors
(3) or bind with the traditional cytoplasmic-associated nuclear steroid receptors (4).
462 S. Marwein et al.

Fig. 14.7 Different potential NEUROACTIVE

mechanisms of action of STEROIDS
neuroactive steroids,
including hormonal steroids in 2 3
the nervous system

1 G proteins, AMPc,
4
IP3, Ca2+, MAPK,
PI3K, Akt, GSK3β

The binding of neuroactive steroids to the membrane and cytoplasmic-associated

receptors regulates intracellular signaling by a series of steps which include G
protein activation, alteration of intracellular levels of cAMP, inositol 1,4,5-
trisphoshate (IP3) and Ca2+ and modulation of kinases activity such as mitogen-
activated protein kinase (MAPK), phosphoinositide-3 kinase (PI3K), and Akt or
glycogen synthase kinase 3b (GSK3b). However, the binding and triggering of
cytoplasmic nuclear receptors by neuroactive steroids (4) result in their dimerization
followed by binding to steroid-responsive elements in the promoter region of
discrete genes and subsequent transcriptional activity (5). Moreover, neuroactive
steroids, modulative ability regarding both the membrane and cytoplasmic transduc-
tion signaling pathway exerts an effect on transcriptional activity (6) (Melcangi and
Mensah-Nyagan 2008).
The rapid neurotransmitter-like effects of steroids such as progesterone and
estrogen have been described to change the activity of neuronal systems through
several types of receptors (Micevych and Mermelstein 2008; Mani and Oyola 2012;
Micevych et al. 2011; Valadez-Cosmes et al. 2016). Signaling pathway stimulation
is usually initiated via the extranuclear or membrane steroid receptors mediated
through the G proteins or other secondary messengers (Boulware et al. 2005;
Zuloaga et al. 2012; Valadez-Cosmes et al. 2016). Nevertheless, classical nuclear
steroid receptors containing the palmitoylation sequences can be translocated to the
plasma membrane to rapidly change cellular action (Mani and Oyola 2012; Meitzen
et al. 2013; Schwartz et al. 2016). These nuclear transcription factors could initiate
their signaling at the plasma membrane level only by direct interactions with other
proteins (Boonyaratanakornkit et al. 2007; Micevych and Mermelstein 2008;
Boulware and Mermelstein 2009). Subsequently, intracellular signaling pathways
which involve effectors (e.g., MAPK, PI3K, Akt) are initiated via the transactivation
of receptors bound to the cell surface (Micevych and Mermelstein 2008; Boulware
and Mermelstein 2009).
14 Hormones and Steroids as Neurotransmitters 463

Neuroactive steroid estrogens trigger faster signaling mechanisms by interacting

with the membrane-bound estrogen receptors, ERα and ERβ (Milner et al. 2001;
Razandi et al. 2004), and on steroid binding, these receptors simultaneously also
trigger intracellular signaling cascades (Chaban et al. 2004; Cheskis 2004; Song
et al. 2005). The estrogen membrane-initiated signaling causes subsequent activation
of cAMP response element binding protein (CREB) to affect transcriptional activity.
For example, estradiol is capable of evoking immediate effects by inducing protein
kinase A activity (Gu et al. 1999) and MAPK pathway in neurons, or protein kinase
A and MAPK/Src pathway in astrocytes (Pawlak et al. 2005; Chen et al. 2014).
Remarkably, the membrane-initiated signaling activity of estradiol can also
be stimulated by the binding of estradiol with the metabotropic glutamate receptors
(Boulware 2005; Boulware et al. 2013; Kuo et al. 2010; Seredynski et al. 2015). In
addition, estrogen-stimulated signaling may also be activated through the
membrane-associated estrogen receptors (mERs) counting the G protein-coupled
estrogen receptor (GPER or GPR30) situated in intracellular membranes. These
receptors also mediate the quick initiation of various signaling pathways by estra-
diol, which include the MAPK pathway (Filardo et al. 2000) and the Akt/PI3K
pathway (Ruiz-Palmero et al. 2013).
Similarly, progesterone can initiate rapid responses in the CNS by triggering
various membrane-bound receptors. These membrane receptors mediate the intra-
cellular signaling pathways via transactivation of Src kinase (Src) (Micevych and
Mermelstein 2008; Boulware and Mermelstein 2009). These receptors are widely
expressed in the neurons and glial cells and they include the progesterone receptor
membrane component-1 (Pgrmc1) and the membrane progesterone receptors, that is,
mPRα, mPRβ, mPRγ, mPRδ and mPRε (Guennoun et al. 2008, 2015; Guerra-Araiza
et al. 2003; Labombarda et al. 2010; Meffre et al. 2013). The cytoplasmic-associated
progesterone receptors (Waters et al. 2008) and the membrane-bound progesterone
receptors (mPRα and Pgrmc) (Labombarda et al. 2003, 2010) widely expressed in
astrocytes also initiate rapid responses in the CNS and mediate the induction of
intracellular signaling pathways via transactivation of Src kinase.
In the case of androgens, the signaling pathway is mediated by initially binding to
the androgen receptors, followed by their interaction with the membrane and cyto-
plasmic signaling proteins (Foradori et al. 2008; Michels and Hoppe 2008), which
eventually leads to an increase in intracellular Ca2+ levels (Gorczynska and
Handelsman 1995) or regulation of MAPK (Cheng et al. 2007; Fix et al. 2004). In
the case of glucocorticoids, studies find that they possess membrane as well as
intracellular actions through the glucocorticoid receptors. This was evident in
primary hippocampal neurons and in hypothalamic slices when rapid activation of
kinase pathways was initiated subsequent to BSA-conjugated glucocorticoids infu-
sion (Qi et al. 2005; Malcher-Lopes et al. 2006; Yang et al. 2013). Similar kinase
pathway activation occurring in intracellular glucocorticoid receptors is not present,
suggesting the regulation of the pathway via other receptors including the
membrane-associated glucocorticoid receptors that also signal via G protein and
protein kinase pathways (Xiao et al. 2005). The intracellular glucocorticoid receptors
464 S. Marwein et al.

can also be trafficked, thereby stimulating rapid membrane-initiated glucocorticoid

signaling (Di et al. 2016).
Furthermore, neuroactive steroids also modulate the ligand-gated ion channels or
G protein-coupled receptors, leading to alteration of the intracellular kinases activity,
which subsequently changes the transcription outlines of the downstream genes, for
example via the cyclic AMP-protein kinase A-cAMP responsive element binding
protein pathway (Wehling 1997; Zakon 1998).

14.5 Physiological Role of Hormones and Steroids in Brain

Function as Neurotransmitters

Neuroactive steroids encompass both endogenous and exogenous steroids that

control brain function by interacting with receptors including the steroid receptors,
ion channels, ligand-gated ion channels and their respective membrane receptors
(Cai et al. 2018). Being neuroactive in nature, these steroids naturally regulate
various physiological functions in the human body (Simerly 2005), which is not
limited to sexual functions, reproductive biology and the development of accessory
reproductive organs, but also affect brain physiology and pathophysiological
functions (Frick et al. 2015; Maeng and Milad 2015; Diamanti-Kandarakis et al.
2017). Under physiological conditions, neuroactive steroids can illustrate significant
modulatory effects on brain-behavioral functions such as behavioral affection, stress,
memory, cognition and emotion (Vallée et al. 1997; Serra et al. 2000; Frye 2001;
Darnaudery et al. 2002; Johansson et al. 2002; Melcangi and Mensah-Nyagan 2008).
They also demonstrate significant roles in the etiology and management of neuro-
logical disorders such as epilepsy, schizophrenia, anxiety, depression, multiple
sclerosis, premenstrual syndrome and other brain function-related disorders
(Bäckström et al. 1983; Landgren et al. 1987; Pisu and Serra 2004; Marx et al.
2006; MacKenzie et al. 2007; Morrow 2007).

14.5.1 Physiological Role of Estrogens as Neurotransmitters

The neuronal activity of estrogens can stimulate various brain-related activities and
behavior. Abundant evidence describing the relationship of estrogens with brain
disorders has been attained through different investigations in female animals or
women. These studies have demonstrated the role of estrogens in brain-related
disorders (Walf and Frye 2006). Women are observed to be susceptible to depression
when there are fluctuations in the concentration of sex hormones, especially estro-
gen, which is believed to be responsible for disorders including post-partum depres-
sion, premenstrual dysphoric disorder, and perimenopausal or postmenopausal
depression (Thorpe et al. 2001). On the other hand, estrogens can display
neuroprotective effects when abundant amounts of endogenous estrogens are pro-
duced within the brain (Carswell et al. 2000; McCullough et al. 2003). Estrogens are
reported to display a vital role in processes such as neuronal plasticity and spine
14 Hormones and Steroids as Neurotransmitters 465

synapse formation (Herrick et al. 2006; McEwen et al. 2012). Additionally, numer-
ous studies have revealed the significant effects of estrogens on cognition (Fortress
and Frick 2014; Vahaba and Remage-Healey 2015; Sheppard et al. 2018). It has also
been found that estrogen shows protective effects on neurons to counteract the
toxicity of amyloid plaques in Alzheimer’s disease patients (Thomas et al. 1999).
However, this finding is contradicted by the results from the Women’s Health
Initiative Memory Study, which reported that combination therapy of estrogen
with progestin increased the possiblity of acquiring dementia in postmenopausal
women (Shumaker et al. 2003; Webber et al. 2005).
Estrogens also exhibit modulation action on dopaminergic signaling via their D1
receptors potentiation activity and D2 (Dopamine 2) receptor antagonizing activity
(Soares et al. 2003; Hedges et al. 2010). Through their antagonistic effects on D2
receptors, estrogens can lessen the severe symptoms of psychotic disorders. On the
other hand, they may intensify addictive behaviors through their agonistic actions on
D1 receptors (Hedges et al. 2010). Estrogens have modulating activity on other
signaling systems, which includes their stimulating effect on glutamatergic activity
(Cyr et al. 2001), and their negative effect on GABAergic activity (Wójtowicz et al.
2008; Wójtowicz and Mozrzymas 2010). These modulating effects of estrogens on
D1 and D2 receptors are believed to trigger their positive stimulation in mood
management, cognition improvement (Amin et al. 2005) and attention (Soares
et al. 2003; Soares and Frey 2010). Other neuroactive steroids belonging to this
class such as estradiol also display their enhancing effects on the development and
care of the central nervous system (McEwen and Alves 1999). This is clearly evident
in the presence of estrogen receptors in the wide array of brain structures (McEwen
and Alves 1999). Studies have shown that estrogen receptors expressed on
GABAergic neurons may play a key role by which estradiol exerts an excitatory
effect on the nervous system (Schultz et al. 2009). Additionally, it is demonstrated
that positive modulation of ERα on GABAergic neurons decreases the release of
GABA, the inhibitory neurotransmitter (Xiao et al. 2003).

14.5.2 Physiological Role of Progestogens as Neurotransmitters

Progestogens such as progesterone exhibit neuronal excitability function directly by

acting at their own receptors or when they are converted into other neuroactive
steroids. Allopregnanolone and pregnenolone are two metabolites of progesterone
and effective allosteric modulators of the GABAA receptor (Djebaili et al. 2005;
Gibson et al. 2007). The neuroactive steroid progesterone and the neuroactive
metabolites with sex steroids including estrogens and testosterone have been linked
to neuronal disorders such as schizophrenia (Koenig et al. 2002; Corcoran et al.
2003; Marx et al. 2011; Melcangi et al. 2011). There are additional studies which
highlight the role of progesterone as a main player in schizophrenia through its direct
interaction with it own receptors and also through an indirect pathway by first being
converted to glucocorticoids or allopregnanolone followed by subsequent binding of
466 S. Marwein et al.

these neuroactive metabolites with specific receptors to elicit their modulating

activity (Marx et al. 2011).
The physiological role of progesterone in brain function and diseases has been
clearly demonstrated by various studies in animal models. For instance, infusion of
exogenous progesterone to traumatic-induced brain injury and ischemia-induced
animal models lessens the lesion volume in the brain and stimulates improvement
in cognition function of those animal models (Djebaili et al. 2005; Gibson et al.
2007). Furthermore, the effect of progesterone has been broadly evaluated by Frye
and colleagues for improvement of depression, anxiety and cognition in animal
models (Frye and Lacey 2000; Frye and Sora 2010; Frye and Walf 2010; Frye
2011). For instance, ovariectomized rats when treated with a high level of proges-
terone show enhanced brain function performance such as improvement in spatial
memory, working memory and avoidance memory (Frye and Lacey 2000). Simi-
larly, progesterone administration shows better performance in cognitive tasks in
intact aged mice as assessed through object recognition, T-maze, water maze,
inhibitory avoidance and contextual fear experimental conditions (Frye and Walf
2008). Even in progesterone receptor knock-out mice, progesterone was still able to
stimulate enhanced performance in various learning and memory tasks, signifying
the possibility of progesterone’s metabolites being responsible for the memory
enhancement effect (Frye and Walf 2010).
Other neuroactive steroids and allopregnanolone (progesterone metabolites)
have a significant influence on brain physiology and its system. Allopregnanolone
has been found to exhibit an inhibitory effect on serotonergic neurons of the raphe
nucleus (Genazzani et al. 2000; Birzniece et al. 2006). Progesterone has also
been described to antagonize sigma-1 receptor activation, thereby inhibiting the
release of norepinephrine (Genazzani et al. 2000; Pluchino et al. 2006). Furthermore,
progestogens act as strong agonists of the GABA-receptor, particularly
allopregnanolone (Bäckström et al. 2011), and are implicated in the decline of
dopaminergic tone in the nucleus accumbens (Quinones-Jenab and Jenab 2010)

14.5.3 Physiological Role of Androgens as Neurotransmitters

Neuroactive steroids including testosterone and DHEA have a great modulating

impact on brain functional activity and brain-related behavior (Carré et al. 2011).
These androgenic neuroactive steroids are found to play an influential role in the
management of disorders such as anxiety and depression, particularly for meno-
pausal women and men suffering from hypogonadism (Seidman 2006). Fluctuations
of testosterone levels have accompanied pathophysiological conditions such as
mood disorders, depression and psychosis (Talih et al. 2007; van Wingen et al.
2011; McHenry et al. 2014). Then again, these neuroactive steroids do exhibit a
neuroprotective role as evident in an investigation which demonstrates that
continuing exposure to these steroids stimulates neurogenesis in the hippocampus
in addition to increased survival of new neurons (Galea et al. 2013). They are also
14 Hormones and Steroids as Neurotransmitters 467

known to play a vital role in the process of synapse formation in the spine (Leranth
et al. 2004; Romeo et al. 2005).
Several studies have demonstrated testosterone’s ability to induce positive mod-
ulatory effects on serotonergic signaling indirectly when it is metabolized to estra-
diol, thereby activating the estrogen receptors (Ebinger et al. 2009). Testosterone
also synergistically enhances the effect of noradrenergic antidepressant agents
through its androgen receptor activating activity (Martı́nez-Mota and Fernández-
Guasti 2004; Ebinger et al. 2009). Similarly, DHEA and its sulfated derivative have
an affinity with a variety of receptors including GABAA, sigma-1 and metabotropic
glutamate receptor (mGluR) (Maninger et al. 2009). Owing to its versatility of action
toward various receptors, DHEA and its sulfated derivative can induce various
synaptic transmissions including GABAergic, dopaminergic, cholinergic and
glutamatergic synaptic transmission (Yoon et al. 2010; Xu et al. 2012). In the
nervous tissues, DHEA is believed to affect neuronal excitability via its modulating
effect on the NMDA receptor (Baulieu 1997) and its positive allosteric modulating
effect on the GABAA receptor (Marx et al. 2006). This is confirmed through an
animal model study which demonstrated that DHEA and its sulfated derivative can
bring about excitation of neuronal function in the nervous system of rodents mainly
by their ability to activate the glutamate and GABA-releasing neurons (Wolf and
Kirschbaum 1999; Meyer et al. 2002; Dong et al. 2007). DHEA also displays
neuroprotective and anti-glucocorticoid effects, thereby aiding in the improvement
of depression, anxiousness, psychotic symptoms and cognitive deficits (Maninger
et al. 2009). DHEA and its sulfated derivative have a protective effect against
NMDA-induced neurotoxicity. The pathophysiological activity of DHEA and its
sulfated derivative also include neuroprotection, enhancing neuronal survival,
neurogenesis and neurite growth (Pluchino et al. 2015). Despite its modulating
effect on various synaptic transmissions and their neurotransmitter production,
DHEA sulfate can also activate the postsynaptic receptors, thereby, encouraging
various significant brain activities, including antidepressant and anxiolytic effects
and enhancement of memory, especially in patients suffering from brain function
disorders such as Alzheimer’s disease and addiction (Kaminska et al. 2000;
Balashov 2010; Dong and Zheng 2012).

14.5.4 Physiological Role of Glucocorticoids as Neurotransmitters

Glucocorticoids such as corticosterone and cortisol are also involved in modulation

of brain activities and various synaptic neurotransmissions. Additionally, their
concentration level has been associated with various pathophysiological conditions
of the brain functions such as stress, cognition, memory and psychotic behavior
(Whitaker et al. 2016; Jentsch et al. 2019; Kinlein et al. 2019). An organism’s
capacity to cope with stressful experiences is dependent on its ability to appropri-
ately engage central and peripheral systems, such as the hypothalamic-pituitary-
adrenal (HPA) axis. The HPA axis is a primary neuroendocrine mediator of neural
and behavioral responses to stress, and dysfunction of this system is linked to
468 S. Marwein et al.

increased risk for developing mental health disorders including anxiety, post-
traumatic stress disorder and depression. Numerous studies has revealed that corti-
costerone pretreatment before stress, as observed in rodent models, helps in proper
functioning of the HPA axis, which would reduce stress and stimulate improved
emotional stability (Whitaker et al. 2016). Glucocorticoids such as cortisol have
been demonstrated to enhance regulatory activity in the ventro-lateral prefrontal
cortex. Research also suggests that delayed cortisol release in response to a stressor
facilitates cognitive emotion regulation processes that might be beneficial for restor-
ing emotional stability in the aftermath of stressful events (Jentsch et al. 2019). In
recent studies it has been found that cortisol concentration level is high in patients
suffering from psychotic disorders including schizophrenia. Cortisol concentration
level has also been found to be closely linked to disorders such as cognitive
dysfunction, mild cognitive impairment and Alzheimer’s disease (Mondelli et al.
2015; Pietrzak et al. 2017; Hubbard and Miller 2019). The positive results of various
investigations advocate the possibility of therapies that target depressing cortisol and
Aβ levels, which can be employed in extenuating cognitive impairment in
Alzheimer’s disease. Additionally, in the case of the commencement of psychosis,
cortisol can act as a predictor of treatment response or can be considered as a target
for the design of new therapeutic agents (Mondelli et al. 2015; Hubbard and Miller
2019).
The physiological role of other neuroactive steroids, for example pregnenolone
and pregnenolone sulfate, also has implications for their role in neuronal excitability
and plasticity. Pregnenolone, the precursor to neuroactive steroid progesterone,
possesses the ability to boost the neurotransmitter activity of GABA and to directly
activate the GABAA receptor, as observed in studies conducted using bovine
preparations (Callachan et al. 1987). Pregnenolone action on GABA receptor can
either bring about prolonged temporal influx of chloride ions or can directly open the
ion channel when there is elevated plasma concentration of pregnenolone. On the
other hand, pregnenolone sulfate has an inhibitory action toward the GABA receptor
and acts as an antagonist, as evidenced in an investigation utilising rodent cell
cultures (Mienville and Vicini 1989). The mechanism of pregnenolone-sulfate
antagonistic activity is due to its ability to cause a decline in the frequency of the
chloride channels opening on the GABA receptor, thereby reducing inhibition of
neuron discharge, ultimately producing an excitatory effect on the nervous system
(Mienville and Vicini 1989).
The neuroactive steroid pregnenolone has also been described to modulate
molecular targets such as sigma1 receptors and the type-1 cannabinoid (CB1)
receptor (Vallée 2016). It has a positive modulating effect on the sigma1 receptors
but exhibits a positive modulative effect on type-1 cannabinoid receptor.
Pregnenolone’s positive modulating activity toward brain activity advocates the
possibility of pregnenolone involvement in processes that are activated through the
CB1 receptor, including anxiety, memory, cognition and reward-related behavior
(Fujiwara and Egashira 2004; Gardner 2005). Some of the neuroactive steroids
which have been marketed or are under investigation for their action against various
neuronal disorders are listed in Table 14.1.
14 Hormones and Steroids as Neurotransmitters 469

Table 14.1 List of marketed and investigational neuroactive steroid drugs for the treatment of
neurological disorders
Sl.
no Drug Indication Brand name
1 Pregnenolone Used for the treatment of Alzheimer’s Pregnenolone, Life-
disease, depression, seizures Flo Pregnenolone,
etc.
2 Allopregnenolone Used for the treatment of postpartum Zulresso
depression
3 DHEA Used for the treatment of Alzheimer’s Intrarosa
disease, schizophrenia
4 Ganaloxone Under development for the treatment of NCT02358538,
(Investigational postpartum depression, uncontrolled NCT03865732
drug) seizures in female children and other rare
genetic epilepsies
5 Zuranolone (SAGE- Under development for the treatment of NCT04007367,
217) major depressive disorder, postpartum NCT02978781,
(Investigational depression, essential tremor, Parkinson’s NCT03000569
drug) disease, insomnia and seizures
6 Mifepristone Under development for the treatment of NCT00867360,
(Investigational psychotic major depression and other NCT00637494,
drug) depressive disorders NCT00146523

14.6 Receptors as Potential Target for Neurological Disorders

14.6.1 Agonists to the Neuroactive Hormone and Steroid Receptors

and Their Biological Activities

Receptors involved in the neuronal signaling of various neuroactive steroids include

the steroid receptors and many other receptors by which the conventional
neurotransmitters exert their function. Thus, agonists to these receptors encompass
molecules that activate any of these receptors, and these agonists have been
employed in the management of various clinical neurological disorders such as
behavioural and biochemical alterations in Parkinson’s disease (Baraka et al.
2011), negative symptoms and cognition suffered by schizophrenic patients (Usall
et al. 2011), post-partum depression (Smith et al. 2007), Dravet syndrome (Hawkins
et al. 2017), essential tremor (ET), major depressive disorder (MDD) (Martinez
Botella et al. 2017), epilepsy (Reddy and Rogawski 2010) and anxiety disorders
(McKernan et al. 2000). Examples of agonists to the neuroactive hormone and
steroid receptors are shown in Fig. 14.8.
Several investigations have reported the neuroprotective effects of estrogenic
compound 17β-estradiol (1). One of the studies was performed on mice administered
with MPTP (1-methyl 4-phenyl-1,2,3,6 tetrahydropyridine) to induce striatal dopa-
mine depletion. The investigated mice were given the estrogenic compounds contin-
uously for a period of 5 days before MPTP treatment and this was continued until the
 470

O
H HO O
CH3 OH N OH
H CF3 O
N S
H
CH3 CH3 CH3 OH F
OH H
F
H O N H H
H H OH
N H
HO H3C O H H O H H H H
Br S
(1) OH O O CH3 (5) HO O HO
(2)
(3) (4) (6) (7)

O
O O O O O O
O O OH
H
H N
O O H H H H N H H N
H H N N H H
S H H
HO O H H H H H H
O H H CN H H
HO H H
O HO H HO O
(8) H HO
(9)
H
(15)
(11) (12) (13)
(10) (14)
OH CF3
O CH(CH3)2
O O OH CH2 CH2CH2CH2CH3 Cl
O H
H H
H H
H H H H N O O
HO HO
HO HO HO HO HO HO
HO HO OH OH
(16) OH OH
(17) (18) (19)
(21) (22)
(20)
(23)
F F
F

F
O
NH F
F

H H
O N
H H
(24)

Fig. 14.8 Examples of agonists to the neuroactive hormone and steroid receptors and their biological activities
S. Marwein et al.
14 Hormones and Steroids as Neurotransmitters 471

14th day. When dopamine markers in the mice were assessed the results showed that
17β-estradiol inclusion in the therapy prevents the depletion of striatal dopamine and
that MTTP could not cause reduction in the dopamine level in mice (Callier et al.
2001; Grandbois et al. 2000). Likewise, studies have reported that unilateral admin-
istration of 6-hydroxydopamine (6-OHDA) in an ovariectomized female rat model
with Parkinson’s disease can induce similar biochemical alteration and abnormal
behavioural patterns to that seen in human Parkinson’s disease (Baraka et al. 2011).
The administration of 17β-estradiol has been reported to prevent these biochemical
and behavioral alterations induced by 6-OHDA in ovariectomized rats, as observed
in an MPTP-induced mouse model (Quesada et al. 2008; Baraka et al. 2011).
Estradiol when given as an adjuvant has been reported to alleviate psychotic-like
behavioural disorders via its neuroprotective ability (Akhondzadeh et al. 2003;
Begemann et al. 2012; Kulkarni et al. 2008a, b, 2011, 2015).
Likewise, the neuroprotective effects of other estrogen receptor modulators such
as the ERα agonist, propyl-pyrazoletriol (PPT) (2) and raloxifene (3), which acts as a
selective estrogen receptor modulator (SERM) on MPTP-treated mice, are also
described (Grandbois et al. 2000; Callier et al. 2001; Bourque et al. 2019). These
two compounds prevented MPTP-induced striatal dopamine depletion in mice when
the drug treatment was continued for 5–14 days. Similarly, a study on a 6-OHDA
lesion-induced rat model of Parkinson’s disease has shown that raloxifene and
compound 2 prevented the induction of biochemical alterations and altered
behavioural function by 6-OHDA (Baraka et al. 2011).
The examination of raloxifene (3) as a selective modulator of the estrogen
receptor and its ability to prevent the estrogenic stimulation of the gonadal tissue
caused by the long-term use of estradiol has been reported (Chlebowski et al. 2009).
Raloxifene’s ability to selectively modulate the estrogen receptors has also been
reported to enhance memory (Yaffe et al. 2005). In a clinical trial using raloxifene as
an adjuvant therapy for schizophrenia (Kulkarni et al. 2010) it was described that
raloxifene stimulates enhancement of the positive symptoms and improvement of the
negative symptoms in postmenopausal women within a dose of 120 mg/day. Con-
sequent investigations with a dose of 60 mg/day of raloxifene as an adjunct
displayed significant enhancement of cognitive ability and improvement in negative
symptoms (Usall et al. 2011).
The synthetic steroid tibolone (4) is considered as a selective tissue estrogenic
activity regulator due to its ability to convert into neuroactive metabolites that could
bind directly to steroid receptors and regulate enzymes needed for its metabolization
into other neuroactive metabolites depending on the tissue involved (Kloosterboer
2004; Reed and Kloosterboer 2004; Cummings et al. 2008). There are reports which
describe the neuroprotective activity of tibolone in neurons (Belenichev et al. 2012;
Pinto-Almazán et al. 2012; Farfán-García et al. 2014). Tibolone has also been
reported to display protective activity in a human astrocyte cell model whereby it
is first metabolized into 3α-hydroxy tibolone and 3β-hydroxy tibolone followed by
the binding of these metabolites to the ERα and ERβ receptors in a human astrocytes.
It thus results in the activation of these receptors to further bring about the desired
neuronal protective activity (Guzman et al. 2007; Avila Rodriguez et al. 2014).
472 S. Marwein et al.

The G protein-coupled estrogen receptor 1 (GPER1) agonist G1 (5) is another

example with neuroprotective properties and has been reported for its efficacy in
improving brain injury in several experimental models. It has been demonstrated that
G1 enhances the level of protein expression of GPER1, which strengthens the
neuroprotection ability of estrogen in the case of spinal cord injury. There are also
reports that describe the ability of G1 to prevent neurons from NMDA and glutamate
toxicities (Lebesgue et al. 2010; Hu et al. 2012; Liu et al. 2012; Tang et al. 2014;
Chen et al. 2015).
The neuroprotective profile of the estrogen agonist ICI182,780 (Fulvestrant) (6)
in rat primary hippocampal neurons, verifying its neuroprotective efficacy against
neurodegenerative symptoms linked with Alzheimer’s disease and other similar
disorders, has been reported. Compound 6 greatly enhances neuron survival in a
concentration-dependent manner when the rat hippocampal neurons are exposed to
excitotoxic glutamate. The neuron survival is evidenced even when exposed to
neurodegenerative-induced amyloid1-42. It has been shown that compound 6 directly
interacts with ion channels and brings about rapid intracellular Ca2+ concentration
([Ca2+]i) oscillations in a similar manner to that of neurons treated with 17-
β-estradiol. Furthermore, compound 6 also stimulates better activation of signal-
regulated kinase 1/2 and Akt (protein kinase B) and greatly augments the production
of spinophilin and Bcl-2. Altogether, these findings deliver a thorough understand-
ing of the agonistic ability of compound 6 toward estrogen receptors in rat hippo-
campal neurons (Zhao et al. 2006).
The role of the neuroactive steroid pregnenolone (7) in the regulation and
improvement of anxiety and depression has been evidenced in several preclinical
trial studies. Pregnenolone could greatly diminish the sedative side effects of diaze-
pam when given together. This significant reduction of sedation suggests a possible
therapeutic advantage of pregnenolone for the management of psychiatric conditions
that could be useful in reversing the unwanted sedative-hypnotic actions of
benzodiazepines (Meieran et al. 2004). Further, data from preclinical studies and
human trial studies propose the beneficial effect of pregnenolone for the manage-
ment of bipolar depression (Marx et al. 2009; Ritsner et al. 2010). Consequently, a
recent clinical study reports the positive result of pregnenolone in alleviation of
depressive symptoms in bipolar disorder patients when exposed to the pregnenolone
therapy for a period of 12 weeks (Brown et al. 2014). Furthermore, pregnenolone is
also known for its ability to increase the synapse strength that ultimately increases
learning and enhances memory retention. Its positive modulating effect on brain
cholinergic synaptic neurotransmission also boosts the learning process and memory
enhancement in aged rats (Bu and Zu 2014).
Pregnenolone sulfate (8) is another excitatory neuroactive steroid that acts as an
NMDA receptor agonist and plays a role in memory-enhancing effects. Pregneno-
lone sulfate has been shown to restore memory impairment in cirrhotic rats as
compared to rats treated with AP5, a competitive antagonist of NMDA receptor,
which shows no significant effect on memory performance. This shows that acute
pregnenolone sulfate therapy could enhance memory in cirrhosis memory deficit
through its NMDA receptor activating effect (Dastgheib et al. 2015).
14 Hormones and Steroids as Neurotransmitters 473

The neuroactive steroid progesterone (9) has also been reported for its
neuroprotective effects and its ability to enhance neuronal survival in males and
females (Wei and Xiao 2013). This activity of progesterone was reported by
Schumacher et al. (2004) whereby they described the neuroprotection action of
progesterone for the dopaminergic neurons in rats. In this study progesterone
displayed protectective activity for the dopaminergic neurons against MPTP-
induced degeneration, an outcome which is closely related to hormonal-induced
weakening of dopaminergic signaling caused by age and the onset of Parkinson’s
disease (Schumacher et al. 2004). Additionally, the beneficial effects of progesterone
for the treatment of multiple sclerosis is evidenced in the experimental autoimmune
encephalomyelitis model. Progesterone exerts its action through its involvement in
repairing of the myelin sheath and by increasing the concentration of endogenous
oligodendrocyte precursor cells (Zawadzka and Franklin 2007).
Progestin (10), a synthetic form of progesterone, is also known for its association
with physiological processes including preservation of myelin sheath integrity,
formation of synapses and neuronal survival (McEwen and Woolley 1994; Mellon
2007; Zhang et al. 2010; Schumacher et al. 2012). Progestin has been reported to
display neuroprotection action in the case of spinal cord and peripheral nerve injury
(Labombarda and Garcia-Ovejero 2014). There are also data which support the
neuroprotective effect of progestin in brain disorders that include demyelinating
disease, motor neuron diseases, epilepsy and depression (Frye and Lacey 2000; Walf
et al., 2006; Deutsch et al. 2013; Li et al. 2013).
Allopregnanolone (11), a metabolic product of progesterone, is a well-known
endogenous neurosteroid with inhibitory action. This neuroactive steroid acts as an
allosteric modulator toward the GABAA receptor and exerts its modulative
properties at the synaptic and extrasynaptic GABAA receptors. Allopregnanolone
is well known for its anticonvulsion property, and numerous experimental animal
models of epilepsy such as the pentylenetetrazol (PTZ)-induced seizure model and
picrotoxin-induced and bicuculline-induced seizure models have helped to establish
this fact (Kaminski et al. 2004; Reddy and Rogawski 2012; Rogawski et al. 2013).
The anticonvulsant activity of allopregnanolone is observed to fully suppress limbic
seizures and status epilepticus in animal models induced by pilocarpine and kainic
acid (Reddy 2010, 2011; Reddy and Rogawski 2012). A phase III clinical trial on the
anticonvulsant activity of allopregnanolone revealed that continuous parenteral
infusion of allopregnanolone formulation represents a better therapy regime for the
management of persistent seizure, a deadly brain condition known as super refrac-
tory status epilepticus (SRSE), that does not respond to usual treatments
(Vaitkevicius et al. 2013; Bialer et al. 2015). Allopregnanolone has also been
reported to have better effects in the management of postpartum depression (PPD)
and essential tremor (ET) in a phase II clinical trial and exploratory study, respec-
tively (Ellenbogen et al. 2016; Kanes et al. 2017a). In the case of postpartum
depression it is observed that with the delivery of the placenta, allopregnanolone
levels decrease rapidly, and this is believed to cause the onset of postpartum
depression in susceptible women (Smith et al. 2007). This is not the case in a healthy
women, as the rapid decline in allopregnanolone levels activates GABA receptor
474 S. Marwein et al.

expression (MacKenzie and Maguire 2013). Studies on the efficacy of brexanolone,

a synthetic form of allopregnanolone, in the management of postpartum depression
present hopeful outcomes. It acts as a positive allosteric modulator of GABAA
receptors. This result can be further testified through a phase II randomized
placebo-controlled trial where brexanolone treatment showed 70% efficacy in
decreasing the severe state of postpartum depression (Kanes et al. 2017b).
The compound SGE-516 (12) is a 1,2,5-triazole analog of allopregnanolone
which can be administered orally while still retaining similar modulative activity
toward the GABAA receptors (Martinez Botella et al. 2015). The 1,2,5-triazole
analog was reported to display comparable pharmacological properties to
allopregnanolone with better aqueous solubility and enhanced pharmacokinetic
profile (Hammond et al. 2017). This potent allosteric modulator of both synaptic
and extrasynaptic GABAA receptors has been reported to exhibit anticonvulsant
activity as demonstrated in a seizure rodent model (Hammond et al. 2017). Further-
more, the anticonvulsant activity of SGE-516 on an experimental epilepsy animal
model employing the Scn1a+/
mouse with symptoms similar to those of Dravet
syndrome has been evaluated for efficacy on hyperthermia-induced seizure. The
results revealed that SGE-516 prevents seizures induced by hyperthermia, reduces
the seizure frequency and increased the survival rate of the Scn1a+/
mice (Hawkins
et al. 2017).
Certain classes of neuroactive steroids such as 5β-nor-19-pregnan-20-one analogs
conjugated with substituted pyrazoles and triazoles at C-21 exhibit strong allosteric
modulation activity for the synaptic and extrasynaptic GABAA receptors. The
pyrazole substituted derivative, 3α-hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-
10 -yl)-19-nor-5β-pregnan-20-one (SAGE-217, 13), is one such example of an allo-
steric modulator of GABAA receptor with brilliant oral pharmacodynamic and
pharmacokinetic properties. SAGE-217, also known as Zuranolone, is an investiga-
tional medication and has undergone a successful phase I clinical study and is
presently animated for a parallel phase II clinical trial to investigate its effectiveness
for the management of essential tremor, postpartum depression and major depressive
disorder (MDD) (Martinez Botella et al. 2017). A phase II clinical trial on SAGE-
217 for its implication toward Parkinson’s disease is also currently ongoing
(NCT02978781 n.d., B245).
Ganaxolone (14) is another example of an allosteric modulator of the GABAA
receptors with comparable properties to those of allopregnanolone. This neuroactive
steroid has been verified for its anticonvulsant potential from ample of supporting
data attained from numerous preclinical studies on epileptic animals such as the
pentylenetetrazole-induced seizure model and the amygdala kindling model (Gasior
et al. 2000; Reddy and Rogawski 2010). The synthetic neuroactive steroid,
ganaxolone, induces its inhibitory action by activating both the synaptic and
extrasynaptic GABAA receptors (Reddy and Rogawski 2010, 2012). Reportedly, a
large number of clinical studies have been conducted to verify the efficacy and safety
of ganaxolone meant for the management of epilepsy. One such study includes the
phase III multicenter, randomized, double-blind, placebo-controlled study of
ganaxolone as an adjunctivant for the treatment of uncontrolled partial-onset
14 Hormones and Steroids as Neurotransmitters 475

seizures in adult patients (NCT01963208 n.d.). This study established safety and
tolerability issues with the revelation of some adverse events such as drowsiness and
fatigue. Another ongoing clinical study on ganaxolone is the phase II multicenter,
open-label, proof-of-concept study for the management of epilepsy in children
(NCT02358538 n.d.).
The neuroactive steroid testosterone (15) acting as an agonist to the androgen
receptors was also reported to have a neuroprotective effect (Moffat et al. 2002). The
Baltimore Longitudinal Study of Aging (BLSA) reported significant results regard-
ing the neuroprotection action of testosterone in the case of deterioration of cognitive
ability in Alzheimer’s disease due to aging. The study reports that elevated endoge-
nous testosterone concentration can stimulate verbal memory enhancement in young
patients which is otherwise absent in elders and in men with testosterone deficiency
(Moffat et al. 2002). Studies also report that low testosterone production in the case
of older men increases the risk for the onset of Parkinson-like disorders (Okun et al.
2006). Accordingly, many investigations have been conducted which have described
the benefit of including testosterone in therapy meant to correct Parkinson disease-
like symptoms due to its ability to directly effect and improve motor symptoms
(Okun et al. 2002). Mitchell and group noted the substantial decrease of resting
tremor and improved motor symptoms in a Parkinson’s patient with testosterone
deficiency only after the administration of testosterone (Mitchell et al. 2006).
The neuroactive steroid DHEA (16) and its role in alleviating depressive
symptoms have been studied widely. Recent studies have revealed that higher
DHEA and DHEA sulfate levels prevent the commencement of depressive
symptoms in both males and females (Veronese et al. 2015). However,
low-plasma level DHEA sulfate was described to alleviate severe depression in
men but not in women (Veronese et al. 2015). Additionally, a longitudinal cohort
study revealed that low levels of DHEA sulfate improve depressive symptoms
irrespective of gender and age (Souza-Teodoro et al. 2016).
DHEA analogs conjugated with spiro-epoxy functional group at C-17 were found
to induce substantial protective activity on neurons. Compounds under this class act
as good positive modulators of ion channel receptors such as the NMDA and
GABAA receptors. The spiro-epoxy steroidal derivatives 17–19 reported by
Calogeropoulou et al. (2009) were found to be the most potent neuroprotective
agents against induced apoptosis when tested in neural PC12 cells. In addition,
treatment of PC12 cells with neuroactive steroid derivatives 17–19 also up-regulates
the production of dopamine from the dopaminergic neurons. A further structure
activity relationship study showed that introduction of an epoxide moiety at C-17, as
in the case of the DHEA analogs 17–19, facilitates their easy transportation across
the cell membrane, whereby they can bind directly to estrogen receptors ERɑ and
ERβ. The structure activity relationship study also revealed that the presence of the
hydroxy group at C-21 in 18 and 19 encourages better hydrogen bond formation and
better interaction with the NMDA or the GABAA receptors that can be credited with
enhancing neuroprotective activity of these spiro-epoxy conjugated steroids
(Calogeropoulou et al. 2009).
476 S. Marwein et al.

Alternative hydroxy derivatives of DHEA (20–22) that are known to bind to the
membrane receptors, NMDA and GABAA receptors with high affinity, were studied
for their neuroprotective effects against hypoxia-induced neurodegeneration in the
cortical neurons. The triol derivatives of DHEA could easily cross any physiological
membrane, then directly bind to the membrane receptors and display significant
neuroprotection of the neurons. Supplementary structure activity relationship studies
revealed that addition of a methylene functional group at C-17 as in the derivative 20
produces less neuroprotective effect, while incorporation of the alkyl moiety at C-17
just as in the derivatives 21 and 22 presented improved dimensional flexibility and
enhanced interaction with NMDA and GABAA receptors, leading to better
neuroprotective effects (Long et al. 2016).
The compound 1OP-2198 or XEN 1101 (undisclosed structure) represents a new
class of neuronal allosteric modulator of the Kv7 (KCNQ) potassium channel. The
Kv7 neuronal channels have been described for their function of slowly activating
and deactivating voltage-gated M-current. Studies validate the role these channels
play in the management of hyperexcitability disorders such as epilepsy (Large et al.
2012). 1OP-2198 has been described to exhibit anticonvulsant effect in the maximal
electroshock seizure, in pentylenetetrazol-induced, picrotoxin-induced, bicuculline-
induced, and 6 Hz seizure animal models (Hoffmann et al. 2017). Studies reported
that the 1OP-2198 mechanism of action is comparable to that of other Kv7 channel
modulators such as retigabine; however, it is more potent than the other modulators
(Roeloffs et al. 2008). 1OP-2198 lessens the state of convulsion, thereby exerting
antiepileptic action through a mechanism which involves opening of the neuronal
Kv7 channels through their direct interaction with the ion channel (Roeloffs et al.
2008).
Neuroactive steroids also exert a protective activity on the diabetic-induced
degeneration of neurons. But these steroidal compounds can trigger endocrine side
effects when administered into the physiological system (Moran et al. 2013; Mehlig
et al. 2014). However, other physiological pathways could be targeted so as to
induce the neuroactive steroid production directly in the nervous tissues specifically;
for example, the liver X receptors (LXRs) could be targeted instead of the conven-
tional steroid receptors. In this regard, the LXR agonist GW3965 (23) was evaluated
and reported to increase the neuroactive steroid level specifically in nervous tissues
such as the cerebral cortex, cerebellum and spinal cord of the diabetic-induced rat
model (Mitro et al. 2012). Studies also demonstrated that the LXR agonist GW3965
stimulates an upsurge production of progesterone metabolites that include
dihydroprogesterone, tetrahydroprogesterone and isopregnanolone, and these
metabolites themselves are potent GABAA receptor modulators. In particular,
isopregnanolone does not interact or modulate the GABAA receptor activity, but it
exhibits an antagonistic effect against the tetrahydroprogesterone-activation of the
GABAA receptor (Bäckström et al. 2005). These findings suggest that LXR agonist
GW3965 can improve neuronal synaptic activity in diabetic patients through its
indirect activation of GABAA receptors (Mitro et al. 2012).
Some of the neuroactive compounds exert their neuronal activity not by their
interaction with any receptors but by targeting the enzymes that are necessary for the
14 Hormones and Steroids as Neurotransmitters 477

biosynthesis of steroids rather than acting directly on the receptors involved in the
neurotransmission functions of the neurotransmitters. For instance, the 5α-reductase
inhibitor dutasteride (24) has been described for its ability to activate dopaminergic
signaling and as a drug beneficial in the management of motor neuron disorders and
other neurodegenerative disorders including Parkinson’s disease (Paba et al. 2011).
Current research shows that dutasteride prevented the MPTP-induced damage of
several dopaminergic markers in male mice (Litim et al. 2015). The study was
conducted on mice which were previously exposed to MPTP so as to reduce the
striatal dopamine in the mice brain up to 50%. However, the MPTP-exposed mice
that received dutasteride showed a significant increase in striatal dopamine and its
metabolite concentrations. Dutasteride prevented MPTP toxicity on dopamine
metabolism in MPTP-treated mice (Litim et al. 2015). The neuroprotective activity
of dutasteride could be facilitated by its 5ɑ-reductase inhibitory action, which in turn
stops the conversion of these steroid precursors into their metabolites, leading to
buildup of the precursors such as progesterone, estrogens and DHEA which are
known to possess a neuroprotective effect against MPTP-induce neurodegeneration
(Bourque et al. 2009; Bourque et al. 2019).

14.6.2 Antagonists to the Neuroactive Hormone and Steroid

Receptors and Their Biological Activities

The antagonists to the hormone and steroid receptors include drugs and derivatives
of various structural functionality, acting on the different receptors, namely, GABAA
receptors (Johansson et al. 2015), NMDA receptors (Hu et al. 2014), progestin
receptors (Sun et al. 2018), AMPA glutamate receptors (French et al. 2012) and
various ion channels including the voltage-gated sodium channels (Bialer et al.
2013), T-type calcium channels (Tringham et al. 2012) and Na(+)-K(+)-2Cl(
)
cotransporter (Löscher, Puskarjov & Kaila 2013) that are involved in the neurotrans-
mitter signaling of the hormone and steroidal neurotransmitter. Through their antag-
onistic effects, they exert various therapeutic effects on various neurological
disorders. Examples of antagonists to the neuroactive hormone and steroid receptors
are shown in Fig. 14.9.
There are a group of neuroactive steroid compounds that exhibit an antagonistic
effect toward the GABAA receptor and these are categorized as the GABAA receptor
modulating steroid antagonists (GAMSA). They selectively inhibit neuroactive
steroid-mediated enhancement of GABA-evoked currents at the GABAA receptor.
These compounds exert an antagonistic action to the activation effect of the GABAA
receptor by the neurosteroid but do not hamper the neurotransmitter GABA to
induce chloride flux via the receptor (Johansson et al. 2016). The most common
example of GAMSA is the neuroactive steroid isoallopregnanolone (25), similar to
allopregnanolone, whereby the difference is in the hydroxyl group orientation at C-3
in the A-ring of the steroid. Its antagonistic action toward the GABAA receptors has
been extensively explored through various experimental models. For instance, the
voltage-clamp model using the recombinant GABAA receptors containing rat
478 S. Marwein et al.

H3C
O OH N OH

H H
H
H H H H
H H
HO HO HO
HO H H
H
(25) (26) (27) (28)

H H
O O
O H H
O
H F H H
H2N
O
H H H
HO
HO
OH
(29) COOH
(30) (31)

CH2
O CH3 H3C
N
HO H3C
OH
O O
HO C (CH2)n C O H
H HO
HO
n=1-6 OH H
O
(32)
(33) (34)

Fig. 14.9 Examples of antagonists to the neuroactive hormone and steroid receptors and their
biological activities

revealed the inhibitory action of isoallopregnanolone toward the GABAA receptor

activation by allopregnanolone (Wang et al. 2002). In the same way,
isoallopregnanolone shows an antagonistic effect toward allopregnanolone-induced
neuronal inhibition of the CA1 pyramidal neurons when it is applied directly to the
pyramidal cell obtained from the hippocampal slices of rats (Wang et al. 2000,
2018). The specificity of the antagonistic effect of isoallopregnanolone was
established through a demonstration whereby it antagonizes allopregnanolone-
induced chloride ion flux even in the presence of GABA, without affecting the
chloride ion uptake induced by GABA alone (Lundgren et al. 2003). Additionally,
isoallopregnanolone treatment was not accompanied by any adverse effects as
reported by the findings of the phase I/II clinical trial on isoallopregnanolone for
the treatment of premenstrual dysphoric disorder (Bixo 2014).
The antagonists of the GABAA receptor modulator include numerous other
3β-hydroxy-steroids (Strömberg et al. 2006; Wang et al. 2002). UC1011 (3β-20-
β-dihydroxy-5ɑ-pregnane) (26) is another GABAA receptor modulating steroid
antagonist which has been reported to antagonize the allopregnanolone activation
of GABA-induced chloride ion uptake as observed in isolated hippocampal and
cortical microsac models (Turkmen et al. 2004). The compound GR3027 (27) also
comes under the category of GABAA receptor modulating steroid antagonists
through its antagonizing effect on tetrahydrodeoxycorticosterone activation of the
14 Hormones and Steroids as Neurotransmitters 479

GABAA receptors as seen in HEK-293 cells (Johansson et al. 2015). Another

example includes 17PA ((3ɑ5ɑ)-17-phenylandrost-16-en-3-ol) (28), which exerts
its effect by selectively antagonizing the effects of allopregnanolone. The
compound17PA was also described to antagonize GABAA receptor activation by
5ɑ-reduced steroids as demonstrated in isolated rat hippocampal neurons and in
homogenized fractions of mouse brain cortex (Mennerick et al. 2004; Kelley et al.
2007).
Fluasterone (DHEF) (29) is yet another compound with possible NMDA receptor
inhibition activity that is testified to be beneficial in the treatment of traumatic brain
injury and in the management of various behavioral symptoms including changes in
affection, memory, coordination and loss of motor function suffered by many
traumatic brain injury patients. In research carried out by Mallik et al. in an animal
model, DHEF significantly improved behavioral recovery after 3 days when given
prior to 12 h post injury (Malik et al. 2003).
The synthetic neuroactive steroid derivative 20-oxo-5β-pregnan-3ɑ-yl-L-
glutamyl-1-ester (30) has been described to possess a strong antagonist effect toward
the NMDA receptor and was reported to be a neuroprotective agent by Kapras and
his co-workers. This derivative by-passes the fast metabolic process catalyzed by
sulfatase enzyme and possesses a improved bioavailability profile in comparison to
the sulfated derivative of pregnanolone. Furthermore, this derivative exerts its
inhibitory effects on the NMDA receptor in a dose-dependent manner (Kapras
et al. 2012).
The metabolite of cholesterol, cholestane-3,5,6-triol (Triol) (31), was also noted
as a major endogenous neuroprotectant by Hu and his colleagues (Hu et al. 2014).
Derivative 31 was found to exhibit a protective effect on the neurons from injury as
observed in in vitro models. In an in vivo animal study, the triol derivative 31
prevents neuronal injury as observed in ischemic-induced rabbits and rats. Different
studies notified that triol treatment greatly decreases cellular calcium ions concen-
tration and directly antagonizes the NMDA receptors, thereby inducing its
neuroprotective effect (Hu et al. 2014).
Most of the therapeutically active compounds of NMDA receptor antagonists
exert activity through their competence in selectively blocking the NMDA receptors.
But it is also known that overexpression of NMDA receptors causes excitotoxicity,
leading to neurological disorders (Zhou and Sheng 2013). Some neuroactive steroids
including pregnanolone sulfate can selectively inhibit the tonically activated NMDA
receptors. With this concept in mind, the development of pregnanolone derivatives
32 (n ¼ 1–6) was achieved by incorporating carboxylic acid functionalities at the
terminal of the alkyl chain with varying carbon number substituted at C-3 of the
parent steroid. These derivatives were evaluated in vivo for their potential to inhibit
the NMDA receptors and the results revealed that the derivative with the longest
chain, pregnanolone hemipimelate (n¼6), exhibits the maximum inhibitory effect
against the tonically activated NMDA receptors with no psychotogenic side effects.
This result offers a better understanding of the action of synthetic neurosteroids on
neuronal function that can be employed in the design of novel neurosteroid-based
ligands (Jiang et al. 2012).
480 S. Marwein et al.

The neuroactive steroid of marine origin c 24-methylenecholestane-3β,5α,6β,19-

tetrol (33) has been explored for its neuroprotective profile on neurons induced with
toxic concentration of glutamate in an animal model. The outcome of the study
represents enhanced neuron survival of cerebellar granule neurons when the neuro-
nal cells are incubated with tetrol. This marine steroid derivative can also attenuate
NMDA-induced intracellular calcium [Ca2+] and inhibit the NMDA currents in
cortical neurons, thus demonstrating the neuroprotective effects (Yan et al. 2015).
RU486 (Mifepristone) (34), an efficient antagonist of progestin receptor (Sun
et al. 2018), has shown therapeutic activity on major psychotic depressions and
meningiomas. It also exhibits protective effects against traumatic neuronal
alterations such as protection of cerebellar Purkinje cells. This agent, which is also
an antiglucocorticosteroid, can be orally administered and has demonstrated anti-
neurosteroidal effects on inappropriate neuroprogesterone and
neuroglucocorticosteroid pathologies (De Nicola et al. 2006; Rakotomamonjy
et al. 2011).

14.7 Future Perspectives

The nervous system can be described as a site of neuroactive steroid production

whereby these steroids target the neurons and glial cells. These hormonal steroids
that are synthesized locally in the neuronal or glial cells, as well as the synthetic
steroids, have been found to exhibit neuronal activity and are involved in various
brain functions through their interaction with different receptors. Their ability to alter
neuronal excitability has been investigated in different clinical trials and some of
these steroids have been used in clinical practice (Giatti et al. 2019). For instance,
estradiol was employed in clinical trials for the treatment of schizophrenia. One of
the clinical trials has reported a dose-finding pilot study on estradiol and its ability to
alleviate schizophrenic symptoms by employing a transdermal implant of estradiol
(Kulkarni et al. 2001), while the other clinical trial has described the activity of
transdermal estradiol at a dose of 100 mcg (Kulkarni et al. 2008a, b). In both studies,
estradiol displayed excellent effectiveness, thus suggesting its usefulness for the
management of psychotic sypmtoms in women suffering from schizophrenia.
Another clinical trial as reported by Kulkarni and his group also represents results
that support the efficacy of adjunctive transdermal estradiol for the treatment of
schizophrenia (Akhondzadeh et al. 2003; Kulkarni et al. 2015). The estradiol
treatment was described to have a positive effect on both male and female patients.
This was presented through a 14-day trial in men suffering from schizophrenia,
whereby patients with abundant estradiol concentration showed rapid recovery from
psychotic symptoms (Kulkarni et al. 2011).
The modulative effect of neuroactive steroids on neuronal function has the drawn
attention of many researchers to study further their effects as well as the effects of
their analogs. Recently, Hogenkamp patented his method that describes the devel-
opment of the novel 17β-heteroaryl-substituted steroid compounds 35–37.
According to his findings, these compounds act as GABAA receptor modulators
14 Hormones and Steroids as Neurotransmitters 481

O OH H2N
O SiMe3
N
N S
N

HO
HO HO

(35) (36) (37)

OH
NO2 CN
O O
MeO MeO
MeO H H
MeO H
H
H H H H H
H H
H H HO HO
O H H
O H
(38) (39) (40) (41)

Fig. 14.10 Examples of patented neuroactive steroids and their biological activities

and are considered valuable in the treatment and/or prevention of various CNS
disorders including epilepsy, cognitive dysfunction, depressive or bipolar disorders,
stroke and multiple sclerosis (Hogenkamp 2014). Similarly, Covey and Robichaud
patented their invention of the neuroactive 19-alkoxy-17-substituted steroids 38–41
for their effectiveness against CNS disorders through their action on the GABA
receptors regulating the excitation of neuron, mood disorder and decreasing stress
level (Covey and Robichaud 2014). Examples of patented neuroactive steroids are
shown in Fig. 14.10.
Neuroactive steroids embodied various protective therapeutic agents against CNS
(central nervous system) and PNS (peripheral nervous system) disorders and
diseases. Nevertheless, attention must be given to other aspects that are associated
with the application of neuroactive steroids. It is important to point out that system-
atic administration of neuroactive steroids can bring about endocrine side effects.
Undeniably, the receptors for neuronal excitability, including the steroid receptors
themselves, are extensively distributed in several nervous tissues. The study describ-
ing the SERMs provides an example of alternative approaches to selectively activate
only those receptors expressed in the nervous tissues. SERMs are molecules that
exert regulation of estrogen receptor and transcriptional activity in specific tissues,
especially in nerve cells, and may not exert any effects or even have an antagonistic
effects toward the estrogen receptors expressed in other cell types (Arévalo et al.
2015). However, the use of synthetic steroids may not fully evoke the desired effects
as the actions of neuroactive steroids depend on their conversion into active
metabolites. Most of the synthetic steroidal receptor ligands cannot be metabolized
in other active metabolites and are ineffective or induce partial effects. This occurs
with medroxyprogesterone acetate whereby it is not converted to the progesterone
metabolites (i.e., dihydroprogesterone and tetrahydroprogesterone) in the CNS as is
natural progesterone (Ciriza et al. 2006).
482 S. Marwein et al.

14.8 Conclusions

The understanding about the physiological activity of neuroactive steroids, the

complex process involved in their metabolism, the complex mechanisms of action
and their multiple roles within the nervous system is still emerging. Thus, further
studies could be an interesting endeavour. It can be noted that neuroactive steroids
are mainly produced within the neuronal tissues and these steroids do exert effects
other than the conventional well-known genomic effects. These steroids, especially
those produced within the brain, also exerts neuronal excitability action, myelin
sheath protection and many other activities related to brain function. They bind to
the neurotransmitter receptors, for example GABAA receptors, the NMDA gluta-
mate receptors, the sigma 1 receptors and the steroid receptors and produce physio-
logical effects within the nervous system. It is also well known that their levels in the
nervous system are affected by pathophysiological conditions, indicating neuroac-
tive steroids as a potential therapeutic intervention. However, fluctuations of steroid
concentration within the brain do not correspond to the peripheral steroid levels.
Therefore, employing neuroactive steroids in the treatment of neuronal disorders
might bring about unanticipated effects on brain steroid levels. Thus, they may cause
more negative effects than positive. Lastly, novel steroidal derivatives that are found
to have neuronal excitability functions may be used to modulate brain steroidogene-
sis and could be a therapeutic possibility that ought to be assessed further to establish
them as future drug candidates.

Acknowledgements The authors gratefully acknowledge Tripura University (a Central Univer-

sity), Suryamaninagar-799022, for providing the library facility to write this manuscript. The
authors express their gratitude to the Ministry of Tribal Affairs, Govt. of India (Awardee
no. 201819-NFST-MEG-01673), UGC, New Delhi ([F.30376/2017(BSR]), CSIR, New Delhi (02
(0329)/17/EMR) and DBT, New Delhi (No.BT/PR24783/NER/95/851/2017) for their research
funding. The authors declare no conflict of interest.

References
Akhondzadeh S, Nejatisafa AA, Amini H, Mohammadi MR, Larijani B, Kashani L, Raisi F,
Kamalipour A (2003) Adjunctive estrogen treatment in women with chronic schizophrenia: a
double-blind, randomized, and placebo-controlled trial. Progr Neuropsychopharmacol Biol
Psychiatry 27(6):1007–1012
Akk G, Shu HJ, Wang C, Steinbach JH, Zorumski CF, Covey DF, Mennerick S (2005)
Neurosteroid access to the GABAA receptor. J Neurosci 25(50):11605–11613
Akk G, Covey DF, Evers AS, Steinbach JH, Zorumski CF, Mennerick S (2007) Mechanisms of
neurosteroid interactions with GABAA receptors. Pharmacol Ther 116(1):35–57
Akk G, Covey DF, Evers AS, Steinbach JH, Zorumski CF, Mennerick S (2009) The influence of the
membrane on neurosteroid actions at GABAA receptors. Psychoneuroendocrinology 34:S59–
S66
Almey A, Filardo EJ, Milner TA, Brake WG (2012) Estrogen receptors are found in glia and at
extranuclear neuronal sites in the dorsal striatum of female rats: evidence for cholinergic but not
dopaminergic colocalization. Endocrinology 153(11):5373–5383
14 Hormones and Steroids as Neurotransmitters 483

Amin Z, Canli T, Epperson CN (2005) Effect of estrogen-serotonin interactions on mood and

cognition. Behav Cogn Neurosci Rev 4(1):43–58
Appelgren LE (1967) Sites of steroid hormone formation. Autoradiographic studies using labelled
precursors. Acta Physiol Scand Suppl 301:1
Aranda A, Pascual A (2001) Nuclear hormone receptors and gene expression. Physiol Rev 81
(3):1269–1304
Arevalo MA, Azcoitia I, Garcia-Segura LM (2015) The neuroprotective actions of oestradiol and
oestrogen receptors. Nat Rev Neurosci 16(1):17–29
Baraka AM, Korish AA, Soliman GA, Kamal H (2011) The possible role of estrogen and selective
estrogen receptor modulators in a rat model of Parkinson’s disease. Life Sci 88(19–20):879–885
Bourque M, Morissette M, Di Paolo T (2019) Repurposing sex steroids and related drugs as
potential treatment for Parkinson’s disease. Neuropharmacol 147:37–54
Bourque M, Dluzen DE, Di Paolo T (2009) Neuroprotective actions of sex steroids in Parkinson’s
disease. Front Neuroendocrinol 30(2):142–157
Bäckström T, Sanders D, Leask R, Davidson D, Warner P, Bancroft J (1983) Mood, sexuality,
hormones, and the menstrual cycle: II. Hormone levels and their relationship to the premenstrual
syndrome. Psychosom Med. https://doi.org/10.1097/00006842-198312000-00004
Bäckström T, Wahlström G, Wahlström K, Zhu D, Wang MD (2005) Isoallopregnanolone; an
antagonist to the anaesthetic effect of allopregnanolone in male rats. Eur J Pharmacol 512
(1):15–21
Bäckström T, Haage D, Löfgren M, Johansson IM, Strömberg J, Nyberg S, Andreen L,
Ossewaarde L, Van Wingen GA, Turkmen S, Bengtsson SK (2011) Paradoxical effects of
GABA-A modulators may explain sex steroid induced negative mood symptoms in some
persons. Neuroscience 191:46–54
Badgaiyan DR (2011) Neurotransmitter imaging: basic concepts and future perspectives. Curr Med
Imaging Rev 7(2):98–103
Balashov AM (2010) Neurosteroids and addictive pathology. Zh Nevrol Psikhiatr Im S S
Korsakova 110(6):107
Balthazart J, Baillien M, Ball GF (2006) Rapid control of brain aromatase activity by glutamatergic
inputs. Endocrinology 147(1):359–366
Baulieu EE (1997) Neurosteroids: of the nervous system, by the nervous system, for the nervous
system. Recent Prog Horm Res 52:1–32
Baulieu EE (1998) Neurosteroids: a novel function of the brain. Psychoneuroendocrinology 23
(8):963–987
Baulieu EE, Robel P, Schumacher M (2001) Neurosteroids: beginning of the story. Int Rev
Neurobiol 46:1–32
Begemann MJ, Dekker CF, van Lunenburg M, Sommer IE (2012) Estrogen augmentation in
schizophrenia: a quantitative review of current evidence. Schizophr Res 141(2–3):179–184
Belelli D, Lambert JJ (2005) Neurosteroids: endogenous regulators of the GABA A receptor. Nat
Rev Neurosci 6(7):565
Belelli D, Herd MB, Mitchell EA, Peden DR, Vardy AW, Gentet L, Lambert JJ (2006) Neuroactive
steroids and inhibitory neurotransmission: mechanisms of action and physiological relevance.
Neuroscience 138(3):821–829
Belenichev IF, Odnokoz OV, Pavlov SV, Belenicheva OI, Polyakova EN (2012) The
neuroprotective activity of tamoxifen and tibolone during glutathione depletion in vitro.
Neurochem J 6(3):202–212
Benarroch EE (2007) Neurosteroids: endogenous modulators of neuronal excitability and plasticity.
Neurology 68(12):945–947
Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T, White HS (2013) Progress report on
new antiepileptic drugs: a summary of the Eleventh Eilat Conference (EILAT XI). Epilepsy Res
103(1):2–30
Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T, White HS (2015) Progress report on
new antiepileptic drugs: a summary of the Twelfth Eilat Conference (EILAT XII). Epilepsy Res
111:85–141. [Allopregnanolone (SAGE-547) injection, p. 91]
484 S. Marwein et al.

Bianchi MT, Macdonald RL (2002) Slow phases of GABAA receptor desensitization: structural
determinants and possible relevance for synaptic function. J Physiol 544(1):3–18
Bianchi MT, Macdonald RL (2003) Neurosteroids shift partial agonist activation of GABAA
receptor channels from low-to high-efficacy gating patterns. J Neurosci 23(34):10934–10943
Birzniece V, Bäckström T, Johansson IM, Lindblad C, Lundgren P, Löfgren M, Olsson T,
Ragagnin G, Taube M, Turkmen S, Wahlström G (2006) Neuroactive steroid effects on
cognitive functions with a focus on the serotonin and GABA systems. Brain Res Rev 51
(2):212–239
Bixo M (2014) Biomarkers for premenstrual dysphoric disorder. GABAA modulating steroid
antagonists—a possible treatment for premenstrual dysphoric disorder. In 44th Annual Meeting
of the International Society of Psychoneuroendocrinology, 19–22 August
Bologa CG, Revankar CM, Young SM, Edwards BS, Arterburn JB, Kiselyov AS, Parker MA,
Tkachenko SE, Savchuck NP, Sklar LA, Oprea TI (2006) Virtual and biomolecular screening
converge on a selective agonist for GPR30. Nat Chem Biol 2(4):207
Boonyaratanakornkit V (2011) Scaffolding proteins mediating membrane-initiated extra-nuclear
actions of estrogen receptor. Steroids 76(9):877–884
Boonyaratanakornkit V, McGowan E, Sherman L, Mancini MA, Cheskis BJ, Edwards DP (2007)
The role of extranuclear signaling actions of progesterone receptor in mediating progesterone
regulation of gene expression and the cell cycle. Mol Endocrinol 21(2):359–375
Boulware MI, Mermelstein PG (2009) Membrane estrogen receptors activate metabotropic gluta-
mate receptors to influence nervous system physiology. Steroids 74(7):608–613
Boulware MI, Weick JP, Becklund BR, Kuo SP, Groth RD, Mermelstein PG (2005) Estradiol
activates group I and II metabotropic glutamate receptor signaling, leading to opposing
influences on cAMP response element-binding protein. J Neurosci 25(20):5066–5078
Boulware MI, Heisler JD, Frick KM (2013) The memory-enhancing effects of hippocampal
estrogen receptor activation involve metabotropic glutamate receptor signaling. J Neurosci 33
(38):15184–15194
Brinton RD, Thompson RF, Foy MR, Baudry M, Wang J, Finch CE, Morgan TE, Pike CJ, Mack
WJ, Stanczyk FZ, Nilsen J (2008) Progesterone receptors: form and function in brain. Front
Neuroendocrinol 29(2):313–339
Brown N, Kerby J, Bonnert TP, Whiting PJ, Wafford KA (2002) Pharmacological characterization
of a novel cell line expressing human α4β3δ GABAA receptors. Br J Pharmacol 136
(7):965–974
Brown ES, Park J, Marx CE, Hynan LS, Gardner C, Davila D, Nakamura A, Sunderajan P, Lo A,
Holmes T (2014) A randomized, double-blind, placebo-controlled trial of pregnenolone for
bipolar depression. Neuropsychopharmacology 39(12):2867
Bu J, Zu H (2014) Effects of pregnenolone intervention on the cholinergic system and synaptic
protein 1 in aged rats. Int J Neurosci 124(2):117–124
Callier S, Morissette M, Grandbois M, Pélaprat D, Di Paolo T (2001) Neuroprotective properties of
17β-estradiol, progesterone, and raloxifene in MPTP C57Bl/6 mice. Synapse 41(2):131–138
Cai H, Zhou X, Dougherty GG, Reddy RD, Haas GL, Montrose DM, Keshavan M, Yao JK (2018)
Pregnenolone-progesterone-allopregnanolone pathway as a potential therapeutic target in first-
episode antipsychotic-naïve patients with schizophrenia. Psychoneuroendocrinology 90:43–51
Callachan H, Cottrell GA, Hather NY, Lambert JJ, Nooney JM, Peters JA (1987) Modulation of the
GABAA receptor by progesterone metabolites. Proc R Soc London Ser B Biol Sci 231
(1264):359–369
Calogeropoulou T, Avlonitis N, Minas V, Alexi X, Pantzou A, Charalampopoulos I, Zervou M,
Vergou V, Katsanou ES, Lazaridis I, Alexis MN (2009) Novel dehydroepiandrosterone
derivatives with antiapoptotic, neuroprotective activity. J Med Chem 52(21):6569–6587
Cardona-Gomez GP, DonCarlos L, Garcia-Segura LM (2000) Insulin-like growth factor I receptors
and estrogen receptors colocalize in female rat brain. Neuroscience 99(4):751–760
Carmeci C, Thompson DA, Ring HZ, Francke U, Weigel RJ (1997) Identification of a gene
(GPR30) with homology to the G-protein-coupled receptor superfamily associated with estro-
gen receptor expression in breast cancer. Genomics 45(3):607–617
14 Hormones and Steroids as Neurotransmitters 485

Carré JM, McCormick CM, Hariri AR (2011) The social neuroendocrinology of human aggression.
Psychoneuroendocrinology 36(7):935–944
Carrer HF, Araque A, Buño W (2003) Estradiol regulates the slow Ca2+-activated K+ current in
hippocampal pyramidal neurons. J Neurosci 23(15):6338–6344
Carswell HVO, Dominiczak AF, Macrae IM (2000) Estrogen status affects sensitivity to focal
cerebral ischemia in stroke-prone spontaneously hypertensive rats. Am J Physiol Heart Circ
Physiol 278(1):H290–H294
Carver CM, Reddy DS (2013) Neurosteroid interactions with synaptic and extrasynaptic GABA A
receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network
excitability. Psychopharmacology (Berl) 230(2):151–188
Cato AC, Nestl A, Mink S (2002) Rapid actions of steroid receptors in cellular signaling pathways.
Sci STKE 2002(138):re9
Chaban VV, Lakhter AJ, Micevych P (2004) A membrane estrogen receptor mediates intracellular
calcium release in astrocytes. Endocrinology 145(8):3788–3795
Charalampopoulos I, Remboutsika E, Margioris AN, Gravanis A (2008) Neurosteroids as
modulators of neurogenesis and neuronal survival. Trends Endocrinol Metab 19(8):300–307
Chen C, Kuo J, Wong A, Micevych P (2014) Estradiol modulates translocator protein (TSPO) and
steroid acute regulatory protein (StAR) via protein kinase A (PKA) signaling in hypothalamic
astrocytes. Endocrinology 155(8):2976–2985
Chen J, Hu R, Ge H, Duanmu W, Li Y, Xue X, Hu S, Feng H (2015) G-protein-coupled receptor
30-mediated antiapoptotic effect of estrogen on spinal motor neurons following injury and its
underlying mechanisms. Mol Med Rep 12(2):1733–1740
Cheng J, Watkins SC, Walker WH (2007) Testosterone activates mitogen-activated protein kinase
via Src kinase and the epidermal growth factor receptor in sertoli cells. Endocrinology 148
(5):2066–2074
Cheng ZX, Lan DM, Wu PY, Zhu YH, Dong Y, Ma L, Zheng P (2008) Neurosteroid dehydroepi-
androsterone sulphate inhibits persistent sodium currents in rat medial prefrontal cortex via
activation of sigma-1 receptors. Exp Neurol 210(1):128–136
Cheskis BJ (2004) Regulation of cell signalling cascades by steroid hormones. J Cell Biochem 93
(1):20–27
Chlebowski RT, Kuller LH, Prentice RL, Stefanick ML, Manson JE, Gass M, Aragaki AK, Ockene
JK, Lane DS, Sarto GE, Rajkovic A (2009) Breast cancer after use of estrogen plus progestin in
postmenopausal women. N Engl J Med 360(6):573–587
Ciriza I, Carrero P, Frye CA, Garcia-Segura LM (2006) Reduced metabolites mediate
neuroprotective effects of progesterone in the adult rat hippocampus. The synthetic progestin
medroxyprogesterone acetate (Provera) is not neuroprotective. J Neurobiol 66(9):916–928
Coleman KM, Smith CL (2001) Intracellular signaling pathways: nongenomic actions of estrogens
and ligand-independent activation of estrogen receptors. Front Biosci 6(1):D1379–D1391
Corcoran C, Walker E, Huot R, Mittal V, Tessner K, Kestler L, Malaspina D (2003) The stress
cascade and schizophrenia: etiology and onset. Schizophr Bull 29(4):671–692
Corpechot C, Robel P, Axelson M, Sjövall J, Baulieu EE (1981) Characterization and measurement
of dehydroepiandrosterone sulfate in rat brain. Proc Natl Acad Sci 78(8):4704–4707
Costa ET, Soto EE, Cardoso RA, Olivera DS, Valenzuela CF (2000) Acute effects of ethanol on
kainate receptors in cultured hippocampal neurons. Alcohol Clin Exp Res 24(2):220–225
Covey D, Robichaud AJ (2014) Washington University in St Louis and Sage Therapeutics Inc,
2014. Neuroactive 19-alkoxy-17-substituted steroids, prodrugs thereof, and methods of treat-
ment using same. U.S. Patent 20140235600
Covey DF, Evers AS, Mennerick S, Zorumski CF, Purdy RH (2001) Recent developments in
structure–activity relationships for steroid modulators of GABAA receptors. Brain Res Rev 37
(1–3):91–97
Cummings SR, Ettinger B, Delmas PD, Kenemans P, Stathopoulos V, Verweij P, Mol-Arts M,
Kloosterboer L, Mosca L, Christiansen C, Bilezikian J (2008) The effects of tibolone in older
postmenopausal women. N Engl J Med 359(7):697–708
486 S. Marwein et al.

Cyr M, Ghribi O, Thibault C, Morissette M, Landry M, Di Paolo T (2001) Ovarian steroids and
selective estrogen receptor modulators activity on rat brain NMDA and AMPA receptors. Brain
Res Rev 37(1–3):153–161
Darbandi-Tonkabon R, Manion BD, Hastings WR, Craigen WJ, Akk G, Bracamontes JR, He Y,
Sheiko TV, Steinbach JH, Mennerick SJ, Covey DF (2004) Neuroactive steroid interactions
with voltage-dependent anion channels: lack of relationship to GABAA receptor modulation
and anesthesia. J Pharmacol Exp Ther 308(2):502–511
Darnaudéry M, Pallarès M, Piazza PV, Le Moal M, Mayo W (2002) The neurosteroid pregnenolone
sulfate infused into the medial septum nucleus increases hippocampal acetylcholine and spatial
memory in rats. Brain Res 951(2):237–242
Dastgheib MONA, Dehpour AR, Heidari M, Moezi L (2015) The effects of intra-dorsal hippocam-
pus infusion of pregnenolone sulfate on memory function and hippocampal BDNF mRNA
expression of biliary cirrhosis-induced memory impairment in rats. Neuroscience 306:1–9
De Nicola AF, Gonzalez SL, Labombarda F, Deniselle MCG, Garay L, Guennoun R, Schumacher
M (2006) Progesterone treatment of spinal cord injury. J Mol Neurosci 28(1):3–15
Deutsch ER, Espinoza TR, Atif F, Woodall E, Kaylor J, Wright DW (2013) Progesterone’s role in
neuroprotection, a review of the evidence. Brain Res 1530:82–105
Dewing P, Boulware MI, Sinchak K, Christensen A, Mermelstein PG, Micevych P (2007) Mem-
brane estrogen receptor-α interactions with metabotropic glutamate receptor 1a modulate female
sexual receptivity in rats. J Neurosci 27(35):9294–9300
Di S, Itoga CA, Fisher MO, Solomonow J, Roltsch EA, Gilpin NW, Tasker JG (2016) Acute stress
suppresses synaptic inhibition and increases anxiety via endocannabinoid release in the
basolateral amygdala. J Neurosci 36(32):8461–8470
Diamanti-Kandarakis E, Dattilo M, Macut D, Duntas L, Gonos ES, Goulis DG, Gantenbein CK,
Kapetanou M, Koukkou E, Lambrinoudaki I, Michalaki M (2017) Mechanisms in endocrinol-
ogy: aging and anti-aging: a combo-endocrinology overview. Eur J Endocrinol 176(6):R283–
R308
Djebaili M, Guo Q, Pettus EH, Hoffman SW, Stein DG (2005) The neurosteroids progesterone and
allopregnanolone reduce cell death, gliosis, and functional deficits after traumatic brain injury in
rats. J Neurotrauma 22(1):106–118
Do Rego JL, Seong JY, Burel D, Leprince J, Luu-The V, Tsutsui K, Tonon MC, Pelletier G, Vaudry
H (2009) Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by
neurotransmitters and neuropeptides. Front Neuroendocrinol 30(3):259–301
Dong Y, Zheng P (2012) Dehydroepiandrosterone sulphate: action and mechanism in the brain. J
Neuroendocrinol 24(1):215–224
Dong LY, Cheng ZX, Fu YM, Wang ZM, Zhu YH, Sun JL, Dong Y, Zheng P (2007) Neurosteroid
dehydroepiandrosterone sulfate enhances spontaneous glutamate release in rat prelimbic cortex
through activation of dopamine D1 and sigma-1 receptor. Neuropharmacology 52(3):966–974
Dubrovsky BO (2005) Steroids, neuroactive steroids and neurosteroids in psychopathology. Progr
Neuropsychopharmacol Biol Psychiatry 29(2):169–192
Dubrovsky B (2006) Neurosteroids, neuroactive steroids, and symptoms of affective disorders.
Pharmacol Biochem Behav 84(4):644–655
Ebinger M, Sievers C, Ivan D, Schneider HJ, Stalla GK (2009) Is there a neuroendocrinological
rationale for testosterone as a therapeutic option in depression? J Psychopharmacol 23
(7):841–853
Ellenbogen A, Raines S, Kanes S (2016) Exploratory trial results for SAGE-547 in essential tremor
(P4. 297)
Ellsworth KP, Azzolina BA, Cimis G, Bull HG, Harris GS (1998) Cloning, expression and
characterization of rhesus macaque types 1 and 2 5alpha-reductase: evidence for mechanism-
based inhibition by finasteride. J Steroid Biochem Mol Biol 66(5–6):271–279
Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240
(4854):889–895
14 Hormones and Steroids as Neurotransmitters 487

Farfán-García ED, Castillo-Hernández MC, Pinto-Almazán R, Rivas-Arancibia S, Gallardo JM,

Guerra-Araiza C (2014) Tibolone prevents oxidation and ameliorates cholinergic deficit induced
by ozone exposure in the male rat hippocampus. Neurochem Res 39(9):1776–1786
Fatehi M, Fatehi-Hassanabad Z (2008) Effects of 17β-estradiol on neuronal cell excitability and
neurotransmission in the suprachiasmatic nucleus of rat. Neuropsychopharmacology 33(6):1354
Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr (2000) Estrogen-induced activation of Erk-1 and
Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation
of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 14
(10):1649–1660
Fix C, Jordan C, Cano P, Walker WH (2004) Testosterone activates mitogen-activated protein
kinase and the cAMP response element binding protein transcription factor in Sertoli cells. Proc
Natl Acad Sci 101(30):10919–10924
Foradori CD, Weiser MJ, Handa RJ (2008) Non-genomic actions of androgens. Front
Neuroendocrinol 29(2):169–181
Fortress AM, Frick KM (2014) Epigenetic regulation of estrogen-dependent memory. Front
Neuroendocrinol 35(4):530–549
French JA, Krauss GL, Biton V, Squillacote D, Yang H, Laurenza A, Kumar D, Rogawski MA
(2012) Adjunctive perampanel for refractory partial-onset seizures: randomized phase III study
304. Neurology 79(6):589–596
Frick KM, Kim J, Tuscher JJ, Fortress AM (2015) Sex steroid hormones matter for learning and
memory: estrogenic regulation of hippocampal function in male and female rodents. Learn Mem
22(9):472–493
Frye CA (2001) The role of neurosteroids and non-genomic effects of progestins and androgens in
mediating sexual receptivity of rodents. Brain Res Rev 37(1–3):201–222
Frye CA (2011) Progesterone attenuates depressive behavior of younger and older adult C57/BL6,
wildtype, and progesterone receptor knockout mice. Pharmacol Biochem Behav 99(4):525–531
Frye CA, Lacey EH (2000) Progestins influence performance on cognitive tasks independent of
changes in affective behavior. Psychobiology 28(4):550–563
Frye CA, Sora I (2010) Progesterone reduces hyperactivity of female and male dopamine trans-
porter knockout mice. Behav Brain Res 209(1):59–65
Frye CA, Walf AA (2008) Progesterone enhances performance of aged mice in cortical or
hippocampal tasks. Neurosci Lett 437(2):116–120
Frye CA, Walf AA (2010) Progesterone enhances learning and memory of aged wildtype and
progestin receptor knockout mice. Neurosci Lett 472(1):38–42
Fu XD, Simoncini T (2008) Extra-nuclear signaling of estrogen receptors. IUBMB Life 60
(8):502–510
Fugger HN, Foster TC, Gustafsson JÅ, Rissman EF (2000) Novel effects of estradiol and estrogen
receptor α and β on cognitive function. Brain Res 883(2):258–264
Fujiwara M, Egashira N (2004) New perspectives in the studies on endocannabinoid and cannabis:
abnormal behaviors associate with CB1 cannabinoid receptor and development of therapeutic
application. J Pharmacol Sci. https://doi.org/10.1254/jphs.fmj04003x2. 0412090007-
0412090007
Grandbois M, Morissette M, Callier S, Di Paolo T (2000) Ovarian steroids and raloxifene prevent
MPTP-induced dopamine depletion in mice. Neuroreport 11(2):343–346
Galea LA, Wainwright SR, Roes MM, Duarte-Guterman P, Chow C, Hamson DK (2013) Sex,
hormones and neurogenesis in the hippocampus: hormonal modulation of neurogenesis and
potential functional implications. J Neuroendocrinol 25(11):1039–1061
Gardner EL (2005) Endocannabinoid signaling system and brain reward: emphasis on dopamine.
Pharmacol Biochem Behav 81(2):263–284
Gasior M, Ungard JT, Beekman M, Carter RB, Witkin JM (2000) Acute and chronic effects of the
synthetic neuroactive steroid, ganaxolone, against the convulsive and lethal effects of pentyl-
enetetrazol in seizure-kindled mice: comparison with diazepam and valproate. Neuropharma-
cology 39(7):1184–1196
488 S. Marwein et al.

Genazzani AR, Stomati M, Morittu A, Bernardi F, Monteleone P, Casarosa E, Gallo R,

Salvestroni C, Luisi M (2000) Progesterone, progestagens and the central nervous system.
Hum Reprod 15(Suppl_1):14–27
Giatti S, Garcia-Segura LM, Melcangi RC (2015) New steps forward in the neuroactive steroid
field. J Steroid Biochem Mol Biol 153:127–134
Giatti S, Melcangi RC, Pesaresi M (2016) The other side of progestins: effects in the brain. J Mol
Endocrinol 57(2):R109–R126
Giatti S, Garcia-Segura LM, Barreto GE, Melcangi RC (2019) Neuroactive steroids,
neurosteroidogenesis and sex. Prog Neurobiol. https://doi.org/10.1016/j.pneurobio.2018.06.007
Gibbs TT, Russek SJ, Farb DH (2006) Sulfated steroids as endogenous neuromodulators.
Pharmacol Biochem Behav 84(4):555–567
Gibson CL, Gray LJ, Bath PM, Murphy SP (2007) Progesterone for the treatment of experimental
brain injury; a systematic review. Brain 131(2):318–328
Girdler SS, Lindgren M, Porcu P, Rubinow DR, Johnson JL, Morrow AL (2012) A history of
depression in women is associated with an altered GABAergic neuroactive steroid profile.
Psychoneuroendocrinology 37(4):543–553
Gorczynska E, Handelsman DJ (1995) Androgens rapidly increase the cytosolic calcium concen-
tration in Sertoli cells. Endocrinology 136(5):2052–2059
Grassi S, Frondaroli A, Dieni C, Dutia MB, Pettorossi VE (2007) Neurosteroid modulation of
neuronal excitability and synaptic transmission in the rat medial vestibular nuclei. Eur J
Neurosci 26(1):23–32
Gu Q, Korach KS, Moss RL (1999) Rapid action of 17β-estradiol on kainate-induced currents in
hippocampal neurons lacking intracellular estrogen receptors. Endocrinology 140(2):660–666
Guennoun R, Meffre D, Labombarda F, Gonzalez SL, Deniselle MG, Stein DG, De Nicola AF,
Schumacher M (2008) The membrane-associated progesterone-binding protein 25-Dx: expres-
sion, cellular localization and up-regulation after brain and spinal cord injuries. Brain Res Rev
57(2):493–505
Guennoun R, Labombarda F, Deniselle MG, Liere P, De Nicola AF, Schumacher M (2015)
Progesterone and allopregnanolone in the central nervous system: response to injury and
implication for neuroprotection. J Steroid Biochem Mol Biol 146:48–61
Guerra-Araiza C, Villamar-Cruz O, Gonzalez-Arenas A, Chavira R, Camacho-Arroyo I (2003)
Changes in progesterone receptor isoforms content in the rat brain during the oestrous cycle and
after oestradiol and progesterone treatments. J Neuroendocrinol 15(10):984–990
Guzman CB, Zhao C, Deighton-Collins S, Kleerekoper M, Benjamins JA, Skafar DF (2007)
Agonist activity of the 3-hydroxy metabolites of tibolone through the oestrogen receptor in
the mouse N20. 1 oligodendrocyte cell line and normal human astrocytes. J Neuroendocrinol 19
(12):958–965
Handa RJ, Pak TR, Kudwa AE, Lund TD, Hinds L (2008) An alternate pathway for androgen
regulation of brain function: Activation of estrogen receptor beta by the metabolite of dihydro-
testosterone, 5α-androstane-3β, 17β-diol. Horm Behav 53(5):741–752
Haage D, Johansson S (1999) Neurosteroid modulation of synaptic and GABA-evoked currents in
neurons from the rat medial preoptic nucleus. J Neurophysiol 82(1):143–151
Hammond RS, Althaus AL, Ackley MA, Maciag C, Botella GM, Salituro FG, Robichaud AJ,
Doherty JJ (2017) Anticonvulsant profile of the neuroactive steroid, SGE-516, in animal
models. Epilepsy Res 134:16–25
Hawkins NA, Lewis M, Hammond RS, Doherty JJ, Kearney JA (2017) The synthetic neuroactive
steroid SGE-516 reduces seizure burden and improves survival in a Dravet syndrome mouse
model. Sci Rep 7(1):15327
Hayden-Hixson DM, Ferris CF (1991) Steroid-specific regulation of agonistic responding in the
anterior hypothalamus of male hamsters. Physiol Behav 50(4):793–799
Hedges VL, Staffend NA, Meisel RL (2010) Neural mechanisms of reproduction in females as a
predisposing factor for drug addiction. Front Neuroendocrinol 31(2):217–231
14 Hormones and Steroids as Neurotransmitters 489

Herrick SP, Waters EM, Drake CT, McEwen BS, Milner TA (2006) Extranuclear estrogen receptor
beta immunoreactivity is on doublecortin-containing cells in the adult and neonatal rat dentate
gyrus. Brain Res 1121(1):46–58
Higo S, Hojo Y, Ishii H, Komatsuzaki Y, Ooishi Y, Murakami G, Mukai H, Yamazaki T,
Nakahara D, Barron A, Kimoto T (2011) Endogenous synthesis of corticosteroids in the
hippocampus. PLoS One 6(7):e21631
Hoffmann E, Wald J, Raines S, Nomikos G, Colquhoun H, Kanes S (2017) The pharmacokinetics
of SAGE-217 in Phase 1 SAD and MAD studies (P3. 008)
Hogenkamp DJ (2014) Novel 17b-heteroaryl-substituted steroids as modulators of gabaa receptors.
U.S. Patent Application 14/166,912
Horishita T, Minami K, Uezono Y, Shiraishi M, Ogata J, Okamoto T, Terada T, Sata T (2005) The
effects of the neurosteroids: pregnenolone, progesterone and dehydroepiandrosterone on mus-
carinic receptor-induced responses in Xenopus oocytes expressing M 1 and M 3 receptors.
Naunyn Schmiedebergs Arch Pharmacol 371(3):221–228
Hu R, Sun H, Zhang Q, Chen J, Wu N, Meng H, Cui G, Hu S, Li F, Lin J, Wan Q (2012) G-protein
coupled estrogen receptor 1 mediated estrogenic neuroprotection against spinal cord injury. Crit
Care Med 40(12):3230–3237
Hu H, Zhou Y, Leng T, Liu A, Wang Y, You X, Chen J, Tang L, Chen W, Qiu P, Yin W (2014) The
major cholesterol metabolite cholestane-3β, 5α, 6β-triol functions as an endogenous
neuroprotectant. J Neurosci 34(34):11426–11438
Hubbard DB, Miller BJ (2019) Meta-analysis of blood cortisol levels in individuals with first-
episode psychosis. Psychoneuroendocrinology. https://doi.org/10.1016/j.psyneuen.2019.03.
014
Huss B, Long NP, Ponce L, Gomez L, Moore CA, Sinchak K (2011) Progesterone rapidly facilitates
sexual receptivity through deactivation of muopioid receptors in the medial preoptic nucleus of
the hypothalamus. In: Society for Neuroscience, Program 391.307, Abstract Viewer/Itinerary
Planner, Washington, DC
Jentsch VL, Merz CJ, Wolf OT (2019) Restoring emotional stability: cortisol effects on the neural
network of cognitive emotion regulation. Behav Brain Res 374:111880
Jiang CS, Guo XJ, Gong JX, Zhu TT, Zhang HY, Guo YW (2012) Synthesis and biological
evaluation of 21-arylidenepregnenolone derivatives as neuroprotective agents. Bioorg Med
Chem Lett 22(6):2226–2229
Johansson IM, Birzniece V, Lindblad C, Olsson T, Bäckström T (2002) Allopregnanolone inhibits
learning in the Morris water maze. Brain Res 934(2):125–131
Johansson M, Agusti A, Llansola M, Montoliu C, Strömberg J, Malinina E, Ragagnin G,
Doverskog M, Bäckström T, Felipo V (2015) GR3027 antagonizes GABAA receptor-
potentiating neurosteroids and restores spatial learning and motor coordination in rats with
chronic hyperammonemia and hepatic encephalopathy. Am J Physiol Gastrointest Liver Physiol
309(5):G400–G409
Johansson M, Strömberg J, Ragagnin G, Doverskog M, Bäckström T (2016) GABAA receptor
modulating steroid antagonists (GAMSA) are functional in vivo. J Steroid Biochem Mol Biol
160:98–105
Joksovic PM, Covey DF, Todorovic SM (2007) Inhibition of T-type calcium current in the reticular
thalamic nucleus by a novel neuroactive steroid. Ann N Y Acad Sci 1122(1):83–94
Jensen EV (1962) On the mechanism of estrogen action. Perspec Biol Med 6(1):47–60
Kelley SP, Alan JK, O’Buckley TK, Mennerick S, Krishnan K, Covey DF, Morrow AL (2007)
Antagonism of neurosteroid modulation of native γ-aminobutyric acid receptors by (3α, 5α)-17-
phenylandrost-16-en-3-ol. Eur J Pharmacol 572(2–3):94–101
Kaminska M, Harris J, Gijsbers K, Dubrovsky B (2000) Dehydroepiandrosterone sulfate (DHEAS)
counteracts decremental effects of corticosterone on dentate gyrus LTP. Implications for
depression. Brain Res Bull 52(3):229–234
490 S. Marwein et al.

Kaminski RM, Livingood MR, Rogawski MA (2004) Allopregnanolone analogs that positively
modulate GABAA receptors protect against partial seizures induced by 6-Hz electrical stimula-
tion in mice. Epilepsia 45(7):864–867
Kanes S, Colquhoun H, Gunduz-Bruce H, Raines S, Arnold R, Schacterle A, Doherty J, Epperson
CN, Deligiannidis KM, Riesenberg R, Hoffmann E (2017a) Brexanolone (SAGE-547 injection)
in post-partum depression: a randomised controlled trial. Lancet 390(10093):480–489
Kanes SJ, Colquhoun H, Doherty J, Raines S, Hoffmann E, Rubinow DR, Meltzer-Brody S (2017b)
Open-label, proof-of-concept study of brexanolone in the treatment of severe postpartum
depression. Human Psychopharmacol 32(2):e2576
Kapras V, Slavickova A, Stastna E, Vyklicky L Jr, Vales K, Chodounska H (2012) Synthesis of
deuterium labeled NMDA receptor inhibitor–20-Oxo-5β-[9, 12, 12-2H3] pregnan-3α-yl-l-
glutamyl 1-ester. Steroids 77(3):282–287
Karki P, Webb A, Zerguine A, Choi J, Son DS, Lee E (2014) Mechanism of raloxifene-induced
upregulation of glutamate transporters in rat primary astrocytes. Glia 62(8):1270–1283
Kelly MJ, Moss RL, Dudley CA (1976) Differential sensitivity of preoptic-septal neurons to
microelectrophoressed estrogen during the estrous cycle. Brain Res 114(1):152–157
Kelly MJ, Rønnekleiv OK, Ibrahim N, Lagrange AH, Wagner EJ (2002) Estrogen modulation of K
+ channel activity in hypothalamic neurons involved in the control of the reproductive axis.
Steroids 67(6):447–456
Kinlein SA, Phillips DJ, Keller CR, Karatsoreos IN (2019) Role of corticosterone in altered
neurobehavioral responses to acute stress in a model of compromised hypothalamic-pituitary-
adrenal axis function. Psychoneuroendocrinology 102:248–255
Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB,
Willson TM, Zetterström RH, Perlmann T (1998) An orphan nuclear receptor activated by
pregnanes defines a novel steroid signaling pathway. Cell 92(1):73–82
Kloosterboer HJ (2004) Tissue-selectivity: the mechanism of action of tibolone. Maturitas
48:30–40
Koenig JI, Kirkpatrick B, Lee P (2002) Glucocorticoid hormones and early brain development in
schizophrenia. Neuropsychopharmacology 27(2):309
Kudwa AE, Michopoulos V, Gatewood JD, Rissman EF (2006) Roles of estrogen receptors α and β
in differentiation of mouse sexual behavior. Neuroscience 138(3):921–928
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996) Cloning of a novel
receptor expressed in rat prostate and ovary. Proc Natl Acad Sci 93(12):5925–5930
Kulkarni J, Riedel A, De Castella AR, Fitzgerald PB, Rolfe TJ, Taffe J, Burger H (2001)
Estrogen—a potential treatment for schizophrenia. Schizophr Res 48(1):137–144
Kulkarni J, de Castella A, Fitzgerald PB, Gurvich CT, Bailey M, Bartholomeusz C, Burger H
(2008a) Estrogen in severe mental illness: a potential new treatment approach. Arch Gen
Psychiatry 65(8):955–960
Kulkarni J, Gurvich C, Gilbert H, Mehmedbegovic F, Mu L, Marston N, Gavrilidis E, de Castella A
(2008b) Hormone modulation: a novel therapeutic approach for women with severe mental
illness. Aust N Z J Psychiatry 42(1):83–88
Kulkarni J, Gurvich C, Lee SJ, Gilbert H, Gavrilidis E, de Castella A, Berk M, Dodd S, Fitzgerald
PB, Davis SR (2010) Piloting the effective therapeutic dose of adjunctive selective estrogen
receptor modulator treatment in postmenopausal women with schizophrenia.
Psychoneuroendocrinology 35(8):1142–1147
Kulkarni J, de Castella A, Headey B, Marston N, Sinclair K, Lee S, Gurvich C, Fitzgerald PB,
Burger H (2011) Estrogens and men with schizophrenia: is there a case for adjunctive therapy?
Schizophr Res 125(2–3):278–283
Kulkarni J, Gavrilidis E, Wang W, Worsley R, Fitzgerald PB, Gurvich C, Van Rheenen T, Berk M,
Burger H (2015) Estradiol for treatment-resistant schizophrenia: a large-scale randomized-
controlled trial in women of child-bearing age. Mol Psychiatry 20(6):695–702
Kuo J, Hamid N, Bondar G, Prossnitz ER, Micevych P (2010) Membrane estrogen receptors
stimulate intracellular calcium release and progesterone synthesis in hypothalamic astrocytes.
J Neurosci 30(39):12950–12957
14 Hormones and Steroids as Neurotransmitters 491

Kushida A, Tamura H (2009) Retinoic acids induce neurosteroid biosynthesis in human glial GI-1
Cells via the induction of steroidogenic genes. J Biochem 146(6):917–923
Krȩżel W, Dupont S, Krust A, Chambon P, Chapman PF (2001) Increased anxiety and synaptic
plasticity in estrogen receptor β-deficient mice. Proc Natl Acad Sci 98(21):12278–12282
Löscher W, Puskarjov M, Kaila K (2013) Cation-chloride cotransporters NKCC1 and KCC2 as
potential targets for novel antiepileptic and antiepileptogenic treatments. Neuropharmacol
69:62–74
Labombarda F, Garcia-Ovejero D (2014) Give progesterone a chance. Neural Regen Res 9
(15):1422
Labombarda F, Gonzalez SL, Deniselle MG, Vinson GP, Schumacher M, De Nicola AF, Guennoun
R (2003) Effects of injury and progesterone treatment on progesterone receptor and progester-
one binding protein 25-Dx expression in the rat spinal cord. J Neurochem 87(4):902–913
Labombarda F, Meffre D, Delespierre B, Krivokapic-Blondiaux S, Chastre A, Thomas P, Pang Y,
Lydon JP, Gonzalez SL, De Nicola AF, Schumacher M (2010) Membrane progesterone
receptors localization in the mouse spinal cord. Neuroscience 166(1):94–106
Laconi MR, Reggiani PC, Penissi A, Yunes R, Cabrera RJ (2007) Allopregnanolone modulates
striatal dopamingergic activity of rats under different gonadal hormones conditions. Neurol Res
29(6):622–627
Lambert JJ, Belelli D, Hill-Venning C, Peters JA (1995) Neurosteroids and GABAA receptor
function. Trends Pharmacol Sci 16(9):295–303
Landgren S, Aasly J, Bäckström T, Dubrovsky B, Danielsson E (1987) The effect of progesterone
and its metabolites on the interictal epileptiform discharge in the cat’s cerebral cortex. Acta
Physiol Scand 131(1):33–42
Lapchak PA, Araujo DM (2001) Preclinical development of neurosteroids as neuroprotective
agents for the treatment of neurodegenerative diseases. Int Rev Neurobiol 46:379–397
Large CH, Sokal DM, Nehlig A, Gunthorpe MJ, Sankar R, Crean CS, VanLandingham KE, White
HS (2012) The spectrum of anticonvulsant efficacy of retigabine (ezogabine) in animal models:
implications for clinical use. Epilepsia 53(3):425–436
Le Mellédo JM, Baker GB (2002) Neuroactive steroids, and anxiety disorders. J Psychiatry
Neurosci 27(3):161–165
Lebesgue D, Traub M, De Butte-Smith M, Chen C, Zukin RS, Kelly MJ, Etgen AM (2010) Acute
administration of non-classical estrogen receptor agonists attenuates ischemia-induced hippo-
campal neuron loss in middle-aged female rats. PLoS One 5(1):e8642
Leonhardt SA, Boonyaratanakornkit V, Edwards DP (2003) Progesterone receptor transcription and
non-transcription signaling mechanisms. Steroids 68(10–13):761–770
Leranth C, Hajszan T, MacLusky NJ (2004) Androgens increase spine synapse density in the CA1
hippocampal subfield of ovariectomized female rats. J Neurosci 24(2):495–499
Li Y, Raaby KF, Sanchez C, Gulinello M (2013) Serotonergic receptor mechanisms underlying
antidepressant-like action in the progesterone withdrawal model of hormonally induced depres-
sion in rats. Behav Brain Res 256:520–528
Litim N, Bourque M, Al Sweidi S, Morissette M, Di Paolo T (2015) The 5α-reductase inhibitor
Dutasteride but not Finasteride protects dopamine neurons in the MPTP mouse model of
Parkinson’s disease. Neuropharmacology 97:86–94
Liu SB, Zhang N, Guo YY, Zhao R, Shi TY, Feng SF, Wang SQ, Yang Q, Li XQ, Wu YM, Ma L
(2012) G-protein-coupled receptor 30 mediates rapid neuroprotective effects of estrogen via
depression of NR2B-containing NMDA receptors. J Neurosci 32(14):4887–4900
Long L, Xu L, Xiao Z, Hu S, Luo R, Wang H, Lu X, Xu Z, Yao X, Zhou L, Long H (2016)
Neurological complications and risk factors of cardiopulmonary failure of EV-A71-related
hand, foot and mouth disease. Sci Rep 6:23444
Lösel R, Breiter S, Seyfert M, Wehling M, Falkenstein E (2005) Classic and non-classic progester-
one receptors are both expressed in human spermatozoa. Horm Metab Res 37(01):10–14
492 S. Marwein et al.

Lundgren P, Strömberg J, Bäckström T, Wang M (2003) Allopregnanolone-stimulated GABA-

mediated chloride ion flux is inhibited by 3β-hydroxy-5α-pregnan-20-one
(isoallopregnanolone). Brain Res 982(1):45–53
Micevych PE, May Wong A, Mittelman-Smith MA (2011) Estradiol membrane-initiated signaling
and female reproduction. Compr Physiol 5(3):1211–1222
MacKenzie SM, Clark CJ, Ingram MC, Lai M, Seckl J, Gomez-Sanchez CE, Fraser R, Connell
JMC, Davies E (2000) Corticosteroid production by fetal rat hippocampal neurons. Endocr Res
26(4):531–535
Maurice T, Grégoire C, Espallergues J (2006) Neuro (active) steroids actions at the
neuromodulatory sigma1 (σ1) receptor: Biochemical and physiological evidence, consequences
in neuroprotection. Pharmacol Biochem Behav 84(4):581–597
Marx CE, Bradford DW, Hamer RM, Naylor JC, Allen TB, Lieberman JA, Strauss JL, Kilts JD
(2011) Pregnenolone as a novel therapeutic candidate in schizophrenia: Emerging preclinical
and clinical evidence. Neurosci 191:78–90
Martinez Botella G, Salituro FG, Harrison BL, Beresis RT, Bai Z, Blanco MJ, Belfort GM, Dai J,
Loya CM, Ackley MA, Althaus AL (2017) Neuroactive steroids. 2. 3α-Hydroxy-3β-methyl-21-
(4-cyano-1 H-pyrazol-10 -yl)-19-nor-5β-pregnan-20-one (SAGE-217): A clinical next-
generation neuroactive steroid positive allosteric modulator of the (γ-aminobutyric acid) A
receptor. J Med Chem 60(18):7810–7819
MacKenzie G, Maguire J (2013) Neurosteroids and GABAergic signaling in health and disease.
Biomol Concepts 4(1):29–42
MacKenzie EM, Odontiadis J, Le Mellédo JM, Prior TI, Baker GB (2007) The relevance of
neuroactive steroids in schizophrenia, depression, and anxiety disorders. Cell Mol Neurobiol
27(5):541–574
Maeng LY, Milad MR (2015) Sex differences in anxiety disorders: interactions between fear, stress,
and gonadal hormones. Horm Behav 76:106–117
Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM (1986) Steroid hormone
metabolites are barbiturate-like modulators of the GABA receptor. Science 232
(4753):1004–1007
Malcher-Lopes R, Di S, Marcheselli VS, Weng FJ, Stuart CT, Bazan NG, Tasker JG (2006)
Opposing crosstalk between leptin and glucocorticoids rapidly modulates synaptic excitation
via endocannabinoid release. J Neurosci 26(24):6643–6650
Malik AS, Narayan RK, Wendling WW, Cole RW, Pashko LL, Schwartz AG, Strauss KI (2003) A
novel dehydroepiandrosterone analog improves functional recovery in a rat traumatic brain
injury model. J Neurotrauma 20(5):463–476
Mameli M, Carta M, Partridge LD, Valenzuela CF (2005) Neurosteroid-induced plasticity of
immature synapses via retrograde modulation of presynaptic NMDA receptors. J Neurosci 25
(9):2285–2294
Mani S, Oyola MG (2012) Progesterone signaling mechanisms in brain and behavior. Front
Endocrinol 3:7
Mani SK, Blaustein JD, Allen JM, Law SW, O’Malley BW, Clark JH (1994) Inhibition of rat sexual
behavior by antisense oligonucleotides to the progesterone receptor. Endocrinology 135
(4):1409–1414
Maninger N, Wolkowitz OM, Reus VI, Epel ES, Mellon SH (2009) Neurobiological and neuropsy-
chiatric effects of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Front
Neuroendocrinol 30(1):65–91
Martinez Botella G, Salituro FG, Harrison BL, Beresis RT, Bai Z, Shen K, Belfort GM, Loya CM,
Ackley MA, Grossman SJ, Hoffmann E (2015) Neuroactive steroids. 1. Positive allosteric
modulators of the (γ-aminobutyric Acid) A receptor: structure–activity relationships of hetero-
cyclic substitution at C-21. J Med Chem 58(8):3500–3511
Martı́nez-Mota L, Fernández-Guasti A (2004) Testosterone-dependent antidepressant-like effect of
noradrenergic but not of serotonergic drugs. Pharmacol Biochem Behav 78(4):711–718
Marx CE, Stevens RD, Shampine LJ, Uzunova V, Trost WT, Butterfield MI, Massing MW, Hamer
RM, Morrow AL, Lieberman JA (2006) Neuroactive steroids are altered in schizophrenia and
14 Hormones and Steroids as Neurotransmitters 493

bipolar disorder: relevance to pathophysiology and therapeutics. Neuropsychopharmacology 31

(6):1249
Marx CE, Keefe RS, Buchanan RW, Hamer RM, Kilts JD, Bradford DW, Strauss JL, Naylor JC,
Payne VM, Lieberman JA, Savitz AJ (2009) Proof-of-concept trial with the neurosteroid
pregnenolone targeting cognitive and negative symptoms in schizophrenia.
Neuropsychopharmacology 34(8):1885
McCullough LD, Blizzard K, Simpson ER, Öz OK, Hurn PD (2003) Aromatase cytochrome P450
and extragonadal estrogen play a role in ischemic neuroprotection. J Neurosci 23
(25):8701–8705
McEwen BS (1991) Non-genomic and genomic effects of steroids on neural activity. Trends
Pharmacol Sci 12:141–147
McEwen BS, Alves SE (1999) Estrogen actions in the central nervous system. Endocr Rev 20
(3):279–307
McEwen BS, Woolley CS (1994) Estradiol and progesterone regulate neuronal structure and
synaptic connectivity in adult as well as developing brain. Exp Gerontol 29(3–4):431–436
McEwen BS, Akama KT, Spencer-Segal JL, Milner TA, Waters EM (2012) Estrogen effects on the
brain: actions beyond the hypothalamus via novel mechanisms. Behav Neurosci 126(1):4
McHenry J, Carrier N, Hull E, Kabbaj M (2014) Sex differences in anxiety and depression: role of
testosterone. Front Neuroendocrinol 35(1):42–57
McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, Farrar S, Myers J,
Cook G, Ferris P, Garrett L (2000) Sedative but not anxiolytic properties of benzodiazepines are
mediated by the GABA A receptor α 1 subtype. Nat Neurosci 3(6):587
Meffre D, Labombarda F, Delespierre B, Chastre A, De Nicola AF, Stein DG, Schumacher M,
Guennoun R (2013) Distribution of membrane progesterone receptor alpha in the male mouse
and rat brain and its regulation after traumatic brain injury. Neuroscience 231:111–124
Mehlig K, Skoog I, Waern M, Jonasson JM, Lapidus L, Björkelund C, Östling S, Lissner L (2014)
Physical activity, weight status, diabetes and dementia: a 34-year follow-up of the population
study of women in Gothenburg. Neuroepidemiology 42(4):252–259
Meieran SE, Reus VI, Webster R, Shafton R, Wolkowitz OM (2004) Chronic pregnenolone effects
in normal humans: attenuation of benzodiazepine-induced sedation. Psychoneuroendocrinology
29(4):486–500
Meitzen J, Luoma JI, Boulware MI, Hedges VL, Peterson BM, Tuomela K, Britson KA,
Mermelstein PG (2013) Palmitoylation of estrogen receptors is essential for neuronal membrane
signaling. Endocrinology 154(11):4293–4304
Melcangi RC, Mensah-Nyagan AG (2008) Neurosteroids: measurement and pathophysiologic
relevance. Neurochem Int 52(4–5):503–505
Melcangi RC, Panzica G, Garcia-Segura LM (2011) Neuroactive steroids: focus on human brain.
Neuroscience 191:1–5
Mellon SH (2007) Neurosteroid regulation of central nervous system development. Pharmacol Ther
116(1):107–124
Mellon SH, Griffin LD (2002) Neurosteroids: biochemistry and clinical significance. Trends
Endocrinol Metab 13(1):35–43
Mennerick S, He Y, Jiang X, Manion BD, Wang M, Shute A, Benz A, Evers AS, Covey DF,
Zorumski CF (2004) Selective antagonism of 5α-reduced neurosteroid effects at GABAA
receptors. Mol Pharmacol 65(5):1191–1197
Meyer DA, Carta M, Partridge LD, Covey DF, Valenzuela CF (2002) Neurosteroids enhance
spontaneous glutamate release in hippocampal neurons POSSIBLE ROLE OF
METABOTROPIC ς1-LIKE RECEPTORS. J Biol Chem 277(32):28725–28732
Micevych PE, Mermelstein PG (2008) Membrane estrogen receptors acting through metabotropic
glutamate receptors: an emerging mechanism of estrogen action in brain. Mol Neurobiol 38
(1):66
Micevych PE, May Wong A, Mittelman-Smith MA (2011) Estradiol membrane-initiated signaling
and female reproduction. Compr Physiol 5(3):1211–1222
494 S. Marwein et al.

Michels G, Hoppe UC (2008) Rapid actions of androgens. Front Neuroendocrinol 29(2):182–198

Midzak A, Rone M, Aghazadeh Y, Culty M, Papadopoulos V (2011) Mitochondrial protein import
and the genesis of steroidogenic mitochondria. Mol Cell Endocrinol 336(1–2):70–79
Mienville JM, Vicini S (1989) Pregnenolone sulfate antagonizes GABAA receptor-mediated
currents via a reduction of channel opening frequency. Brain Res 489(1):190–194
Milner TA, McEwen BS, Hayashi S, Li CJ, Reagan LP, Alves SE (2001) Ultrastructural evidence
that hippocampal alpha estrogen receptors are located at extranuclear sites. J Comp Neurol 429
(3):355–371
Mitchell E, Thomas D, Burnet R (2006) Testosterone improves motor function in Parkinson’s
disease. J Clin Neurosci 13(1):133–136
Mitro N, Cermenati G, Giatti S, Abbiati F, Pesaresi M, Calabrese D, Garcia-Segura LM, Caruso D,
Melcangi RC (2012) LXR and TSPO as new therapeutic targets to increase the levels of
neuroactive steroids in the central nervous system of diabetic animals. Neurochem Int 60
(6):616–621
Mitsushima D, Takase K, Funabashi T, Kimura F (2007) Gonadal steroid hormones maintain the
stress-induced acetylcholine release in the hippocampus: simultaneous measurements of the
extracellular acetylcholine and serum corticosterone levels in the same subjects. Endocrinology
149(2):802–811
Moffat SD, Zonderman AB, Metter EJ, Blackman MR, Harman SM, Resnick SM (2002) Longitu-
dinal assessment of serum free testosterone concentration predicts memory performance and
cognitive status in elderly men. J Clin Endocrinol Metabol 87(11):5001–5007
Mondelli V, Ciufolini S, Belvederi Murri M, Bonaccorso S, Di Forti M, Giordano A, Marques TR,
Zunszain PA, Morgan C, Murray RM, Pariante CM (2015) Cortisol and inflammatory
biomarkers predict poor treatment response in first episode psychosis. Schizophr Bull 41
(5):1162–1170
Monnet FP, Maurice T (2006) The σ1 protein as a target for the non-genomic effects of neuro
(active) steroids: molecular, physiological, and behavioral aspects. J Pharmacol Sci. https://doi.
org/10.1254/jphs.cr0050032. 0602100001-0602100001
Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit-Singh CJ, Willson TM, Collins JL, Kliewer
SA (2000) St. John’s wort induces hepatic drug metabolism through activation of the pregnane
X receptor. Proc Natl Acad Sci 97(13):7500–7502
Moran C, Phan TG, Chen J, Blizzard L, Beare R, Venn A, Münch G, Wood AG, Forbes J,
Greenaway TM, Pearson S (2013) Brain atrophy in type 2 diabetes: regional distribution and
influence on cognition. Diabetes Care 36(12):4036–4042
Morohaku K, Pelton SH, Daugherty DJ, Butler WR, Deng W, Selvaraj V (2014) Translocator
protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis.
Endocrinology 155(1):89–97
Morrow AL (2007) Recent developments in the significance and therapeutic relevance of neuroac-
tive steroids—introduction to the special issue. Pharmacol Ther 116(1):1–6
Mortensen M, Patel B, Smart TG (2012) GABA potency at GABAA receptors found in synaptic
and extrasynaptic zones. Front Cell Neurosci 6:1
Murphy DD, Cole NB, Greenberger V, Segal M (1998) Estradiol increases dendritic spine density
by reducing GABA neurotransmission in hippocampal neurons. J Neurosci 18(7):2550–2559
Melcangi RC, Garcia-Segura LM, Mensah-Nyagan AG (2008) Neuroactive steroids: State of the art
and new perspectives. Cell Mol Life Sci 65(5):777–797
NCT01963208 (n.d.) Phase 3 study of adjunctive ganaxolone in adults with drug-resistant partial
onset seizures and open-label extension
NCT02358538 (n.d.) A multicenter, open-label proof-of-concept trial of ganaxolone in children
with pcdh19 female pediatric epilepsy and other rare genetic epilepsies
NCT02978781 (n.d.) A study to evaluate SAGE-217 in subjects with essential tremor
NCT03000569 (n.d.) A study to evaluate SAGE-217 in subjects With Parkinson’s disease
Nguyen TV, Ducharme S, Karama S (2017) Effects of sex steroids in the human brain. Mol
Neurobiol 54(9):7507–7519
14 Hormones and Steroids as Neurotransmitters 495

Niitsu T, Iyo M, Hashimoto K (2012) Sigma-1 receptor agonists as therapeutic drugs for cognitive
impairment in neuropsychiatric diseases. Curr Pharm Des 18(7):875–883
Oakley RH, Cidlowski JA (2013) The biology of the glucocorticoid receptor: new signaling
mechanisms in health and disease. J Allergy Clin Immunol 132(5):1033–1044
Ogden KK, Traynelis SF (2011) New advances in NMDA receptor pharmacology. Trends
Pharmacol Sci 32(12):726–733
Okun MS, Walter BL, McDonald WM, Tenover JL, Green J, Juncos JL, DeLong MR (2002)
Beneficial effects of testosterone replacement for the nonmotor symptoms of Parkinson disease.
Arch Neurol 59(11):1750–1753
Okun MS, Fernandez HH, Rodriguez RL, Romrell J, Suelter M, Munson S, Louis ED, Mulligan T,
Foster PS, Shenal BV, Armaghani SJ (2006) Testosterone therapy in men with Parkinson
disease: results of the TEST-PD Study. Arch Neurol 63(5):729–735
Orchinik M, Murray TF, Moore FL (1991) A corticosteroid receptor in neuronal membranes.
Science 252(5014):1848–1851
Österlund MK, Witt MR, Gustafsson JÅ (2005) Estrogen action in mood and neurodegenerative
disorders. Endocrine 28(3):235–241
Paradiso K, Sabey K, Evers AS, Zorumski CF, Covey DF, Steinbach JH (2000) Steroid inhibition
of rat neuronal nicotinic α4β2 receptors expressed in HEK 293 cells. Mol Pharmacol 58
(2):341–351
Pluchino N, Luisi M, Lenzi E, Centofanti M, Begliuomini S, Freschi L, Ninni F, Genazzani AR
(2006) Progesterone and progestins: Effects on brain, allopregnanolone and β-endorphin. J
Steroid Biochem Mol Biol 102(1–5):205–213
Paba S, Frau RC, Godar S, Devoto P, Marrosu F, Bortolato M (2011) Steroid 5α-reductase as a
novel therapeutic target for schizophrenia and other neuropsychiatric disorders. Curr Pharm Des
17(2):151–167
Papadopoulos V, Fan J, Zirkin B (2018) Translocator protein (18 kDa): an update on its function in
steroidogenesis. J Neuroendocrinol 30(2):e12500
Paul SM, Purdy RH (1992) Neuroactive steroids. FASEB 6:2311–2322
Pietrzak RH, Laws SM, Lim YY, Bender SJ, Porter T, Doecke J, Ames D, Fowler C, Masters CL,
Milicic L, Rainey-Smith S (2017) Plasma cortisol, brain amyloid-β, and cognitive decline in
preclinical Alzheimer’s disease: a 6-year prospective cohort study. Biol Psychiatry Cogn
Neurosci Neuroimaging 2(1):45–52
Pinto-Almazán R, Calzada-Mendoza CC, Campos-Lara MG, Guerra-Araiza C (2012) Effect of
chronic administration of estradiol, progesterone, and tibolone on the expression and phosphor-
ylation of glycogen synthase kinase-3β and the microtubule-associated protein tau in the
hippocampus and cerebellum of female rat. J Neurosci Res 90(4):878–886
Pisu MG, Serra M (2004) Neurosteroids and neuroactive drugs in mental disorders. Life Sci 74
(26):3181–3197
Pluchino N, Drakopoulos P, Bianchi-Demicheli F, Wenger JM, Petignat P, Genazzani AR (2015)
Neurobiology of DHEA and effects on sexuality, mood and cognition. J Steroid Biochem Mol
Biol 145:273–280
Pratt WB, Toft DO (2003) Regulation of signaling protein function and trafficking by the hsp90/
hsp70-based chaperone machinery. Exp Biol Med 228(2):111–133
Prossnitz ER, Arterburn JB, Smith HO, Oprea TI, Sklar LA, Hathaway HJ (2008) Estrogen
signaling through the transmembrane G protein–coupled receptor GPR30. Annu Rev Physiol
70:165–190
Pappas TC, Gametchu B, Watson CS (1995) Membrane estrogen receptors identified by multiple
antibody labeling and impeded-ligand binding. FASEB J 9(5):404–410
Pathirathna S, Brimelow BC, Jagodic MM, Krishnan K, Jiang X, Zorumski CF, Mennerick S,
Covey DF, Todorovic SM, Jevtovic-Todorovic V (2005) New evidence that both T-type
calcium channels and GABAA channels are responsible for the potent peripheral analgesic
effects of 5α-reduced neuroactive steroids. Pain 114(3):429–443
Pedram A, Razandi M, Sainson RC, Kim JK, Hughes CC, Levin ER (2007) A conserved
mechanism for steroid receptor translocation to the plasma membrane. J Biol Chem 282
(31):22278–22288
496 S. Marwein et al.

Pawlak J, Karolczak M, Krust A, Chambon P, Beyer C (2005) Estrogen receptor-α is associated

with the plasma membrane of astrocytes and coupled to the MAP/Src-kinase pathway. Glia 50
(3):270–275
Pérez-Neri I, Montes S, Ojeda-López C, Ramírez-Bermúdez J, Ríos C (2008) Modulation of
neurotransmitter systems by dehydroepiandrosterone and dehydroepiandrosterone sulfate:
Mechanism of action and relevance to psychiatric disorders. Prog Neuro-Psychopharmacol
Biol Psychiatry 32(5):1118–1130
Quesada I, Tudurí E, Ripoll C, Nadal A (2008) Physiology of the pancreatic α-cell and glucagon
secretion: Role in glucose homeostasis and diabetes. J Endocrinol 199(1):5–19
Qi AQ, Qiu J, Xiao L, Chen YZ (2005) Rapid activation of JNK and p38 by glucocorticoids in
primary cultured hippocampal cells. J Neurosci Res 80(4):510–517
Quesada A, Romeo HE, Micevych P (2007) Distribution and localization patterns of estrogen
receptor-β and insulin-like growth factor-1 receptors in neurons and glial cells of the female rat
substantia nigra: localization of ERβ and IGF-1R in substantia nigra. J Comp Neurol 503
(1):198–208
Quinones-Jenab V, Jenab S (2010) Progesterone attenuates cocaine-induced responses. Horm
Behav 58(1):22–32
Rogawski MA, Loya CM, Reddy K, Zolkowska D, Lossin C (2013) Neuroactive steroids for the
treatment of status epilepticus. Epilepsia 54(s6):93–98
Rakotomamonjy J, Levenes C, Baulieu EE, Schumacher M, Ghoumari AM (2011) Novel protective
effect of mifepristone on detrimental GABAA receptor activity to immature Purkinje neurons.
FASEB J 25(11):3999–4010
Ramirez VD, Zheng J (1996) Membrane sex-steroid receptors in the brain. Front Neuroendocrinol
17(4):402–439
Razandi M, Pedram A, Merchenthaler I, Greene GL, Levin ER (2004) Plasma membrane estrogen
receptors exist and functions as dimers. Mol Endocrinol 18(12):2854–2865
Reddy DS (2003) Pharmacology of endogenous neuroactive steroids. Crit Rev Neurobiol
15:197–234
Reddy DS (2010) Neurosteroids: endogenous role in the human brain and therapeutic potentials.
Prog Brain Res 186:113–137. Elsevier
Reddy D (2011) Role of anticonvulsant and antiepileptogenic neurosteroids in the pathophysiology
and treatment of epilepsy. Front Endocrinol 2:38
Reddy DS, Rogawski MA (2010) Ganaxolone suppression of behavioral and electrographic
seizures in the mouse amygdala kindling model. Epilepsy Res 89(2–3):254–260
Reddy DS, Rogawski MA (2012) Neurosteroids—Endogenous regulators of seizure susceptibility
and role in the treatment of epilepsy. In Jasper’s Basic Mechanisms of the Epilepsies [Internet].
4th edition. National Center for Biotechnology Information (US).
Reed MJ, Kloosterboer HJ (2004) Tibolone: a selective tissue estrogenic activity regulator
(STEAR). Maturitas 48:4–6
Remage-Healey L (2014) Frank Beach Award Winner: steroids as neuromodulators of brain circuits
and behavior. Horm Behav 66(3):552–560
Rhodes ME, Harney JP, Frye CA (2004) Gonadal, adrenal, and neuroactive steroids’ role in ictal
activity. Brain Res 1000(1–2):8–18
Ritsner MS, Gibel A, Shleifer T, Boguslavsky I, Zayed A, Maayan R, Weizman A, Lerner V (2010)
Pregnenolone and dehydroepiandrosterone as an adjunctive treatment in schizophrenia and
schizoaffective disorder: an 8-week, double-blind, randomized, controlled, 2-center, parallel-
group trial. J Clin Psychiatry 71(10):1351–1362
Rodriguez MÁ, Garcia-Segura LM, Cabezas R, Torrente D, Capani F, Gonzalez J, Barreto GE
(2014) Tibolone protects T98G cells from glucose deprivation. J Steroid Biochem Mol Biol
144:294–303
Roeloffs R, Wickenden AD, Crean C, Werness S, McNaughton-Smith G, Stables J, McNamara JO,
Ghodadra N, Rigdon GC (2008) In vivo profile of ICA-27243 [N-(6-chloro-pyridin-3-yl)-3,
14 Hormones and Steroids as Neurotransmitters 497

4-difluoro-benzamide], a potent and selective KCNQ2/Q3 (Kv7. 2/Kv7. 3) activator in rodent

anticonvulsant models. J Pharmacol Exp Ther 326(3):818–828
Romeo RD, McCarthy JB, Wang A, Milner TA, McEwen BS (2005) Sex differences in hippocam-
pal estradiol-induced N-methyl-D-aspartic acid binding and ultrastructural localization of estro-
gen receptor-alpha. Neuroendocrinology 81(6):391–399
Rossetti MF, Cambiasso MJ, Holschbach MA, Cabrera R (2016) Oestrogens and progestagens:
synthesis and action in the brain. J Neuroendocrinol 28(7). https://doi.org/10.1111/jne.12402
Rudolph LM, Cornil CA, Mittelman-Smith MA, Rainville JR, Remage-Healey L, Sinchak K,
Micevych PE (2016) Actions of steroids: new neurotransmitters. J Neurosci 36
(45):11449–11458
Ruiz-Palmero I, Hernando M, Garcia-Segura LM, Arevalo MA (2013) G protein-coupled estrogen
receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal
neurons. Mol Cell Endocrinol 372(1–2):105–115
Rupprecht R (1997) The neuropsychopharmacological potential of neuroactive steroids. J Psychiatr
Res 31(3):297–314
Rupprecht R (2003) Neuroactive steroids: mechanisms of action and neuropsychopharmacological
properties. Psychoneuroendocrinology 28(2):139–168
Rupprecht R, Holsboer F (1999) Neuroactive steroids: mechanisms of action and
neuropsychopharmacological perspectives. Trends Neurosci 22(9):410–416
Rupprecht R, Reul JM, Trapp T, van Steensel B, Wetzel C, Damm K, Zieglgänsberger W, Holsboer
F (1993) Progesterone receptor-mediated effects of neuroactive steroids. Neuron 11(3):523–530
Rupprecht R, di Michele F, Hermann B, Ströhle A, Lancel M, Romeo E, Holsboer F (2001)
Neuroactive steroids: molecular mechanisms of action and implications for
neuropsychopharmacology. Brain Res Rev 37(1–3):59–67
Scharfman HE, MacLusky NJ (2006) The influence of gonadal hormones on neuronal excitability,
seizures, and epilepsy in the female. Epilepsia 47(9):1423–1440
Schiess AR, Partridge LD (2005) Pregnenolone sulfate acts through a G-protein-coupled σ1-like
receptor to enhance short term facilitation in adult hippocampal neurons. Eur J Pharmacol 518
(1):22–29
Schultz KN, Silke A, Hu M, Bennett AL, Kennedy RT, Musatov S, Toran-Allerand CD, Kaplitt
MG, Young LJ, Becker JB (2009) Viral vector-mediated overexpression of estrogen receptor-α
in striatum enhances the estradiol-induced motor activity in female rats and estradiol-modulated
GABA release. J Neurosci 29(6):1897–1903
Schumacher M, Guennoun R, Robert F, Carelli C, Gago N, Ghoumari A, Deniselle MCG, Gonzalez
SL, Ibanez C, Labombarda F, Coirini H (2004) Local synthesis and dual actions of progesterone
in the nervous system: neuroprotection and myelination. Growth Horm IGF Res 14:18–33
Schumacher M, Guennoun R, Stein DG, De Nicola AF (2007) Progesterone: therapeutic
opportunities for neuroprotection and myelin repair. Pharmacol Ther 116(1):77–106
Schumacher M, Hussain R, Gago N, Oudinet JP, Mattern C, Ghoumari A (2012) Progesterone
synthesis in the nervous system: implications for myelination and myelin repair. Front Neurosci
6:10
Schwartz N, Verma A, Bivens CB, Schwartz Z, Boyan BD (2016) Rapid steroid hormone actions
via membrane receptors. Biochim Biophys Acta 1863(9):2289–2298
Seidman SN (2006) Normative hypogonadism and depression: does ‘andropause’exist? Int J Impot
Res 18(5):415
Selvaraj V, Stocco DM (2015) The changing landscape in translocator protein (TSPO) function.
Trends Endocrinol Metab 26(7):341–348
Selvaraj V, Stocco DM, Tu LN (2015) Minireview: translocator protein (TSPO) and steroidogene-
sis: a reappraisal. Mol Endocrinol 29(4):490–501
Seredynski AL, Balthazart J, Ball GF, Cornil CA (2015) Estrogen receptor β activation rapidly
modulates male sexual motivation through the transactivation of metabotropic glutamate recep-
tor 1a. J Neurosci 35(38):13110–13123
498 S. Marwein et al.

Serey C, Huss B, Chuon T, Mahavongtrakul M, Sinchak K (2014) The signaling pathways of

progesterone receptor, Src kindase, and dopamine D1 receptor converge in the arcuate nucleus
of the hypothalamus to facilitate lordosis. In Society for Neuroscience, Neuroscience Meeting
Planner, Program, vol. 167
Serra M, Pisu MG, Littera M, Papi G, Sanna E, Tuveri F, Usala L, Purdy RH, Biggio G (2000)
Social isolation-induced decreases in both the abundance of neuroactive steroids and GABAA
receptor function in rat brain. J Neurochem 75(2):732–740
Shannon EE, Purdy RH, Grant KA (2005a) Discriminative stimulus effects of 5.6 mg/kg
pregnanolone in DBA/2J and C57BL/6J inbred mice. Alcohol 37(1):35–45
Shannon EE, Porcu P, Purdy RH, Grant KA (2005b) Characterization of the discriminative stimulus
effects of the neuroactive steroid pregnanolone in DBA/2J and C57BL/6J inbred mice. J
Pharmacol Exp Ther 314(2):675–685
Sheppard PAS, Koss WA, Frick KM, Choleris E (2018) Rapid actions of oestrogens and their
receptors on memory acquisition and consolidation in females. J Neuroendocrinol 30(2):e12485
Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK, Hendrix SL, Jones BN III,
Assaf AR, Jackson RD, Kotchen JM (2003) Estrogen plus progestin and the incidence of
dementia and mild cognitive impairment in postmenopausal women: the Women’s Health
Initiative Memory Study: a randomized controlled trial. JAMA 289(20):2651–2662
Simerly RB (2005) Wired on hormones: endocrine regulation of hypothalamic development. Curr
Opin Neurobiol 15(1):81–85
Sinchak K, Wagner EJ (2012) Estradiol signaling in the regulation of reproduction and energy
balance. Front Neuroendocrinol 33(4):342–363
Sinchak K, Dalhousay L, Sanathara N (2015) Orphanin FQ-ORL-1 regulation of reproduction and
reproductive behavior in the female. Vitam Horm 97:187–221. Academic Press
Singh M, Su C, Ng S (2013) Non-genomic mechanisms of progesterone action in the brain. Front
Neurosci 7:159
Smith SS, Woolley CS (2004) Cellular and molecular effects of steroid hormones on CNS
excitability. Cleve Clin J Med 71(2):s4
Smith SS, Shen H, Gong QH, Zhou X (2007) Neurosteroid regulation of GABAA receptors: focus
on the α4 and δ subunits. Pharmacol Ther 116(1):58–76
Soares CN, Frey BN (2010) Challenges and opportunities to manage depression during the
menopausal transition and beyond. Psychiatr Clin North Am 33(2):295–308
Soares CN, Poitras JR, Prouty J (2003) Effect of reproductive hormones and selective estrogen
receptor modulators on mood during menopause. Drugs Aging 20(2):85–100
Song RX, Zhang Z, Santen RJ (2005) Estrogen rapid action via protein complex formation
involving ERα and Src. Trends Endocrinol Metab 16(8):347–353
Souza-Teodoro LH, de Oliveira C, Walters K, Carvalho LA (2016) Higher serum dehydroepian-
drosterone sulfate protects against the onset of depression in the elderly: findings from the
English Longitudinal Study of Aging (ELSA). Psychoneuroendocrinology 64:40–46
Strömberg J, Haage D, Taube M, Bäckström T, Lundgren P (2006) Neurosteroid modulation of
allopregnanolone and GABA effect on the GABA-A receptor. Neuroscience 143(1):73–81
Strous RD, Maayan R, Weizman A (2006) The relevance of neurosteroids to clinical psychiatry:
from the laboratory to the bedside. Eur Neuropsychopharmacol 16(3):155–169
Sun S, Cai J, Tao W, Wu L, Tapas C, Zhou L, Wang D (2018) Comparative transcriptome profiling
and characterization of gene expression for ovarian differentiation under RU486 treatment. Gen
Comp Endocrinol 261:166–173
Szego CM, Davis JS (1967) Adenosine 3’,5’-monophosphate in rat uterus: acute elevation by
estrogen. Proc Natl Acad Sci U S A 58(4):1711
Saldanha CJ, Remage-Healey L, Schlinger BA (2011) Synaptocrine signaling: Steroid synthesis
and action at the synapse. Endocr Rev 32(4):532–549
Tuem KB, Atey TM (2017) Neuroactive steroids: Receptor interactions and responses. Front
Neurol 8:442
14 Hormones and Steroids as Neurotransmitters 499

Talih F, Fattal O, Malone D (2007) Anabolic steroid abuse: psychiatric and physical costs. Cleve
Clin J Med 74(5):341
Tang YT, Hu T, Arterburn M, Boyle B, Bright JM, Emtage PC, Funk WD (2005) PAQR proteins: a
novel membrane receptor family defined by an ancient7-transmembrane pass motif. J Mol Evol
61(3):372–380
Tang H, Zhang Q, Yang L, Dong Y, Khan M, Yang F, Brann DW, Wang R (2014) GPR30 mediates
estrogen rapid signaling and neuroprotection. Mol Cell Endocrinol 387(1–2):52–58
Thomas T, Rhodin JA, Sutton ET, Bryant MW, Price JM (1999) Estrogen protects peripheral and
cerebral blood vessels from toxicity of Alzheimer peptide amyloid-beta and inflammatory
reaction. J Submicrosc Cytol Pathol 31(4):571–579
Thomas P, Pang Y, Dong J, Groenen P, Kelder JD, De Vlieg J, Zhu Y, Tubbs C (2007) Steroid and
G protein binding characteristics of the seatrout and human progestin membrane receptor α
subtypes and their evolutionary origins. Endocrinology 148(2):705–718
Thorpe L, Whitney DK, Kutcher SP, Kennedy SH (2001) Clinical guidelines for the treatment of
depressive disorders. VI Special populations. Can J Psychiatry 46:63S–76S
Todorovic SM, Pathirathna S, Brimelow BC, Jagodic MM, Ko SH, Jiang X, Nilsson KR, Zorumski
CF, Covey DF, Jevtovic-Todorovic V (2004) 5β-reduced neuroactive steroids are novel voltage-
dependent blockers of T-type Ca2+ channels in rat sensory neurons in vitro and potent
peripheral analgesics in vivo. Mol Pharmacol 66(5):1223–1235
Toft D, Gorski J (1966) A receptor molecule for estrogens: isolation from the rat uterus and
preliminary characterization. Proc Natl Acad Sci U S A 55(6):1574
Torres JM, Ortega E (2003) DHEA, PREG and their sulphate derivatives on plasma and brain after
CRH and ACTH administration. Neurochem Res 28(8):1187–1191
Tringham E, Powell KL, Cain SM, Kuplast K, Mezeyova J, Weerapura M, Eduljee C, Jiang X,
Smith P, Morrison JL, Jones NC (2012) T-type calcium channel blockers that attenuate thalamic
burst firing and suppress absence seizures. Sci Transl Med 4(121):121ra19
Turkmen S, Lundgren P, Birzniece V, Zingmark E, Backstrom T, Johansson IM (2004) 3beta-
20beta-dihydroxy-5alpha-pregnane (UC1011) antagonism of the GABA potentiation and the
learning impairment induced in rats by allopregnanolone. Eur J Neurosci 20:1604–1612
Usall J, Huerta-Ramos E, Iniesta R, Cobo J, Araya S, Roca M, Serrano-Blanco A, Teba F, Ochoa S
(2011) Raloxifene as an adjunctive treatment for postmenopausal women with schizophrenia: a
double-blind, randomized, placebo-controlled trial. J Clin Psychiatry 72(11):1552
van Wingen GA, Ossewaarde L, Bäckström T, Hermans EJ, Fernández G (2011) Gonadal hormone
regulation of the emotion circuitry in humans. Neuroscience 191:38–45
Vahaba DM, Remage-Healey L (2015) Brain estrogen production and the encoding of recent
experience. Curr Opin Behav Sci 6:148–153
Vaitkevicius H, Ng M, Moura L, Rosenthal E, Westover MB, Rosand J, Rogawski MA, Reddy K,
Cole AJ (2013) Successful allopregnanolone treatment of new onset refractory status epilepticus
(NORSE) syndrome: first in man experience. Epilepsia 54(suppl 6):106–124
Valadez-Cosmes P, Vázquez-Martínez ER, Cerbon M, Camacho-Arroyo I (2016) Membrane
progesterone receptors in reproduction and cancer. Mol Cell Endocrinol 434:166–175
Vallée M (2016) Neurosteroids and potential therapeutics: focus on pregnenolone. J Steroid
Biochem Mol Biol 160:78–87
Vallée M, Mayo W, Darnaudéry M, Corpéchot C, Young J, Koehl M, Le Moal M, Baulieu EE,
Robel P, Simon H (1997) Neurosteroids: deficient cognitive performance in aged rats depends
on low pregnenolone sulfate levels in the hippocampus. Proc Natl Acad Sci 94
(26):14865–14870
Vega-Vela NE, Osorio D, Avila-Rodriguez M, Gonzalez J, García-Segura LM, Echeverria V,
Barreto GE (2017) L-type calcium channels modulation by estradiol. Mol Neurobiol 54
(7):4996–5007
Veronese N, De Rui M, Bolzetta F, Zambon S, Corti MC, Baggio G, Toffanello ED, Crepaldi G,
Perissinotto E, Manzato E, Sergi G (2015) Serum dehydroepiandrosterone sulfate and incident
depression in the elderly: the Pro. VA study. Am J Geriatr Psychiatry 23(8):863–871
500 S. Marwein et al.

Walf AA, Frye CA (2006) A review and update of mechanisms of estrogen in the hippocampus and
amygdala for anxiety and depression behavior. Neuropsychopharmacology 31(6):1097
Wang MD, Bäckström T, Landgren S (2000) The inhibitory effects of allopregnanolone and
pregnanolone on the population spike, evoked in the rat hippocampal CA1 stratum pyramidale
in vitro, can be blocked selectively by epiallopregnanolone. Acta Physiol Scand 169
(4):333–341
Wang M, He Y, Eisenman LN, Fields C, Zeng CM, Mathews J, Benz A, Fu T, Zorumski E,
Steinbach JH, Covey DF (2002) 3β-hydroxypregnane steroids are pregnenolone sulfate-like
GABAA receptor antagonists. J Neurosci 22(9):3366–3375
Wang Y, Wang Y, Chen Z (2018) Double-edged GABAergic synaptic transmission in seizures: the
importance of chloride plasticity. Brain Res. https://doi.org/10.1016/j.brainres.2018.09.008
Waters EM, Torres-Reveron A, McEwen BS, Milner TA (2008) Ultrastructural localization of
extranuclear progestin receptors in the rat hippocampal formation. J Comp Neurol 511(1):34–46
Webber KM, Casadesus G, Marlatt MW, Perry G, Hamlin CR, Atwood CS, Bowen RL, Smith MA
(2005) Estrogen bows to a new master: the role of gonadotropins in Alzheimer pathogenesis.
Ann N Y Acad Sci 1052(1):201–209
Wehling M (1997) Specific, nongenomic actions of steroid hormones. Annu Rev Physiol 59
(1):365–393
Wei J, Xiao GM (2013) The neuroprotective effects of progesterone on traumatic brain injury:
current status and future prospects. Acta Pharmacol Sin 34(12):1485
Whitaker AM, Farooq MA, Edwards S, Gilpin NW (2016) Post-traumatic stress avoidance is
attenuated by corticosterone and associated with brain levels of steroid receptor co-activator-1
in rats. Stress 19(1):69–77
Wójtowicz T, Mozrzymas JW (2010) Estradiol and GABAergic transmission in the hippocampus.
Vitam Horm 82:279–300. Academic Press
Wójtowicz T, Lebida K, Mozrzymas JW (2008) 17β-estradiol affects GABAergic transmission in
developing hippocampus. Brain Res 1241:7–17
Wolf OT, Kirschbaum C (1999) Actions of dehydroepiandrosterone and its sulfate in the central
nervous system: effects on cognition and emotion in animals and humans. Brain Res Rev 30
(3):264–288
Xiao L, Jackson LR, Becker JB (2003) The effect of estradiol in the striatum is blocked by ICI
182,780 but not tamoxifen: pharmacological and behavioral evidence. Neuroendocrinology 77
(4):239–245
Xiao L, Qi A, Chen Y (2005) Cultured embryonic hippocampal neurons deficient in glucocorticoid
(GC) receptor: a novel model for studying nongenomic effects of GC in the neural system.
Endocrinology 146(9):4036–4041
Xu Y, Tanaka M, Chen L, Sokabe M (2012) DHEAS induces short-term potentiation via the
activation of a metabotropic glutamate receptor in the rat hippocampus. Hippocampus 22
(4):707–722
Yaffe K, Krueger K, Cummings SR, Blackwell T, Henderson VW, Sarkar S, Ensrud K, Grady D
(2005) Effect of raloxifene on prevention of dementia and cognitive impairment in older
women: the Multiple Outcomes of Raloxifene Evaluation (MORE) randomized trial. Am J
Psychiatry 162(4):683–690
Yagishita T, Kushida A, Tamura H (2012) Vitamin D3 enhances ATRA-mediated neurosteroid
biosynthesis in human glioma GI-1 cells. J Biochem 152(3):285–292
Yan M, Liu AL, Zhou SJ, Tang LP, Ou YQ, Yin W, Chen XY, Su XW, Qiu PX, Huang YJ, Zhang
JX (2015) Characterization of a synthetic steroid 24-keto-cholest-5-en-3β, 19-diol as a
neuroprotectant. CNS Neurosci Ther 21(6):486–495
Yang S, Roselli F, Patchev AV, Yu S, Almeida OF (2013) Non-receptor-tyrosine kinases integrate
fast glucocorticoid signaling in hippocampal neurons. J Biol Chem 288(33):23725–23739
Yoon SY, Roh DH, Seo HS, Kang SY, Moon JY, Song S, Beitz AJ, Lee JH (2010) An increase in
spinal dehydroepiandrosterone sulfate (DHEAS) enhances NMDA-induced pain via
14 Hormones and Steroids as Neurotransmitters 501

phosphorylation of the NR1 subunit in mice: involvement of the sigma-1 receptor. Neurophar-
macology 59(6):460–467
Zakon HH (1998) The effects of steroid hormones on electrical activity of excitable cells. Trends
Neurosci 21(5):202–207
Zawadzka M, Franklin RJ (2007) Myelin regeneration in demyelinating disorders: new
developments in biology and clinical pathology. Curr Opin Neurol 20(3):294–298
Zhang Z, Yang R, Zhou R, Li L, Sokabe M, Chen L (2010) Progesterone promotes the survival of
newborn neurons in the dentate gyrus of adult male mice. Hippocampus 20(3):402–412
Zhao L, O’Neill K, Brinton RD (2006) Estrogenic agonist activity of ICI 182,780 (Faslodex) in
hippocampal neurons: implications for basic science understanding of estrogen signaling and
development of estrogen modulators with a dual therapeutic profile. J Pharmacol Exp Ther 319
(3):1124–1132
Zheng P (2009) Neuroactive steroid regulation of neurotransmitter release in the CNS: action,
mechanism and possible significance. Prog Neurobiol 89(2):134–152
Zhou Q, Sheng M (2013) NMDA receptors in nervous system diseases. Neuropharmacology
74:69–75
Zuloaga DG, Yahn SL, Pang Y, Quihuis AM, Oyola MG, Reyna A, Thomas P, Handa RJ, Mani SK
(2012) Distribution and estrogen regulation of membrane progesterone receptor-β in the female
rat brain. Endocrinology 153(9):4432–4443
Pharmacology of Neuropeptides:
Substance P, Vasoactive Intestinal Peptides, 15
Neuropeptide Y, Calcitonin Peptides
and Their Receptors

Nabil A. Nimer, Najlaa S. Ismael, Ruwaida W. Abdo, Sura Y. Taha

Alkhammas, and Qutaiba A. Alkhames Aga

Abstract

Neuropeptides are responsible for the regulation of various biological activities

and mediate various regulatory mechanisms associated with every organ system.
They control and regulate communication between endocrine and nervous
systems and are also involved in signaling between cells found in the central
and peripheral nervous system. Certainly, they can modulate immunomodulation,
neuroprotection, and physiological homeostasis (e.g., feeding behavior, balance
of water, blood pressure, breakdown of glucose, cognition, stress response, and
pain) by working as peptide hormones.

Keywords

Calcitonin peptide · Neuropeptides · Neuropeptide tyrosine · Neuropeptide Y ·

Substance P · Tachykinins · Vasoactive intestinal polypeptides

Abbreviations

CD Circular dichroism
CNS Central nervous system
DAG Diacylglycerol
GDP Guanosine diphosphate
GLP Glucagon-like peptide
GPCRs G protein-coupled receptors
GTP Guanosine triphosphate
IL Interleukin

N. A. Nimer (*) · N. S. Ismael · R. W. Abdo · S. Y. Taha Alkhammas · Q. A. Alkhames Aga

Faculty of Pharmacy, Philadelphia University, Amman, Jordan
e-mail: [email protected]

# Springer Nature Singapore Pte Ltd. 2020 503

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_15
504 N. A. Nimer et al.

IP3 Inositol triphosphate

NF-κB Nuclear factor kappa B
NK1R Neurokinin one receptor
NK2R Neurokinin two receptor
NK3R Neurokinin three receptor
NKA Neurokinin A
NKB Neurokinin B
NMR Nuclear magnetic resonance
PACAP Pituitary adenylate cyclase activity peptide
PHI Peptide histidine isoleucine
PRL Pituitary prolactin
SP Substance P
TAC Tachykinin
TNF Tumor necrosis factor
VIP Vasoactive intestinal polypeptide

15.1 Introduction

Neuropeptides are molecules similar to a protein, which are created and discharged
by neurons. They regulate the secretory tract and act on neural substrates (Russo
2017). Holmgren and Jensen defined neuropeptide as a peptide discharged by neuron
as a signaling molecule. This signaling molecule will impact other excitable cells as
a modulator or a transmitter (and actually affecting its own cells sometimes)
(Belzung et al. 2006). A considerable point is that neuropeptide is not only just a
peptide existing in neurons but also acts as a medium of communication within the
cells, which means it stimulates other cells through high-affinity binding toward
particular targets (receptors). These molecules are articulated in peripheral nerves
like sensory and motor nerves and also in the central nervous system (CNS).

15.2 Definition of the Neuropeptides

In terms of pleiotropic potential, the concept was best demonstrated by Candace

Pert, who stated: “Similar to the change in the feelings, the peptides mixture are
regulated around the body as well as the brain. This leads them to alter the chemistry
throughout the body.” In the general sense, neuropeptides are basically transmitters,
which are made up of amino acids, where a peptide bond combines them. In terms of
size, they are quite large and hold amino acids in the range of 3–36. These are
released into the synaptic cleft together with other neurotransmitters, extracted from
the large 90 amino acids, i.e., inactive precursors. Bioactive peptide is produced
when the eradication of an individual sequence occurs through neuropeptide ante-
cedent. The same bioactive neuropeptide is also produced in few neuropeptide
precursor peptides, in various forms. These are synthesized into the cell part of the
neuron, following its lumen sequestration as well as transition to the axon, during the
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 505

Rough
endoplasmic Golgi Synapse
Nucleus
reculum apparatus

Axonal transport Receptor

Gene

Precursor Mature Precursor Precursor breakdown and

mRNA Release
mRNAS protein liberaon of neuropepde

Fig. 15.1 Neuropeptide synthesis

Table 15.1 Origin and examples of neuropeptides

Origin of neuropeptides Examples
Pituitary peptides PRL, vasopressin, GH, TSH, ACTH, α-MSH, β-endorphin, and
oxytocin peptides creating an impact on the brain and gut
Hypothalamic releasing GIHH (somatostatin pituitary peptides, RH and LRH)
hormones
Impact of peptides on the Insulin, nerve GF, CCK, subs P, leucine enkephalin, brain-related
gut and brain neurotrophic factors, methionine enkephalin, neurotensin,
glucagon from other tissues
From other tissues Bradykinin, sleep peptides, ag-II, carnosine, calcitonin

processing events such as signal peptide cleavage. These are placed in a large dense-
core vesicles (LDCVs). Subsequent to the LDCV exocytosis, the re-internalization
of the LDCV components occurs in the component part of the membrane. Thus, no
neuropeptide is re-used in the synapse. Generally, neuropeptides are released at low
cytosolic Ca concentrations, though LDCV exocytosis is usually stimulated by Ca
ions, whereas the same ion may be utilized for exocytosis when derived through
other important sources such as transmembrane internal stores (Fig. 15.1;
Table 15.1).
Along with it, neuropeptide also imposes certain alteration on behavior. The
alteration in the peptide effect behavior is demonstrated by the gene manipulation
experiments either by transgene mutations or viral gene transfer, exhibiting changes
in the behavioral traits which are part of the expression of peptide receptor (Ludwig
2011). This shows that the peptidergic neurons in the brain do not determine the
behavior but the peptide distribution. The human neurological disorder, such as
depression, autism spectrum disorder, schizophrenia, and social anxiety, has been
associated with vasopressin and oxytocin. In addition, there are neuropeptides
involved in appetite regulation, mood disorder, and libido disorder, which are
included in the peptide-based therapies.
506 N. A. Nimer et al.

15.3 Research Focusing on Neuropeptides and Opportunities

for Drug Discovery

In the last 40 years, neuropeptides and their receptors with similarity have been
steadily increasing. At first, the isolation of peptides from the brain and guts can be
observed (e.g., substance P, somatostatin). This can be done by targeted mining in a
particular region that includes orexin in the brain, cortistatin, etc., as well as G
protein-coupled receptors that are deorphanized (GPCRs including ghrelin receptors
and orexin). Moreover, the accomplishment of the Human Genome Project can also
be seen (Schalla and Stengel 2018). The distribution of neuropeptides is being
regionally restricted in the central and peripheral nervous system. The signaling of
neuropeptides and their receptors is more distinct in terms of spatial than signaling
with the classical as well as low-molecular-weight neurotransmitters. These
neurotransmitters seemed to be widely expressed due to the assumption that drugs
that act on neuropeptide receptors possess more selective actions pharmacologically
with a few side effects. However, drugs reacting on glutamatergic, cholinergic,
monoaminergic, and GABAergic systems are less selective pharmacologically.
The functions of neuropeptides include neurotransmitting and acting as growth
factors. They are observed in the hormones found in the endocrine system such as
glial cells; they also act as the messengers in the immune system. When the nervous
system faces any complications (e.g., by stress, injury, or drug abuse), these
neuropeptides seemed to be important. This leads to a massive number of
opportunities to produce new drugs to treat disorders belonging to the nervous
system. As a result of this, subtypes of receptor agonists and certain antagonists as
well as the substance P receptor (neurokinin-1) were discovered. This has proved its
clinical efficiency while treating depression and in chemotherapy induction. Addi-
tionally, several other neuropeptides are under clinical trials for different indications
(Hoyer and Bartfai 2012).
These receptors are assumed to contribute to the discovery of potential drugs
linked to various neurological disorders. According to the researchers, there is a dire
need for antiepileptic drugs (AEDs) which act in several ways than the drugs already
present in the market. A large number of AEDs block the sodium channels to act and
also increase the GABAergic transmission. The therapeutic options are also
expanded by a completely new generation of AEDs; however, these are not as
effective as the older drugs (Hanaya and Arita 2016). To these medications, patients
having mesial temporal epilepsy (mTLE) seemed to be the most pharmacoresistant
(Pati and Alexopoulos 2010). With the aim of rectifying the dilemma, finding the
new mechanism of AEDs is not the only need of neuropharmacologists but to also
focus on the information shared on the pathophysiology of epilepsy. Moreover,
several mechanisms can be observed during the complex procedure of
epileptogenesis; therefore, new compounds should be identified ideally that can
target the other pathways simultaneously. While studying the pathogenesis of
epilepsy, a number of neuropeptides including somatostatin, galanin, and neuropep-
tide Y were briefly studied. However, these neuropeptides are poor and are seen as
the main hurdle for developing the brain drug due to their poor ability to penetrate
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 507

Neuroprotection An-inf lammatory

Protection from neuronal Inflammation is believed
death and/or to play an important part
neuronal and network dysfunction in epileptogenesis

Neurogenesis Ghrelin Anconvulsant

Regulates adult Attenuates seizures in
hippocampal neurogenesis different acute models of
epileptic seizures
Crosses BBB
Majority of neuropeptides
are unable to cross BBB–major
bottleneck for brain drug development

Fig. 15.2 Benefits of ghrelin

the blood-brain barrier (BBB) (Robertson et al. 2011). The discovery of ghrelin in
1999 brought great excitement to all scientists (Chen et al. 2017). It has been
produced both in peripheral and central terms in the field of epilepsy (Fig. 15.2).
While comparing the well-established anticonvulsant neuropeptides, ghrelin is
recognized to possess a large number of advantages. This neuropeptide has
the ability to cross the BBB while also possessing the characteristic to affect the
physiological processes. The physiological procedures range from the
neuroprotective properties and potent anti-inflammatory along with BBB protection
and integrated hippocampal neurogenesis (Portelli et al. 2012). Besides, various
research studies indicated that physiological procedures are affected negatively
during the process of epileptogenesis (Coremans et al. 2010; Vezzani et al. 2011;
Marchi et al. 2012; Parent and Kron 2012; Vezzani et al. 2013). The recent phase III
clinical trial of ghrelin receptor agonist JMV1843, which highlighted the lack of
hormone among humans, seems to be completed, and the drugs were observed to be
well effective. A company Aeterna Zentaris is successfully developing this drug
now. Moreover, through clinical trials, orexin receptor seemed to be developed and
progressed to treat sleeping disorders. Almorexant completed the phase III clinical
trials (Hoever et al. 2012) and serves as unrecognized orexin receptor antagonist
(Owen et al. 2009). The production of this drug has stopped to a greater extent, as
provided through a brief review of data that was achieved through clinical studies.
This further served as an avenue to demonstrate the advantages of almorexant
(Actelion 2011). The next drug includes suvorexant (MK-4305) which has also
accomplished the phase III clinical trials to identify its effects on different sleeping
patterns (Cox et al. 2010). Based on the classical system of neurotransmitter,
majority of the drugs were approved for treating obesity, while the potential ability
of the system which provides important side effects was ignored. Lorcaserin
(Belviq), a 5-HT2C receptor, seems to receive FDA approval recently for treating
508 N. A. Nimer et al.

obesity in selected obese patients. Weight loss can be seen to be greater than 5% by
placebo-subtraction which was achieved by 27.2%. Maintenance of weight loss by
following the same treatment is observed in 68% of patients (Fidler et al. 2011).
However, the impact of lorcaserin cannot be understood completely specifically for
body weight. However, by mediating it with hypothalamic POMC, activation can be
accomplished (Halford et al. 2007). Mild side effects are revealed in the clinical trials
of lorcaserin while treating for a headache (Smith et al. 2010).
Neuropeptides are also recognized as the molecules that are significant in deter-
mining neurological disorders such as epilepsy and depression. The integration of
the peptides as pharmacological components serves as a great proposition given its
decreased level of toxicity related to the functioning of the metabolism, which also
escalates its potency. Reflecting on an in-depth understanding of the neurological
disorder, it has been found that their use in the therapeutic methods has remained
limited particularly for the treatment of clinical problems. The main contributing
factor for their deficiency of delivering adequate results in the clinical treatment is
their intact peptide delivery to particular regions of the brain, which is essential for
treating the neurological disorder. Generally, the delivery of the drug, which is
derived from the peptides, is confined to two factors: the first includes the issues
concerning the general bioavailability, while the second deals with providing infor-
mation regarding blood-brain barriers. Several factors have been recognized in
creating an impact over pharmaceutical bio-presence of the brain. The factors
comprise the distribution, which takes place in the cardiovascular space, its total
volume, the disappearance of the half-life, as well as the drug capacity for causing
the biological effect by reaching the target. All these factors contribute differently to
each of the drugs, and in terms of the neuropharmaceutical peptide, this serves as a
likely problem, which can be associated with the delivery of the drug.
Depending on the distribution of the cardiovascular system, the component can
be divided into three fractions, namely, protein, blood cells, and protein free fraction
(PFF). In the latter category, the existence of peptide drugs is usually achieved when
it is being transported to the brain. The dynamic equilibrium is maintained by these
three components for their peptide distribution. Generally, the core objective of this
equilibrium is to evolve peptide in PFF, as it is in continuous depletion due to the
action of the metabolic enzymes, its excretion as well as its uptake. Moreover, the
cardiovascular compartment is of great importance as numerous
neuropharmaceutical peptide areas are available in the analogous form of the
endogenous peptides along with its capacity to interact with two other carriers
such as proteins and peptidases. Moreover, it must be emphasized that the level,
which these comprise, can be regulated by enzymes, as well as proteins as the change
in the disease takes place along with the variation in species. Insulin-integrate
growth factor (IGF)-1 that is usually comprised of approximately 70 amino acids
is further assimilated into the binding locations found within the plasma; each
location however has its own different constant for binding. One of the
IGF-binding proteins has demonstrated changes due to its IGF level regulation as
well as changing disease state. The efficiency of the peptide is affected by various
endogenous peptides, which are present in the serum as well as in the blood vessels.
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 509

Example includes the peptide of amino peptidase A, which exists in the serum, to
beat the high level in pregnant women than non-pregnant women. The presence of
BBB also sets parameters for the peptide delivery to the brain. It is because it exists
at the brain micro-vascular capillaries at the endothelial cell level. The resistance
exists because of its integration of the increased level of resistance from the
transendothelial electrical resistance, i.e., 2000 cm in comparison with the peripheral
vessels where it is 3–30 cm. Moreover, the number of endothelial cells in BBB is
low, which reduces the vesicular transportation. BBB also causes neuropeptide
degradation due to its inclusion of the proteolysis enzymes encompassing amino
peptidase A, amino peptidase M, and angiotensin-converting enzyme. In addition,
alkaline phosphatase, glutamyl, transpeptidase, and monoamine oxidase enzymes
are also present in it, which increase the micro-vessel level in the brain and may or
may not be expressed in the peripheral vessels at low levels. It also contributes to the
significant role in the brain homeostasis as well as in several transport systems
allowing the entrance of the substance toward the brain, where large substrates are
present in each of these systems inclusive of several peptides, which can be
transported actively.
Peptide holds relatively strong potential for therapeutic regulation for prevention
against gene and treatment, though it remains limited as a drug due to various
obstacles. In addition, the drug formation of peptide specifically developed for the
disorders related to the central nervous system has remained limited, as a result of
BBB. The peptide drug development and its delivery are being enhanced, which
improves the delivery of the drugs. Previously it was not possible, such as the
concept of the particularly directed antibody vectors as well as glycosylation as its
effect is not only confined to the enhancement of the pharmacological profile of the
drugs but also increases the transportation of the BBB to a particular region in the
brain. The drug delivery of the neuro-peptide can be demonstrated by the utilization
of the vector-mediated delivery, which is a promising area for improving the peptide
targeting, along with gene, where the possibility of success remains high. The
application of these methods is of great assistance in chemotherapeutic treatment
of disease related to the chronic CNS such as Parkinson disease, along with the
effective delivery of neuroprotective agents in the case of acute disorders such as
strokes. To enhance the bio-distribution of the brain, glycosylation is found to be an
effective approach, which improves the stability and mitigated clearance as well as
enhance the transport of the BBB. The enhancement of the opioid peptide related to
the analgesia serves as a great area for recognizing the potential engraved in the
glycosylation strategy. The strategy is also reflected as a source, which can improve
the drug administration for a long period of time, for treating depression or patients
suffering from chronic pain. It is projected that peptide-based CNS drug may prove
to be a composite of more similar methods, of which its discovery through more
research and development in this area continues.
510 N. A. Nimer et al.

15.4 Classification of Neuropeptides

It is often tricky to classify these types of peptides with the discovery of endocrine
peptides and neuropeptides, as well as their area of production and their target. This
gives rise to various approaches to the classification of neuropeptide; some classifi-
cation of neuropeptide are illustrated below (Table 15.2).

15.4.1 Hypothalamic Hormones

The hypothalamus is a part of the brain containing various neurons responsible for
the release of numerous hormones. It is located just above the brainstem, below the
thalamus. The hypothalamus is present in all vertebrates and in humans, and its size
almost the same as that of an almond. The hypothalamus is involved in various
regulatory mechanisms and other related functions of the autonomic nervous system;
it produces and releases neuropeptides and neurohormones. Due to the presence of
receptors, there is an increased interest in extra-pituitary actions of these
neurohormones in several non-pituitary tissues. Furthermore, the presence of

Table 15.2 Classification and examples of neuropeptides

Classification of neuropeptides
Pituitary hormones • LH: luteinizing hormone
• β-Endorphin
• PRL: prolactin
• GH: growth hormone
• FSH: follicle-stimulating hormone
• TSH: thyrotropin [thyroid-stimulating hormone]
• αMSH: α-melanocyte-stimulating hormone
• ACTH: adrenocorticotropic hormone
Neurohypophyseal peptides • Oxytocin
• Vasopressin
Circulating peptides • Angiotensin
• Bradykinin
Hypothalamic releasing factors • GHRH: growth hormone that is used to release another
hormone
• Somatostatin
• CRH: corticotropin-discharging hormones
• TRH: thyrotropin-releasing hormone
• GnRH: gonadotropin-discharging hormone
Neuronal and endocrine • Vasopressin
peptides • Oxytocin
Opiate peptides • Dynorphin
• Leu-enkephalin
• Met-enkephalin
• β-Endorphin
GI and pancreas peptides • Glucagon
• PP: pancreatic polypeptide
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 511

receptors in immune cells suggests a paracrine or autocrine role throughout the

immune system (Quintanar and Guzman-Soto 2013). These kinds of peptides are
usually called hypothalamic or releasing hormones, which accelerate or restrain the
release of pituitary hormones. The function of the hypothalamus includes regulating
the temperature of the body, parenting and attachment behaviors, hunger, thirst,
sleep, tiredness, and the circadian clock. The released peptides are discharged into
the blood through the network of capillaries, which route quickly to portal veins
present in the second capillary bed located in the pituitary gland anterior lobe, where
it exerts its effects. The neuropeptides are secreted in periodic spurts, and that is the
reason why replacement hormone therapy with the help of these hormones fails
because they do not work unless the replacements are done with spurts as well. It is
important for notifying the number of identified neuropeptides, which far exceeds
the number of classical neurotransmitters (Siegel 1999).

15.4.2 The Pituitary Gland and the Related Hormones

The pituitary gland is placed beneath the brain, in the midline pocket of fossa, which
is a small space or cavity in the sphenoid bone. This cavity is also called the “sella
turcica” or the “Turkish Chair”; it is a saddle-figured cavity found in the sphenoid
bone of the human skull and also in other Hominidae skulls, which are the great
family of primitive apes, chimpanzees, gorillas, and also orangutans. The pituitary
gland in humans is made up of two lobes, namely, posterior and anterior lobes.
However, the posterior lobe includes one third of the given area, while the anterior
lobe is based on two thirds of the area. The plain radiography has been replaced with
the availability of modern radiological techniques of the sella turcica to examine
hypothalamo-pituitary abnormalities (Tekiner et al. 2015).
The posterior lobe of the pituitary gland is a lump located underneath the
hypothalamus on the bottom of the brain. The neurons present in the hypothalamus
gland are launched directly to the posterior lobe which are associated with the
pituitary gland. Approximately 100,000 axons are projected from the hypophyseal
nerve tract. The nerve terminals and axons from hypothalamic neurons constitute the
posterior lobe of pituitary gland. The hormones stored in the terminals are secreted
by electrical excitation. Moreover, the altered astrocytes which are also called
pituicytes surround the nerve terminals. The modified astrocytes, known as
pituicytes, surround the nerve terminals, which assume to have a significant role in
the local control of hormone release (Nussey and Whitehead 2013). Various
hormones are produced by pituitary cells and secreted into the bloodstream affecting
other organs of the body. It is also referred to as “the master gland” because it
releases various kinds of peptide hormones which regulate the functions of many
other mechanisms.
The pituitary gland is responsible for sensing the needs of the body, where it
sends signals to different organs and glands throughout the body to regulate their
function and maintain an appropriate environment after sensing. The hormones
secreted by pituitary gland act as messengers after being released in the bloodstream.
512 N. A. Nimer et al.

These messengers are responsible for transmitting information from the pituitary
gland to distant cells, regulating their activity. Spontaneous electrical activity is
enough for driving the intracellular calcium concentration exceeding the threshold
for stimulus transcription and stimulus secretion coupling in some cells. The func-
tion of these action potentials is to retain the cells with cytosolic calcium in a
response state near the threshold phase (Stojilkovic et al. 2010). Some of the
pituitary gland hormones have been indicated below with their functions:

• Prolactin—acts on the breasts and stimulates the mammary glands to

produce milk.
• Other hormones are secreted to stimulate the adrenal gland, thyroid gland, testes,
and ovaries that in turn stimulate other hormones.
• The hormones released by the pituitary gland are responsible for controlling
metabolism, sexual maturation, blood pressure, reproduction, growth, and other
vital physical processes and functions.

15.4.3 Tachykinins

The tachykinin peptides are related to an extended family of neuropeptide, which is

found in a large number of species in both mammals and amphibians. The name
tachykinins is derived from their ability to timely stimulate the contraction of the gut
tissues. Its family can be distinguished by C-terminal alignment of “Phe-X-Gly-Leu-
Met-NH2.” Here, X represents the aromatic amino acid. For instance, neurokinin A
(NKA), neurokinin B (NKB), first neuropeptide discovered in mammals, and sub-
stance P (SP) are included in these neurotransmitters (Majkowska-Pilip et al. 2019).
The contraction of the bladder muscle and gut, secretion of saliva in mammals, and
hypotension are caused by tachykinin peptides. The preprotachykinin genes that are
involved in encoding the precursor proteins are intertwined randomly to create
diverse set of peptides. The processing of the precursor protein is done with the
protease assistance to create smaller peptide units. The neurokinins are also included
in the family of tachykinin including pysalaemin, neurokinin B, substance P, and
eledoisin. Initially, the porcine spinal cord is the source that was used to derive
neurokinin A and B. The neurokinin receptors that include NK1 and NK3 and
neurokinins (neurokinin A, neurokinin B, and substance P) are articulated exten-
sively in the nucleus of the solitary tract (NST), where they are a part of the central
regulation of visceral mechanism. Hematopoietic regulation is the function of
neurokinin A, while neurokinin B is involved in mediating the pain of transmission.
Moreover, the structure of neurokinin A is very identical to the structure of
substance P, and it also has nearly similar biological activities like substance
P. However, neurokinin A is a potent bronchoconstrictor, and it is created by a
complicated nervous system that is found in the gut (Majkowska-Pilip et al. 2019).
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 513

15.4.4 “Novel” Neuropeptides

The latest advancement in neuropeptides technology is used in proteomics and

genomics, which has led to the discovery of neuropeptides and also newly found
functional peptides which are present in the neuron systems of various species as
well as humans. The exploitation of nanoscale chromatography merged with mass
spectrometry has even enabled the structural identification of neuropeptides in a
variety of lower-level species, which was not previously possible. A number of
bioactive peptides comprise a proline residue in the second or third position from the
N-terminus. This aspect also supports the peptide at the N-terminus from peptidase
activity. Across the animal kingdom, all these structural attributes can be widely
reported in neuropeptide sequences (Hayakawa et al. 2019). As peptides produced in
the nervous system function as both communicators and regulators of various
biological processes, they are essential in identifying how these peptides function
and how they are created. It is also important to identify the structure of naturally
produced neuropeptides to interpret the ways in which neuropeptide precursors
emerge and how they operate, as faulty production of neuropeptides or inaccurate
cell signaling can cause organism dysfunctionality and even death.

15.4.5 Putative Neuropeptides

Putative neuropeptides refer to the neuropeptide precursors and encoding genes that
are attained through a biologically active process. Not all members of the cerebellin
family meet the definition of a neuropeptide; in particular, the regulated discharge
has not been recognized for each member of this family. The names of the genes are
thus abbreviated using the standard nomenclature for genes and their given localiza-
tion in the human genome. Neuropeptides and their receptors are concerned in
behaviors, specifically in mammals, which include sleep and feeding. Neuropeptide
functions are yet not understood, despite their clear roles in synaptic behavior and
signaling (Nathoo et al. 2001).

15.5 Tachykinin-Related Peptides: Substance P

The tachykinin family is made up of approximately 12 amino acid peptides

characterized by a carboxyl terminus, represented as Phe–X–Gly–Leu–Met–NH2.
Here X can be referred to as branched aliphatic particle (Schöppe et al. 2019;
Pennefather et al. 2004). Tachykinin further involves different peptides like
neurokinin B (NKB), substance P (SP), neurokinin B (NKB), and endokinins that
are structurally related to peptides. The peptides are found in abundance and are
expressed in the cell system in the body (Lorente et al. 2018) and thus help in
regulating multiple cell activities including activation of postsynaptic receptor and
neurotransmitter release (Chi et al. 2018). In particular, they serve as agonists with
different binding forces for three different receptors of neurokinin (NK1R, NK2R,
514 N. A. Nimer et al.

and NK3R) that belong to the grand categories of G protein-coupled receptors

(GPCRs) (Yin et al. 2018). Therefore, NK1R represents a complicated structure of
neuropeptide (Steinhoff et al. 2014; Schöppe et al. 2019). Lorente et al. (2017)
indicated that the peptides belonging to the category of tachykinin play a fundamen-
tal role in various psychological processes of different mechanisms including gas-
trointestinal, respiratory, immune and nervous system and dermal and urogenital
system. This further creates a significant impact on nociception pain, muscle con-
tractility, gastrointestinal proliferations, and epithelial secretions. These peptides are
further involved in generating various health-related problems including chronic
pains and inflammations, functional disparities of the urinary bladder and intestine,
cancer injuries, bronchial asthma, etc.
In mammals, substance P was identified as the prime member of the tachykinin
family. The given peptide was first discovered in 1931 from the intestine and brain of
a horse and thus was provided in a study that discusses the impact of intestinal
contractility (Chi et al. 2018). Such pieces of evidence are significant in identifying
the role and significance of NK1R and SP in the process of generating pain and
neurogenic inflammation in the intestinal muscles (Lorente et al. 2018). The study
further went on to provide the major cause of peripheral vasodilation.

15.5.1 Synthesis and Degradation of Substance P

In various cases, tachykinins are extracted in the form of an enzyme from the
existing proteins. This helps in the formation of products that are biologically active
and functional. The neuropeptides can be achieved through the alternate processing
of three significant TAC genes. The genes are further encoded by preprotachykinin
genes, where the preprotachykinin A (TAC1) gene helps in encoding the sequences
of neurokinin A (NKA) and SP. In contrast to the two, the third category (TAC3)
helps in the uniformity of neurokinin B (Kalina et al. 2018). Besides, α-amidation is
generated through the bio-activation of SP that is integrated through α-amidating
monooxygenase and peptidylglycine. The α-amidation is functionally limited to
provide α-amidated peptides. Other enzymes such as the angiotensin and endopepti-
dase help in altering or minimizing the impact of α-amidation (Scholzen and Luger
2004; Mendlewicz et al. 2005).
The degradation and synthesis of substance P is shown in Fig. 15.3. Plasma levels
of SP in humans ranges between 30 pg/ml and 500 pg/ml (Bondy et al. 2003; Lee
et al. 1997; Reynolds et al. 1988).

15.5.2 Structure of Substance P

Schank and Heilig (2017) illustrated substance P as an 11 amino acid undecapeptide.

However, the sequence of the given amino acid is provided as H-Arg-Pro-Lys-Pro-
Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (Chang et al. 1971). SP shares a carboxyl
terminal with other tachykinins in the same family; however, it differs from those
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 515

Neurokinin A

TAC1
Substance P
(Inactive)
Peptidylglycine α-amidating monooxygenase

Substance P
(NK1R)
(Active)
Angiotensin-converting enzyme Neutral endopeptidase
Carboxyl Terminal Enzyme C4

Degradation Amino
Terminal

Fig. 15.3 Synthesis and degradation of substance P

Fig. 15.4 Molecular structure of substance P (Lu et al. 2015)

tachykinins in terms of its pathophysiologic activity and receptor subtype affinities

(Lu et al. 2015; Chi et al. 2018). The molecular formation of substance P is
illustrated in Fig. 15.4.

15.5.3 Substance P Receptors

Substance P binds to NK1R, NK2R, and NK3R receptors in target tissues (Yin et al.
2018), having the highest affinity to NK1R; the biological effects of SP are thus
mainly mediated through this receptor (Chi et al. 2018).
There are two isoforms of NK1R, which are derived from differences in the
tachykinin receptor gene and are different depending on the cytoplasmic tails of
the C-terminal. The number of amino acids is also different, as one of the categories
consists of 407 amino acids. The second, i.e., the truncated receptor, is based on
516 N. A. Nimer et al.

Fig. 15.5 Full-length

isoform NK1R

311 receptors. The bonding power of SP is ten times higher for the receptor available
in full length in comparison to the truncated receptor (Chi et al. 2018). The isoforms
are different depending on their impact over the metabolic functions of cell and thus
result in separate pathways (Steinhoff et al. 2014; Lai et al. 2008). The long isoform
takes just 1–2 min to phosphorylate extracellular-regulated protein kinases, while
this cellular response is not observed until considerable time, at least 20 min, has
elapsed after exposure to SP in cells expressing truncated NK1R (Chi et al. 2018).
The mediation of complete NK1R helps in cyclic adenosine monophosphate
(cAMP), while Ca2+ helps in the formation of binding protein. It further helps in
increasing the mechanism of gene transmission. The independent mechanism of Ca2+
helps in signaling the truncated NK1R of the C-terminal (Lai et al. 2008).
The formation of the transmembrane domains is initiated through a NK1R
polypeptide chain that is functional by seven times providing seven different trans-
membrane domains. It is further connected to extracellular and intracellular loops
(i.e., EL1, EL2, and EL2 and C1, C2, C3). However, the extracellular terminal is
found in amino (N) terminal of the receptor, while intracellular terminal is located at
the carboxyl (C) terminal of the receptor. G protein interacts with cytoplasmic
regions of the receptor, especially around loop C3, connecting the fifth and sixth
transmembrane domains. The binding sites of antagonists and agonist are found in
the second and third transmembrane domains (Chi et al. 2018). Figure 15.5
illustrates the layout of full-length NK1R.

15.5.4 Signaling Pathways Integrated Through Tachykinins

and NK1R

Different types of G proteins (Gαs, Gαi, Gα12/13, Gα0, Gαq) that are attached to the
receptor are functional in demonstrating the intracellular pathways through
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 517

substance P (Garcia-Recio and Gascon 2015). Besides, the replacement of guanosine

diphosphate (GDP) on α-subunit takes place through the activation of G protein. The
process however is integrated through the allocation of SP at extracellular surface.
This helps in releasing the Gα and Gβγ units through the separation of G protein. The
process further modulates different enzymes to liberate the secondary source of
messengers such as DAG and IP3. These secondary messengers are responsible
for further metabolic activities in the cell. The terminal tail of the cytoplasmic
receptors comprises various threonine residues. However, the hydroxyl (-OH)
groups of the residues can be phosphorylated and are connected to the diminished
receptor G protein to promote receptor endocytosis (von Zastrow 2018).

15.5.5 Physiological and Pathological Role of Substance P

Tachykinin-related peptides such as SP participate in multiple important physiolog-

ical processes, and its major purpose is to regulate different functions related to
gastrointestinal, urogenital, immune, and nervous system. It further includes the
mechanism of epithelial secretion, inflammations, and contractility of muscles along
with proliferation (Lorente et al. 2018). The molecular bases of different processes
that are related to the human pathologies include the mechanism of SP-NK1R that
helps in maximizing the amount of serum. Moreover, a significant contribution in
various pathological activities including chronic or acute inflammation such as
functional disparities of urinary bladder and intestine can be provided through
receptors that are found in different cells (Lorente et al. 2018). Other pathological
activities include infectious diseases (respiratory syncytial receptors and acquired
immune deficiency syndrome), migraine, cancer, anxiety, depression, etc. (Campbell
et al. 2006).

15.5.5.1 Central Nervous System

The central and peripheral systems are rich with the distribution of receptor and SP
(Satake and Kawada 2006). SP is however located at unmyelinated primary sensory
neurons of the nervous system. It is further released through the brain stem and the
spinal cord; this helps in developing the slow excitatory potential in the target
neurons. The sensory fibers further help in the transmission of nociception and
noxious stimuli while releasing different enzymes and peptides such as glutamate
and substance P. Therefore, SP serves as a fundamental functional position in terms
of binding NK1R and functions as the transmitter of neuronal sensors that helps in
providing different responses to stress-related problems. It is further important in
performing other functions, as it is located at the central nervous system that is not
associated with pain pathways. In addition, SP/NK1R system plays a role in the
pathophysiology of depression and anxiety disorders, epilepsy, and migraine (Yin
et al. 2018; Wang et al. 2017; Chi et al. 2018; Gray 2018).
In various cases, SP serves as a neuromodulator that is functional in the transmis-
sion of sensory neurons associated with anxiety, stress, emesis, and depression.
Other important regions of the brain (area postrema and the nucleus tractus
518 N. A. Nimer et al.

solitarius) help in providing spontaneous responses related to vomiting (Garcia-

Recio and Gascon 2015).

15.5.5.2 Cardiovascular System

The cardiovascular cell system consists of substance P that helps in creating a
significant impact over neurophysiological activities that include systematic regula-
tion of heart rate. The information however is provided through different activities,
such as blood pressure monitoring, stimulation of cholinergic neurons, and elasticity
of vessels. It further plays a crucial role in atrial myocardial electrophysiology, and
intact SP levels protect against the development of atrial fibrillation. Besides, the
frequent occurrence of atrial fibrillation helps in reducing the SP levels. It also helps
patients that have undergone coronary bypass surgery in generating the atrial
fibrillation that is also initiated through the low levels of SP serum (Veldkamp
et al. 2018a). Other pieces of evidence provide the implication of SP in different
stress responses integrated through reperfusion, ischemia, and cardiovascular
disorders. In the past, SP has been implicated in inflammation, angiogenesis, and
the transmission of pain (Eliska et al. 2016).
In various studies of rabbits, potassium currents that are mediated through the
activation of NK3R by SP help in increasing the duration of potential functions. In
contrast, ventricular action potential duration was unaffected by NK3R activation.
The stimulation of NK3R helps in extending the overall duration of atrial repolariza-
tion of the rabbit’s heart, and as a result, the duration of arrhythmia is decreased. A
similar case was observed when the functions are implied in the human system. This
shows that the stimulation of NK3R helps in the representation of anti-arrhythmic
concepts including the non-functionality of atrial fibrillation (Veldkamp et al. 2018a;
b).
SP played a beneficial role in the improvement of left ventricular ejection fraction
recovery after myocardial infarction and led to a reduction of infarct size, reducing
inflammation and ischemia-reperfusion injury after acute myocardial infarction (Sim
et al. 2018). The findings are associated with the porcine model of myocardial
infarction.

15.5.5.3 Gastrointestinal Tract

The enteric nervous system serves as the fundamental source of SP, which is also
present in the immune and enterochromaffin cells of gastrointestinal tract mucosa
(Holzer 2013). It is mainly functional in the gastrointestinal tract; therefore, it can be
perceived that changes occurring in SP might create a significant impact in different
gastrointestinal functions. In cases where SP is incorporated through different
functions including inflammation and other pathological processes, the resulting
impact over gastrointestinal functions is usually high (Vilisaar and Arsenescu
2016; Zalecki 2019). The open distribution of NK2R suggests that the substance is
majorly involved in ensuring smooth functioning of human colonic activities (Jaafari
et al. 2008).
In addition, SP and its receptors are highly functional in managing smooth
regulation of the stomach muscles through the enteric nerve. Other major responses
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 519

include acute antral ulcerations of the stomach tissue. The increasing amount of
SP-immunoreactive Auerbach’s response in the porcine stomach helps in proving
the significant value of the given neurons for neuroplasticity, since the inflammatory
problems are highly responsive to ulcer diseases. This, however, promoted the
indirect functioning of SP in the gastric nerve pathways and submucosal neurons
to provide a massive supply of pyloric sphincters in various animals with antral
ulcerations. This indicates that majority of the issues in the given regions are often
connected to inefficient functioning of SP providing no significant contribution in
regulating the myenteric neurons of the stomach (Zalecki 2019).

15.5.5.4 Musculoskeletal System

The skeletal growth period also includes various functions of SP, provided in the
form of homeostasis of cartilage, and autocrine functions along with the regulation
of the physiological mechanism of chondrocytes (Grässel and Muschter 2017).
Various subclasses of SP including the receptors and neuropeptides are further
represented by chondrocytes and other bone cells. SP further helps in increasing
the proliferation of chondrocytes, indicating anabolic effects (Opolka et al. 2012;
Grässel and Muschter 2017). Besides, the formation rate of bones is further affected
through the reduction or loss of SP. The information thus shows that SP further helps
in regulating the osteoclast differentiation that significantly affects the remodeling of
bone (Wang et al. 2009). The function is common in the different cell types
including the mesenchymal stem cells of bone marrow, osteoblasts, B or T
lymphocytes, and synovial fibroblastic cells (Nilsson et al. 1985; Liu et al. 2007).
In murine studies, costal chondrocytes express SP and NK1Rs, and SP stimulation
dose-dependently potentiates the chondrocyte proliferation rate and induces the
formation of focal adhesion contacts (Niedermair et al. 2014).
Neuropeptides are also significant in the pathogenesis of osteoarthritis and osteo-
porosis. The pathogens are important in creating a negative influence over the
microstructure of bone and may cause severe pain (Xiao et al. 2016).
Additional mechanical load increases the expression of NK1R and endogenous
production of SP, promoting cartilage degradation (Pirosa et al. 2018). The stimula-
tion of synovial cells and SP helps in releasing the immune-regulatory cytokines and
inflammatory mediators (Campbell et al. 2006). This suggests that SP serves as an
important mediator of inflammation acting by stimulating the secretion of prosta-
glandin E2, reactive oxygen species, IL-1β, TNF-α, and SP. Thus, it further leads to
cartilage degradation, when it is incorporated in chondrocytes. The functionality of
SP along with the associated receptors was significantly high in chondrocytes and
extracellular matrix. The following functions were integrated through low exercise,
which helps in providing significance to its role that helps in providing fundamental
response of chondrocytes to the mechanical functionality. Blockades of SP signaling
using NK1R antagonists were similarly found to prevent chondrocyte responses to
mechanical stimulation (Grässel and Muschter 2017).
520 N. A. Nimer et al.

15.5.5.5 Respiratory System

Respiratory diseases such as cystic fibrosis, asthma, and chronic obstructive pulmo-
nary disease are stimulated through the involvement of substance P (Atanasova and
Reznikov 2018). In asthmatic airways, mucus content was found to be positively
related to SP expression, with NK1Rs also elevated based on extensive expression
detected on goblet cells (Chu et al. 2000). Patients suffering from chronic obstructive
pulmonary disease were provided with the maximum concentration of SP (Tian et al.
2000).
Restoring SP-mediated signaling by means of tachykinin agonists has shown to
be an ineffective strategy in cystic fibrosis. As per the pieces of evidence, it is
suggested that the deformity of cystic fibrosis transmembrane serves as the major
cause of the undue functionality of the glandular secretion that is mediated through
SP. The cystic fibrosis transmembrane plays a crucial role in the absorption and
secretion of transepithelial salt, which is integrated through different epithelial
tissues (Ianowski et al. 2008).

15.5.5.6 Immune System

SP and other neuropeptides, when released through sensory nerves and other
inflammatory cells, usually result in mast cells (Black 2002; Rosa and Fantozzi
2013) and further modulate the interactions between the immune and nervous
systems and trigger neurogenic inflammation (Manak et al. 2010).
In most of the immune disorders, substance P plays a fundamental role in
stimulating the mast cells that are often functional in pro-inflammatory activities
and thus plays various roles to regulate the immune system (Amin 2012). This
produces allergic, immune, and inflammatory responses. Mast cell stimulation
occurs either through high-affinity NK1R or low-affinity Mas-related G protein-
coupled receptors (Zikou et al. 2018). The stimulated mast cells play an active role in
allergic and non-allergic disorders based on the secretion of histamine and tryptase
alongside cytokines and chemokines (Petra et al. 2018a; b). Interleukin (IL)-33
belongs to the IL-1 family of cytokines, which helps in increasing the functionality
of mast cells by involving SP. This however results in the secretion of tumor necrosis
factor (TNF) (Zikou et al. 2018). Besides, cytokine interleukin (IL)-31 also serves as
an important factor for various inflammatory diseases that are related to pruritus,
including the mastocytosis and atomic dermatitis. Besides, various mast cells that are
regulated through both allergic and non-allergic triggers help in the secretion of
tryptase, histamine, chemokines, and cytokines (Petra et al. 2018a, b). Besides, SP is
functional in improving the production of inflammatory cytokine (TNF-α, IL-1, and
IL-6). The process is integrated through immune cells including macrophages that
are activated due to the nuclear characteristic of kappaB (NF-κB) (Manak et al.
2010).
SP and its receptor (NK-1R) also participate in HIV infection of monocyte-
derived macrophages. HIV infections are found in human immune cells, and SP
shows a significant association, since the HIV replicated through the isolated
mononuclear phagocytes is often augmented through SP, whereas the HIV infection
found in the cell is regulated through the nonpeptide SP antagonist (CP-96,345) (Lai
et al. 2001).
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 521

The SP/NK1R system potentiates numerous pathways involved in the prolifera-

tion and formation of cancerous colonic epithelium cells from normal cells (Koon
et al. 2004), and truncated -NK1R contributes to colorectal adenoma growth, with
possible participation in adenoma-carcinoma growth (Gao and Wang 2017).

15.5.6 NK1R Antagonist Pharmacology and Therapeutics

The crystal structure of human NK1R bound to a high-affinity antagonist reveals the
molecular basis of antagonist interactions and offers information toward understand-
ing ligand binding selectivity for this pharmacologically therapeutically important
family of GPCRs (Yin et al. 2018). Recently, SP and NK1R have gained attention
based on the development of complex NK1R antagonists that have many promising
therapeutic indications for issues such as depression, pain, and emesis (Campbell
et al. 2006). It has been shown that NK1R is significant in creating the impact on
different activities of the brain including emesis centers. Aprepitant which is an oral
antagonist is proved to be an impactful agent in preventing acute and delayed
chemotherapy-induced nausea and vomiting (Manak et al. 2010; Muñoz and
Coveñas 2013; Schank and Heilig 2017; Yin et al. 2018). Maximum exposure of
aprepitant is important in creating a significant impact over the physiological
activities that include the unbalance metabolism of cholesterol and cell trafficking,
inflammation, and apoptosis. Plasma levels of SP decrease when treated with
aprepitant due to a significant impact of over 176 plasma proteins and other
pathways that are important for metabolisms including lipid metabolism and inflam-
mation. The agent is further related to a significant increase in HDL cholesterol. Also
significant impact is provided to different metabolic and hematologic markers,
which are further helpful in restoring the baseline levels after 30 days of aprepitant
treatment (Spitsin et al. 2017).
The ability of aprepitant treatment is significant in coping with the blood-brain
complexities; its approval as drug to reduce chemotherapy complexities provided by
FDA helps in giving it a significant status in treating infections related to human
immunodeficiency virus (Manak et al. 2010). Cases that include the NK1R as an
antagonist help in suppressing cholesterol that in return provides significant
macrophages. This helps in protecting the metabolism from atherosclerosis (Spitsin
et al. 2017).
Other oral NK1R antagonists, including netupitant and rolapitant, cross the blood-
brain barrier and bind brain NK1R. They are highly selective and have no affinity for
serotonin, dopamine, or corticosteroid receptors. However, netupitant (300 mg) is
available only in a combination formulation with palonosetron (0.5 mg).
Fosaprepitant is an intravenous pro-drug of aprepitant that is converted within
30 min after infusion (McQuaid 2018). These NK1R antagonists are safe and well
tolerated, and most adverse events are minor and well tolerated, such as headaches
(Muñoz and Coveñas 2013), fatigue, and dizziness (McQuaid 2018).
An increasing number of studies on NK1R antagonist have been conducted which
focuses on psychiatric processes and disorders like depression, stress, and anxiety.
522 N. A. Nimer et al.

The clinical developments, however, indicated the failure of the given treatment.
Several studies in the given framework have indicated the importance of taking
NK1R drug as it has impact on the treatment of stress-related problems. Despite
greater efforts, studies remain unsuccessful in increasing the usage of drugs among
the given populations. The study further demonstrated that the inefficiency of NK1R
antagonists in treating alcohol addictions is due to the insufficient presence of
receptors. As a result of this, several developments are being made on the functions
of NK1R to complete the target of providing new pharmacotherapeutics in treating
addiction problems (Schank and Heilig 2017).
With regard to pain transition, SP receptor antagonists do not block responses to
certain types of pain, though they can modify some responses where glutamate,
which is often extracted through SP, provides a greater significance in eradicating
pain (Gray 2018). As a therapeutic agent for treating epilepsy, NK1R antagonists
reduce kainic acid-induced seizure activity, though their therapeutic potential for
treating epilepsy has not yet been fully exploited (Wang et al. 2017; Chi et al. 2018).
Additionally, human NK1R modulators have shown promise in clinical trials for
migraine and depression (Yin et al. 2018). In particular, there is a greater similarity
found between SP receptor antagonist MK-86 and paroxetine due to their anxiolytic
and anti-depressant effect that is usually attained in treating patients with unipolar
disorder (Kramer et al. 1998). The models related to the chronic obstructive pulmo-
nary disease have significantly provided decrease in inflammation in the respiratory
tract when treated through tachykinin receptors (De Swert et al. 2009). The insuffi-
cient evidence provided in this regard demonstrates that the antagonists are impor-
tant to minimize the airway responses while improving the function among patients
with asthma (Ramalho et al. 2011).
In terms of addressing tumor genesis, there is also evidence to indicate that NK1R
antagonists may have value in treating patients with colorectal adenomas (Gao and
Wang 2017).

15.6 VIP-Glucagon Family: Vasoactive Intestinal Polypeptide

(VIP)

Vasoactive intestinal polypeptide (VIP) belongs to superfamily of glucagon that are

similar to neuro and endocrine peptides. VIP further consists of 28 amino acids.
Furthermore, the given peptides are present in the human body and are expressed
through their functionality. Also, they are important in performing significant
biological functions in the form of muscle relaxation, secretion of electrolytes and
water in intestine, and cerebral and coronary artery vasodilation along with increased
myocardial contractile performance (Dickson and Finlayson 2009; Henning 2013).
At an initial level, VIP was characterized as the intestinal hormone, which
represents its isolation from digestive tract, while acting to be fully functional in
the intestinal tract for the secretion of electrolytes. Later, it was shown that VIP is
extensively distributed in multiple types of tissue as a neurotransmitter. Besides, the
structural formation of VIP occurs when released from different sources including
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 523

the endocrine cells, the digestive tract, and the central nervous system. VIP is further
important as it regulates secretion and blood flow in both the intestine and pancreas.
This helps in increasing the gastrointestinal motility due to the display of similar
effects in urological, respiratory, and cardiovascular system. Reports provided for
this neuropeptide have recently identified a wider range of activities, highlighting its
neuroprotective and immunological effects (Dickson and Finlayson 2009; Hisato
Igarashi et al. 2011). The ligands along with the Class II G protein-coupled receptors
have a great therapeutic potential, as suggested by clinical data along with similar
investigations (Chapter et al. 2010). Still, it is important to analyze the developments
of applications in various ongoing disease and pathological disorders, along with the
structural and functional value of neuropeptides and its receptors (Hisato Igarashi
et al. 2011). The existing information related to VIP and its receptors involves
several pharmacological and clinical findings that are relevant to the existing
developments in the given problem. Therefore, the following sections provide a
detailed description about the given idea.

15.6.1 Structure of Vasoactive Intestinal Polypeptide

As mentioned earlier, VIP acts as a linear peptide that is composed of 28 different

amino acids. The molecular weight of VIP is 3326 which is provided by neural
tissues; however, the sequence of amino acid is illustrated in Fig. 15.6. VIP is based
on the structural family that includes various neuropeptides and hormones such as
glucagon, growth hormones, secretin, helodermin, glucagon peptide 1 and 2, etc.
However, the most homologous peptide to VIP is pituitary adenylate cyclase-
activating peptide (PACAP), based on 70% identity at the amino acid level (Vaudry

VIP

PACAP

VPAC1 VPAC2 PAC1

-Neuroprotecon -Regulates circadian -Mediates light-induced behavior

rhythm and gene expression
-Regulates producon of -Facilitates glucose homeostasis
inflammatory cytokines -Regulates producon of
-Contribute to insulin inflammatory cytokine
sensivity

Fig. 15.6 Overview of VIP and PACAP and their receptors

524 N. A. Nimer et al.

1
2
Ser
His Nh2 N-terminus
3
Asp

4
Ala 5
Val 6
Phe β-bends

Thr
8
21
Asp 22
9 Lys 20
Tyr
Asp 23 Lys
19
10
Leu Val
Tyr 24
Asn
11 Thr 18 Ala

12 17
25 Arg
α-helix
Ser Met
13 16
Leu 14 15
Ile Gln

26 Leu Arg Lys

27 Asn
28

Fig. 15.7 Helical conformation of the secondary structure of VIP

et al. 2009; White et al. 2010). Figure 15.6 demonstrates the high degree of the
amino acid sequences homology shared by both VIP and PACAP peptides.
Analyses of VIP using nuclear magnetic resonance spectroscopy (NMR)
provided a helical conformation with α-helix and two β-bends provided at the
given residues of 11–26 and 2–5 and 1–10, respectively. The helical conformation
is located at the N-terminus as indicated in Fig. 15.7. The two domains including the
C-terminal and N-terminal are important, as they play a significant role in managing
bio-activity and receptor recognition (Igarashi et al. 2011).
In 1995, cloning and mapping of a VIP gene was identified in humans and is
placed at chromosome 6q25. The structural formation of a human VIP gene is based
on 7 exons that are incorporated with 6 introns along with the spans of 9 kb. The
gene is further significant in providing the two peptides that are biologically active
and highly functional. The formation occurred through the incorporation of
170 amino acid preproteins tailored in a proteolytical manner. The two resulting
peptides are known as peptide histidine methionine (PHM) and VIP (Dickson and
Finlayson 2009; Dorsam et al. 2011).
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 525

15.6.2 Vasoactive Intestinal Polypeptide Receptors

VIP is based on two definite receptors including VPAC1 and VPAC2 that are
mutually connected through PACAP and VIP to a certain extent. The given receptors
are important and thus belong to a class B group of the G protein that are further
coupled through seven different transmembrane receptors. It further consists of
approximately 437–459 residues of amino acids followed by a long chain of
N-terminal (Laburthe et al. 2007; Vaudry et al. 2009). The availability of
N-terminal with a structural representation of N-cap indicates that it is highly
involved in the stimulation of receptors and could be implicated in providing
important developments in terms of treatment design that targets different
membranes of B GPCRs and VPAC receptors (Neumann et al. 2008). However,
the α-helical conformation is usually indicated through the binding of peptides along
with certain specific receptors (Laburthe et al. 2007; Vaudry et al. 2009).
The examination regarding the distribution patterns of VPAC1 and VPAC2 is
provided in abundance. The allocation of VPAC1 specific to certain regions of the
central nervous system includes the piriform cortex, putamen, choroid plexus, lateral
amygdaloid nucleus, etc. Besides, the functionality of VPAC1 is provided in
kidneys, small intestine, liver, mucosa of the stomach, prostate gland, spleen,
lymphocytes, mammary glands, and many other tissues. Contrary to this, VPAC2
is extensively found in smooth muscles of various organs and vascular walls that
specifically include the large intestinal mucosa, heart, retina, pancreas, adrenal
medulla, alveolar epithelium, testis, and adipose tissue. VPAC2 when functioning
in the central nervous system is usually found in the cerebral cortex, amygdala,
hippocampus, olfactory brain, paraventricular cortex, and thalamus (Hisato Igarashi
et al. 2011).
VIP and PACAP receptors are stimulated by several endogenous peptides that
vary in their affinities such as VIP, PACAP-38, PACAP-27, peptide histidine
methionine amide (PHM), peptide histidine isoleucine amide (PHI), and peptide
histidine valine (PHV). Similar binding strengths have been identified in VIP and
PACAP by both receptors. However, PACAP27 and PACAP38 with 1000-fold
reflected maximum affinity in comparison to VIP that serves as the antagonist of
isoforms found in the PAC1 receptor (Henning 2013). VIP and PACAP are impor-
tant in maintaining the physiological mechanism of several systems that contribute
to important pharmacological effects in the human body as described in the sections
below.

15.6.3 Mechanisms of Physiological Action of Vasoactive Intestinal

Polypeptide

The peripheral and central nervous systems and gastrointestinal tract are comprised
of VIP neuropeptides that are found in abundance. The given neuropeptide is crucial
in functioning as a neurotransmitter and neuromodulator for different physiological
functions including ion transportation, neuronal functionality, and mucosal
secretions. It further helps in vasodilation in endocrine, cardiovascular, pancreatic,
526 N. A. Nimer et al.

Table 15.3 Some actions of VIP in different biological systems

Biological
system Effect
Cardiovascular It helps in coronary and peripheral vasodilation and thus creates a positive
system impact in terms of chronotropic and inotropic effects
Respiratory Homeostasis of the respiratory system and exerts a potent vasodilatory and
system bronchodilatory effect
Endocrine It helps in the regulation of pituitary functions such as releasing of growth
system hormones, FSH, T3 and T4 thyroid, PRL, ACTH, adrenal and testosterone,
TSH, vasopressin, and PRL
Pancreas It releases hormone from the pancreas and further serves as a stimulator to
insulin
Immune system It controls the homeostasis of the immune system. Furthermore, it is
indicated as a potent anti-inflammatory factor

respiratory, and urological systems. VIP neuropeptide is highly important as it

regulates the mucosal inflammatory response, promotes gastric acid secretion,
moderates hemodynamic regulations, etc. (Vu et al. 2015; Takei et al. 2016).
Some of these physiological activities are summarized in Table 15.3.
VIP basically mediates three proven major physiological effects including (Abba
Kastin 2013):

• Relaxation of blood vessels and smooth muscles by inducing the release of nitric
oxide (NO)
• Enhancement of electrolytes and diaresis by increasing urine volume and enhanc-
ing fractional excretion of sodium, chloride, and potassium
• Regulating the neuroendocrine and endocrine functions

The vasodilatory impact generated by VIP in vascular muscles is based on the

ability of VIP to provide increased concentration of vascular cyclic AMP. This helps
in triggering the process of protein kinase A and thus promotes the sequestration of
Ca2+ through sarcoplasmic reticulum. Besides, it is important to maximize the
extrusion of Ca2+ into the extracellular space. According to previous studies,
vasorelaxant impact of VIP is based on endothelium that is found in the aorta,
uterine artery, and intrapulmonary artery. It is further mediated through the func-
tioning of lipoxygenase found in the aorta of rat. Other important components
include nitric oxide that helps in the activation of guanylyl cyclase that is usually
found in the uterine artery of humans (Henning and Sawmiller 2001). Several pieces
of evidence indicated that neuropeptide VIP is significant in regulating the cardiac
rhythm of the heart. The abnormality of the cardiac function results in cardiac
pathology. Therefore, VIP serves as the major tool in regulating the physiological
process of the heart that includes the suprachiasmatic nucleus that helps in regulating
the cardiovascular function (Schroeder et al. 2010).
Immunocytochemical evidence suggests that VIP functions as a vagal neurotrans-
mitter in pancreatic nerve fibers and plays a potential role in triggering the release of
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 527

hormones from the pancreas. It is further important in increasing the flow of blood
through vasodilation (Dickson and Behrman 2013; Chandra and Liddle 2015). VIP
and its close relative PACAP also promote insulin and glucagon release through
several ways, such as the secretion of insulin through cAMP coupling metabolism,
extracellular signal-regulated kinase (ERK) pathway along with signaling of P13K,
β-adrenoceptors. Others include the mobilization of Ca2+ that is integrated through
the intracellular stores of the gastrin-releasing peptide (Röder et al. 2016).
Studies further indicated that the anterior pituitary and hypothalamus are used to
secrete and synthesize VIP. Also, it helps in making accurate regulations of the
pituitary functions including FSH, LH, TSH, and prolactin, T3 and T4 thyroid, and
gonadal and adrenal hormones, in response to acute inflammation provided through
LPS (Bik et al. 2004). Immunoreactive VIP that is found in the hypothalamus and
pituitary usually increases due to the treatment provided through estrogen and
adrenalectomy. Moreover, the immunoreactive VIP decreases followed by the
hyperprolactinemic states. During the duration of sexual maturation and lactation,
the levels of VIP mRNA in the hypothalamus are significantly high. Results however
provided the physiological role of pituitary and hypothalamic VIP gene expression
in relation to its fundamental function as neuroendocrine hormone. Moreover, VIP
stimulates the release of luteotropic hormone called pituitary prolactin (PRL) from
the pituitary at different levels that can easily be obtained from the hypophyseal-
portal blood, while including other pituitary functions, such as release of growth
hormone, ACTH, and vasopressin, and stimulates the release of catecholamine from
the adrenal medulla.
The control of the PRL secretion is integrated through the endogenous VIP found
at multiple locations. This further helps in maintaining firm interaction with other
moderators of the PRL secretion that include prostaglandins, cholecystokinin, sero-
tonin, and oxytocin (Chaiseha et al. 2010; Blanco et al. 2013).
VIP serves as the fundamental regulator of neuropeptide that is extensively
distributed in both peripheral and central nervous system. The representation of
the significant type of endogenous sponsors to the formation and regulation of the
immune variances in various CNS immune organ, further helps in controlling acute
inflammation in various peripheral immune organs (Ganea et al. 2014). Provides an
important recognition to VIP in terms of serving as a valuable anti-inflammatory
factor in adaptive and innate immunity. The production of pro-inflammatory
cytokines and chemokines from dendritic cells, microglia, and macrophages is
integrated through VIP. The process is highly common in innate immunity. Also,
VIP diminishes the expression of costimulatory molecules on the antigen-presenting
cells and consequently decreases the activation of T cells that are highly specific to
antigens. In the case of adaptive immunity, VIP promotes the responses provided by
Th2 and helps in reducing the Th1 type of pro-inflammatory responses (González-
Rey et al. 2004).
VIP is significantly functional in the homeostasis of the respiratory system and
employs a potent vasodilatory and bronchodilatory consequences in the system. It
further encourages the secretion of mucous that is headed through the tracheobron-
chial submucosal glands. Through several clinical trials and research studies, it can
528 N. A. Nimer et al.

be concluded that VIP-inhaled agonists are significantly functional in respiratory

therapy (Mathioudakis et al. 2013).

15.6.4 Role of Vasoactive Intestinal Polypeptide in Pathogenesis

of Disease

Vasoactive intestinal polypeptide has a significant biological role in health and

disease and may serve as an important determinant in the pathogenesis of
neurodevelopmental, allergic, gastroesophageal, and osteoarthritis diseases. Several
studies indicated that up-regulation of VIP can counteract the effect of
pro-inflammatory stimuli and is valuable in minimizing the pain in osteoarthritis
(OA) which is a chronic and degenerative bone disease that is commonly represented
through disabilities in aged population. The important knowledge regarding VIP is
that it is effective in preventing the damage of chronic cartilages along with
remodeling activities of the joints. Through various pieces of evidence, it is indicated
that VIP is regulated at minimal levels in the synovial fluids of OA. This helps in
increasing the level of production related to pro-inflammatory cytokines that are
significant in contributing toward OA pathogenesis (Jiang et al. 2016).
Past studies have shown that VIP is important in the pathogenesis of bronchial
obstruction throughout the period of exacerbation of the disease (increase in the
severity of a disease or its signs and symptoms). Elevated levels of VIP and other
neuropeptides were found in the serum of asthmatic patients during exacerbation
period of the disease, indicating its important clinical significance in asthmatic
patients (Semernik and Lebedenko 2015). Recent review provides an updated
understanding on the functions and the value of VIP in treating various allergic
diseases including asthma, allergic rhinitis, and dermatitis. The immunomodulatory
impact of VIP is reflected in the mononuclear leukocytes of the peripheral blood.
Moreover, various subjects suffering from diseases such as asthma and allergic
rhinitis were found to have various neuropeptides such as morphine, SP, VIP, and
ACTH. The symptoms were only prominent in the form of VIP in both allergic and
normal subjects. Other than this, the current evidence is significant in providing
practical information of the integration of VIP in various skin inflammatory
disorders reflecting the pathophysiological background of such diseases. The partic-
ipation was evaluated through improvised proliferation and keratinocyte production
of cytokine. The pathogenesis of inflammatory dermatosis indicates the significance
of cytokine structures around keratinocytes (Verma et al. 2017).
The most recurrent gastrointestinal disorder, i.e., the gastroesophageal reflux
disease (GERD), is developed through acid reflux and heartburn. GERD pathogene-
sis is also developed through the increasing levels of VIP serum. Relaxants that are
facilitated through VIP often have a significant impact on lower esophageal sphinc-
ter (LEP), which interacts with the formation of harmful acids. These acids in return
increase the level of nitric oxide (NO) that is responsible for providing low pressures
for LEP (Kassim et al. 2002).
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 529

Recently, in vivo test was used to test the interneurons that function through VIP
and are mostly found in post-natal development of the cortical circuits. The removal
of the early postnatal ErbB4 helped in dysregulation of the VIP interneurons. In
some cases, it appears during the growth period which is visible in creating
digressions in their functioning. It further leads to high dysregulations in the
relationship between cortical, temporal, and state dependence. The data however
provides valuable insight about the function of VIP interneurons in the formation of
cortical circuit and is also important in creating a significant impact over the
pathophysiology of neurodevelopmental disorders (Batista-Brito et al. 2017).

15.6.5 Pharmacology of Vasoactive Intestinal Polypeptide

Vasoactive intestinal peptide belongs to the superfamily of various peptides that are
structurally related and thus includes growth hormone-releasing hormones, secretin,
gastric inhibitory peptide, glucagon, and glucagon-related peptides.
The peptide is important as it exerts its action through VPAC1 and VPAC2
secretin receptor-like. Progress in characterizing the functions of these receptors
has been delayed due to the insufficient availability of drugs. Literatures identify
several antagonists and agonists related to the VIP receptors. [Ala11,22,28] VIP and
[Lys15, Arg16, Leu27] VIP (1–7)/GRF (8–27)-NH2 are certain agonists of the VPAC1
receptor and PG 97–269 is a selective antagonist. The most selective identified
VPAC2 agonist is Ro 25–1392; but on the other hand, myristoylation provides a
-K-K-G-G-T sequence of the amino-terminus for VIP (1–26) [K (12)]. It further
provides the extended carboxyl-terminal of Ro 25–1553 that is a specific antagonist
of VPAC2 receptor antagonist. The molecule provides a VPAC2 receptor antagonist
that is found in humans and is widely present (Moreno et al. 2000; Dickson et al.
2006; Harmar et al. 2012).
Cardiopulmonary system highly reflects the activities of VIP through various
pharmacological functioning that includes; anti-inflammatory actions of, potent
improved blood circulation within the region of lungs and heart, potent airway and
dilatory actions, etc. (Wu et al. 2011). Since VIP functions as a neurotransmitter, a
significant impact is created in the form of vasodilatory and potent bronchodilatory
functioning. Also, it helps in the secretion of mucus through submucosal glands. VIP
functions as immunomodulator, for instance, in suppressing the humoral immune
response, inhibition of bronchial and vascular inhibition, and diminishing the
extracts of cigarette smoke resulting in the loss of L2 alveolar cells. Recent investi-
gation indicates a potential therapeutic role of a novel stabilized inhaled VIP agonist
with minimized side effects dealing with a variety of lung diseases such as pulmo-
nary hypertension, cystic fibrosis, asthma, chronic obstructive pulmonary diseases,
etc. (Mathioudakis et al. 2013).
VIP plays a fundamental role followed by various functions such as the regulation
and control of timing of circadian rhythms, memory, and learning along with various
stress responses. The present study relates VPAC2 receptor in providing vulnerabil-
ity to schizophrenia. VIP, in various peripheral regions, plays a significant role in
530 N. A. Nimer et al.

dealing with inflammation and immunity controls and the emancipation of

catecholamines from the adrenal medulla and functioning as a co-transmitter in
both sensory and autonomic neurons (Harmar et al. 2012).
The endogenous VIP is further important in controlling the composition of body
mass and secretion of important hormones that regulate the overall metabolism. VIP
plays a significant role in maintaining body weight and reducing fat mass. This
indicates that VIP may play a crucial role in treating issues related to obesity by
moderating the appetite and body mass (Vu John et al. 2013). Important roles are
played by VIP and PACAP in controlling inflammation and immunity, the release of
catecholamines, and the control of pancreatic insulin secretion and as co-transmitters
in sensory and autonomic neurons (Harmar et al. 2012).

15.6.6 Vasoactive Intestinal Polypeptide: Therapeutics

By providing a firm consideration toward pharmacotherapeutics and drug formation,

the present study focused on the function of Class II G protein-coupled receptors
(GPCRs) to provide a significant knowledge regarding the development of new
therapeutic drugs. The development of information in the study involved recent
clinical findings. Ligands including VIP help in stimulating the given receptors and
are characterized as effective therapeutic targets to develop treatment methods
regarding neurological disorders such as autism spectrum disorder and Alzheimer’s
and Parkinson’s disease (White et al. 2010). The development of the drug may
provide important treatments for stroke, sleep disorder, age-related loss of memory,
and neurodegenerative disorders. Other than this, VIP is classified as an effective
source of treatment for various cardiopulmonary disorders like PAH, asthma, and
COPD. Clinical application of VIP has been limited in the past for a number of
reasons, including its short plasma half-life and difficulty in routes of administration.
The development of long-acting VIP analogs, in combination with appropriate drug
delivery systems, may provide clinically useful agents for the treatment of PAH,
asthma, and COPD (Wu et al. 2011). Despite all the advantages of VIP as a
promising candidate in pharmacotherapeutic targeting, VIP is still regarded as highly
reactive to peptidases (DPP-4) that is found in most tissues. Thus, the therapeutic
impact of VIP can be identified through high-dose injections, when provided
multiple times (Pezzilli 2006).
VIP is highly functional in exhibiting the neuroprotective response against
various cell deaths and serves as the inflammatory modulator for the inflammatory
immune responses. It further offers promise as a therapeutic approach for the
treatment of Alzheimer’s disease and has been regarded as an efficient treatment
for neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Here,
the first is a growing disease that has affected more than 35 million people world-
wide and is diagnosed at the aging phases of life. The disease consists of fibrillar
β-amyloid (Aβ) in the brain senile plaques, which is later activated and thus becomes
functional through the release of pro-inflammatory chemokines and cytokines from
microglia and astrocytes. This initiates the inflammatory progression, resulting in
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 531

neurodegeneration over time. In a recent study, VIP was shown to effectively limit
injurious stimulation of Aβ-concentrated microglia and helps in discharging the
neurotoxins including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric
oxide (NO). Thus, VIP is crucial in preventing the neuronal cell death leading toward
AD pathology in the brain (Delgado et al. 2008; Querfurth and LaFerla 2010).
Publications have resulted in providing significant relationship between
variations of gene encoding including schizophrenia susceptibility and VPAC2
receptor. These results have generated some enthusiasm in the field of new antipsy-
chotic drug developments (Levinson et al. 2011).
The suppression of Th1 immune response along with the activation of T cells is
integrated through VIP, where it serves as the anti-inflammatory mediator to provide
immune balance. In the present era, VIP acts as a therapeutic agent for various
autoimmune diseases such as ulcerative colitis, multiple sclerosis, and rheumatoid
arthritis (Pezzilli 2006).
Current literature reviews show that vasoactive intestinal peptide receptors
(VIPRs) are over functional in some malignant tumors. Also, they provided infor-
mation related to the progression of several malignancies. The analogs of the agents
are labeled through radionuclide and are used for imaging of tumor receptor.
The images are important in visualizing the VIPR surface of protein functioning.
The images were visualized via in vivo test that provides information regarding the
properties of molecular drugs when used for treating tumor. Besides, the relationship
between VIPRs and angiogenesis and malignant transformation is important in the
treatment of cancer (Bo Tang et al. 2014).
The function of VIP specifically in the gastrointestinal tract indicates that
neurotransmitters and its receptors help in dealing with several disorders related to
the secretion of gastric acid along with their impact on gastrointestinal motility
including functional bowel syndrome (Mawe and Hoffman 2013).

15.7 Neuropeptide Y and Related Peptides: Neuropeptide

Tyrosine (NPY)

Deficiency of nutrients (or caloric restriction) can accelerate the orexigenic peptide
neuropeptide Y (NPY) and autophagy in cortical and hypothalamic neurons
(Catalani et al. 2017). The NPY plays a very essential role in various physiological
functions like energy homeostasis, cognition, and neuroprotection and neurogenesis,
intake of food, stress response, and circadian rhythm; therefore, it serves as an
important neuropeptide that is widely present in the brain (Diaz-delCastillo et al.
2018).
A distinguished hypothalamic neural cell in rat and hypothalamic neuronal cell
line in mouse enhance the flux of neuronal autophagy, which is exhibited by the
study of LC3-II turnover, the rise in the number of autolysosomes and
autophagosomes, and decline in the amount of p62. This impact is exercised by
the triggering of Y1 and Y5 receptors. The protein kinase stimulation such as PKA,
532 N. A. Nimer et al.

PI3K, and ERK1/2-MAPK by NPY results in the signaling of track linked with the
induction of autophagy. By in vivo over manifestation of NPY in the arcuate
nucleus, the flux of NPY-induced stimulation was verified in the hypothalamus of
mice. Furthermore, in cortical neurons of rat, the autophagy is accelerated by NPY
(i.e., the decline of p62 expression and growth of LC3-II) possibly by the prevention
of mTOR activity. In mice, which are nourished with a diet containing a high level of
fats, the removal of AMPK activity within the arcuate nucleus of hypothalamus
reduced NPY expression and autophagy, hence decreasing body weight and intake
of food as a result. Therefore, in the hypothalamic cell lines, increased levels of
NPY, the stimulation of AMP-activated protein kinase, and regulating of mTOR
signaling result in the induction of autophagy. In both, the NPY levels and
autophagy keep declining with age; research is being carried out to enhance the
level of NPY and increase autophagy including caloric restriction, as a result of
which it was recommended to develop defending effects that postpone deficiencies
related to prolonged existence. By regulating hypothalamic autophagy, one might
also be able to eliminate the prospects of obesity and other metabolic disorders
associated with aging.
Lastly, the NPY exercises a neuroprotective effect on the cerebellum and striatum
of spinocerebellar ataxia type 3 of two mouse models, which is a disorder
symbolized by autophagy defects. Therefore, it was recommended that this action
might be associated with the stimulation of the clearance process of protein like
autophagy; however, more data is required to support this hypothesis or assumption.
Generally, the NPY ability to prolong neuro-degeneration by activation of
autophagy as an approach to a free abnormal and misfolded protein that results in
neurodegenerative disease is necessary to be studied in detail.

15.7.1 Composition of Neuropeptide Tyrosine Receptors

Neuropeptide Y (NPY) was first discovered in 1982 and was found in the porcine
brain. The information suggested that formation of neuropeptide Y is possible
through the amalgamation of 36 amino peptides and thus is generally functional in
the peripheral nervous system (PNS) and central nervous system (CNS) of
mammals. This neuropeptide comprises pancreatic polypeptide (PP) and peptide
YY (PYY) as it shares its amino acid sequence homology in 70% and 50%,
respectively. The similarity of the sequence in terms of their molecules represents
them in a U-shaped molecule, which is formed in a structure similar to the hairpin,
along with a tertiary structure comprising of an amphiphilic α-helix, which are
joined by the β-turn with a type II helix and similar to polypro line. This structure
of the neuropeptide represents the tridimensional structure, which constitutes peptide
affinity and efficacy connecting them to their several receptors. The NPY biosynthe-
sis translates the molecule of the pre-pro-NPY in which translocation occurs directly
with the endoplasmic reticulum, where the mitigation of the peptide takes place.
Through the prohormone convertases, cleaving of the pro-NPY precursor takes place
in the NPY along with its C-terminal NPY peptide (CPN), where the NPY peptide
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 533

goes through two truncations, i.e., through a peptidyl glycine alpha-amidating mono
oxygenase and a carboxypeptidase for the NPY maturation as well as its activation.
The amidation of the C-terminal serves as the final characterization of the molecule,
which impedes the degradation and takes place through the action of the
carboxypeptidases. With NPY immense consistent distribution of the CNS and
PNS, there are several biological impacts as well as brain disorder, which encompass
disorder like obesity, anorexia, depression, drug addiction, itching, pain, and energy
homeostasis (Loh et al. 2015; Gonçalves et al. 2015; Farzi et al. 2015; Bourane et al.
2015; Arcourt et al. 2017).

15.7.2 Mechanisms of the Physiological Action of Neuropeptide

Tyrosine

The physiological action pertaining to the neuropeptide tyrosine is more prominent

in the up-regulation of the peripheral injury of the nerve that provides cell bodies in
various diameters. These cell bodies are associated with the neurons of the primary
sensors, which innervate with the spinal cord dorsal horn. Its two receptors, i.e., Y1R
and Y2R, are located in DRG in the form of CGRP—positive neurons which
indicate the mediating role of NPY in nociception. This is due to NPY supplies in
a physical circuit, which permits the effect of autocrine or paracrine NPY on the
modulation of the pain. The Y1R receptor is found in neurons, which are small and
medium, whereas the location of the Y2R is generally found in large and medium
neuronal somas. The use of the optogenetic method for Y2R neuron targeting shows
that the Y2R is mostly expressed as a peptidergic A-fiber subset in the activation of
the nociceptors, which is done through the mechanical stimulus belonging to the
noxious range (Arcourt et al. 2017). In addition, 40% of the positive neurons of the
NPY is located in the DRG, followed by the mechanical injury of the nerve, which
also indicates Y1R as well as Y2R. The exertion of NPY actions has remained
controversial, but some expressed it as pro-nociceptive, while some categorized it as
anti-nociceptive in the functional form. Studies have suggested that the differences
in action are based on the deliverance path. Moreover, the peripheral administration
of NPY is pronociceptive. The subcutaneous injection of the peptide or Y2R agonist
and subsequent sciatic nerve injury intensifies the hyperalgesia both mechanically
and thermally. However, divergent results are obtained when the Y1R agonist is
used for administration in a similar manner, which leads to the improvement of
mechanical hyperalgesia, whereas it is low for thermal hyperalgesia. The NPY
receptor has been observed to have a mediating effect on the pronociceptive,
which is part of the sympathetic terminals of the nerve, following the increase of
the affinity or intercellular effect on the injury of the nerve, and its expression. In
addition, the expression of the de novo NPY synthesis is located at the dorsal root
ganglia along with the terminal regions of the spinal cord, where the neurons inhibit
nociceptive pathway where it serves as the compensatory mechanism for adaptation
in response to increase in the excessive excitatory signaling. The two NPY receptors
such as Y1R and Y2R are viewed to form a mediating effect, which is primarily
534 N. A. Nimer et al.

related to the spinal cord in the dorsal horn and encompasses the likely targets for the
drug in the treatment of chronic pain.

15.7.3 Functional Value of Neuropeptide Tyrosine

in the Pathogenesis of Disease

Concerning the pathogenesis of a disease, the characterization of NPY of the

cardiovascular system is primary. In line with norepinephrine, the involvement of
NPY is extensive in the regulation of cardiovascular sympathetic responses. The
presence of NPY is significant in the heart and is also recognized as a cardiac peptide
at the time of its isolation and sequencing. The plasma levels of NPY are in
correlation with the activity of the sympathetic nervous system, which is observed
in various pathophysiological situations, i.e., during exercise, whether mild or
heavy; the activation of the sympathetic nervous system is related to the reduced
sympathetic nervous system responses, as well as in heart-related disease or pheo-
chromocytoma patients. At the moment, the NPY also shares a link with coronary
artery disease (CAD) such as the single nucleotide polymorphisms of the NPY
genome which is related to the human CAD and more significantly on the first-
onset patients. Moreover, the role of NPY is also related to that of the gastrointestinal
(GI) tract involving adaptation to diet, electrolyte balance, intestinal growth, water
uptake, and gastric emptying.

15.7.4 Pharmacology of Neuropeptide Tyrosine

In recent times, the association of neuropeptide tyrosine (NPY) has been found to be
in neurological responses related to ethanol and drug abuse, which is highlighted
from genetic, pharmacological, and molecular indication (Ciafrè et al. 2016). The
pharmacology of neuropeptide tyrosine has initially been examined from the periph-
eral sympathetic control of the neurons related to the blood vessels, spleen, heart, as
well as vas deferens. The release of the NPY largely takes place when the stimulation
constitutes of high frequency or activation of the strong sympathetic reflex. NPY has
a particular receptor mechanism, which is present at the pre-junction level as well as
post-junction levels, where the existence of an enormous amidated C-terminal
portion of NPY is required to bind the receptor and results in vasoconstrictor effects
as well as the inhibition of cyclic AMP formation. Moreover, the release of the NPY
is also impacted by various pharmacological agents encompassing clonidine, gua-
nethidine, yohimbine, nicotine, angiotensin, angiotensin II, and desipramine.
Among the NPY receptors, the Y5 receptor constitutes the pharmacological profile
particularly for the peripheral tissues, though its mRNA expression is greatly
confined to the CNS (Dan Larhammar et al. 2015).
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 535

15.7.5 Neuropeptide Tyrosine Receptors

The fragments of the NPY C-terminal serve as the source, which characterizes the
NPY receptors. It is due to its NPY and PYY fragment imitation of the responses
which are given by the NPY such as in the twitch responses of the pre-junctional
inhibition as it occurred in rat vas deferens; however, they are not observed in
vasoconstriction of guinea pig iliac vein. In the context of the NPY receptor, it has
been suggested that Y1 are the receptors that are activated by holopeptides, whereas
Y2 are the receptors that are activated by the fragment of the C-terminal and
holopeptides. Although the synthesis of the various C-terminal fragments occurs,
the NPY and PYY of the 3–36, 13–36, and 18–36 fragments are often utilized
irrespective of their obvious benefit between these fragments. The discrimination of
the receptors such as Y1 and Y2 is done using the NPY C-terminal fragment, though
these are not only confined to the Y2 selection as Y5 receptors can also be activated
using the same concentration. It should be noted that the selection of the receptors is
based on the difference between the availability of tools and the substantial differ-
ence concerning the affinity of the receptor in relation to their cognate ligands which
prevails between the species. The recognition of the receptor is based on the various
combination of the complementary agents which are used. The application of this
condition is more momentous for in vivo, whereas the less characterization of the
compounds occurs in contrast to in vitro.

15.7.6 Neuropeptide Tyrosine: Therapeutics

The neuropeptide tyrosine is significantly used for the therapeutic of various

diseases. One of such disease is a gastrointestinal disease where NPY receptor
performs in the capacity of the modulators in cases of constipation, diarrheas, as
well as IBD. The use of the NPY agonist in the position of a non-specific activating
agent is said to assist in IBD treatment. This idea gains further momentum to the fact
that the application of the therapeutic agent does not require integration in the blood
stream. For instance, the earlier study by Litvak et al. (1999) demonstrated that in the
case of diarrhea, the presence of two stable PYY peptide analogs is found
(BIM-43073D and BIM-43004C), which resulted in increase in the absorption of
water when dogs were treated with these two analogs. This further indicates that the
health of the gut is promoted by the assimilation of a selective Y1R agonist. On the
contrary, there is another fact that the use of Y1 receptor knockout mice which
comprises lower IBD susceptibility also highlights the fact that the use of the
antagonist is more effectual in constipation and IBD treatment. This elucidates the
use of Y2R and Y4R receptors in overcoming the discrepancy and finding more
treatment.
In addition, the use of NPY is also linked to the administration of chronic pain.
This is due to the NPY receptors’ heterogeneity, their application site, as well as the
various chronic pain models for the chronic pain, which allow the NPY to exert
various effects. The use of central NPY contributes to the production of analgesia
536 N. A. Nimer et al.

which causes mediating influence on the spinal as well as supraspinal level (Taylor
et al. 2014). In the chronic pain modulation, the role of NPY along with its Y1R and
Y2R receptors is found to be crucial, where the NPY receptors can also be regarded
as a target for a drug where they are used for devising new drug design. However,
various problems exist, which are difficult to overcome with the use of this targeted
treatment. The route of administration of these receptors, such as the NPY agonists
which are peripherally applied, results in the behavior that emerges due to a halt, and
it appears that the analgesic effect occurs only when the peptide or drug formulation
takes place following the blood-brain barrier (BBB). However, the lumbar intrathe-
cal drug delivery is often found to be linked with some patients’ sort of discomfort
and inconvenience, which can be mitigated through increasing the treatment effi-
ciency as well as persistence of its effects, which are still lacking. Another difficulty
associated with the determination of the severe pain in NPY agonist when found in
clinical practices is linked with the deficiency of the non-peptidic small molecule of
the NPY receptor agonists (Mittapalli and Roberts 2014). To treat these cases,
different strategies are adopted such as targeted agonist delivery by using the therapy
related to the nanoparticles or gene (Chandrasekaran 2013).

15.8 Calcitonin Peptides and Others

Huang et al. (2006) initially discovered calcitonin (CT) which is a kind of polypep-
tide hormone (Masi and Brandi 2007). This identification occurred when the regu-
latory hormone for calcium level was being discovered. This peptide is able to
reduce the blood calcium level through the direct inhibition of the mediation of the
bone resorption, which also improves the kidney exertion of calcium. There are
32 amino acid residues present in the single chain of human CT peptide, which has
its molecular mass as 3418 Da. The cysteines are linked through the disulfide bridge,
which connects them from positions 1 to 7 to form a ring structure that includes
seven amino acids located at the amino terminus. Moreover, the CT precursor,
pre-procalcitonin (PreProCT), withholds 141 amino acids (Fig. 15.8).
The stem cells from neural crest as well as the rostral are transported into the
ultimo-branchial glands of lower vertebrate animals and into the para-follicular cells
in humans. The C cells found in the ultimo-branchial glands of lower vertebrates and
in the thyroid gland of mammals produce calcitonin (CT) and release them into
circulation, which works in a similar manner to other hormones in the body. Through
this advance movement, it is assumed that concentration of C cells is required in
ultimo-branchial body and thyroid gland (Johansson et al. 2015). However, it is
often noticed that some C cells might not be able to make their way forward toward
the thyroid and end up in the extra-thyroidal tissues during their forward movement
(Giovanella et al. 2013). Calcitonin is considered as endogenous controller of
calcium homeostasis and protects the skeleton especially in “calcium stresses” by
acting primarily on the bone. Calcitonin also acts directly on gastrointestinal and
kidney secretion activity by creating an indirect impact on the central nervous system
(CNS) to lessen the pain. Studies have found that the role of calcitonin is not limited
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 537

a. Human CALC-I gene

5’ I II III IV V VI 3’
0
0
0 TRANSCRIPTION, MATURATION
0 SPLICING AND POLYADENYLATION
0
0
0

Calcitonin I precursor
77777 Calcitonin II precursor CGRP I- precursor

b. Human CALC-II gene

I II III IV V VI

CGRP II- precursor

b. Human CALC-III gene b. Human CALC-IV gene

I II I II III

CT: CGRP Amylin precursor

Fig. 15.8 Human calcitonin gene family (Source: Wimalawansa 2010)

to intrinsic analgesic effects on CNS and thus creates a significant impact over
various neuronal activities, like moderating them directly on their existing location
or indirectly moderating them by a process which is yet to be explained on other
locations. Presently, the main indications for using calcitonin in therapies are
disorders, which include high-bone turnover osteoporosis, acute pancreatitis, osteo-
porosis linked to pain or metastases of bone, hypercalcemia, Sudeck’s atrophy, and
Paget’s disease (osteitis deformans). Many kinds of calcitonin are commonly con-
sumed such as synthetic human calcitonin (SCT), analog (Elcatonin), synthetic
salmon calcitonin (SCT) also known as salcatonin, natural porcine calcitonin
(PCT), and synthetic eel calcitonin (ECT). One of the problems with its treatment
is that the only imaginable way for its administration is through an injection,
although other ways for its dosage are being explored.
538 N. A. Nimer et al.

15.8.1 Structure of Calcitonin

During the evolution, calcitonin has been well preserved. Calcitonin from 9 distinc-
tive species has been recognized with the identification of 12 different sequences.
The remains of six unchangeable amino acids are found at the amino terminal, while
two others are obtained through the end of the carboxyl terminal of the peptide
molecule.
Moreover, at C-terminal, all the calcitonins have 1–7 disulfide bridge and proline
amide. For the osteoclast inhibitory functions and for hypocalcemic bioactivity, all
the 32 amino acids are needed. The substitution of certain amino acids during the
synchronization of synthetic calcitonin (like synthetic eel-CT) helps in increasing
their lifetime to a certain extent by struggling against degradation, hence improving
their circulatory half-life and its biological function. The method involves certain
risks as it may result in allergenicity. Calcitonin is prepared as a precursor molecule.
Before the release of the fully developed form of biologically active calcitonin,
various C-terminal amidation and post-translational changes, such as cleavage, take
place. Glucagons, theophylline, cholecystokinin, dibutyryl cyclic AMP, gastrin, and
cations like Mg2+ and Ca2+ accelerate the secretion of calcitonin from C cells. The
impact of calcitonin over osteoclasts can be represented through dibutyryl cyclic
AMP. However, the dose dependency to eliminate the cyclic AMP is integrated
through calcitonin.
The quantification of plasma i-CT levels (i.e., for hypertension) is being utilized for
diagnostic and inspection tests for C cell hyperplasia for the diagnosis of
pre-malignant and malignant disorders that are related to C cell (i.e., patients’ families
suffering through medullary thyroid carcinoma (MCT)) (Wimalawansa 2010). Cur-
rently, there is the availability of various diagnostic-stimulation tests for the diagnosis
of MTC like an infusion of pentagastrin and injecting calcium (Fig. 15.9).

Fig. 15.9 Human calcitonin amino acid sequence (Source: Wimalawansa 2010)
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 539

15.8.2 Mechanisms of the Physiological Action of Calcitonin

The calcitonin physiological concentration is expected to have an energizer effect to

limit the resorption of osteoclastic bone. Physiologically, the function of calcitonin is
to sustain skeletal form throughout the time of calcium stresses like pregnancy,
lactation, and growth (i.e., when the skeleton is required to be sustained). During
lactation and pregnancy, the calcium is released into the milk, and the retention of
calcium by the fetus happens at the cost of prolactin and the skeleton of the mother
(i.e., activation through parathyroid hormone-related protein (PTHrP)). Therefore, in
these conditions, it is mandatory to provide solutions that may act against the
regulatory impact to secure the mineral contents of maternal skeletal. The release
of calcitonin is expected to have an impact by regulating unnecessary resorption of
bone. Estrogen and testosterone, which are sex steroid hormones, activate calcitonin
production by C cells.
Though the calcitonin’s primary function is to regulate calcium homeostasis,
which acts mainly on bones, it also has a direct effect on the gastrointestinal tract and
kidneys. The receptors of calcitonin are abundantly present in osteoclasts. Other than
this, the brain, kidney, and hypothalamus are other sources of these receptors.
Calcitonin is further evident in creating both direct and indirect impact on CNS
and is involved in regulating the neuromodulatory functions and pain. However, the
osteoclast serves as the basic cell that is targeted for calcitonin with more than a
million calcitonin receptors. The osteoclast withdraws its pseudopodia under just a
few minutes of the organization of calcitonin, and as a result, it diminishes the
resorption function of bone, cell size, and motility. The cells become steady for
hours and stop bone resorption. Throughout bone excavation, the osteoclasts release
acids (HCl) and enzymes (like acid phosphatases and metalloproteinases), which
later leads to several pits in the bone matrix through hydrolysis. The information thus
revealed that physiological integration of plasma calcitonin is significant in
performing functions related to osteoclast (Fig. 15.10).

15.8.3 Role of Calcitonin in the Pathogenesis of Disease

Calcitonin has been observed to play a significant role in the treatment of pathogenic
diseases. The effectiveness of calcitonin is well-established for providing treatment
to patients who are suffering from Paget disease. The raised Pagetic bone turnover
indices are decreased when the calcitonin is administered along with the subcutane-
ous injection of about 50% (Pondel 2000).
The symptoms related to the Paget disease are also reduced with the use of CT
which encompass disease comprising pain in the bones and complications related to
the neurology comprising nerve root compression, headache, and spinal stenosis.
Along with it, osteoporosis is characterized as the systemic skeletal disease which
comprises low bone mass as well as bone tissue deterioration which result in fragility
in the bone along with fracture susceptibility. The decrease in the levels of estrogen
which takes place at menopause results in increase resorption of the osteoclastic
540 N. A. Nimer et al.

Man Rat S-1 S-2 S-3 Eel *Chiek Bov Pore Ovi *Man2
1 Cys - - - - - - - - - Tyr
2 Gly - Ser Ser Ser Ser Ala Ser Ser Ser Ser
3 Asn - - - - - Ser - - - -
4 Leu - - - - - - - - - -
5 Ser - - - - - - - - - -
6 Thr - - - - - - - - - -
7 Cys - - - - - - - - - -
8 Met - Val - Val Val Val Val Val Val Leu
9 Leu - - - - - - - - - Gln
10 Gly - - - - - - Ser Ser Ser -
11 Thr - Lys Lys Lys Lys Lys Ala Ala Ala -
12 Tyr - Leu Leu Leu Leu Leu - - - -
13 Thr - Ser Ser Ser Ser Ser Trp Trp Trp Leu
14 Gln - - - - - - Lys Arg Lys -
15 Asp - Glu - - Glu Glu - Asn - Tyr
16 Phe Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu
17 Asn - His His His His His - - - Lys
18 Lys - - - - - - Asn Asn Asn Asn
19 Phe - Leu Leu Leu Leu Leu Tyr - Tyr -
20 His - Gln Gln Gln Gln Gln - - - -
21 Thr - - - - - - Arg Arg Arg Met
22 Phe - Tyr - - Tyr Tyr - - Tyr -
23 Pro - - - - - - Ser Ser Ser -
24 Gln - Arg Arg Arg Arg Arg Gly Gly Gly Gly
25 Thr - - - - - - Met Met Met Ile
26 Ala Ser Asn Asn Asn Asp Asp Gly Gly Gly Asn
27 Ile - Thr Thr Thr Val Val Phe Phe Phe Phe
28 Gly - - - - - - - - - -
29 Val - Ser Ala Ala Ala Ala Pro Pro Pro Pro
30 Gly - - - - - Glu Glu Glu Glu Gln
31 Ala - Thr Val Val Thr Thr Thr Thr Thr Ile
32 Pro - - - - - - - - - -

Fig. 15.10 Predicted calcitonin sequences of amino acid

bone, up surging the overall loss of the skeletal bone mass. In addition, CT is also
recognized as the steady inhibitor related to the osteoclast-mediated bone resorption
which makes it effective to be used in the therapeutic treatment related to
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 541

osteoporosis. The osteoclastic bone resorption which is inhibited by the calcitonin

reduces serum calcium (antagonized parathyroid hormone).

15.8.4 Pharmacology of Calcitonin

The inhibition of bone resorption which emerges as a result of calcitonin causes a

direct inhibitory influence on the osteoclast. The use of calcitonin results in the
mitigation of the osteoclast number, when it is provided for some months. Though
the mitigation of the osteoclast number is not apparent, it occurs as a result of the
influence of calcitonin on osteoclasts and has an independent influence on associated
precursor cells. The consequence of this effect is observed to be crucial for treating
various diseases related to bone metabolism. The impact of calcitonin regarding the
lowering of calcium is not observed in the average individual due to its overall slow
bone turnover rate. Thus, calcitonin administration causes either minor or no influ-
ence on the plasma calcium present in an average individual.
Though when bone turnover is high such as in children, or in various disease
conditions, i.e., hypercalcemic statuses, Paget’s disease, and more, following calci-
tonin regulation, decrease in calcium level in the circulatory system can occur.
Moreover, the pharmacological activities of calcitonin are also reported for the
gastrointestinal (GI) system, where it leads to increase in intestinal secretion of
sodium, water, and chloride as well as inhibition of acid secretion and gastric
emptying. Furthermore, it is also linked with the secretion of various gastrointestinal
regulatory peptides, encompassing insulin, motilin, pancreatic glucagons, pancreatic
polypeptide, gastrin, and possibly gastric inhibitory peptide. Moreover, the secretion
of pituitary hormones is also inhibited by calcitonin, which constitutes growth
hormones, thyroid-stimulating hormone, as well as luteinizing hormone.

15.8.5 Calcitonin Receptors

The CT activities are regulated with the use of calcitonin receptors which possess
high affinity. The calcitonin receptors are a member of G protein peptides of seven
transmembranes (Masi and Brandi 2007). All the members of the family have
identical structure along with seven other transmembranes spanning domain G
protein-coupled receptors. The cloning of porcine CTR (pCTR) cDNA was initially
done in the year 1991 by Lin et al. (1991). The characterization of the receptor is
based on its constituent of the extracellular long NH2-terminal domain. It shares
similarity with the parathyroid hormone which is linked with peptide receptor as well
as secretin receptor. Later, pCTR gene cloning showed that its length is about 70 kb
which also contains a minimum of 14 axons, in which 12 axons were inclusive of
protein. The cloning of the human CTR was done from ovarian cancer cell line, i.e.,
BIN-67. The different isoforms of CTR are the outcome of the gene splicing change
which has been indicated in various species of animals comprising divergent
transcripts of tissue expression as well as divergent signaling properties. Concerning
542 N. A. Nimer et al.

the large tumor cell of the bone, two kinds of isoforms have been explained, where
the first comprises similar design like GC-10, which is divergent from the earlier
explained human ovarian cancer CTR gene in the 50 region as it is devoid of the
71-bp segment while being almost similar to the 30 region. However, the second is
the CTR cDNA variant of the large human tumor cell, which is highlighted as GC-2,
devoid of the 71-bp 50 insert, though it comprises 48 encoded nucleotides part in the
first intercellular domain. The difference in the expression of CTR isoforms may be
the result of the biological regulatory mechanism which provides responses to the
calcitonin. The significant shift in the CTR isoforms can be explained as the
responsiveness of the variable related to calcitonin of the patients who have
increased turnover of the metabolic bone ailment. Moreover, the outlying of the
CTR gene is based on the chromosome 7q21.3.

15.8.6 Calcitonin: Therapeutics

With the several bone disorders, patients are being treated with calcitonin for the last
four decades, characterized on the basis of people that have high bone resorption
issues. The therapeutic function of calcitonin can be indicated through the patients
with disorders such as hypercalcemic states, i.e., bony metastases, Paget’s disease,
pain in osteoporotic fractures, toxicity of vitamin D, Sudeck’s atrophy, and osteopo-
rosis with high bone turnover. Until a few years ago, injection was the only possible
means of management. On the contrary, dosage forms are being produced, and a
calcitonin nasal spray is now commercially available using other routes
(Wimalawansa 2010). It seems difficult to administer calcitonin orally as it is a
peptide. However, in line with research studies into several delivery systems as well
as the increased improvisation of calcitonin molecule, it is highly probable to
administer the analogs of calcitonin orally. The process of oral administration can
be integrated through the buccal mucosal route. Bone pain that includes vertebral
fractures/osteolysis because of neoplasms is observed to be an indication for calci-
tonin therapy. Calcitonin seems to be successful for patients with Sudeck’s atrophy
and algoneurodystrophy. These are the syndromes caused by several factors such
as reflex dystrophy, post-traumatic osteoporosis, and iatrogenic neuropathy. More-
over, apart from the developed uses of calcitonin, the advantageous outcomes of
using calcitonin can also be observed, especially in the prevention of osteoporosis.
In case of osteoporosis that is associated with pregnancy, immobilization, and
increased bone catabolism, osteogenesis imperfecta, controlling loss of bone while
administering prednisone and heparin and severe renal insufficiency and excessive
osteoclastic activity is associated with these mentioned disorders. Calcitonin is
observed to be an additive therapy, although the growth of bone mass should be
increased when it comes to the ideal treatment for osteoporosis. Calcitonin and
bisphosphonates are important in controlling bone loss. In various osteoporosis
patients, albeit, that type of calcitonin serves as a less functional agent that is used
to provide stability to bone mass. In different types of osteoporosis, it seems to have
the greatest advantages when enhanced resorption is considered to be a feature. It has
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 543

also been employed for post-menopausal osteoporosis. For effective prevention of

bone loss, it has been observed that calcitonin dose of 50 IU twice a week is really
effective. Moreover, the hormone replacement therapy (HRT) along with calcitonin
seems to be effective. Calcitonin is highly efficient in preventing bone loss. How-
ever, its effectiveness in reducing osteoporosis seems to be less than bisphosphonate.
Calcitonin has been used in the treatment of complex issues related to Paget’s
disease of the bone over the last 40 years. The osteoclastic activities are important to
provide the moderate effects of calcitonin, whereas the long-term response can be
seen with the decrease of osteoclasts. A symptomatic response can be observed in
patients having Paget’s disease under 1–2 weeks’ therapy, and the response could be
increased after 12 weeks. Until maximum symptomatic relief, the therapy seems to
be continued till at least the next 6 months. Calcitonin can be useful (e.g., a dose of
100 IU that is provided through injections) for patients with radiological evidence of
an osteolytic lesion. It is only when osteolytic lesion can be seen in the bone that it
becomes a burden due to extensive body weight which may result in fracture in the
future. Calcitonin along with intravenously administered bisphosphonate will accel-
erate the process of recovery. Patients having skeletal metastatic disease can also be
treated with calcitonin that is secondary to malignancy. s-CT works as an analgesic
when calcitonin is injected in the subarachnoid space. It seems the main location for
the action is within the CNS, whereas the mechanism is still not understood when it
is analgesic. By using a number of mechanisms, calcitonin and its analgesic effects
have been suggested. A direct, as well as indirect, action can be observed by the
interference with the neurotransmitters that include prostaglandin, serotonin, and
peripheral action. All these can be mediated through hindrance of inflammatory
cytokines as well as chemical factors. There is an association between bone pain and
hormone-sensitive tumors that reveals the great response along with some lung
tumors that secrete ectopic hormones. These sensitive tumors include breast, thyroid,
and prostate. Moreover, a possibility can be seen for the effects of calcitonin on the
release of β-endorphins. Calcitonin can also be combined with calcitonin gene-
related peptide receptors (CGRP) found in the hypothalamus of CNS. Suggestions
are given when CGRP along with its receptors are distributed in the dorsal view of
the spinal cord and brain. The suggestions include the involvement of processes of
pain sensation. In several studies, calcitonin has been shown to interact with CGRP
and the associated sites in the kidney and nervous system that involved the hypo-
thalamus. Therefore, in cases where the calcitonin doses are required, CGRP
neuromodulatory receptors serve as highly functional in CNS. This happens specifi-
cally in the hypothalamic region that alters the sensory neurotransmission.

15.9 Conclusion

Neuropeptide proteolytic processing contributes significantly in terms of its regu-

latory function along with the resulting fragment peptide which emerges due to the
degradation of the enzyme exerting vital physiological roles. The generation of
proteolytic processing is not limited to the biologically inactive fragments but also
544 N. A. Nimer et al.

expands to those fragments which modulate and at times counteract the parent
peptide response. Generally, the integration of the peptide fragments is frequent
for those receptors which are unidentified by the parent peptides. This chapter
explains the concepts related to the tachykinins, VIP-glucagon family,
neuropeptide Y, and calcitonin peptides which are present with the bioactive degra-
dation processes. The chapter demonstrates their related mechanisms of physiologi-
cal action, pathogenesis of the disease, pharmacology, receptors, and therapeutics.
The chapter provides insight that these neuropeptides can further be explored for
their utilization in drugs to improve the efficiency of the drugs as well as drug-like
development of the substance, which can serve as a rich source of development of
new pharmaceuticals.

References
Actelion (2011) Actelion and GSK discontinue clinical development of Almorexant [press release].
Available at: http://www.actelion.com/en/investors/media-releases/index.page?
newsId¼1483135. Accessed 3 Feb 2019
Amin K (2012) The role of mast cells in allergic inflammation. Respir Med 106(1):9–14. https://doi.
org/10.1016/j.rmed.2011.09.007. Epub 2011 Nov 22
Arcourt A, Gorham L, Dhandapani R, Prato V, Taberner FJ, Wende H, Gangadharan V,
Birchmeier C, Heppenstall PA, Lechner SG (2017) Touch receptor-derived sensory information
alleviates acute pain signaling and fine-tunes nociceptive reflex coordination. Neuron 93
(1):179–193
Atanasova KR, Reznikov LR (2018) Neuropeptides in asthma, chronic obstructive pulmonary
disease and cystic fibrosis. Respir Res 19(1):149. https://doi.org/10.1186/s12931-018-0846-4
Batista-Brito R, Vinck M, Ferguson KA, Chang JT, Laubender D, Lur G, Mossner JM, Hernandez
VG, Ramakrishnan C, Deisseroth K, Higley MJ, Cardin JA (2017) Developmental dysfunction
of VIP interneurons impairs cortical circuits. Neuron 95(4):884–895.e9
Belzung C, Yalcin I, Griebel G, Surget A, Leman S (2006) Neuropeptides in psychiatric diseases:
an overview with a particular focus on depression and anxiety disorders. CNS Neurol Disord
Drug Targets 5(2):135–145
Bik W, Wolinska-Witort E, Chmielowska M, Baranowska-Bik A, Rusiecka-Kuczalek E,
Baranowska B (2004) Vasoactive intestinal peptide can modulate immune and endocrine
responses during lipopolysaccharide-induced acute inflammation. Neuroimmunomodulation
11(6):358–364
Black PH (2002) Stress and the inflammatory response: a review of neurogenic inflammation. Brain
Behav Immun 16:622–653
Blanco EJ, Carretero-Hernández M, García-Barrado J, Iglesias-Osma MC, Carretero M, Herrero JJ,
Rubio M, Riesco JM, Carretero J (2013) The activity and proliferation of pituitary prolactin-
positive cells and pituitary VIP-positive cells are regulated by interleukin 6. Histol Histopathol
28(12):1595–1604
Bondy B, Baghai TC, Minov C, Schüle C, Schwarz MJ, Zwanzger P, Rupprecht R, Möller HJ
(2003) Substance P serum levels are increased in major depression: preliminary results. Biol
Psychiatry 53:538–542
Bourane S, Duan B, Koch SC, Dalet A, Britz O, Garcia-Campmany L, Kim E, Cheng L, Ghosh A,
Ma Q, Goulding M (2015) Gate control of mechanical itch by a subpopulation of spinal cord
interneurons. Science 350(6260):550–554
Campbell DE, Raftery N, Richard Tustin III, Tustin NB, DeSilvio ML, Cnaan A et al (2006)
Measurement of plasma-derived substance P: biological, methodological, and statistical
considerations. Clin Vaccine Immunol 13(11):1197–1203
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 545

Catalani E, De Palma C, Perrotta C, Cervia D (2017) Current evidence for a role of neuropeptides in
the regulation of autophagy. Biomed Res Int. https://doi.org/10.1155/2017/5856071
Chaiseha Y, Kang SW, Leclerc B, Kosonsiriluk S, Sartsoongnoen N, El Halawani ME (2010)
Serotonin receptor subtyopes influence prolactin secretion in the turkey. Gen Comp Endocrinol
165(1):170–175
Chandra R, Liddle RA (2015). Regulation of pancreatic secretion. Pancreapedia: Exocrine pancreas
knowledge base. https://doi.org/10.3998/panc.2015.38`
Chandrasekaran B (ed) (2013) Intelligence as adaptive behavior: an experiment in computational
neuroethology, vol 6. Academic Press, Cambridge
Chang MM, Leeman SE, Niall HD (1971) Amino-acid sequence of substance P. Nat New Biol 232
(29):86–87
Chapter MC, White CM, DeRidder A, Chadwick W, Martin B, Maudsley S (2010) Chemical
modification of class II G protein-coupled receptor ligands: frontiers in the development of
peptide analogs as neuroendocrine pharmacological therapies. Pharmacol Ther 125(1):39–54
Chen SR, Chen H, Zhou JJ, Pradhan G, Sun Y, Pan HL, Li DP (2017) Ghrelin receptors mediate
ghrelin-induced excitation of agouti-related protein/neuropeptide Y but not
pro-opiomelanocortin neurons. J Neurochem 142(4):512–520
Chi G, Huang Z, Li X, Zhang K, Li G (2018) Substance P regulation in epilepsy. Curr
Neuropharmacol 16(1):43–50
Chu HW, Kraft M, Krause JE, Rex MD, Martin RJ (2000) SP and its receptor neurokinin
1 expression in asthmatic airways. J Allergy Clin Immunol 106:713–722
Ciafrè S, Fiore M, Ceccanti M, Messina MP, Tirassa P, Carito V (2016) Role of neuropeptide
Tyrosine (NPY) in ethanol addiction. Biomed Rev 27:27–39
Coremans V, Ahmed T, Balschun D, D’Hooge R, DeVriese A, Cremer J, Cremer H (2010)
Impaired neurogenesis, learning and memory and low seizure threshold associated with loss
of neural precursor cell survivin. BMC Neurosci 11(1):2
Cox CD, Breslin MJ, Whitman DB, Schreier JD, McGaughey GB, Bogusky MJ, Roecker AJ,
Mercer SP, Bednar RA, Lemaire W, Bruno JG (2010) Discovery of the dual orexin receptor
antagonist [(7 R)-4-(5-chloro-1, 3-benzoxazol-2-yl)-7-methyl-1, 4-diazepan-1-yl] [5-methyl-2-
(2 H-1, 2, 3-triazol-2-yl) phenyl] methanone (MK-4305) for the treatment of insomnia. J Med
Chem 53(14):5320–5332
De Swert KO, Bracke KR, Demoor T, Brusselle GG, Joos GF (2009) Role of the tachykinin NK1
receptor in a murine model of cigarette smoke-induced pulmonary inflammation. Respir Res
10:37
Delgado M, Nieves V, Gonzalez-Rey E (2008) Vasoactive intestinal peptide protects against
β-amyloid-induced neurodegeneration by inhibiting microglia activation at multiple levels.
Glia 56(10):1091–1103
Diaz-delCastillo M, Woldbye DP, Heegaard AM (2018) Neuropeptide Y and its involvement in
chronic pain. Neuroscience 387:162–169
Dickson PV, Behrman SW (2013) Management of pancreatic neuroendocrine tumors. Surg Clin N
Am 93:675–691
Dickson L, Finlayson K (2009) VPAC and PAC receptors, “from ligands to function”. Pharmacol
Ther 121(3):294–316
Dickson L, Aramori I, McCulloch J, Sharkey J, Finlayson K (2006) A systematic comparison of
intracellular cyclic AMP and calcium signalling highlights complexities in human VPAC/PAC
receptor pharmacology. Neuropharmacology 51(6):1086–1098
Dorsam GP, Benton K, Failing J, Batra S (2011) Vasoactive intestinal peptide signaling axis in
human leukemi. World J Biol Chem 2(6):146–160
Eliska M, Peter K, Magdalena CD (2016) Role of substance P in the cardiovascular system.
Neuropeptides 58:41–51
Farzi A, Reichmann F, Holzer P (2015) The homeostatic role of neuropeptide Y in immune function
and its impact on mood and behaviour. Acta Physiol 213(3):603–627
546 N. A. Nimer et al.

Fidler C, Elmelund Christensen T, Gillard S (2011) Hypoglycemia: an overview of fear of

hypoglycemia, quality-of-life, and impact on costs. J Med Econ 14(5):646–655
Ganea D, Hooper KM, Kong W (2014) The neuropeptide vasoactive intestinal peptide: direct
effects on immune cells and involvement in inflammatory and autoimmune diseases. Acta
Physiol (Oxf) 213(2):442–452
Gao X, Wang Z (2017) Difference in expression of two neurokinin-1 receptors in adenoma and
carcinoma from patients that underwent radical surgery for colorectal carcinoma. Oncol Lett 14
(3):3729–3733
Garcia-Recio S, Gascon P (2015) Biological and pharmacological aspects of the NK1-receptor.
Biomed Res Int 2015:495704–495714. https://doi.org/10.1155/2015/495704
Giovanella L, Verburg FA, Imperriali M, Valaberga S, Trimboli P, Ceriani L (2013) Comparison of
derum calcitonin and procalcitonin in detecting medullary thyroid carcinoma among patients
with thyroid nodules. Clin Chem Lab Med 51(7):1477–1481
Gonçalves J, Martins J, Baptista S, Ambreòsio AF, Silva AP (2015) Effects of drugs abuse on the
central neuropeptide Y system. Addict Biol 21(4):755–765
González-Rey E, Fernández-Martin A, Chorny A, Delgado M (2004) Therapeutic approaches of
vasoactive intestinal peptide as an immunomodulatory cytokine. Inmunol 23(4):348–363
Grässel S, Muschter D (2017) Peripheral nerve fibers and their neurotransmitters in
osteoarthritispathology. Int J Mol Sci. 18(5):931
Gray JA (2018) Introduction to the pharmacology of CNS drugs. In: Katzung BG (ed) Basic and
clinical pharmacology, 14th edn. McGraw Hill, New York, p 379
Halford JC, Harrold JA, Boyland EJ, Lawton CL, Blundell JE (2007) Serotonergic drugs. Drugs 67
(1):27–56
Hanaya R, Arita K (2016) The new antiepileptic drugs: their neuropharmacology and clinical
indications. Neurol Med Chir 56(5):205–220
Harmar AJ, Fahrenkrug J, Gozes I, Laburthe M, May V, Pisegna JR, Vaudry D, Vaudry H,
Waschek JA, Said SI (2012) Pharmacology and functions of receptors for vasoactive intestinal
peptide and pituitary adenylate cyclase-activating polypeptide: IUPHAR review 1. Br J
Pharmacol 166(1):4–17
Hayakawa E, Watanbe H, Menschaert G, Holstein TW, Baggerman G, Schoofs L (2019) A
combined strategy of neuropeptide predictions and tandem mass spectrometry identifies evolu-
tionarily conserved ancient neuropeptided in the sea anemone Nematostella vectensis. BioRxiv,
593384
Henning RJ (2013) Handbook of biologically active peptides, 2nd edn. Elsevier, Amsterdam
Henning RJ, Sawmiller DR (2001) Vasoactive intestinal peptide: cardiovascular effects. Cardiovasc
Res. 9(1):27–37
Hoever P, Dorffner G, Beneš H, Penzel T, Danker-Hopfe H, Barbanoj MJ, Pillar G, Saletu B,
Polo O, Kunz D, Zeitlhofer J (2012) Orexin receptor antagonism, a new sleep-enabling
paradigm: a proof-of-concept clinical trial. Clin Pharmacol Ther 91(6):975–985
Holzer P (2013) Tachykinins. In: Kastin A (ed) Biologically active peptide castro intestinal, 2nd
edn. Academic Press, Cambridge, p 1131
Huang CLH, Sun L, Moonga BS, Zaidi M (2006) Molecular physiology and pharmacology of
calcitonin. Cell Mol Biol 52(3):33–43
Hoyer D, Bartfai T (2012) Neuropeptides and neuropeptide receptors: drug targets, and peptide and
non-peptide ligands: a tribute to prof. Dieter Seebach. Chem Biodivers 9(11):2367–2387
Ianowski JP, Choi JY, Wine JJ, Hanrahan JW (2008) SP stimulates CFTR-dependent fluid secretion
by mouse tracheal submucosal glands. Pflugers Arch 457:529–537
Igarashi H, Fujimori N, Ito T, Nakamura T, Oono T, Nakamura K, Suzuki K, Jensen RT,
Takayanagi R (2011) Vasoactive intestinal peptide (VIP) and VIP receptors—elucidation of
structure and function for therapeutic applications. Int J Clin Med 2011(2):500–508
Jaafari N, Hua G, Adélaïde J, Julé Y, Imbert J (2008) Expression of the tachykinin receptor mRNAs
in healthy human colon. Eur J Pharmacol 599(1–3):121–125
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 547

Jiang W, Wang H, Li YS, Luo W (2016) Role of vasoactive intestinal peptide in osteoarthritis. J
Biomed Sci 23(1):63
Johansson E, Andersson L, Ornros J, Carlsson T, Ingeston-Carlsson C, Jansson S, Parillo L,
Zoppoli P, Barilla GO, Altschuler DL, Padula D, Lickert H, Fagman H, Nilsson M (2015)
Revising the embryonic origin of thyroid C cells in mice and humans. Development 142
(20):3519–3528
Kalina R, Gladkikh I, Dmitrenok P, Chernikov O, Koshelev S, Kvetkina A, Kozlov S,
Koslovskaya E, Monastyrnaya M (2018) New APETx-like peptided from sea anemone
Heteractus crispa modulate ASIC1a channels. Peptides 104:41–49
Kassim SK, ElTouny M, ElGuinaidy M, Abd ElMoghni MA, Abd ElMohsen A (2002) Serum
nitrates and vasoactive intestinal peptide in patients with gastroesophageal reflux disease. Clin
Biochem 35(8):641–646
Kastin A (2013) Handbook of biologically active peptides, 2nd edn. Academic Press, Cambridge,
pp 966–973
Koon HW, Zhao D, Na X, Moyer MP, Pothoulakis C (2004) Metalloproteinases and transforming
growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation
and proliferation in human colonocytes. J Biol Chem 279:45519–45527
Kramer MS, Cutler N, Feighner J, Shrivastava R, Carmen J, Sramek JS, Scott A (1998) Distinct
mechanism foir antidepressant activity by blockade of central substance P receptors. Science
281(5383):1640–1645
Laburthe M, Couvineau A, Tan V (2007) Class II G protein-coupled receptors for VIP and PACAP:
structure, models of activation and pharmacology. Peptides 28(9):1631–1639
Lai JP, Ho WZ, Zhan GX, Yi Y, Collman RG, Douglas S (2001) Substance P antagonist (CP-96,
345) inhibits HIV-1 replication in human mononuclear phagocytes. Proc Natl Acad Sci U S A
98:3970–3975
Lai JP et al (2008) Differences in the length of the carboxyl terminus mediate functional properties
of neurokinin-1 receptor. Proc Natl Acad Sci U S A 105:12605–12610. https://doi.org/10.1073/
pnas.0806632105
Larhammar D, Beck-Sickinger A, Colmers WF, Cox HM, Doods HN, Herzog H, Michel CM,
Quirion R, Schwartz T, Westfall T (2015) Neuropeptide Y receptors, introduction. Last
modified on 10/08/2015. Accessed 01 Feb 2019. IUPHAR/BPS Guide to PHARMACOLOGY,
http://www.guidetopharmacology.org/GRAC/FamilyIntroductionForward?familyId¼46
Lee FY, Lin HC, Tsai YT, Chang FY, Lu RH, Hou MC, Li CP, Chu CJ, Wang SS, Lee SD (1997)
Plasma SP levels in patients with liver cirrhosis: relationship to systemic and portal hemody-
namics. Am J Gastroenterol 92(11):2080–2084
Levinson DF, Duan J, Oh S, Wang K, Sanders AR, Shi J, Zhang N, Mowry BJ, Olincy A, Amin F
(2011) Copy number variants in schizophrenia: confirmation of five previous findings and new
evidence for 3q29 microdeletions and VIPR2 duplications. Am J Psychiatry 168:302–316
Lin H, Harris T, Flannery M, Aruffo A, Kaji E, Gorn A, Kolakowski L, Lodish H, Goldring SR
(1991) Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science 254
(5034):1022–1024
Litvak DA, Iseki H, Evers BM, Greely GH, Hellmich MR, Iwase K (1999) Characterization of two
novel proabsortive peptide YY analogs, BIM-43073 and BIM-43004C. Dig Dis Sci 44
(3):643–648
Liu J, Hu JH, Zhu QG, Li FQ, Wang J, Sun HJ (2007) Effect of matrine on the expression of
substance P receptor and inflammatory cytokines production in human skin keratinocytes and
fibroblasts. Int Immunopharmacol 7(6):816–823
Loh K, Herzog H, Shi YC (2015) Regulation of energy homeostasis by the NPY system. Trends
Endocrinol Metab 26(3):125–135
Lorente L, Martín MM, Pérez-Cejas A, Ferreres J, Solé-Violán J, Labarta L, Díaz C, Jiménez A
(2017) Sustained low serum substance P levels in non-surviving septic patients. Int J Mol Sci 18
(7):1531. https://doi.org/10.3390/ijms18071531
548 N. A. Nimer et al.

Lorente L, Rodriguez ST, Sanz P, Pérez-Cejas A, Padilla J, Díaz D et al (2018) Patients with high
serum substance P levels previously to liver transplantation for hepatocellular carcinoma have
higher risk of one-year liver transplantation mortality. Oncotarget 9(30):21552–21559
Lu CT, Jin RR, Jiang YN, Lin Q, Yu WZ, Mao KL, Tian FR, Zhao YP, Zhao YZ (2015) Gelatin
nanoparticle-mediated intranasal delivery of substance P protects against 6-hydroxydopamine-
induced apoptosis: an in vitro and in vivo study. Drug Des Devel Ther 9:1955–1962
Ludwig M (2011) Are neuropeptides brain hormones? J Neuroendocrinol 23(4):381–382
Majkowska-Pilip A, Halik PK, Gniazdowska E (2019) The significance of NK1 receptorligands and
their application in targeted radionuclide tumour therapy. Pharmaceutics 11(9):443
Manak MM, Moshkoff DA, Nguyen LT, Meshki J, Tebas P, Tuluc F, Douglas SD (2010) Anti-
HIV-1 activity of the neurokinin-1 receptor antagonist aprepitant and synergistic interactions
with other antiretrovirals. AIDS (London, England) 24(18):2789–2796
Marchi N, Granata T, Ghosh C, Janigro D (2012) Blood–brain barrier dysfunction and epilepsy:
pathophysiologic role and therapeutic approaches. Epilepsia 53(11):1877–1886
Masi L, Brandi ML (2007) Calcitonin and calcitonin receptors. Metabolism 4(2):117–122
Mathioudakis AG, Chatzimavridou-Grigoriadou V, Evangelopoulou E, Mathioudakis G (2013)
Vasoactive intestinal peptide inhaled agonists: potential role in respiratory therapeutics.
Hippokratia 17(1):12–16
Mawe GM, Hoffman JM (2013) Serotonin signalling in the gut – functions, dysfunctions and
therapeutic targets. Nat Rev Gastroenterol Hepatol 10(8):473–486
McQuaid KR (2018) Drugs used in the treatment of gastrointestinal diseases. In: Katzung BG
(ed) Basic and clinical pharmacology, 14th edn. McGraw Hill, New York, p 1105
Mendlewicz J, Oswald P, Claes S, Massat I, Souery D, Van Broeckhoven C, Del-Favero J (2005)
Patient-control association study of substance P-related genes in unipolar and bipolar affective
disorders. Int J Neuropsychopharmacol 8(4):505–513
Mittapalli GK, Roberts E (2014) Ligands of the neuropeptide Y Y2 receptor. Res Gate 24:430–441
Moreno D, Gourlet P, De Neef P, Cnudde J, Waelbroeck M, Robberecht P (2000) Development of
selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC
(2) receptor. Peptides 21(10):1543–1549
Muñoz M, Coveñas R (2013) Safety of neurokinin-1 receptor antagonists. Expert Opin Drug Saf 12
(5):673–685. https://doi.org/10.1517/14740338.2013.804059]
Nathoo AN, Moeller RA, Westlund BA, Hart AC (2001) Identification of neuropeptide-like protein
gene families in Caenorhabditis elegans and other species. PNAS 98(24):14000–14005
Neumann JM, Couvineau A, Murail S, Lacapere JL, Marc NJ (2008) Class-B GPCR activation: is
ligand helix-capping the key? Trends Biochem Sci 33(7):314–319
Niedermair T, Kuhn V, Doranehgard F, Stange R, Wieskötter B, Beckmann J et al (2014) Absence
of substance P and the sympathetic nervous system impact on bone structure and chondrocyte
differentiation in an adult model of endochondral ossification. Matrix Biol 38:22–35
Nilsson J, von Euler AM, Dalsgaard C-J (1985) Stimulation of connective tissue cell growth by
substance P and substance K. Nature 315(6014):61–63
Nussey SS, Whitehead SA (2013) Endocrinology: an integrated approach. CRC Press,
Philadelphia, PA
Opolka A, Straub RH, Pasoldt A, Grifka J, Grassel S (2012) Substance P and norepinephrine
modulate murine chondrocyte proliferation and apoptosis. Arthritis Rheum 64:729–739
Owen RT, Castaner R, Bolos J, Estivill C (2009) Almorexant dual orexin OX1/OX2 antagonist
treatment of sleep disorders. Drugs Future 34(1):5–10
Parent J, Kron M (2012) Jasper’s basic mechanisms of the epilepsies - neurogenesis and epilepsy,
4th edn. National Center for biotechnology information (US), Bethesda, MD
Pati S, Alexopoulos AV (2010) Pharmacoresistant epilepsy: from pathogenesis to current and
emerging therapies. Cleve Clin J Med 77(7):457–567
Pennefather JN, Lecci A, Candenas ML et al (2004) Tachykinins and tachykinin receptors: a
growing family. Life Sci 74:1445–1463
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 549

Petra AI, Tsilioni I, Taracanova A, Alexandra Katsarou-Katsari M, Theoharides TC (2018a)

Interleukin 33 and interleukin 4 regulate interleukin 31 gene expression and secretion from
human laboratory of allergic diseases 2 mast cells stimulated by substance P and/or immuno-
globulin E. Allergy Asthma Proc 39(2):153–160
Petra AI, Tsilioni I, Taracanova A, Katsarou-Katsari A, Theoharides TC (2018b) Interleukin 33 and
interleukin 4 regulate interleukin 31 gene expression and secretion from human laboratory of
allergic diseases 2 mast cells stimulated by substance P and/or immunoglobulin E. Allergy
Asthma Proc 39(2):153–160
Pezzilli R (2006) Early antibiotic treatment in acute pancreatitis: more news. J Pancreas 7
(4):435–437
Pirosa A, Gottardi R, Alexander PG, Rocky S (2018) Tuan engineering in-vitro stem cell-based
vascularized bone models for drug screening and predictive toxicology. Stem Cell Res Ther
9:112
Pondel M (2000) Calcitonin and calcitonin receptors: bone and beyond. Int J Exp Pathol 81
(6):405–422
Portelli J, Michotte Y, Smolders I (2012) Ghrelin: an emerging new anticonvulsant neuropeptide.
Epilepsia 53(4):585–595
Querfurth HW, LaFerla FM (2010) Alzheimer’s disease. N Engl J Med 362(4):329–344
Quintanar JL, Guzman-Soto I (2013) Hypothalamic neurohormones and immune responses. Front
Integr Neurosci 7:56. https://doi.org/10.3389/fnint.22013
Ramalho R, Soares R, Couto N, Moreira A (2011) Tachykinin receptors antagonism for asthma: a
systematic review. BMC Pulm Med 11:41
Reynolds WJ, Chiu B, Inman RD (1988) Plasma SP levels in fibrositis. J Rheumatol 15
(12):1802–1803
Robertson CR, Flynn SP, White HS, Bulaj G (2011) Anticonvulsant neuropeptides as drug leads for
neurological diseases. Nat Prod Rep 28(4):741–762
Röder PV, Wu B, Liu Y, Han W (2016) Pancreatic regulation of glucose homeostasis. Exp Mol
Med 48(3):219
Rosa AC, Fantozzi R (2013) The role of histamine in neurogenic inflammation. Br J Pharmacol
170:38–45
Russo AF (2017) Overview of neuropeptides: awakening the senses? Headache 57:37–46
Satake H, Kawada T (2006) Overview of the primary structure, tissue-distribution, and functions of
tachykinins and their receptors. Curr Drug Targets 7(8):963–974
Schalla M, Stengel A (2018) Phoenixin—A pleiotropic gut-brain peptide. Int J Mol Sci 19(6):1726
Schank JR, Heilig M (2017) Substance P and the neurokinin-1 receptor: the new. In: Thiele TE
(ed) The role of neuropeptides in addiction and disorders of excessive consumption, 1st edn.
Academic Press, Cambridge, pp 151–175
Scholzen TE, Luger TA (2004) Neutral endopeptidase and angiotensin-converting enzyme – key
enzymes terminating the action of neuroendocrine mediators. Exp Dermatol 13(l4):22–26
Schöppe J, Ehrenmann J, Klenk C, Rucktooa P, Schütz M, Doré AS, Plückthun A (2019) Crystal
structures of the human neurokinin 1 receptor in complex with clinically used antagonists. Nat
Commun 10(1):17. https://doi.org/10.1038/s41467-018-07939-8
Schroeder A, Loh DH, Jordan MC, Roos KP, Colwell CS (2010) Circadian regulation of cardio-
vascular function: a role for vasoactive intestinal peptide. Am J Physiol Heart Circ Physiol 300
(1):H241–H250
Semernik O, Lebedenko A (2015) The role of vasoactive intestinal peptide in regulation of
bronchial tone in children with asthma during the period of exacerbation. Eur Respir J 46
(suppl 59):PA4194
Siegel DJ (1999) The developing mind: toward a neurobiology of interpersonal experience.
American Psychological Association. Guilford Press, New York
Sim DS, Kim W, Lee KH, Song HC, Kim JH, Park DS et al (2018) Cardioprotective effect of
substance P in a porcine model of acute myocardial infarction. Int J Cardiol 271:228–232
550 N. A. Nimer et al.

Smith SR, Weissman NJ, Anderson CM, Sanchez M, Chuang E, Stubbe S, Behavioral Modification
and Lorcaserin for Overweight and Obesity Management (BLOOM) Study Group (2010)
Multicenter, placebo-controlled trial of lorcaserin for weight management. N Eng J Med 363
(3):245–256
Spitsin S, Tebas P, Barrett JS, Pappa V, Kim D, Taylor D, Evans DL, Douglas SD (2017)
Antiinflammatory effects of aprepitant coadministration with cART regimen containing ritona-
vir in HIV-infected adults. JCI insight 2(19):e95893. https://doi.org/10.1172/jci.insight.95893.
Advance online publication
Steinhoff MS, von Mentzer B, Geppetti P, Pothoulakis C, Bunnett NW et al (2014) Tachykinins and
their receptors: contributions to physiological control and the mechanisms of disease. Physiol
Rev 94(1):265–301
Stojilkovic SS, Tabak J, Bertram R (2010) Ion channels and signaling in the pituitary gland. Endocr
Rev 31(6):845–915
Takei Y, Ando H, Tsutsui K (2016) Handbook of hormones: comparative endocrinology for basic
and clinical research. The Japan Society for Comparative Endocrinology; The University of
Tokyo, Chiba, Tokyon
Tang B, Yong X, Xie R, Li Q-W, Yang S-M (2014) Vasoactive intestinal peptide receptor-based
imaging and treatment of tumors. Int J Oncol 44(4):1023–1031
Taylor BK, Fu W, Kuphal KE, Stiller C-O, Winter MK, Corder GF, Urban JH, McCarson KE,
Marvizon JC (2014) Inflammation enhances Y1 receptor signaling, neuropeptide Y-mediated
inhibition of hyperglasia, and substance P release from primary afferent neurons. Neuroscience
256:178–194
Tekiner H, Acer N, Kelestimur F (2015) Sella turcica: an anatomical, endocrinological, and
historical perspective. Pituitary 18(4):575–578
Tian L, Cai L, Kang J (2000) Elevated substance P content in sputum and plasma in patients with
COPD and its relationship with FEV1/FVC. Zhonghua Jie He He Hu Xi Za Zhi 23:138–140
Vaudry D, Falluel-Morel A, Bourgault S, Basille M, Burel D, Wurtz O, Fournier A, Chow BK,
Hashimoto H, Galas L, Vaudry H (2009) Pituitary adenylate cyclase-activating polypeptide and
its receptors: 20 years after the discovery. Pharmacol Rev 61(3):283–357
Veldkamp MW, Geuzebroek GSC, Baartscheer A, Verkerk AO, Schumacher CA et al (2018a)
Neurokinin-3 receptor activation selectively prolongs atrial refractoriness by inhibition of a
background K+ channel. Nat Commun 9:4357. https://doi.org/10.1038/s41467-018-06530-5
Veldkamp MW, Geuzebroek G, Baartscheer A, Verkerk AO, Schumacher CA, Suarez GG, Berger
WR, Casini S, van Amersfoorth S, Scholman KT, Driessen A, Belterman C, van Ginneken A, de
Groot JR, de Bakker J, Remme CA, Boukens BJ, Coronel R (2018b) Neurokinin-3 receptor
activation selectively prolongs atrial refractoriness by inhibition of a background K+ channel.
Nat Commun 9(1):4357
Verma AK, Manohar M, Upparahalli Venkateshaiah S, Mishra A (2017) Neuroendocrine cells
derived chemokine vasoactive intestinal polypeptide (VIP) in allergic diseases. Cytokine
Growth Factor Rev 38:37–48
Vezzani A, French J, Bartfai T, Baram T (2011) The role of inflammation in epilepsy. Nat Rev
Neurol 7:31–40
Vezzani A, Friedman A, Dingledine RJ (2013) The role of inflammation in epileptogenesis.
Neuropharmacology 69:16–24
Vilisaar J, Arsenescu RI (2016) Roles of substance P in gastrointestinal functions and neuroimmune
interactions. In: Constantinescu C, Arsenescu R, Arsenescu V (eds) Neuro-immuno-gastroen-
terology. Springer, Cham, pp 53–73
von Zastrow M (2018) Drug receptors & pharmacodynamics. In: Katzung BG (ed) Basic and
clinical pharmacology, 14th edn. McGraw Hill, New York, pp 20–41. Gray
Vu J, Larauche M, Fores M, Luong L, Norris J, Oh S, Li-Jung L, Waschek J, Pisegna J, Germano P
(2015) Regulation of apetite, biody composition and metabolic hormones by vasoactive intesti-
nal polypeptide (VIP). J Mol Neurosci 56(2):377–387
15 Pharmacology of Neuropeptides: Substance P, Vasoactive Intestinal Peptides,. . . 551

Wang L, Singh M, Bonewald LF, Detamore MS (2009) Signalling strategies for osteogenic
differentiation of human umbilical cord mesenchymal stromal cells for 3D bone tissue engi-
neering. J Tissue Eng Regen Med 3:398–404
Wang XF, Ge TT, Fan J, Yang W, Cui RJ (2017) The role of substance P in epilepsy and seizure
disorders. Oncotarget. 8(44):78225–78233
White CM, Ji S, Cai H, Maudsley S, Martin B (2010) Therapeutic potential of vasoactive intestinal
peptide and its receptors in neurological disorders. CNS Neurol Disord Drug Targets 9
(5):661–666
Wimalawansa SJ (2010) Calcitonin: history, physiology, pathophysiology and therapeutic
applications. https://doi.org/10.1016/B978-0-12-374602-300053-5
Wu D, Lee D, Sung YK (2011) Prospect of vasoactive intestinal peptide therapy for COPD/PAH
and asthma: a review. Respir Res 12(1):45
Xiao J, Yu W, Wang X, Wang B, Chen J, Liu Y, Li Z (2016) Correlation between neuropeptide
distribution, cancellous bone microstructure and joint pain in postmenopausal women with
osteoarthritis and osteoporosis. Neuropeptides 56:97–104
Yin J, Chapman K, Clark LD, Shao Z, Borek D, Xu Q et al (2018) Rosenbaum crystal structure of
the human NK1 tachykinin receptor. Proc Natl Acad Sci U S A 115(52):13264–13269
Zalecki M (2019) Gastric ulcer induced changes in substance P and Nk1, Nk2, Nk3 receptors
expression in different stomach localizations with regard to intrinsic neuronal system.
Histochem Cell Biol 151(1):29–42
Zikou A, Sioka C, Alexiou GA, Fotopoulos A, Voulgaris S, Argyropoulou MI (2018) Radiation
Necrosis, pseudoprogression, pseudoresponse, and tumor recurrence: imaging challenges for
the evaluation of treated gliomas. Contrast Media Mol Imaging 2018:1–6
Neuropeptides and Neurotransmission
16
Anindita Mondal Gantait, Yazan A. Bataineh, Hiba Salim Surchi,
Arunava Gantait, G. Tulja Rani, Paramita Paul, Sarah Falah Kokaz,
Bilal A. Al-Jaidi, Puneet Kumar, Saumen Karan,
and Tanushree Singha

Abstract

Over the past four decades, many neuropeptides, that is, 3–100 amino-acid-long
polypeptides, have been identified in the central nervous system and the periph-
eral nervous system which can act on either neural substrates such as neurons and
glial cells or other target cells. Neuropeptides mediate neuronal communication
by acting on neuropeptide receptors. Neuropeptide receptors include over
44 receptor families, of which most are G protein-coupled receptors.
Neuropeptides and their cognate receptors are involved in various physiological
and pathophysiological functions, such as pain regulation, blood pressure, body

A. M. Gantait (*) · G. T. Rani

Malla Reddy Pharmacy College, Hyderabad, Telangana, India
Y. A. Bataineh · H. S. Surchi · S. F. Kokaz
Faculty of Pharmacy, Philadelphia University, Amman, Jordan
A. Gantait
Dr. Reddy’s Laboratories Limited, IPDO, Hyderabad, Telangana, India
P. Paul
Department of Pharmaceutical Technology, University of North Bengal, Raja Rammohunpur, Dist-
Darjeeling, West Bengal, India
B. A. Al-Jaidi
Faculty of Pharmacy, Philadelphia University—Jordan, Amman, Jordan
Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Yarmouk
University, Irbid, Jordan
P. Kumar
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India
S. Karan · T. Singha
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India

# Springer Nature Singapore Pte Ltd. 2020 553

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_16
554 A. M. Gantait et al.

temperature, feeding behavior, reproduction, sleep, learning and memory. There-

fore, neuropeptide transmission is an attractive area for drug discovery in several
therapeutic areas, including inflammatory conditions, epilepsy and psychiatric
diseases targeting neuropeptide receptors. This chapter elaborates and expounds
on the role of various small neuropeptides such as substance P (SP), vasoactive
intestinal peptides (VIP), neuropeptide Y (NPY) and endogenous opioid
neuropeptides, along with their physiology, pathophysiology and pharmacology.
This chapter also describes the various neuropeptide receptor agonists and
antagonists and their possible roles in the treatment of various disorders.

Keywords
Neuropeptide · VIP · Substance P · NPY · Endorphin · Enkephalin · Dynorphin

Abbreviations

5-HT Serotonin
ACE Angiotensin-converting enzyme
Ach Acetylcholine
ACTH Adrenocorticotrophic hormone
a-MSH Melanocyte-stimulating hormone
CAMP Cyclic Adenosine Monophosphat
CGRPs Calcitonin gene-related peptides
CINV Chemotherapy induced nausea and vomiting
CNS Central Nervous System
DAG Diacylglycerol
DOR δ opioid receptor
EPI Epinephrine
GABA Gamma amino butyric acid
GIP GPCR-interacting proteins
GLP-1 Glucagon-like peptide-1
GPCR G protein-coupled receptors
HA Histamine
IBD Inflammatory bowel disease
IP3 Inositol triphosphate
KOR κ opioid receptor
MAPK Mitogen-activated protein kinase
MEC Moderately emetogenic chemotherapy
MOR μ-opioid receptors
NE Noradrenaline
NK Neurokinin
NKA Neurokinin A
16 Neuropeptides and Neurotransmission 555

NMDA N-methyl-D-aspartate
NPY Neuropeptide Y
PACAP Pituitary adenylate cyclase-activating polypeptide
PC2 Pro-protein convertase-2
PENK-A Proenkephalin-A
PHM Peptide histidine methionine
POMC Precursor proopiomelanocortin
PP Pancreatic polypeptide
PPT-A PreprotachykininA
PYY Peptide YY
RAMPs Receptor activity modifying proteins
SP Substance P
VIP Vasoactive intestinal peptides

16.1 Introduction

Research on the underlying cellular and molecular mechanisms involved in various

activities of the brain composes a leading area in neuroscience that started long
before (Fan and Markram 2019). Neuropeptides are small protein-like molecules that
act as neuronal signaling molecules (Burbach 2011). These help the neurons to
communicate with one another and regulate various types of biological actions and
mediate many regulatory functions involving all organ systems, particularly brain
function such as food intake, social behavior, memory and learning, reproduction
and analgesia (Russo 2017). They are responsible for cellular signaling in the CNS,
PNS and the endocrine system (Catalani et al. 2017). Interestingly, humans have a
broad collection of neuropeptides that can influence a multitude of activities. To
date, there are over 100 neuropeptides that have been identified and it is predicted
that many more are present that are yet to be identified from the over 1000 predicted
peptides encoded by the genome (Russo 2017). The neurotransmitters are involved
in the transmission of electrical stimuli between nerve cells and mediate neuron-to-
neuron communication transmission. Neuropeptides and neurotransmitters are quite
different when monitoring their mode of communication in the nervous system.
Neurotransmitters are generally smaller than neuropeptides. Interestingly,
neurotransmitters may be a single amino acid, for example L-Glu, NMDA and
gamma aminobutyric acid (GABA), or any other molecules with small size such
as noradrenaline (NE), epinephrine (EPI), serotonin (5-HT), acetylcholine (ACh) or
histamine (HA). A signaling molecule is called a neuropeptide when it is compiled
with more than three amino acids (Purves et al. 2001). Thus the present chapter
describes the general overview of various small neuropeptides such as substance P
(SP), vasoactive intestinal peptides (VIP), neuropeptide Y (NPY) and other
neuropeptides and their related physiology, pathophysiology and pharmacology
(Fieber 2017).
556 A. M. Gantait et al.

16.2 Neuropeptides and Neurotransmission

Neurotransmitters are endogenous substances that are released from the neuron.
These molecules are responsible for the transmission of information on chemical
synapses. Neurotransmitters are released from the presynaptic axonal membrane to
the synaptic cleft and bind to the receptors present in the postsynaptic membrane of
other neurons and conduct signal transfer across the synapse. A molecule can be
considered a neurotransmitter if it fulfills the following criteria. It must present in the
vesicles along with the suitable enzymes that help in their synthesis and breakdown.
The substance should be released from the nerve ending in a chemically or
pharmacologically identifiable form as an effect of presynaptic nerve stimulation.
They must have action in the synapse and its actions must be prevented by receptor
blockers. Thus the neurotransmitters are chemicals that are released from the neuron
on nerve stimulation by generation of action potential and communicate with the
muscle, other organs and other neurons (Purves et al. 2001; González-Espinosa and
Guzmán-Mejía 2014; Fieber 2017).
The process of neurotransmission takes place in the synaptic cleft of the nervous
system composed of the basic cells called neurons. Neurons, in spite of having
unimaginable variation in their structure and functions, possess many common
features among them, and they do the work of cellular communication (Patri 2019;
Lodish et al. 2000). The communication between two neurons starts when electrical
impulse or action potential is carried in only one direction by the long slender
projection called the axon (Hormuzdi et al. 2004). The action potential reaches the
axon terminal but cannot cross the synaptic space and thus triggers the neurotrans-
mitter release from their storage vesicles present in synaptic terminals (presynaptic
membrane) into a space known as the synapse. The neurotransmitters then bind with
special proteins called the receptors present in the postsynaptic membrane of another
neuron, which triggers the action potential to move toward the cell body to the axon
of the postsynaptic neuron (Lodish et al. 2000). The neurotransmitters then release a
related message from the receptor into the synaptic space. Some of the
neurotransmitters degraded by the local enzymes are taken back by the transporter
proteins present in the presynaptic membrane (Lodish et al. 2000; Forehand 2009).
The neurotransmitters that are taken back are repackaged into a vesicle which is
released again when an action potential reaches the axon terminal. The entire process
is repeated when an action potential reaches the axon terminal (Forehand 2009).

16.3 Substance P

Substance P (SP), alongside Neurokinin A (NKA) and Neurokinin B (NKB) and

others, is a member of the tachykinin family, which is known to be one of the largest
neuropeptide families. It has marked biological activity in different systems across
species. It was originally identified in the early years of the twentieth century in the
course of investigating the tissue distribution of acetylcholine by researchers
experimenting on a rabbit’s isolated intestine, though the substance was initially
16 Neuropeptides and Neurotransmission 557

extracted from equine horse brain and gut tissues (V Euler and Gaddum 1931). This
substance was later named Substance P, P as in the powder obtained during the
procedure. Substance P is an undecapeptide, composed of a chain of 11 amino acid
residues (Arg-Pro-Lys-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2). It shares the same
carboxyl terminal sequence, Phe-(Phe or Val)-Gly-Leu-Met-NH2, with the rest of
the tachykinin family members; however, it has a net positive charge. The
N-terminus holds the positively charged residues, while the C-terminus is confined
to hydrophobic residues, rendering it an amphiphilic peptide. The biological actions
are mediated by neurokinin (NK) G protein-coupled receptors acting on smooth
muscle contraction, vasodilation, nociception and modulation of inflammatory
responses (Mashaghi et al. 2016a, b).

16.3.1 Physiology

16.3.1.1 Biosynthesis and Metabolism

Substance P is encoded by the TAC1 gene, which is found in the central nervous
system, peripheral afferent sensory neurons, cardiovascular system and other body
parts. The gene expresses four mRNA isoforms (α, β, γ and δ), α being more
abundant in the brain and β and γ in peripheral tissues, all generating substance P
(Dehlin and Levick 2014). The peptide precursor was designated as
preprotachykinin A (PPT-A), which in turn is cleavaged by the actions of proteases.
The C-terminus undergoes amidation executed by the enzyme peptidyl-
Gly-α-amidatingmonoxygenase, using glycine as an amide donor. Produced peptide
fragments are then bound by a pair of basic amino acids (Lys-Arg) on both sides. The
synthesis occurs in ribosomes and is then packed into large dense-core storage
vesicles and transported to nerve endings for enzymatic processing and converted
into the active form and stored in vesicles ready for release (Nakanishi 1987;
Steinhoff et al. 2014). A number of enzymes are responsible for the metabolism of
substance P, including substance-P-degrading enzyme, angiotensin-converting
enzyme (ACE) and endopeptidases (Matsas et al. 1984), among many others.

16.3.1.2 Action on Target Tissues

Neurokinin (NK) receptors belong to the class I rhodopsin-like G protein-coupled
receptors family, consisting of seven hydrophobic transmembrane domains,
connected by extracellular and intracellular loops and coupled to G proteins. Sub-
stance P exerts its effects as a neurotransmitter and neuromodulator principally by
subsequent activation of a second messenger system. It binds majorly to the NK1
receptor, which is most abundant in body tissues. However, it also activates the NK2
and NK3 receptors in certain conditions, that is, high peptide concentration and
receptor availability. The peptide has two distinguishable binding sites: one part is
inserted in to the hydrophobic ligand binding site, while the remainder interacts with
amino acid residues on the extracellular sites of the receptor. C-terminal of the
receptor holds threonine and serine amino acids, which are sites of phosphorylation
558 A. M. Gantait et al.

of the GPCR. This results in the activation of phospholipase C which catalyzes

the hydrolysis of phosphatidylinositol and the production of inositol triphosphate
(IP3) and diacylglycerol (DAG), elevating cytosolic calcium concentration. Then
phospholipase A induces an increase in arachidonic acid movement while adenylyl
cyclase invokes cAMP accumulation. The rise in the intracellular calcium concen-
tration then activates a calcium-dependent chloride channel that leads to cell
response. It has been proposed that SP can employ negative feedback via activation
of autoreceptors. This finding is particularly substantial concerning inflammation
and nociception, and the possibility of designing a drug affecting its functions
(Takeda et al. 1992; Malcangio and Bowery 1999; Yin et al. 2018).

16.3.2 Pathophysiology

Due to its wide distribution in the body, substance P has numerous physiological
implications in the CNS, cardiovascular, respiratory, gastrointestinal systems and
modulation of the immune response. Additionally, it plays a critical role in the
regulation of the excitability of dorsal horn nociceptive neurons (Christofi 2018). It
is associated with the regulation of mood disorders, anxiety, stress, neurotoxicity and
pain. In the gastrointestinal tract, SP and other tachykinins act as neurotransmitters
that regulate motor activity, secretion of ions and vascular functions. Therefore, the
elevation of the neuropeptide or its receptor has been linked to several chronic
pathological conditions such as inflammatory disorders (e.g. irritable bowel syn-
drome) (Sohn et al. 2013), Alzheimer’s disease (Severini et al. 2016), Parkinson’s
disease (Thornton and Vink 2015), and mood and mental disorders. Nerve fibers
producing substance P were detected both in atrial and ventricular myocardia, where
efferent and afferent sensory functions of the peripheral neurons innervating the
heart and coronary arteries are mediated by tachkykinins and calcitonin gene-related
peptides (CGRPs). It has been pointed out that substance P possesses binding sites
surrounding coronary arteries and cardiac fibers and on mitral and tricuspid valves. It
has negative inotropic and chronotropic effects due to an increase in the activity of
cholinergic neurons, leading to a weaker heart rate and a change in the force of
contractions (Mistrova et al. 2016). SP and its receptors’ distribution in the brain was
first described in the 1970s. It has been demonstrated that high levels of SP
immunoreactivity have been spotted in areas associated with regulation of stress
and anxiety reactions, mainly in the hippocampus, amygdala and some areas of the
hypothalamus. In these aforementioned regions, the peptide coexists in the same
neuron terminal with various neurotransmitters and neuropeptides such as glutamate,
GABA, histamine, acetylcholine, dopamine and serotonin (Ebner and Singewald
2006).
Substance P initiates the expression of various known immunological chemical
messengers. Primary lymphoid organs such as the bone marrow and thymus and
secondary ones such as the spleen and lymph nodes have been observed to be
innervated by neurons containing SP. This suggests that substance P may act as a
16 Neuropeptides and Neurotransmission 559

mediator crosslinking the nervous and immune systems. Activated T lymphocytes

are shown to express PPT-A, thus producing the neuropeptide, which may act in an
autocrine fashion to regulate T cell proliferation (Mashaghi et al. 2016a, b). It has
been demonstrated that substance P contributes to pain transmission and neurogenic
inflammation, both mediated through the NK1 receptor. Pain-transmitting receptors
are present in the dorsal horn of the spinal cord. SP is released from the peripheral
terminals of the primary afferent nerves after peripheral nerve injury, contributing to
the induction of neuropathic pain.

16.3.3 Pharmacology of Substance P

Different organs and multiple neurotransmitters are involved in the response to

emetic triggers. Afferent nerve impulses transmitting from the cerebral cortex,
chemoreceptor trigger zone and vagal afferent fibers of the gastrointestinal
(GI) tract travel to the vomiting center, which is located in the medulla oblongata
of the nervous system. Efferent impulses then travel from the vomiting center to
the abdominal muscles, salivation center and respiratory center, causing vomiting.
Other areas of the CNS are also involved, such as the limbic and vestibular systems
due to the states of vertigo, motion sickness and pain. Predominant neurotransmitter
and neuropeptide receptors involved in this kind of signaling include serotonin
(5-HT3) receptors, neurokinin-1 (NK-1) receptors, histamine and dopamine
receptors. Available and effective antiemetic agents target these receptors, usually
in combination, targeting more than one receptor at once. Chemotherapeutic agents
and anesthetics stimulate the release of these neurotransmitters and induce nausea
and vomiting. Chemotherapy induced nausea and vomiting (CINV) has five
categories. Acute-onset CINV can be triggered a few hours (within 24 h) after
chemotherapy initiation. Delayed CINV occurs after the acute phase after >24 h
(peak in 2–3 days, and can last up to 1 week). Some CINV patients develop
‘anticipatory nausea and vomiting’, occurring prior to administration of a chemo-
therapeutic agent and attributed to the adverse memory of prior CINV. Breakthrough
emesis is a type of vomiting that occurs within 5 days after the prophylactic or rescue
use of an antiemetic agent. Refractory emesis is defined as vomiting which occurs
after chemotherapy administration in subsequent chemotherapy sessions where
antiemetic prophylaxis has failed in earlier sessions. Age, sex, dose and
emetogenicity are risk factors for developing CINV; for example, females and
younger patients are at a higher risk, and a higher chemotherapy dose is also a
relevant factor. Neurokinin-1 (NK-1) receptor antagonists represent the newest class
of antiemetic agents that are effective for the prevention of chemotherapy-induced
nausea and vomiting (Aapro et al. 2016; Grunberg et al. 2011; Hesketh 2008)
(Fig. 16.1).
560 A. M. Gantait et al.

Higher CNS center

Chemotherapy
Medulla oblongata

Chemoreceptor trigger zone Vomiting center

Increased afferent input to the chemoreceptor trigger zone and vomiting center

In small intestine it leads to cell damage

as well as activation of vagus and splanchnic nerves

Fig. 16.1 Proposed pathway of chemotherapy-induced emesis

16.3.4 Neurokinin-1 Receptor Antagonists

16.3.4.1 Aprepitant/Fosaprepitant

Pharmacodynamics and Indications

Aprepitant is a highly selective and potent NK-1 receptor blocker, thus alleviating
the emetic effects of substance P. It has a low affinity for NK-2 and NK-3 receptors
and little or negligible affinity for 5-HT3 and dopamine receptors. Fosaprepitant is a
phosphoryl prodrug of aprepitant, is water-soluble and is converted to its active form
30 min after intravenous administration via phosphatases. Aprepitant crosses the
blood brain barrier (BBB) and occupies NK-1 receptors, thereby antagonizing
the effects of substance P in the CNS and periphery. The drug has been approved
for the prevention of nausea and vomiting in patients receiving moderately
emetogenic chemotherapy (MEC) and highly emetogenic chemotherapy (HEC),
for example cisplatin-containing, regimens. There have been significantly higher
rates of response when given in combination with a 5-HT3 receptor antagonist
(e.g. ondansetron) in addition to a corticosteroid (e.g. dexamethasone) for delayed-
onset CINV (Zhang 2019; Aapro et al. 2016) (Fig. 16.2).

16.3.4.2 Pharmacokinetics

Administration and Dosing

Aprepitant is administered orally and intravenously, while fosaprepitant is available
only intravenously. It is commercially available under the trade name Emend®,
which contains a nanoparticle form of the drug with a diameter below 200 nm and
16 Neuropeptides and Neurotransmission 561

F F
F F

O F
O O F
N N F
O
HN O O N
F N HO
H N
P
F F N
HO F
F
F NH
F
O
Aprepitant Fosaprepitant

Fig. 16.2 Structures of aprepitant and fosaprepitant

is coated and encapsulated. A dose of 125 mg of aprepitant is given on the first day,
followed by 80 mg once daily. Onset of action is 1 h when administered orally, with
peak plasma level at 4 h. Aprepitant has a bioavailability of 60–70% after oral
administration, indicating a low liver first pass extraction.

Metabolism and Excretion

Metabolism is mainly mediated by cytochrome 450 enzymes, especially CYP3A4,
and less by CYP1A2 and CYP2C9. Therefore, aprepitant can reduce the plasma
concentrations of drugs metabolized by these isoenzymes. The apparent biological
half-life (t1/2) is 9–13 h, with a total clearance of 60–85 ml/min (Roos et al. 2017).

16.3.5 Rolapitant

16.3.5.1 Pharmacodynamics and Indications

Rolapitant is a novel, orally active, selective, high-affinity, competitive NK-1 recep-
tor antagonist, with a prolonged half-life of approximately 180 h. One advantage of
rolapitant over aprepitant and other antiemetic agents is that it does not interact with
(inhibit or induce) CYP3A4, which is involved in the metabolism of numerous
drugs. Therefore, rolapitant is unlikely to interact with drugs metabolized through
this enzyme. However, the drug has shown moderate activity on CYP2D6, acting as
a reversible inhibitor (Wang et al. 2018). Rolapitant was globally approved for use in
2015 in combination with other antiemetics, for delayed-onset emesis associated
with initial or repeat courses of emetogenic cancer chemotherapy. It is usually
administered in combination with a 5-HT3 antagonist and a corticosteroid (Rapoport
et al. 2016; Syed 2015).
562 A. M. Gantait et al.

Fig. 16.3 Structure of O

rolapitant F
F F
HN

NH
O
F

F
H F

Rolapitant

16.3.5.2 Pharmacokinetics

Administration and Dosing

Rolapitant is well absorbed from the gut when taken orally, with or without
the presence of food. The recommended dose of the drug is 180 mg, taken about
1–2 h before initiating chemotherapy. It can be detected in the plasma 30 min after
oral intake. Rolapitant has an extended t1/2, reaching 160–180 h, which is longer than
the half-life of other NK-1 antagonists. Therefore, a single dose of the drug is
sufficient to prevent the risk of developing delayed-onset phase nausea and vomiting
and also improves the patient’s compliance with treatment.

Metabolism and Excretion

Rolapitant is mainly metabolized by CYP3A4 to form its metabolite M19
(C4-pyrrolidine-hydroxylated rolapitant), and a number of insignificant inactive
metabolites. However, in animal studies most of the drug is excreted unmetabolized,
mainly by the liver (14% in urine, 73% in feces) over 6 weeks. The pharmacokinetic
parameters of rolapitant are not significantly affected in patients with mild or
moderate hepatic and renal impairment, and thus dosage adjustments are not
required (Glass et al. 2019, Syed 2015, Wang et al. 2018) (Fig. 16.3).

16.4 Vasoactive Intestinal Peptide (VIP)

Vasoactive intestinal peptide (VIP) is the most abundant neuropeptide in the gut,
controlling intestinal motility and water and electrolyte secretion. VIP belongs to the
glucagon/secretin superfamily; it was isolated from porcine small intestine and
shown to have a similar amino acid sequence than glucagon and secretin (Said and
Mutt 1970; Mutt and Said 1974). VIP not only functions as a peptide neurotransmit-
ter in the gastrointestinal tract but also in the central and peripheral nervous systems.
16 Neuropeptides and Neurotransmission 563

16.4.1 Physiology

16.4.1.1 Biosynthesis and Action

VIP contains 28-amino acid neuropeptide, sharing a 68% homology with the
PACAP, that is, pituitary adenylate cyclase-activating polypeptide, which is also a
neuropeptide (Vu et al. 2015). A common ancestral gene is responsible for the
synthesis of both VIP and PACAP. Breakdown of a certain pre-pro-peptide precur-
sor by the actions of peptidases (endoplasmic reticulum peptidase) obtains pro-VIP,
which undergoes further post-translational modifications resulting in the production
of two peptides, vasoactive intestinal peptide and peptide histidine methionine
(PHM). VIP triggers biological responses through VPAC1, VPAC2, PAC1 and
PAC2, that is, all are G protein-coupled receptors which are available in the central
nervous system, the heart and other tissues. VPAC receptors were cloned in the last
decade of the twentieth century, revealing the existence of a new G protein-coupled
receptor subfamily named Class B or Class II GPCR. Members of this subfamily
share the general structural scheme, having 7-transmembranous helices (TM I to TM
VII), interconnected by intracellular and extracellular loops (Couvineau et al. 2013)
(Fig. 16.4).
Other family members include glucagon, secretin, CGRP, glucagon-like peptide-
1 (GLP-1), growth hormone-releasing factor and several others. Stimulation of VIP
receptors subsequently leads to an increase in cytosolic concentrations of cAMP and
calcium by coupling with adenylyl cyclase through G proteins. In addition, it has
been reported that the neuropeptide also binds to non G protein receptors called
‘accessory proteins or GPCR-interacting proteins (GIP)’, which play an important
role in various functions including control of targeting, trafficking and signaling of

C122 K127

N28
N24
NH2 G116
Y22

N-ted

Q135
D107 F6

F0

Fig. 16.4 3D structural model of VPAC1 receptor showing different binding pocket for VIP.
(Reprinted with permission from Couvineau et al. 2013)
564 A. M. Gantait et al.

GPCRs (Bockaert et al. 2004). VIP participates in a variety of biological and

pathological processes including metabolic processes, smooth muscle relaxation,
acting as a cytokine-like peptide, and control of neuronal and endocrine cells, and
also plays important roles in some cancers, modulation of immune responses and the
circadian rhythm.

16.4.2 Pathophysiology

According to experimentation, certain disease conditions such as hypertension,

obesity, diabetes and hypothyroidism are responsible for the reduction of
VIP-elicited adenylyl cyclase activity (Said and Rattan 2004 & Said et al. 1996).
The role of VIP as a vasodilator and free-radical scavenger in the reduction of
myocardial ischemia and reperfusion injury has been established by several preclini-
cal studies (Said 1967). Thus depletion of Myocardial VIP may lead to cardiomyop-
athy and myocardial fibrosis.
VIP is an important mediator for gut smooth muscle relaxation and also respon-
sible for stimulation of water, electrolyte, enzyme and mucus secretion. It thus
increases the propulsion of chyme in the gut (Furness et al. 1995; Lelievre et al.
2007). VIP and its receptors play an important role in the control of ovarian
folliculogenesis (Bruno et al. 2011). VIP deficiency plays an important role in the
pathogenesis of asthma, pulmonary arterial hypertension and cystic fibrosis (Said
1989; Szema et al. 2006 & Said et al. 2007).

16.4.3 Pharmacology

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating

peptide (PACAP) are important structurally related neuropeptides in the peripheral
and central nervous systems. VIP and PACAP are involved in many pathophysio-
logical processes related to the digestive tract, cardiovascular system, airways,
reproductive system, immune system, endocrine glands and brain (Vaudry and
Laburthe 2006; Sherwood et al. 2000). Even though VIP and/or PACAP used to
play an important role in the treatment of inflammatory and neurodegenerative
diseases, due to their short half-lives, they are no longer used for therapeutic
purposes (Delgado et al. 2004; Gomariz et al. 2006; Gozes et al. 1999). PACAP
and VIP have quite similar affinity (high affinity) for the classical VIP receptors.
There are three different types of receptors for VIP and PACAP which were revealed
by receptor cloning in 1990. VIP and PACAP both have high affinity for the two
receptors, VPAC1 and VPAC2 (Harmar et al. 1998& Laburthe et al. 2002). Both
VPAC1 and VPAC2 are G protein-coupled receptors (GPCRs) which stimulate both
by either activation of adenylyl cyclase (AC) through Gs protein or activation of
phospholipase C (PLC) through Gq and/or Gi/Go protein. The receptor activity-
modifying proteins (RAMPs) determine the balance between coupling to AC versus
PLC. These VPAC1 and VPAC2 receptors belong to the class B or Class II family of
16 Neuropeptides and Neurotransmission 565

Table 16.1 Agonists and antagonists of VPAC1 and VPAC2 receptors

VPAC1 VPAC2
Agonist [Ala11,22,28]VIP Ro 25–1392
Antagonist PG 97-269 Unavailable
References Nicole et al. (2000); Gourlet et al. (1997) Xia et al. (1997)

GPCRs. These class B or class II receptors for peptides bear low sequence
homologies with other members of the superfamily of GPCRs. VPAC receptors
show several common properties with other class II GPCRs such as a large
N-terminal extracellular domain (>120 residues) with 10 highly conserved amino
acids including six cysteines and several potential N-glycosylation sites. Secretin,
helodermin, GRF and PHM (peptide histidine methionineamide), the other members
of the VIP-secretin structural family, can also bind to VPAC1 or VPAC2 receptors
with less affinity (Laburthe et al. 2002). Agonists and antagonists of VPAC1 and
VPAC2 receptors are described in Table 16.1.

16.5 Neuropeptide Y

The neuropeptide Y family comprises three polypeptides activating four distinct

receptors, including neuropeptide Y (NPY), peptide YY (PYY) and pancreatic
polypeptide (PP). NPY is one of the most expressed neuropeptides in the central
and peripheral nervous systems, whereas PYY and PP are considered neuroendo-
crine hormones. NPY was isolated and identified for the first time from porcine
hypothalamus brain tissue in 1982 by using a novel chemical assay that allowed
the detection of peptides with amidated C-terminals. Afterwards, human NPY was
isolated from adrenal-medullary pheochromocytoma tissue. Neuropeptide Y is a
36-amino acid α-amidated peptide (Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-
Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-
Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2) acting on G protein-coupled receptors. All
three peptides have 36-amino acid residues and play vital roles in neural and humoral
communication between body tissues. It was named neuropeptide Y because it
contains many tyrosine (Y) residues in its structure and to distinguish it from PYY
that possesses a very similar structure to NPY. The coexistence of NPY with classic
neurotransmitters (e.g. GABA and glutamate) has been frequently observed
(Tatemoto 1982).

16.5.1 Physiology

16.5.1.1 Biosynthesis and Receptors

Neuropeptide Y is synthesized in the central nervous system and is expressed
preferentially in interneurons. Abundant quantities of NPY receptors can be found
in numerous regions of the brain. The biological and physiological activities of NPY
566 A. M. Gantait et al.

are mediated by at least five types of receptors that have been identified, Y1, Y2, Y4,
Y5 and Y6; however, Y6 has not been assigned to any biological function. Addi-
tional distinguished receptor subtypes exist in other species. The Y receptors belong
to the G protein-coupled receptor family, exhibiting its effects through inhibiting
cAMP-dependent kinase. NPY plays vital roles in the gut-brain axis
communications, by being synthesized in the brain and acting locally in a paracrine
fashion in the GIT through its receptors to regulate motility and electrolyte secretion.
It thus regulates appetite and metabolism (Cox 2007).

16.5.2 Pathophysiology

The local action of NPY-like peptides after their exocytotic release depends on their
concentration, receptor selectivity, the expression of Y receptors and the presence of
specific peptidases influencing half-life (von Hörsten et al. 2004). The potential roles
of NPY in the etiology and pathophysiology of mood and anxiety disorders, as well
as related alcohol use disorders, have been extensively studied. Thus, modulation of
NPY-ergic activity within the CNS, via ligands aimed at different receptor subtypes,
may be an attractive target for treatment development for affective disorders, as well
as for alcohol use disorders (Thorsell and Mathé 2017). NPY has been found to
stimulate intake of food, preferably carbohydrate intake. It decreases latency to eat,
increases the drive to eat and delays satiety by augmenting meal size (Beck 2006).
The interactions between the immune system, that is, immune cells and gut
neurohormones, especially NPY, play an important role in producing inflammation
in inflammatory bowel disease (IBD). Thus, NPY may be targeted to diminish
the inflammation in IBD (El-Salhy and Hausken 2016). The significance of
investigating the underlying pathophysiological mechanisms of arteriosclerotic car-
diovascular disease and the role of NPY in this regard is great. It is involved in the
pathogenesis of arteriosclerosis via aggravating endothelial dysfunction and growth
of vascular smooth muscle cells, by the formation of foam cells and platelet
aggregation (Zhu et al. 2016; Sun et al. 2017). NPY, a major peripheral vascular
contractive neurotransmitter, interacts with its receptors and is thus associated
with the pathology and development of diabetes. It further contributes to diabetes-
induced cardiovascular disease by promoting the proliferation of endothelial cells
and vascular fibrosis (Sun et al. 2017). The effects of NPY on different species are
described in Table 16.2.

16.5.3 Pharmacology

NPY is one of the most effective orexigenic peptides that are mostly found in the
brain (Beck 2006; Hofmann et al. 2019). NPY acts via four different types of
functional G protein-coupled receptors (GPCRs) known as Y1, Y2, Y4 and Y5
(Table 16.3). All of these NPY receptors act via coupling to Gi and thus by inhibiting
the synthesis of cAMP. Y1 is mainly distributed to the brain, that is, anterior
16 Neuropeptides and Neurotransmission 567

Table 16.2 Effect of NPY on different species

Species NPY effects on different target Reference
Human 1. Reduction of vasodilation and plasma Baraniuk et al. (1992); Lacroix
Exudation. et al. (1996); Fujiwara et al.
2. Reduction of nasal airway resistance and (1993)
mucus production.
3. Inhibition of cholinergic component airways.
Dog Vasoconstriction. Laitinen et al. (1987)
Guinea pig Inhibition of cholinergic component. Stretton and Barnes (1988)

Table 16.3 NPY receptors and their possible role

Receptor Y1 Y2 Y4 Y5
Agonist Angiogenesis, Angiogenesis, Decrease Anticonvulsant,
actions anxiolysis, cellular anticonvulsant, feeding, circadian rhythm
proliferation, circadian anxiogenesis, gastrointestinal regulation,
rhythm regulation, cellular regulation. increase feeding.
endocrine regulation, proliferation,
increase feeding, decrease
sedative, feeding,
vasoconstriction. gastrointestinal
motility and
secretion.

thalamus, cerebral cortex, medial geniculate and amygdala; peripheral nervous

system, that is, superior cervical and dorsal root ganglion; and in peripheral blood
vessels, that is, intra-myocardial, colonic and renal blood vessels.
In contrast, the Y2 receptor is distributed in the central nervous system, that is,
hypothalamus, lateral septum, hippocampus and amygdala, peripheral nervous sys-
tem, intestine, certain blood vessels, hypothalamus, lateral septum, hippocampus
and amygdala. Y2 agonists administered centrally can increase BP whereas stimula-
tion of the central Y1 receptor decreases BP. NPY shows its effect on angiogenesis
and circadian rhythms via Y2. In comparison to Y1 and Y2, Y4 has limited
distribution in the brain. However, Y4 is present in skeletal muscle, thyroid gland,
heart, stomach, small intestine, adrenal medulla and nasal mucosa. Y5 mRNA
receptor is extensively distributed in the CNS and the periphery, and it is present
in the intestine, ovary, testes, prostate, spleen, pancreas, kidney, skeletal muscle,
liver, placenta and heart. NPY-inhibited LH release and regulation of seizures and
brain excitability are mediated via the Y5 receptor (Gehlert 2009).
The effects on feeding are mediated through at least two receptors, the Y1 and Y5
receptors. The NPY system that regulates feeding in dietary-induced obesity is
generally located in the hypothalamus (Beck 2006). In arteriosclerotic cardiovascu-
lar disease, increased peripheral NPY was found to be involved in the pathophysio-
logical process of atherosclerosis by affecting the vascular endothelial dysfunction,
the proliferation of vascular smooth muscle cells, the local inflammatory response
of plaques, the formation of foam cells, and activation and aggregation of platelets.
568 A. M. Gantait et al.

The role of NPY in the manifestation of atherosclerotic cardiovascular disease

through the central and/or peripheral nervous system was detected. Increased NPY
was related to dyslipidemia, hypertension, obesity, diabetes, impaired glucose toler-
ance and smoking, which are all risk factors for arteriosclerotic cardiovascular
disease (Zhu et al. 2016).

16.6 Other Neuropeptides

16.6.1 Opioid Neuropeptides

The endogenous opioids system includes numerous peptides that are widely
distributed throughout the human body, all acting as ligands for opioid receptors
including endorphins, enkephalins, dynorphins and, most recently discovered,
endomorphins, all of which are biologically active products that are released at the
synaptic terminals of opioidergic neurons. Classical opioid receptors are divided into
three subtypes, the μ receptor (MOR), δ receptor (DOR) and κ receptor (KOR), all
belonging to the GPCR family, and consisting of highly homologous
7-transmembranous helices linked with extracellular peptide loops (Li et al. 2012).
Opioid neurotransmission appears to affect many CNS functions, such as
nociception, cardiovascular regulation, respiratory rate, neuroendocrine activity,
aggressive, locomotive, pleasure and sexual behaviours, and learning and memory.

16.6.2 Endorphins

Endorphins are natural endogenous opioid neuropeptides, are one of the major
products of the precursor proopiomelanocortin (POMC) and are secreted by the
anterior pituitary gland through the hypothalamus in response to certain physiologi-
cal triggers such as strenuous physical exercise, stress and pain, and they resemble
opiates in their ability to produce analgesic effects and inhibit transmission of pain
signals. Other active products of this precursor include adrenocorticotrophic hor-
mone (ACTH) and a-melanocyte-stimulating hormone (a-MSH). The word endor-
phin is contracted from the words Endogenous and morphine. Four types of
endorphins are produced in the human body, α-, β-, γ- and σ- endorphins, each
having different numbers and types of amino acids in their molecules, between
16 and 31 amino acid residues in each peptide molecule (Shrihari 2017; Li et al.
2012).
Endorphins are widely distributed in many parts of the body, mainly in the
pituitary glands as well as in the brain. β-endorphin is the most potent and abundant,
in terms of natural pain relief, and is present in the neurons of both the central and
peripheral nervous system. They are released during pain or stress, and are
associated with sexual and maternal behavior. Additionally, endorphins have been
found to be associated with states of pleasure including emotions brought about by
laughter, intercourse, love and even appetizing food (Sprouse-Blum et al. 2010).
16 Neuropeptides and Neurotransmission 569

Endorphins mediate their actions mainly through μ-opioid receptors (MOR), μ

referring to morphine. μ-receptors are found presynaptically in various brain regions
and act by inhibiting neurotransmitter release, for instance by inhibiting the release
of the inhibitory neurotransmitter GABA that decreases the inhibition of dopamine
pathways, and consequently leading to increased dopamine release. This process
leads to deviant synaptic pliability, which causes addiction. Moreover, the afore-
mentioned binding of endorphins to their receptors also triggers chemical processes
that prevent the release of substance P, among other tachykinins, which is one of the
substances that participate in the conveyance of pain. MORs not only modify
transmission and perception of nociceptive stimuli, but are also associated with
opioid-induced bowel dysfunction, reduction in the respiratory rate in response to
high CO2 levels and abuse liability due to the manifestation of tolerance and
dependence (Li et al. 2012;Dalayeun et al. 1993). From a clinical standpoint, the
ideal opioid-based drug would be one that provides rapid pain relief while producing
minimal physiologic or psychologic side effects. Use of endogenous peptides as
drugs has remained a challenge, since peptides do not cross the blood-brain barrier
(BBB), and are quickly degraded in the bloodstream prior to delivery to their sites of
action in the brain.
MORs are targeted in clinical use, and several μ-receptor agonists and antagonists
have been developed. DAMGO is a pentapeptide derived from the endogenous
δ-receptor’s ligand enkephalin. It is a well-known compound, having its pharmacol-
ogy assessed in vitro and in vivo, and is frequently used as a reference substance. It
is highly selective for the μ-receptor with an affinity approximately 1000 times
higher than for the δ-opioid receptor. Several studies have proven than DAMGO
is 20 times more potent than morphine against nociception when administered
intracerebroventricularly to mice, while it is comparable to morphine when
administered subcutaneously or intravenously. Despite its effective pharmacological
properties, DAMGO shows very limited CNS effects when administered systemi-
cally (Lindqvist et al. 2016). Another agonist, fentanyl, is a phenylpiperidine-related
synthetic opioid with a very high affinity toward μ-receptors. It is fast acting and
approximately 50 to 100 times more potent morphine, with a good solubility and
a relatively low molecular weight. Sufentanil and hydromorphone are μ-opioid
receptor agonists clinically used in anaesthesia and postoperative analgesia.
Hydromorphone possesses a high affinity to both μ-opioid and δ-opioid receptors,
while sufentanil is a highly μ-receptor-selective agonist (Yang et al. 2018). Although
the μ-opioid receptor agonists induce strong analgesic effects and sedation, it is not
undeniable that they also cause side effects such as respiratory depression, constipa-
tion and euphoria. Subsequent chronic MOR agonism promotes opioid tolerance and
physical dependence.
Antagonists of the μ-opioid receptor are also available, such as naloxone and
naltrexone that can suppress central endogenous opioid receptor systems. Naltrex-
one is a long-acting competitive antagonist, which is suitable for oral administration,
and has been studied and used as an adjunctive in opioid addiction management
programs. In morphine-dependent subjects, naltrexone was found to be more potent
than nalorphine and twice as potent as naloxone. Naloxone is typically administered
570 A. M. Gantait et al.

in cases of opioid overdose to mitigate bodily response to the opioid (Glanz et al.
2018; Gonzalez and Brogden 1988). Another example is alvimopan, as it has a high
affinity for MOR, but low systemic absorption. High concentrations of opioid
receptors can be found throughout the gastrointestinal tract, and stimulation of
these receptors by opioid analgesics has a direct local effect on bowel function and
motility (Vaughan-Shaw et al. 2012). Reports have shown that alvimopan accelerates
the gastrointestinal recovery period and therefore it is indicated for patients
undergoing radical cystectomy for bladder cancer, which is associated with delayed
gastrointestinal recovery that prolongs hospital stay (Cheryl T. Lee et al. 2014).
Other MOR antagonists include levallorphan and nalmefene, the latter being clini-
cally used to reduce alcohol dependence (Clément Palpacuer et al. 2015). In com-
parison to chronically morphine-treated mice, repeated peptidomimetic-treated mice
developed less analgesic tolerance and/or physical dependence. Analysis of in vitro
and in vivo data has permitted structure activity relationships to guide further
discovery and chemical synthesis of opioid analgesics that suggest improved
clinical use.

16.6.3 Enkephalins

Enkephalins are pentapeptides first discovered and isolated from porcine brain
tissues in 1975, involved in the regulation of pain. There are two forms of
enkephalins, Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) containing the amino acid
leucine, and Met-enkephalin (Tyr-Gly-Gly-Phe-Met) containing methionine
(Comb et al. 1982), both generated from a common precursor proenkephalin-A
(PENK-A) by proteolytic enzymes. Leu-enkephalin and met-enkephalin are
metabolized by several enzymes called enkephalinases that include endopeptidases,
aminopeptidases and angiotensin-converting enzyme (Thanawala et al. 2008),
among others. Both enkephalin forms exert their effects through the δ-opioid
receptor (DOR) that belongs to the GPCR family. Met-enkephalin is found mainly
in the adrenal medulla and the brain acting as a neurotransmitter/neuromodulator and
as an active regulator of cell proliferation. Like μ-receptors, δ-opioid receptor
signaling investigations have primarily focused on mechanisms of opioid analgesia
(Al-Hasani and Bruchas 2011). Despite MOR-based analgesics being potent and
efficient in alleviating acute severe pain, they are ineffective in treating chronic pain
syndromes, which is why the attention has been driven to other receptors,
particularily δ-receptors, showing to be potential targets for developing novel opiate
analgesics for chronic pain management. Nonetheless, initiating tolerance is a
limitation for their use as long-acting opioid analgesics (Charfi et al. 2015).
DPDPE is one of the early synthetic DOR agonists developed, and is structurally
similar to met-enkephalin (Bilsky et al. 1995). SNC80 is the first non-peptidic-
selective DOR ligand developed and it successfully produced antidepressant, anxio-
lytic and analgesic effects, but its use was limited due to causing convulsions when
administered in high doses (Dripps et al. 2018). Other agonists include BMS986187,
a potent δ-opioid receptor-positive allosteric modulator that enhances the affinity of
leu-enkephalin to the δ-opioid receptor, yet it has a low potency due to limited
16 Neuropeptides and Neurotransmission 571

receptor phosphorylation and ultimately low receptor internalization and a slower

onset of desensitization (Stanczyk et al. 2019). Many other DOR agonists have been
developed, but they have not been used clinically due to limited data or considerable
side effects, but are currently being used in scientific research, such as BU48 (Broom
et al. 2002), BW373U86, KNT127, DPI125 (Yi et al. 2017) and TAN67 (Min et al.
2017). Peptide compounds do not cross the blood-brain barrier, and therefore
naltrindole, a non-peptide antagonist analogue of enkephalin, was developed. It is
a very potent, selective δ-receptor antagonist, used for biomedical research (Granier
et al. 2012; Werling et al. 1989). Another DOR antagonist is buprenorphine, an
effective medication in the maintenance treatment of opioid dependence, particularly
heroin, maintaining people in treatment at any dose above 2 mg. Buprenorphine was
approved for medical use in the 1980s, and it is heavily prescribed by health care
professionals. However, despite its potency and efficiency, it is associated with
many adverse effects, such as those accompanying opioid use, including nausea
and vomiting, dizziness, drowsiness, impairments in cognitive performance and
withdrawal symptoms (Strand et al. 2019). It has been proposed that activation of
MOR with concurrent antagonism of the δ-receptors could attenuate the develop-
ment of opioid tolerance and dependence.

16.6.4 Dynorphins

Endoproteolytic cleavage of the precursor prodynorphin by pro-protein convertase-

2 (PC2) enzyme produces multiple biologically active peptides including
dynorphin A, dynorphin B, big dynorphin, α- and β-neo-endorphins and
leumorphin, composed of high numbers of hydrophobic and basic amino acid
residues, mainly lysine and arginine (Chavkin 2013; Seidah et al. 1998). Following
formation, they are stored in dense-core vesicles in neuron endings, which need
prolonged stimuli to release their contents into the synaptic cleft (Drake et al. 2007).
Dynorphins bind preferentially to κ-receptors (KOR) with minimum affinity to
MOR and DOR subtypes. κ-receptors belong to the G protein-coupled receptor
(GPCR) superfamily and have two subtypes, K1 and K2; however, only one has
been cloned. Stimulation of the receptor by kappa opioids causes conformational
changes in the 7-transmembranous helices of the receptor, leading to Gα subunit
dissociation from Gβγ dimer and inhibition of adenylyl cyclase, and thus down-
stream cAMP production. The Gβγ dimer modulates the conductance of Ca+2 and
reduces voltage-gated channel openings, for example K+ channels. KOR agonists
activate kinase cascades, for example GPCR kinases (GRK), as well as members of
the mitogen-activated protein kinase (MAPK) family. Chronic KOR agonist activa-
tion results in an upregulation of adenylyl cyclase, and this may contribute to
desensitization, tolerance and physical dependence (Bruchas and Chavkin 2010).
Dynorphin and its κ-receptors are highly expressed and distributed throughout brain
regions presynaptically and mainly in the hypothalamus, hippocampus, midbrain,
medulla and spinal cord. Dynorphins act as modulators of pain response, maintain
appetite and body weight and intervene in circadian rhythm control (Anderson et al.
2019).
572 A. M. Gantait et al.

16.7 Conclusion

This chapter describes some of the different classes of neuropeptides. The biosyn-
thesis and metabolism with their physiology, pathophysiology and pharmacology
have been described. Several agonists and antagonists of these peptide receptors
continue to show great success in the development of therapeutic agents for the
treatment of a variety of disorders, which include cardiovascular, epilepsy, immune,
psychiatric, substance abuse and body weight disorder.

References
Aapro M, Hesketh PJ, Jordan K, Gralla RJ, Rossi G, Rizzi G, Palmas M (2016) Safety of an oral
fixed combination of netupitant and palonosetron (NEPA): pooled data from the phase II/III
clinical program. Oncologist 21(4):494–502
Al-Hasani R, Bruchas MR (2011) Molecular mechanisms of opioid receptor-dependent signaling
and behavior. Anesthesiology 115(6):1363–1381
Anderson RI, Lopez MF, Griffin WC, Haun HL, Bloodgood DW, Pati D, Boyt KM, Kash TL,
Becker HC (2019) Dynorphin-kappa opioid receptor activity in the central amygdala modulates
binge-like alcohol drinking in mice. Neuropsychopharmacology 44(6):1084–1092
Baraniuk JN, Silver PB, Kaliner MA, Barnes PJ (1992) Neuropeptide Y is a vasoconstrictor in
human nasal mucosa. J Appl Physiol 73(5):1867–1872
Beck B (2006) Neuropeptide Y in normal eating and in genetic and dietary-induced obesity. Philos
Trans R Soc Biol Sci 361(1471):1159–1185
Bilsky EJ, Calderon SN, Wang T, Bernstein RN, Davis P, Hruby VJ, McNutt RW, Rothman RB,
Rice KC, Porreca F (1995) SNC 80, a selective, nonpeptidic and systemically active opioid delta
agonist. J Pharmacol Exp Ther 273(1):359–366
Bockaert J, Roussignol G, Becamel C, Gavarini S, Joubert L, Dumuis A, Fagni L, Marin P (2004)
GPCR-interacting proteins (GIPs): nature and functions. Biochem Soc Trans 32(5):851–855
Broom DC, Jutkiewicz EM, Folk JE, Traynor JR, Rice KC, Woods JH (2002) Nonpeptidic δ-opioid
receptor agonists reduce immobility in the forced swim assay in rats.
Neuropsychopharmacology 26(6):744–755
Bruchas MR, Chavkin C (2010) Kinase cascades and ligand-directed signaling at the kappa opioid
receptor. Psychopharmacology 210(2):137–147
Bruno JB, Matos MH, Chaves RN, Figueiredo JR (2011) Involvement of vasoactive intestinal
peptide (VIP) of ovarian physiology. Anim Reprod 8(3/4):51–57
Burbach JP (2011) What are neuropeptides? Methods Mol Biol 789:1–36
Catalani E, De Palma C, Perrotta C, Cervia D (2017) Current evidence for a role of neuropeptides in
the regulation of autophagy. Biomed Res Int 2017:1–10
Charfi I, Audet N, BagheriTudashki H, Pineyro G (2015) Identifying ligand-specific signalling
within biased responses: focus on δ opioid receptor ligands. Br J Pharmacol 172(2):435–448
Chavkin C (2013) Dynorphin–still an extraordinarily potent opioid peptide. Mol Pharmacol 83
(4):729–736
Christofi FL (2018) TRPV1 sensory neurons and enteric glia in ENS link tachykinins to
Neuroinflammation and nociception. Cell Mol Gastroenterol Hepatol 6(3):354–355
Comb M, Seeburg PH, Adelman J, Eiden L, Herbert E (1982 Feb) Primary structure of the human
met-and Leu-enkephalin precursor and its mRNA. Nature 295(5851):663–666
Couvineau A, Tan Y-V, Ceraudo E, Laburthe M (2013) Strategies for studying the ligand binding
site of GPCRs. Methods Enzymol 520:219–237
Cox HM (2007) Peptide YY: a neuroendocrine neighbor of note. Peptides 28(2):345–351
16 Neuropeptides and Neurotransmission 573

Dalayeun JF, Nores JM, Bergal S (1993) Physiology of β-endorphins. A close-up view and a review
of the literature. Biomed Pharmacother 47(8):311–320
Dehlin HM, Levick SP (2014) Substance P in heart failure: the good and the bad. Int J Cardiol 170
(3):270–277
Delgado M, Pozo D, Ganea D (2004) The significance of vasoactive intestinal peptide in
immunomodulation. Pharmacol Rev 56(2):249–290
Drake CT, Chavkin C, Milner TA (2007) Opioid systems in the dentate gyrus. Prog Brain Res
163:245–814
Dripps IJ, Boyer BT, Neubig RR, Rice KC, Traynor JR, Jutkiewicz EM (2018) Role of signalling
molecules in behaviours mediated by the δ opioid receptor agonist SNC80. Br J Pharmacol. 175
(6):891–901
Ebner K, Singewald N (2006) The role of substance P in stress and anxiety responses. Amino Acids
31(3):251–272
El-Salhy M, Hausken T (2016) The role of the neuropeptide Y (NPY) family in the pathophysiology
of inflammatory bowel disease (IBD). Neuropeptides 55:137–144
Fan X, Markram H (2019) A brief history of simulation neuroscience. Front Neuroinform 13:32
Fieber LA (2017) Neurotransmitters and neuropeptides of invertebrates. In: The Oxford handbook
of invertebrate neurobiology. Oxford University Press, Oxford
Forehand CJ (2009) The action potential, synaptic transmission, and maintenance of nerve function.
In: Rhoades RA, Bell DR (eds) Medical physiology: principles for clinical medicine. Lippincott
Williams & Wilkins, A Wolters Kluwer Business, Philadelphia, pp 38–64
Fujiwara H, Kurihara N, Hirata K, Ohta K, Kanazawa H, Takeda T (1993) Effect of neuropeptide Y
on human bronchus and its modulation of neutral endopeptidase. J Allergy Clin Immunol 92
(1):89–94
Furness JB, Young HM, Pompolo S, Bornstein JC, Kunze WA, McConalogue K (1995)
Plurichemical transmission and chemical coding of neurons in the digestive tract. Gastroenter-
ology 108(2):554–563
Gehlert, D. R. (2009). Neuropeptide Y (NP Y) and its receptors. In: Encyclopedia of neuroscience.
Academic Press, Cambridge. pp. 837–842
Glanz JM, Narwaney KJ, Mueller SR, Gardner EM, Calcaterra SL, Xu S, Breslin K, Binswanger IA
(2018) Prediction model for two-year risk of opioid overdose among patients prescribed chronic
opioid therapy. J Gen Intern Med 33(10):1646–1653
Glass SM, Leddy SM, Orwin MC, Miller GP, Furge KA, Furge LL (2019) Rolapitant is a reversible
inhibitor of CYP2D6. Drug Metab Dispos 47(6):567–573
Gomariz RP, Juarranz Y, ABAD C, Arranz A, Leceta J, Martinez C (2006) VIP–PACAP system in
immunity: new insights for multitarget therapy. Ann N Y Acad Sci 1070(1):51–74
Gonzalez JP, Brogden RN (1988) NaltrexoneDrugs 35(3):192–213
González-Espinosa C, Guzmán-Mejía F (2014) Basic elements of signal transduction pathways
involved in chemical neurotransmission. In: Identification of neural markers accompanying
memory. Elsevier, Amsterdam, pp 121–133
Gourlet P, De Neef P, Cnudde J, Waelbroeck M, Robberecht P (1997) In vitro properties of a high
affinity selective antagonist of the VIP1 receptor. Peptides 18(10):1555–1560
Gozes I, Fridkin M, Hill JM, Brenneman DE (1999) Pharmaceutical VIP: prospects and problems.
Curr Med Chem 6:1019–1034
Granier S, Manglik A, Kruse AC, Kobilka TS, Thian FS, Weis WI, Kobilka BK (2012) Structure of
the δ-opioid receptor bound to naltrindole. Nature 485(7398):400–404
Grunberg S, Chua D, Maru A, Dinis J, DeVandry S, Boice JA, Hardwick JS, Beckford E, Taylor A,
Carides A, Roila F (2011) Single-dose fosaprepitant for the prevention of chemotherapy-
induced nausea and vomiting associated with cisplatin therapy: randomized, double-blind
study protocol—EASE. J Clin Oncol 29(11):1495–1501
Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P,
Said SI, Sreedharan SP, Wank SA (1998) Nomenclature of receptors for vasoactive intestinal
peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP). Pharmacol Rev
50:265–270
574 A. M. Gantait et al.

Hesketh PJ (2008) Chemotherapy-induced nausea and vomiting. N Engl J Med. 358

(23):2482–2494
Hofmann S, Bellmann-Sickert K, Beck-Sickinger AG (2019) Chemical modification of neuropep-
tide Y for human Y1 receptor targeting in health and disease. Biol Chem 400(3):299–311
Hormuzdi SG, Filippov MA, Mitropoulou G, Monyer H, Bruzzone R (2004) Electrical synapses: a
dynamic signaling system that shapes the activity of neuronal networks. Biochim Biophys Acta
1662(1–2):113–137
Laburthe M, Couvineau A, Marie JC (2002) VPAC receptors for VIP and PACAP. Recept
Channels 8(3–4):137–153
Lacroix JS, Ricchetti AP, Morel D, Mossimann B, Waeber B, Grouzmann E (1996) Intranasal
administration of neuropeptide Y in man: systemic absorption and functional effects. Br J
Pharmacol 118:2079–2084
Laitinen LA, Laitinen MVA, Widdicombe JG (1987) Parasympathetic nervous control of tracheal
vascular resistance in the dog. J Physiol 385:135–146
Lee CT, Chang SS, Kamat AM, Amiel G, Beard TL, Fergany A, Karnes RJ, Kurz A, Menon V,
Sexton WJ, Slaton JW (2014) Alvimopan accelerates gastrointestinal recovery after radical
cystectomy: a multicenter randomized placebo-controlled trial. Eur Urol 66(2):265–272
Lelievre V, Favrais G, Abad C, Adle-Biassette H, Lu Y, Germano PM, Cheung-Lau G, Pisegna JR,
Gressens P, Lawson G, Waschek JA (2007) Gastrointestinal dysfunction in mice with a targeted
mutation in the gene encoding vasoactive intestinal polypeptide: a model for the study of
intestinal ileus and Hirschsprung's disease. Peptides 28(9):1688–1699
Li Y, Lefever MR, Muthu D, Bidlack JM, Bilsky EJ, Polt R (2012) Opioid glycopeptide analgesics
derived from endogenous enkephalins and endorphins. Future Med Chem 4(2):205–226
Lindqvist A, Rip J, van Kregten J, Gaillard PJ, Hammarlund-Udenaes M (2016) In vivo functional
evaluation of increased brain delivery of the opioid peptide DAMGO by Glutathione-PEGylated
liposomes. Pharm Res 33(1):177–185
Lodish H, Berk A, Zipursky SL et al (2000) Section 21.4, Neurotransmitters, Synapses, and Impulse
Transmission. In: Molecular cell biology, 4th edn. W. H. Freeman, New York, NY. Available
from: https://www.ncbi.nlm.nih.gov/books/NBK21521/
Malcangio M, Bowery NG (1999) Peptide autoreceptors: does an autoreceptor for substance P
exist? Trends Pharmacol Sci 20(10):405–407
Mashaghi A, Marmalidou A, Tehrani M, Grace PM, Pothoulakis C, Dana R (2016a) Neuropeptide
substance P and the immune response. Cell Mol Life Sci 73(22):4249–4264
Mashaghi S, Abbaspourrad A, Weitz DA, van Oijen AM (2016b) Droplet microfluidics: a tool for
biology, chemistry and nanotechnology. TrAC Trends Anal Chem 82:118–125
Matsas R, Kenny AJ, Turner AJ (1984) The metabolism of neuropeptides.The hydrolysis of
peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11.
Biochem J 223(2):433–440
Min JW, Lü L, Freeling JL, Martin DS, Wang H (2017) USP 14 inhibitor attenuates cerebral
ischemia/reperfusion-induced neuronal injury in mice. J Neurochem 140(5):826–833
Mistrova E, Kruzliak P, Dvorakova MC (2016) Role of substance P in the cardiovascular system.
Neuropeptides 58:41–51
Mutt V, Said SI (1974) Structure of the porcine vasoactive intestinal Octacosapeptide: the amino-
acid sequence. Use of Kallikrein in its determination. Eur J Biochem 42(2):581–589
Nakanishi SH (1987) Substance P precursor and kininogen: their structures, gene organizations, and
regulation. Physiol Rev 67(4):1117–1142
Nicole P, Lins L, Rouyer-Fessard C, Drouot C, Fulcrand P, Thomas A, Couvineau A, Martinez J,
Brasseur R, Laburthe M (2000) Identification of key residues for interaction of vasoactive
intestinal peptide with human VPAC1 and VPAC2Receptors and development of a highly
selective VPAC1Receptor agonist alanine scanning and molecular modeling of the peptide. J
Biol Chem 275(31):24003–24012
16 Neuropeptides and Neurotransmission 575

Palpacuer C, Laviolle B, Boussageon R, Reymann JM, Bellissant E, Naudet F (2015) Risks and
benefits of nalmefene in the treatment of adult alcohol dependence: a systematic literature
review and meta-analysis of published and unpublished double-blind randomized controlled
trials. PLoS Med 12(12):e1001924
Patri M (2019) Synaptic transmission and amino acid neurotransmitters. In: Neurochemical basis of
brain function and dysfunction. IntechOpen, London
Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A-S, McNamara JO (eds) (2001)
Neuroscience, 2nd edn. Sinauer Associates, Sunderland, MA
Rapoport B, Schwartzberg L, Chasen M, Powers D, Arora S, Navari R, Schnadig I (2016) Efficacy
and safety of rolapitant for prevention of chemotherapy-induced nausea and vomiting over
multiple cycles of moderately or highly emetogenic chemotherapy. Eur J Cancer 57:23–30
Roos C, Dahlgren D, Berg S, Westergren J, Abrahamsson B, Tannergren C, Sjögren E, Lennernäs
H (2017 Aug 10) In vivo mechanisms of intestinal drug absorption from
aprepitantnanoformulations. Mol Pharm 14(12):4233–4242
Russo AF (2017) Overview of neuropeptides: a wakening the senses? Headache 57:37–46
Said SI (1967) Vasoactive substances in the lung. In: Proceedings of tenth Aspen emphysema
conference, Aspen, CO, June 7–10, 1967. U.S. Public Health Service Publication, vol. 1787,
pp. 223–228
Said SI (1989) Vasoactive intestinal polypeptide and asthma (editorial). New Engl J Med
320:1271–1273
Said SI, Mutt V (1970) Potent peripheral and splanchnic vasodilator peptide from normal gut.
Nature 225(5235):863–864
Said SI, Rattan S (2004) The multiple mediators of neurogenic smooth muscle relaxation. Trends
Endocrinol Metab 15:189–191
Said SI, Berisha HI, Pakbaz H (1996) Excitotoxicity in lung: N-methyl-D-aspartate-induced, nitric
oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by
inhibitors of poly (ADP-ribose) polymerase. Proc Natl Acad Sci U S A 93:4688–4692
Said SI, Hamidi SA, Dickman KG, Szema AM, Lyubsky S, Lin RZ, Jiang YP, Chen JJ, Waschek
JA, Kort S (2007) Moderate pulmonary arterial hypertension in male mice lacking the vasoac-
tive intestinal peptide gene. Circulation 115(10):1260–1268
Seidah NG, Day R, Marcinkiewicz M, Chretien M (1998) Precursor convertases: an evolutionary
ancient, cell-specific, combinatorial mechanism yielding diverse bioactive peptides and
proteins. Ann N Y Acad Sci 839(1):9–24
Severini C, Petrella C, Calissano P (2016) Substance P and Alzheimer’s disease: emerging novel
roles. Curr Alzheimer Res 13(9):964–972
Sherwood NM, Krueckl SL, McRory JE (2000) The origin and function of the pituitary adenylate
cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr Rev 21(6):619–670
Shrihari TG (2017) Endorphins on cancer: a novel therapeutic approach. J Carcinog Mutagen 8:298
Sohn W, Lee OY, Lee SP, Lee KN, Jun DW, Lee HL, Yoon BC, Choi HS, Sim J, Jang K-S (2013)
Mast cell number, substance P and vasoactive intestinal peptide in irritable bowel syndrome
with diarrhea. Scand J Gastroenterol 49(1):43–51
Sprouse-Blum AS, Smith G, Sugai D, Parsa FD (2010) Understanding endorphins and their
importance in pain management. Hawaii Med J 69(3):70–71
Stanczyk MA, Livingston KE, Chang L, Weinberg ZY, Puthenveedu MA, Traynor JR (2019) The
δ-opioid receptor positive allosteric modulator BMS 986187 is a G-protein-biased allosteric
agonist. Br J Pharmacol 176(11):1649–1663
Steinhoff MS, von Mentzer B, Geppetti P, Pothoulakis C, Bunnett NW (2014) Tachykinins and
their receptors: contributions to physiological control and the mechanisms of disease. Physiol
Rev 94(1):265–301
Strand MC, Vindenes V, Gjerde H, Mørland JG, Ramaekers JG (2019) A clinical trial on the acute
effects of methadone and buprenorphine on actual driving and cognitive function of healthy
volunteers. Br J Clin Pharmacol 85(2):442–453
576 A. M. Gantait et al.

Stretton CD, Barnes PJ (1988) Modulation of cholinergic neurotransmission in guinea-pig trachea

by neuropeptide Y. Br J Pharmacol 93(3):672–678
Sun WW, Zhu P, Shi YC, Zhang CL, Huang XF, Liang SY, Song ZY, Lin S (2017) Current views
on neuropeptide Y and diabetes-related atherosclerosis. Diab Vasc Dis Res 14(4):277–284
Syed YY (2015) Rolapitant: first global approval. Drugs 75(16):1941–1945
Szema AM, Hamidi SA, Lyubsky S, Dickman KG, Mathew S, Abdel-Razek T, Chen JJ, Waschek
JA, Said SI (2006) Mice lacking the VIP gene show airway hyperresponsiveness and airway
inflammation, partially reversible by VIP. Am J Phys Lung Cell Mol Phys 291(5):L880–L886
Takeda Y, Blount P, Sachais BS, Hershey AD, Raddatz R, Krause JE (1992) Ligand binding
kinetics of substance P and neurokinin a receptors stably expressed in Chinese hamster ovary
cells and evidence for differential stimulation of inositol 1, 4, 5-trisphosphate and cyclic AMP
second messenger responses. J Neurochem 59(2):740–745
Tatemoto K (1982) Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc Natl
Acad Sci 79(18):5485–5489
Thanawala V, Kadam VJ, Ghosh R (2008) Enkephalinase inhibitors: potential agents for the
management of pain. Curr Drug Targets 9(10):887–894
Thornton E, Vink R (2015) Substance P and its tachykinin NK1 receptor: a novel neuroprotective
target for Parkinson’s disease. Neural Regen Res 10(9):1403
Thorsell A, Mathé AA (2017) Neuropeptide Y in alcohol addiction and affective disorders. Front
Endocrinol 8:178
V Euler US, Gaddum JH (1931) An unidentified depressor substance in certain tissue extracts. J
Physiol 72(1):74–87
Vaudry H, Laburthe M (2006) VIP, PACAP, and related peptides. From gene to therapy. Annu N Y
Acad Sci 1070:1–633
Vaughan-Shaw PG, Fecher IC, Harris S, Knight JS (2012) A meta-analysis of the effectiveness of
the opioid receptor antagonist alvimopan in reducing hospital length of stay and time to GI
recovery in patients enrolled in a standardized accelerated recovery program after abdominal
surgery. Dis Colon Rectum 55(5):611–620
von Hörsten S, Hoffmann T, Alfalah M, Wrann CD, Karl T, Pabst R, Bedoui S (2004) PP, PYY and
NPY: synthesis, storage, release and degradation. In: Neuropeptide Y and related peptides.
Springer, Berlin, pp 23–44
Vu JP, Goyal D, Luong L, Oh S, Sandhu R, Norris J, Parsons W, Pisegna JR, Germano PM (2015)
PACAP intraperitoneal treatment suppresses appetite and food intake via PAC1 receptor in mice
by inhibiting ghrelin and increasing GLP-1 and leptin. Am J Physiol Gastrointest Liver Physiol
309(10):G816–G825
Wang PX, Zhao GN, Ji YX, Zhang P, Zhang XJ, Gong J, Zhao LP, Yan ZZ, Yin M, Jiang Z, Shen
LJ (2018) Wang et al. reply. Nat Med 24(6):700–701
Werling LL, McMahon PN, Portoghese PS, Takemori AE, Cox BM (1989) Selective opioid
antagonist effects on opioid-induced inhibition of release of norepinephrine in Guinea pig
cortex. Neuropharmacology 28(2):103–107
Xia M, Sreedharan SP, Bolin DR, Gaufo GO, Goetzl EJ (1997) Novel cyclic peptide agonist of high
potency and selectivity for the type II vasoactive intestinal peptide receptor. J Pharmacol Exp
Ther. 281(2):629–633
Yang H, Wang W, Romano KA, Gu M, Sanidad KZ, Kim D, Yang J, Schmidt B, Panigrahy D,
Pei R, Martin DA (2018) A common antimicrobial additive increases colonic inflammation and
colitis-associated colon tumorigenesis in mice. Sci Transl Med 10(443):eaan4116
Yi SP, Kong QH, Li YL, Pan CL, Yu J, Cui BQ, Wang YF, Wang GL, Zhou PL, Wang LL, Gong
ZH (2017) The opioid receptor triple agonist DPI-125 produces analgesia with less respiratory
depression and reduced abuse liability. Acta Pharmacol Sin 38(7):977–989
Yin J, Chapman K, Clark LD, Shao Z, Borek D, Xu Q, Wang J, Rosenbaum DM (2018) Crystal
structure of the human NK1 tachykinin receptor. Proc Natl Acad Sci U S A 115
(52):13264–13269
16 Neuropeptides and Neurotransmission 577

Zhang ZY (2019) Volume of distribution: a relevant, possibly overlooked pharmacokinetic param-

eter in drug development. Curr Trends Pharma Clinical Trials 2(1):180014. Copyright# 2019
Zhi-Yi Zhang and Ashley Milton. Current Trends in Pharmacology and Clinical Trials Letter to
Editor; 2(1)
Zhu P, Sun W, Zhang C, Song Z, Lin S (2016) The role of neuropeptide Y in the pathophysiology of
atherosclerotic cardiovascular disease. Int J Cardiol. 220:235–241
Pharmacology of Gasotransmitters (Nitric
Oxide and Carbon Monoxide) and Their 17
Action

Rohitas Deshmukh, Ranjit K. Harwansh, Nabamita Bandyopadhyay,

Shantanu Bandopadhyay, and Puneet Kumar

Abstract

In the last decades, gasotransmitters have gained attention for their crucial role
in pathophysiological and cellular functions. Gasotransmitters are gaseous
mediators of the cellular signaling and biological responses from one end to the
other. The isoform of these gaseous signaling molecules includes NO (nitric
oxide), CO (carbon monoxide), and H2S (hydrogen sulphide), collectively called
gasotransmitters. The diverse role of these gasotransmitters in cell and molecular
biology as well as biochemical processing has been well validated through
several scientific and clinical studies. The biosynthesis, interaction, and move-
ment of gasotransmitters inside the cellular systems are critical especially in terms
of their pharmacological response. These gaseous molecules are very toxic and
hazardous to human health at higher concentrations but at lower levels they may
be considered as therapeutic agents. They can easily diffuse through all the cell
membrane and act on their targets for generating pharmacological responses. Due
to its gaseous nature, the cellular interactions at the target sites are complex and
make it a critical task for researchers to understand. The mode of action and
molecular pathways are still under the exploratory phase. Apart from their toxic
nature, there are several pharmacological activities such as cardioprotective,

R. Deshmukh · R. K. Harwansh
Institute of Pharmaceutical Research, GLA University, Mathura, Uttar Pradesh, India
N. Bandyopadhyay
Molecular Biology Division, National Institute of Malarial Research (NIMR), Dwarka, Delhi, India
S. Bandopadhyay (*)
Faculty of Pharmacy, Naraina Vidya Peeth Group of Institutions, Kanpur, Uttar Pradesh, India
P. Kumar
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, India
Department of Pharmaceutical Sciences and Technology, Maharaja Ranjit Singh Punjab Technical
University, Bathinda, Punjab, India

# Springer Nature Singapore Pte Ltd. 2020 579

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_17
580 R. Deshmukh et al.

antihypertensive, smooth muscle relaxant, vasodilator, antithrombotic, antitumor,

etc. that have been reported for these gaseous molecules. This gaseous family
has become a promising area for multidisciplinary research in order to establish
their importance in relation to disease and health. The perspective of these
gasotransmitters is required to validate the clinical translational and future inves-
tigation following clear cut understanding of their pharmacological properties.
This book chapter deals with the biosynthesis, pharmacological application and
clinical utility of gaseous molecules mainly NO and CO.

Keywords
Natural gases · Novel bioactive molecules · ENOG · Signal transduction · Heme
oxygenase · Biomarker

Abbreviations

AD Alzheimer’s Disease
ARDS Acute respiratory distress syndrome
BP Blood pressure
C/EBP CCAATT-enhancer-binding protein
CaM Calcium-binding messenger protein calmodulin
cGMP 3,5-Cyclic guanosine monophosphate
CO Carbon monoxide
CO2 Carbon dioxide
CoHb Carboxyhemoglobin
COPD Chronic obstructive pulmonary disease
CORMs CO releasing molecules
CP Cisplatin
CVD Cardiovascular disease
CYT2E1 Cytochrome P450 2E
DCM Dichloromethane
ED Erectile dysfunction
EDHF Endothelium-derived hyper polarizing factor
EDRF Endothelium-derived relaxing factor
ENOG European Network on Gasotransmitters
eNOS Endothelial NOS
ER Endoplasmic reticulum
GTN Glyceryltrinitrate
GTP Guanosine5-triphosphate
GTT Glucose tolerance test
H 2S Hydrogen sulfide
Hb Hemoglobin
HIF Hypoxia inducible factor
HO Heme oxygenase
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 581

HO-1 Heme oxygenase-1

HO-2 Heme oxygenase-2
HUVECs Human umbilical vein endothelial cells
iNOS Inducible NOS
MAPK Mitogen activated protein kinases
MI Myocardial infarction
nNOS Neuronal NOS
NO Nitric oxide
NOS Nitric oxide synthase
O 2_ Peroxide radical
OH• Hydroxyl radical
ONOO Peroxynitrite
PAH Pulmonary arterial hypertension
PI3K Phosphatidyl inositol 3-kinase
PI3K Phosphatidylinositol-3 kinase
PKC Protein kinase C
PPARc Peroxisome proliferator-activated receptor-c
RAS Renin–angiotensin system
ROS Reactive oxygen species
S• Superoxide radical
sGC Soluble guanylatecyclase enzyme
SiRNA Small interfering RNA
TB Tuberculosis
TNF Tumor necrosis factor
VEGF Vascular endothelial growth factor
VSMC Vascular smooth muscle cell

17.1 Introduction

Environmental gases like hydrogen sulfide (H2S), carbon monoxide (CO), carbon
dioxide (CO2), nitric oxide (NO), etc. are treated as toxicants and health hazards.
Apart from being hazardous to health, studies have shown these natural gases have
some health benefits (through cellular and molecular mechanisms), following which
they are considered gasotransmitters. The term gasotransmitter was postulated in the
early twenty-first century after their biological importance in our body as gaseous
signaling molecules was recognized. These signaling molecules became the thrust
area of research to establish their molecular pathways at the cellular level during the
last decade. The human health benefits of gasotransmitters have been explored
through scientific investigation worldwide (Wang 2014; Sukmansky and Reutov
2016; Kolluru et al. 2017).
The fact about these gasotransmitters is that they are highly toxic at higher
concentration but at low concentration they may work as a gaseous signaling
molecule and produce specific biological activities (Andreadou et al. 2015). The
biological role of NO was first discovered in the year 1987, after the identification of
582 R. Deshmukh et al.

vasodilation by acetylcholine mimic response through the vascular endothelium of

mammals. Firstly, in biological systems NO is released endogenously as a signaling
molecule (Lowenstein et al. 1994). After understanding the cellular interaction and
signaling of NO, CO, H2S, as well as ammonia have been included as new members
of the gasotransmitters family. Moreover, in the near future methane could be the
next analog of this family (Wang 2014).
Gasotransmitters are categorized as novel bioactive molecules on the basis of the
following criteria:

• Tiny molecules of gaseous family

• Can easily diffuse through biological membrane or cell membrane
• Should not interact with receptors
• Production and regulation via enzymes only
• Its properties may be exogenously modulated
• Should have specific targets on the molecular as well as cellular level (signal
transduction) but not induced by any chemical messengers

A major promising area of research on gasotransmitters concerns the following

concepts:

• Collective interaction of different gaseous molecules

• Targeting of common cells or targets by various gaseous molecules
• Recognition of place of cellular targets and production place of gasotransmitters

These concepts definitely help understand the relevance of NO, CO, H2S, and
others in signal transduction in the human body. They could be novel therapeutic
lead molecules for treatment of several human health problems through scientific
validation. Reportedly, gasotransmitters can effortlessly move across biological
membranes by interacting with a hydrophobic layer of the membrane. For example,
in an animal study it has been observed that normally gaseous molecules move
through biological membranes by using transporter proteins like aquaporin-1. It can
help low molecular weight gases such as NO, CO2 and NH3 to cross cell membranes
easily (Garcia-Mata and Lamattina 2013). The overview of biosynthesis of
gasotransmitters along with their role on vascular system is schematically
represented in Fig. 17.1.
This chapter discusses the role, pathophysiological consequences, biochemistry,
and clinical application of gasotransmitters, particularly that of NO and CO. It will
help us understand the importance of these gasotransmitters as therapeutic bioactive
molecules or drug candidates in association with already established drugs for
enhancing their efficacy against various diseases.
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 583

ENZYME SUBSTRATE
L-arginine Heme L-cysteine

eNOS, nNOS iNOS HO-2 HO-1, HO-3 CBS CSE

GASOTRANSMITTERS
NO CO H2 S

L-citruline Biliverdin, Fe+3 L-serine, pyruvate + NH

Fe2+ guanylate cyclase Fe2+ guanylate cyclase KATP

R-SN, ONOO- HS-NO

Fig. 17.1 Synthesis and major vascular effects of the gasotransmitters

17.2 Gaseous Signaling Molecules

In the recent past, the importance of different gaseous signaling molecules to their
target sites and related biological responses have been clearly defined and validated
through integrated/scientific approaches. There are various types of gaseous
molecules like NO, CO, CO2, H2S, and so on, which have been proven as gaseous
signaling molecules by transduction the information from one cell to other cells as
biological responses which are endogenously produced (Szabo 2010; Ryter and
Choi 2013; Paul and Snyder 2018a, b). The basic features of gasotransmitters and
neurotransmitters have been explained in Table 17.1.
NO was recognized as the first endogenously produced gaseous molecule. In
various mammal cells, NO was synthesized from L-arginine as substrate by different
analogs of nitric oxide synthase (NOS). Discovery of NO as a novel biological

Table 17.1 Difference between gasotransmitters and neurotransmitters

Mode of action Gasotransmitters Neurotransmitters
Release Cytoplasm release Exocytotic vesicle
Reuptake No Yes
Removal Nonenzymatic like oxidation, Enzyme dependent
mechanism scavenging, methylation, etc.
Revert direction Bidirectional Pre to postsynaptic membrane
(single direction)
Membrane receptors Not required Required
584 R. Deshmukh et al.

gasotransmitter in mammals facilitated in better understanding of signal transduction

in cells in order to modulate their functions. Apart from its diverse biological
importance, NO is known as an endothelium-derived relaxing factor (EDRF)
(Farrugia and Szurszewski 2014; Zhao et al. 2015).
Another isoform of the gaseous signaling molecule is CO. Actually, it is a heme
metabolite, produced by an inducible enzyme, i.e., heme oxygenase-1 and -2 (HO-1
and HO-2), which is constitutively released. In most cases the biological features of
CO are similar to NO; for example, it exhibits as a smooth muscle relaxant (vascular
tissues), a vasodilator (slows down the blood pressure in case of hypertension) and
cardioprotective (in case of ischemic or reperfusion heart disease) (Motterlini and
Foresti 2017).
Another endogenous gasotransmitter is H2S, also known as the rotten-egg gas,
mainly expressed in mammalian cells. It is produced from L-cysteine and homocys-
teine after enzymatic reaction of various enzymes, including 3-mercaptopyruvate
sulfurtransferase, cystathionine β-synthase, and cystathionine γ-lyase. It has numer-
ous health benefits through transducion of signals (neurotransmission) and regula-
tion of the various physiological tasks in the body (neuromodulation). H2S is well
known for its cognition improvement through enhancing memory, nociception, and
learning function. It is considered an endothelium-derived hyper polarizing factor
(EDHF) (Polhemus and Lefer 2014; Paul and Snyder 2018a; b).
Of lates, continuous research on these gaseous signaling molecules has
established their diverse biological role in the body. Apart from their traditional
function as signaling substance, they have been identified as cellular regulators.
These molecules can produce different biological functions at a particular site
through multifarious chemical interactions with cells, proteins, as well as
metabolites. With smart technique and engineering technology, these gaseous
molecules are being developed as therapeutic agents that can be administered inside
the body and release gases in a sustained fashion (Cheng and Rong 2017).

17.3 European Network on Gasotransmitters (ENOG)

The ENOG has two major goals for research and regulation of gasotransmitters in
European countries. The first goal is to enhance research on different gaseous
molecules such as NO, CO, H2S, etc. in order to improve their competitiveness,
quality, and efficacy in the development of therapeutic agent against various
diseases. The dissemination of information on gasotransmitters among the
European groups has been through various modes like research problems, skills,
knowledge sharing, expertise, and gasotransmitter biosynthesis. The interactions of
gasotransmitters with their targets for producing cellular effects are also described by
the ENOG among their team. ENOG also help to share the information in multidis-
ciplinary research group through updating and providing the idea, knowledge and
training about gasotransmitters through synthetic chemistry, computational drug
design and pharmacology approaches for establishing gasotransmitters as drug
candidates (Papapetropoulos et al. 2015).
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 585

Table 17.2 Function of ENOG working groups for promotion and development of
gasotransmitters
ENOG groups Functions
Group 1 Molecular control of gasotransmitter production and signaling
Group 2 Gasotransmitters in disease
Group 3 Chemistry and in vitro pharmacology of gasotransmitter-modifying molecules
Group 4 Evaluation of gasotransmitter-modifying agents in animal models of disease

Another important and integral goal of ENOG is as follows (Papapetropoulos

et al. 2015):

• Dissemination of idea, knowledge, skill, and experience shared among different

participating members, ENOG members, and global community.
• Knowledge sharing and application about novel drug (therapeutic agent) devel-
opment against disease. In this context, the small-medium enterprises will benefit
with expertise in NO and CO based advanced drug delivery systems.
• To assist and properly guide the young scientist to continue biomedical research
in the current topic.

The different ENOG working groups for different purposes are mentioned in
Table 17.2.

17.4 Nitric Oxide (NO) as Signaling Molecules

NO was invented in 1980 as a first gaseous signaling molecule. It has an important

role in biological chemistry through understanding its cytotoxicity and the signal
transduction process. Especially in the fields of immunology, neuroscience, and
physiology, NO has been recognized as a physiological key element for several
pharmacological functions of the human body. In the year 1992, Robert F. Furchgott
and his group received the Nobel Prize for discovering NO as a physiological
signaling molecule, particularly for the cardiovascular system. Different physiologi-
cal pathways of NO have been identified such as neurotransmission, vascular
permeability, synaptic plasticity, renal function, platelet aggregation and adhesion,
senescence, and hepatic metabolism. The molecular level activity of NO has also
been observed through tumor suppression and host immunity (Miller and Megson
2007; Nagpure and Bian 2016; Mir and Maurya 2018).
In the recent past, NO has become the most investigated and famous
gasotransmitter throughout the world. NO is generally produced by conversion of
L-arginine to L-citrulline through the enzymatic action of NOS. According to the
functionality, NOS has been subcategorized as eNOS (endothelial NOS), iNOS
(inducible NOS), and nNOS (neuronal NOS). eNOS is an active moiety generally
found in platelets and endothelial cells. iNOS is a highly contributing enzyme for
586 R. Deshmukh et al.

generating NO in immune responses and inflammations, while nNOS as the name

indicates, is mainly identified in neuronal cells (Luo et al. 2014; Shefa et al. 2017).
The diverse kinds of NO-associated biological activities have been reported via
modulating and transducing the signals in neuronal cells, maintaining the tone of
vessels, pro-angiogenic and immune response. In eNOS knockout mouse, several
incidences like poor blood pressure, hypertension, and atherosclerosis have been
accounted, although, their action on thrombotic conditions was not seen in the same
animal. Moreover, endogenous NO has been reported to interfere with platelet
action, but not to its response. NO can also inhibit the function of GPIIb/IIIa
fibrinogen with platelets, P-selectin expression, and 5-HT secretion (Truss and
Warner 2011a; b; Poulos and Li 2017).
Acute inflammatory response leads to maintenance of tissue homeostasis by a
self-regulating process, while chronic inflammatory response involves long range of
progression of diseases such as arthritis, neurodegenerative problems, atherosclero-
sis, cancers, etc. Alleviated level of endogenous NO can affect the aforementioned
inflammatory responses. This endothelium produced gasotransmitter is accountable
for the regulation of tonicity of the vascular system. Its action is not limited to this,
but it has been associated with several pathological and physiological processes.
Particularly, in case of normal physiology, nNOS and eNOS are responsible to exert
the actions while in injury cases iNOS is responsible. These NOS groups are not only
involved in NO generation but also in the production of different molecules like
transition metals, peroxynitrite (ONOO) and S-nitrosothiols through the process of
oxidation or reduction. Generally, NO exhibits its action through sGC (soluble
guanylatecyclase enzyme) and resultant intermediate includes GTP (guanosine5-
triphosphate) and cGMP (3,5-cyclic guanosine monophosphate). During platelet
aggregation inhibition and inflammatory cell apoptosis regulation, NO can work
through cGMP-independent pathway. Additionally, the translation or transcription is
modulated by NO via obstructing the signaling pathways, including
phosphatidylinositol-3 kinase (PI3K), G-proteins, mitogen-activated protein kinases
(MAPK), glyceraldehyde dehydrogenase and RAS (Renin–angiotensin) system
(Socco et al. 2017; Wang et al. 2019).

17.4.1 Biosynthesis of NO

Biologically, NO is synthesized from L-arginine by catalytic activities of NOS

enzymes, heme containing proteins (Forstermann and Sessa 2012). The resultant
intermediate product includes NGhydroxy-L-arginine and L-citrulline during NO
generation. The regulation of NO synthesis through macrophages has been under-
stood clearly by L-arginine uptake by the pathway of inducible L-arginine-NO
(Kovacevic et al. 2017). The generation of NOS enzymes based NO via peritoneal
macrophages and monocyte-derived macrophages has been studied in knock out
C57BL6 mice (Moncada and Higgs 1993; Neilly et al. 1994; Venketaraman et al.
2003). The detailed schematic of NO biosynthesis is shown in Fig. 17.2.
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 587

Fig. 17.2 Biosynthesis of L-Arginine

nitric oxide (NO) NH2

H
H 2N N OH

NH O2 1M NADPH O

N G-Hydroxyl- L-Arginine

NH2

H
H2N N OH

N O
O2 0.5M NADPH
HO

Citrulline NH2

H
H2 N N OH

O O
NO.
Nitric Oxide

eNOS forms NO in the blood vessel lining that results in vasodilation of smooth
muscle through activation of sGC and cGMP (Wen et al. 2018). It leads to the
constant flow of blood and regulation of blood pressure. Moreover, NO has a
significant role as an anticoagulant by inhibiting platelet and clot formation. But
nNOS-mediated NO has a crucial role in neuronal cell signaling process. In case of
iNOS, it is broadly distributed in the body and contributes in an intrinsic immune
system of the body. During microbial infection/invasion, NOS enzymes are highly
expressed in smooth muscle, macrophages and hepatocyets, resulting in the release
of NO at the microgram level. NO is also found in infected cells along with ROS
(reactive oxygen species) like peroxide (O2
), superoxide (S•), and hydroxyl (OH•)
588 R. Deshmukh et al.

radical. It has a certain role in reducing the microbial loads through natural immune
response systems (Doroszko et al. 2018).

17.4.2 Activity at Receptor Levels

The eNOS is an endothelial constitutive enzyme involved in catalytic activity that

regulates cell signaling through Ca2+ and calcium-binding messenger protein cal-
modulin (CaM). The NO is produced after interaction of eNOS with Ca2+-CaM
complex. NO has the ability to diffuse cell membranes and can interact with targeted
intracellular molecular sites of the cells (Piazza et al. 2018). NO can react with the
heme substance sGC causing conversion of GTP to cGMP, which regulates the
various pharmacological activities (smooth muscle vasorelaxation through the signal
transduction cascade) (Forstermann 2010).
It has been proved that sGC, i.e., the prosthetic heme group are responsible for
NO generation. However, if this heme group is eliminated the NO is not formed.
There are diverse kinds of biological activities reported for NO, which includes
wound healing, host defense, immune response, penile erection, angiogenesis, tumor
suppression, bronchodilation, vasodilation, platelet aggregation, regulation of vas-
cular tone, stem cell proliferation, hormone secretion, hemoglobin (Hb) delivery of
oxygen, inflammation regulation, pulmonary hypertension, gastrointestinal mobil-
ity, septic shock, gene and neuronal communication, glaucoma and neural degener-
ation, and smooth muscle cell replication neurotransmission (Fleissner and Thum
2011). These biological effects of NO mainly depend upon the sGC activation and
cGMP formation via GTP. Although, other cGMP pathways of NO like
nitrotyrosine, nitrite and nitrate have also been established (Omar et al. 2016).

17.4.3 Role in Biological Systems

Biological activities mainly depend on different NOS enzymes and their location.
There are three subtypes of NOS in mammals: (1) eNOS is responsible for CVS,
(2) nNOS is accountable for the brain or nervous system, and (3) iNOS is liable for
innate immune response (Lee et al. 2017). The NO has a specific role in CVS
through Ca2+-dependent NOS. There are several chemical substances like acetyl-
choline, ATP, bradykinin, and other agents that can stimulate the flux of calcium
known as NOS agonists and can modulate the NO synthesis in vascular endothelial
cells. Likewise, physical factor/agonists, viz., electrical current, light, electromag-
netic fields, acupuncture, flow, and shear stress can stimulate the NO synthesis in
vascular endothelial tissues (Arzumanian et al. 2003; Piazza et al. 2018).
About 90% consumption of NO is taken up by the blood, resulting in inhibition of
platelet and thrombus formation. The rest of the NO diffuses to the vein and arteries
(smooth muscle), and activates a cascade of effects like smooth muscle relaxation.
These effects are directly proportional to the vasodilation that results in reduced
blood pressure. The CVS sustains the NO level along with blood flow. The level of
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 589

NO is dependent upon the rate of blood flow. As the blood flow increases, endothe-
lial tissues increasingly secrete NO to attain a constant level in the blood stream
(Riddell and Owen 1999; Vanhoutte and Gao 2013).
In case of pathological events like atherosclerosis, the NO level is reduced due to
blockade of cholesterol, resulting in vasoconstriction (Li et al. 2014). This abnor-
mality of CVS leads to decreased blood flow and causes increased blood pressure
(hypertension) (Forstermann et al. 2017).
In this context, nitroglycerin is generally used for cardiac disease like hyperten-
sion, which undergoes metabolic transformation and forms NO in CVS endothelial
tissues where it produces vasorelaxation and increases the blood flow in smooth
muscle including atherosclerotic arteries, thereby improving cardiovascular
functions. The NO has a certain role as an anticoagulant by preventing platelet and
blood clot formation in cardiac arteries. If absent there may be chances of coronary
thrombosis, which is a main cause of heart stroke. Thus, NO has a potent gaseous
signaling molecule with an important role in human life (Rochette et al. 2013; Zang
et al. 2018).

17.4.4 Clinical Utility

The irregular NO generation via different NOS enzymes or signaling cells are related
to several pathological conditions in the organs. In many disease states, NO
metabolites like peroxyl nitrite are raised. The role of NO in various diseases is
discussed in the following.

17.4.4.1 Cardiovascular Disease

NO has a crucial role in cardioprotection against the sequence of cardiovascular
disease (CVD) like diabetes, hypertension, and hypercholesterolemia. The main
cause of CVD is atherosclerosis, which leads to dysfunction of endothelial vascular
system. NO provides protection to the heart from these uncomplicated pathological
conditions via regulation of vascular tone and blood pressure (BP) as well as
reduction in the proliferation of smooth muscle cell by inhibiting leukocyte adhesion
and clotting formation (platelet aggregation) (Naseem 2005). In case of cardiovas-
cular disease (CVD), especially in pulmonary hypertension and ischemic heart
disease, the traditional treatment involves application of NO donors (Munzel and
Daiber 2018).

17.4.4.2 Diabetes Mellitus

Type I diabetes is also associated with vascular problems of retinopathy, nephropa-
thy, and hypertension. These factors are largely responsible for the high death rate in
type I diabetic patient. NO is an endothelium-derived relaxing factor that plays an
important role in the regulation of altered NO activity and vascular tone in insulin-
dependent diabetic condition. However, certain mechanism of NO in diabetic
vasculopathy remains challenging (Traub and Van Bibber 1995).
590 R. Deshmukh et al.

Adela et al. (2015) reported that the high level of NO was noted with increased
concentration of glucose in serum, which may be due to the activation of endothelial
cell and thereby enhanced NO production. Thus, hyperglycemia enhances NO
generation in Type-2 diabetes (Adela et al. 2015). Moreover, NO exhibits an
important role in the progression and regulation of vascular disease (diabetes and
its complication), which is generally affected by the ROS and NOS (van den Born
et al. 2016).

17.4.4.3 Erectile Dysfunction

Erectile dysfunction (ED) is a common problem in men due to altered relaxation
of corpus cavernosum smooth muscle and thereby reduced blood supply to penis.
ED is associated with several factors including age, socioeconomical condition,
depression, hypertensions, diabetes, etc. Erectile function (flaccidity and erection)
of the penis is performed by the smooth muscle tone. The flaccidity and erection is
maintained by sympathetic-contractile factors and parasympathetic-based smooth
muscle relaxation factors, respectively. NO has been recognized as the prime
vasoactive chemical mediator, noncholinergic and nonadrenergic neurotransmitter
responsible for penile erection and relaxation through smooth muscle. nNOS and
eNOS mediated NO is released from the nerves (nonadrenergic and noncholinergic
parasympathetic) and endothelial tissues in the corpus cavernosum and blood vessels
of the penis (Cartledge et al. 2001).
The NO exhibits its mechanism of action through 30 ,50 -cyclic guanosine
monophosphate (cGMP) pathway, cGMP maintains the function of intracellular
contractile proteins and calcium channels for relaxation and erection of penis
(Burnett 2006; Dashwood et al. 2011). For ED treatment, sildenafil and analogues
are generally used to increase the blood flow in nitrergic nerves of the penis by
inhibiting the phosphodiesterase and cGMP-induced vasodilation (Al Omar et al.
2016).

17.4.4.4 Respiratory Disease

NO plays a crucial role in maintenance of the respiratory system including vascular
smooth muscle, neurotransmission, lung cell, and airway. NO is highly exploited as
therapeutic agent for regulation of respiratory disorder. Release of NO is variable
and depends on the different enzymes and ROS, which can help in the generation of
NO (Antosova et al. 2017).
In case of neonatal respiratory distress syndrome, persistent pulmonary hyperten-
sion, and broncho-pulmonary dysplasia, inhaled NO exhibits significant effect to the
patients. Treatment may be improved in conjugation with NO simulators and drugs
like prostaglandins to produce synergism therapeutic effect. Along with the treat-
ment approach, potentiating the therapeutic effect, NO donor could be effective. It is
most important to develop such a type of delivery technique for respiratory system
that can modulate the release of NO in a controlled manner and reduce side effects by
decreasing the formation of unwanted by-products (Akter et al. 2016).
In case of respiratory tract problems like asthma and chronic obstructive pulmo-
nary disease (COPD), the exhaled NO is used as a biomarker for therapeutic
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 591

purposes, particularly during the treatment of acute respiratory distress syndrome

(ARDS). Nowadays, anti-inflammatory and analgesic treatment involves using of
NO-NSAIDs, isoxazolopyrazoles and nitroso derivatives based on hydroxyimoyl
chloride) for potentiating therapeutic effect. This combination system reduces
gastrointestinal irritation and bleeding by providing cytoprotection to the mucosal
lining of the stomach. Thus, NO can be used as a clinical agent for treatment of
diseases (Abdelall et al. 2017).

17.4.4.5 Angina Pectoris

The role of NO in regulation of various physiological functions including angina
pectoris has been established as biological marker. Promising in gaseous signaling
molecules, there is importance in the evolution of these markers for clinical uses. NO
has good vasodilation activity, due to which it is used as a clinical agent in case of
angina pectoris. NO exhibits biological activities through cell permeability, inflam-
mation, vasodilation, vascular and platelet function. The underlying mechanism of
action of NO is based on hemodynamic (vasoldilation of arteries) and
non-hemodynamic activities (nitroglycerine treatment) in dual dependent and inde-
pendent ways. For instance, glyceryltrinitrate (GTN), SNP, and molsidamine are
used for the treatment of angina (Quillon et al. 2015; Divakaran and Loscalzo 2017).
It can also be used in the treatment of cardiac problems like pulmonary hypertension
and cardiac failure. The wide application of organic nitrate is limited due to
development of tolerance (Divakaran and Loscalzo 2017).

17.4.4.6 Myocardial Infarction and Atherosclerosis

Heart problems like myocardial infarction (MI) and atherosclerosis (ASC) are due to
inflammation and fat-deposited arterial blockade, respectively, which has become
challenging throughout the globe. An inflammatory biomarker, C-reactive protein
(CRP), has a significant role in prediction of MI, coronary artery disease (CAD), and
other heart problems. CRP can exert its activity by inhibiting the endothelial cell NO
generation through deactivating eNOS. Low level of NO can produce its effect by
stimulating cell apoptosis and inhibiting (CRP based) angiogenesis in endothelial
cell (Fordjour et al. 2015).
Blood vessel problems like restenosis (abnormal narrowing of an artery or valve)
can also be treated through NO by inhibition of platelet and cell proliferation. It has
been reported that dietary organic nitrates based NO may also have a role in
inhibition of platelet aggregation after conversion into nitrites inside the body.
Both types of function of NO, i.e., vasodilation of smooth muscle and inhibition
of coagulants/platelets facilitate useful management of heart diseases including
myocardial infarction (MI) and atherosclerosis. Thus, there is the future scope for
development of newer kind of clinical agents that produce a prolonged release of NO
for management of CVD (Bohlen 2015; Fadel 2017).
In case of atherosclerosis, the NO and its relation to inflammation is justified.
It has been established that this disease is linked with inflammatory process and
leads to the production of plaque. The inflammatory cells i.e., macrophages and
monocytes inducing proliferation of plaque may be considered as a major factor for
592 R. Deshmukh et al.

producing disease(s). In this context, apoptotic cells are taken up by phagocytes

without producing any unwanted conditions of pro-inflammatory response. With
understating of this NO phenomenon in relation to inflammation, it has been
observed that apoptosis can be a pathway to relapse of plaque. Thus, it can be
predicted that NO has a specific role in controlling the problems associated with
inflammatory response, but its function is limited to the concentration only at local
milieu, the timing and administering route can also affect (Baran et al. 2004).
The main pathway behind MI and ASC is NO/cGMP. This pathway plays a
crucial role in different pathophysiological conditions like MI and atherosclerosis.
Apart from diverse kind of genes and enzymes which involve in NO production and
cGMP, is linked with MI risk and CAD (Wobst et al. 2015).

17.4.4.7 Inflammation
There is a inherent relation between NO mediated apoptosis and inflammation which
in turns depend upon the concentration level at the target site. iNOS produced higher
concentrations of NO cause apoptosis (a programmed cell death) while eNOS and
nNOS mediated lower level of NO can produce cytoprotective activity. For example
ONOO species which may be considered for apoptotic determining process,
although the specific function of ONOO in inflammatory cell apoptosis is still
un-cleared. Hence the concept of NO-apoptosis is applicable in inflammatory
conditions (Lee et al. 2017).
It has been studied that activated macrophages triggers the sensitive tumors
(murine) and thus initiating NO based apoptosis in both activated anti-tumor
T cells and normal tumor cells. Therefore, NO mediated macrophage induces
apoptosis (death of nearby cells) that raises the elimination of apoptotic cells results
in facilitating the resolution phase of inflammatory environment. These inflamed
cells lead to cancerous due to oxidant enriched surroundings, after sometimes these
phenomena cause relentless and self-responsive oxidative stress due to generation of
reactive oxygen species (ROS) and reactive nitrogen species (RNS) (MacMicking
et al. 1997a, b; Baran et al. 2004).

17.4.4.8 Cancer
In negative perspective, some of the NO related activities is related to oxidative
stress in various cancers including melanoma. At low level of NO (<100 nM), it can
produce the metastasis, proliferation and angiogenesis while at higher levels of
400–500 nM it may cause cytotoxicty and cell death (apoptosis). Therefore, NO
donors have been considered as a promising treatment approach of different cancers.
NO-NSAIDs are good example of this approach that NO-NSAIDs have been
reported to be effective in arresting the metastasis and tumor proliferation as
compared to pure drug itself (Abdelall et al. 2017).
NO shows anticancer property against colon cancer (HCT116 cancer cell) by
inhibiting cell proliferation and production (Olah et al. 2018). In case of melanoma,
NO mediates its effect via enzymatic process and free radical production. Thereby,
NO can help in formation and proliferation of melanoma by a bunch of mechanisms
involving inhibition of apoptosis and immune cells, alteration of angiogenic
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 593

processes, activation of tumor-associated macrophages and stimulation of

pro-tumorigenic cytokines (Yarlagadda et al. 2017).
NOS2-based NO has been identified as a key driver for progression of breast
cancer that is pioneering in the finding of novel targets which may be therapeutically
effective for the treatment of estrogen receptor-negative (ER-) disease (Basudhar
et al. 2017). No has wide dual distinct role in tumor or cancer proliferation and
progression but sometimes it prevents tumor initiation/proliferation and act as an
anticancer agent. NO has diverse kind of cellular functions through different process
involving regulation of metabolic, posttranslational, and transcriptional functions
(Keshet and Erez 2018).

17.4.4.9 Tuberculosis
iNOS-based NO has a crucial role in host defense systems against Mycobacterium
tuberculosis in tuberculosis (TB) infection. TB has an internal complex
environments to combat the therapeutic agent, resist and counter the effects of
NO, RNS and ROS in intracellular surrounding of bacteria. The bacterial
proteosome in TB is responsible for resistance against host-mediated nitrosative
and oxidative stress by deprivation of stress-induced protein damage. It has been
reported that TB genes (noxR1 and noxR3) have protection ability against oxidative
molecules (ROS, RNS, and NO) and their harmful events, i.e., apoptosis and
necrosis (Bhat et al. 2017; Braverman and Stanley 2017a, b).
The TB bacteria can change nitrates to nitrites in case of hypoxia, which favors
anaerobic environment and dormancy stage for TB. Recent reports suggest that
NO is an important gas transporter for host defense mechanism and diversified
pathophysological consequences of Mycobacterium tuberculosis infections. NO
could be explored along with anti-tubercular drugs for enhancement of their efficacy.
The quest for developing highly effective anti-tubercular agents and vaccines are
utmost required for prevention of TB (Chinta et al. 2016a, b).

17.4.4.10 Alzheimer’s Disease

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the
persistent loss of neuronal behavior with aging in people. In AD patients, memory
and other mental function is destroyed progressively. AD is the common mental
illness worldwide (Heneka et al. 2013). NO is considered as a gaseous messenger in
the mind (CNS, central nervous system) and its effect in memory recognition is well
explored. Particularly NOS inhibitors and NO donors are involved in recognition of
object memory. The role of NO CNS is well studied and it can be considered as a
potential agent for destruction of memory as well as cognition but its potential
neurotoxicity cannot be neglected (Pitsikas 2015).
Cifuentes et al. (2017) reported that impaired NO synthesis aggravates the
progression of AD in APPPS1 mice (Cifuentes et al. 2017). Austin and Katusic
(2016) reported that the NO has a crucial role in neuronal tissue by promoting p25
production and tau phosphorylation in condition of improper functioning of eNOS.
In this order, overexpressed condition of amyloid precursor protein (APP) and β-site
APP-cleaving enzyme 1 (BACE1) are seen with loss of eNOS in brain tissue of
594 R. Deshmukh et al.

mice. Evidence suggests for establishing new concept for understanding of the
molecular mechanism of NO with endothelial impairment in the context of cognitive
decline and AD progression. Moreover, endothelial NO is recognized as a protector
of healthy mind and key agent establishing activity between CNS and cerebrovas-
cular system (Katusic and Austin 2014; Austin and Katusic 2016).

17.5 Carbon Monoxide (CO) as Signaling Molecules

CO is an atmospheric gas and was considered as an industrial chemical by-product.

In industry, it is produced by the combustion of fossil fuels and the burning of
tobacco (MacMicking et al. 1997a, b; Fadel 2017). It is considered an enemy to
human health as it is known to cause death by asphyxiation. At a higher concentra-
tion it binds to hemoglobin 200 to 300 times stronger as compared to oxygen and
forms carboxyhemoglobin (CoHb). CoHb cannot bind to oxygen, thus reducing the
oxygen carrying capacity of Hb leading to toxicity and hypoxia in the human body
resulting in the death. In addition, it can interact with many intracellular proteins
which leads to toxicity in various body cells. Owing to this harmful effect, this gas
was labeled a “silent killer” for over 100 years (Li et al. 2009; Truss and Warner
2011a, b).
In the 1960s it was discovered that the metabolism of heme by heme oxygenase
(HO) can endogenously produce CO in the body. At that time, people were unable to
realize the implication of this finding. As such, endogenous synthesis of CO was
considered as a metabolic waste product of heme degradation (Chinta et al. 2016a, b;
Braverman and Stanley 2017a, b). But the endogenous synthesis initiated the
concept that normal CO levels can control various functions of the body like
NO. It was in 1987, Brune and Ullrich discovered the similarity of CO with NO in
activating the soluble guanylate cyclase in the mammalian system. Very soon in
1993, Synder and his associates explained the physiological function of CO, which
was found to be similar to that of NO (Verma et al. 1993).
This observation finally paved the way for the subsequent finding of other
functions of CO. Since then the physiological importance of CO as a signaling
molecule has been established and it is presently counted among the three
gasotransmitters along with NO and H2S. The gaseous nature of CO allows it to
pass freely through the plasma membrane without the aid of specialized transport
systems toward the targeted cell, thus allowing the CO to quickly impact the cellular
function and behavior, which is important for physiological regulation. Signaling of
CO inside the cells can control various physiological functions, mainly in the
nervous and cardiovascular systems. Till date, CO has been found to have effective
anti-proliferative, anti-inflammatory, and anti-apoptotic properties (Wu and Wang
2005a, b; Wallace et al. 2015).
CO is gaseous in nature and has no taste, color, and odor. It is a diatomic oxide of
carbon formed by partial oxidation of carbon-containing molecules. CO is chemi-
cally stable with a molecular weight of 28.01-Da with carbon and oxygen atoms
attached by a triple bond (Wu and Wang 2005a; b; Heinemann et al. 2014). The
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 595

solubility of CO in water is 2.3 mL/100 ml at 20  C, and without considerable energy

input it does not react with water. The specific gravity of CO in gaseous form is
1.250 g/L at 0  C. In order to calculate the molarity in the gas phase,
1 ppm ¼ 1.25 mg/m
3 (44.6 nM) at 25  C (Von Burg 1999). Owing to its
lipophilicity, it is able to freely cross the lipidic bilayer of the plasma membrane.
CO is a relatively stable neutral molecule because it does not contain free electrons
and therefore, it is not as reactive as the other gasotransmitters.

17.5.1 Biosynthesis of CO

Before the discovery and establishment of the concept of gasotransmitters, the

scientific community was aware of the fact that the human body can endogenously
synthesize CO (Coburn et al. 1963a; b). In 1966, Tenhunen et al. reported that
degradation of senescent RBC results in the formation of CO. Later on, after about
two decades, it was reported that the enzyme HO is accountable for the formation of
CO by breaking down toxic heme as a protective mechanism (Tenhunen et al. 1968).
The biosynthesis of CO takes place in cells and tissues various through endogenous
metabolic procedures and the rate of synthesis in humans is estimated to be 0.42 ml/
h (Sjostrand 1952; Coburn et al. 1963a; b).
In blood, the level of COHb is accountable for the endogenous production of
CO. It increases during environmental pollutions and in smokers (up to 10%). About
86% of endogenous CO synthesis occurs through heme metabolism, whereas the
residual part arises from cytochrome P450-dependent metabolism of xenobiotics or
lipid oxidation processes (Wu and Wang 2005a; b; Owens 2010).

17.5.1.1 Biosynthesis by Heme Degradation

The major pathway for the production of CO is through HO catalyzed heme
metabolism. This catabolism primarily takes place in the reticulo-endothelial system
constituting of spleen and liver. It was Sjostrand who first revealed the formation of
endogenous CO via oxidation of the α-methene bridge carbon of heme. Tenhunen
et al. reported the involvement of HO enzymes in the degradation of the heme to
form CO (Tenhunen et al. 1968).
The biosynthesis requires heme as substrate and HO processes the heme through
three oxidation cycles, each requiring molecular oxygen. HO along with cytochrome
P450 reductase in the presence of NADPH reduces the iron center of the bound heme
molecule, thus allowing the binding of oxygen to the reduced iron. The second
electron from NADPH activates the oxygen to form a peroxo-intermediate that
attacks the heme ring. Thus, α-meso-hydroxy-heme is formed from the first oxida-
tion cycle. In the second cycle, CO is released before the formation of verdoheme
intermediate. In the final cycle, biliverdin IXα (FeIII) is formed, which further
dissociates to free biliverdin and Fe (II), with the involvement of additional
NADPH. Thus, three reaction products are generated in heme degradation process:
CO, ferrous iron, and biliverdin-IX (blue-green pigment). The iron is recycled and
biliverdin reductase converts the biliverdin-IX into bilirubin-IX (yellow pigment),
596 R. Deshmukh et al.

Fig. 17.3 Biosynthesis of CO by hemoglobin degradation mechanism of red blood cells catalyzed
by heme oxygenase (HO) (Adapted from Fig. 2 of Wu and Wang 2005a, b)

which is excreted from the body via urinary pathway (Fig. 17.3). During injury, lysis
of RBC takes place and deoxygenated Hb is formed, which is observed as a dark
red/purple color formation. Later on a green tinge appears at injury site due to
oxidation of heme to form biliverdin. This pigment is gradually converted to
bilirubin causing a yellow coloration (Johnson et al. 2003).
HO is reported to have three isoforms, i.e., inducible HO-1 and two constitutive
forms, namely, HO-2 and HO-3. The HO-1 has low levels of expression in most
tissues under physiologic conditions except spleen. HO-1, known as the stress
protein (i.e., hsp32) is a redox-sensitive response protein. HO-I activity is
up-regulated in oxidative stress like inflammatory reaction or disease-like
conditions. Furthermore, HO-1 is up-regulated in case of cellular stress and therefore
found to be a vital antioxidant enzyme. Depending on the nature of the stimuli and
cell types, the induction of HO-1 can be moderated by various signaling pathways
like protein kinase C (PKC), cAMP-dependent mechanisms, Ca2+-calmodulin-
dependent protein kinase and the phosphoinositol pathways (Durante et al. 1997;
Immenschuh et al. 1998; Terry et al. 1999; Wu and Wang 2006). In addition, it has
been reported that mitogen-activated protein kinases (ERK and P38) and tyrosine
phosphorylation induce HO-1 in some tissues (Alam et al. 2000; Chen and Maines
2000).
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 597

HO-2 is constitutively expressed under basal conditions in most human tissues

and predominantly in the brain and testes. At a low level, it is also expressed in liver,
myenteric plexus of the gut, distal nephron segments, and endothelium. HO-2 is
mainly responsible for basal HO activity and thus CO production. It is activated by
calcium-calmodulin and casein kinase 2 in neurons. Similar to NO and H2S, CO is
vastly concentrated in endothelial layer of blood vessels, which regulates the vascu-
lar tone (Wang 1998; Abraham et al. 2003; Boehning et al. 2003; Abraham and
Kappas 2008).
Earlier, HO-3 was considered a pseudo gene, but following its cloning from the
rat brain it was observed to be an inactive paralogue. The HO-3 protein has little HO
activity and therefore its activity is uncertain and not much is known about the HO-3.
It is found in the liver, heart, spleen, thymus, prostate, kidney, testis, and brain
(McCoubrey Jr. et al. 1997; Hayashi et al. 2004).

17.5.1.2 Cytochrome P450

Cytochrome P450–dependent metabolism of certain xenobiotics sometimes leads to
the formation of CO as a by-product. During the metabolism of dichloromethane
(DCM), and methylene chloride, an increase in COHb levels in blood has been
observed to result in the formation of CO. In addition, oxidative dechlorination of
DCM by cytochrome P450 2E1 (CYT2E1) also results in the production of CO. The
depletion of hepatic glutathione (GSH) from DCM metabolism also increased CO
production. The CO that is formed by the above mechanism forms a complex with
cytochrome P450 and inactivate them (D. Stewart et al. 1972; Takehito and
Yoshifumi 1988; Amsel et al. 2001).

17.5.1.3 Lipid Peroxidation

Toxicological studies have revealed that inhalation of CO causes lipid peroxidation
in the rat brain by neutrophil recruitment. Moreover, in vitro studies indicate that CO
can be formed as a by-product of lipid peroxidation processes. Arachov et al. also
demonstrated microsomal NADPH- and Fe(II)-dependent lipid oxidation also
originates CO. This CO production occurred in the presence of HO inhibitors and
was eliminated by inhibitors of cytochrome P450 2B4 (R Thom 1990; Archakov
et al. 2002).

17.5.2 Catabolism of Endogenous Carbon Monoxide

The catabolism of the CO takes place by three mechanisms, i.e., expiration, scav-
enging, and oxidation, which are discussed below.

17.5.2.1 Expiration
CO is majorly expelled from the systemic production via lungs. CO diffuses through
the alveolar-capillary membrane, which is mainly dependent on the amount of
hemoglobin in the pulmonary capillaries and alveolar gas volume (Untereiner
et al. 2012). The concentration of CO in the alveoli is determined by the partial
598 R. Deshmukh et al.

pressure of oxygen and CO as these two gases compete for binding of same iron site.
Nowadays, CO is used to measure the heme metabolism and level of bilirubin.
Moreover, the level of CO is also used clinically for the determination of jaundice in
infants (Stevenson et al. 1994; Okuyama et al. 2001).

17.5.2.2 Scavenging
In normal physiological conditions, HO catalyzed heme degradation mechanism is
the major source of endogenously generated CO in our body. The body store of CO
is a combination of CO inhaled from the environment and xenobiotics metabolized
by hepatocytes (Kubic and Anders 1978). About 80% of CO binds with Hb present
in RBC and forms as COHb and other heme proteins which are accountable for the
remaining CO load (Coburn 1970). The CO body stores are transferable. During
hypoxia, it is reported that CO moves from blood to tissue where CO binds to heme
proteins (Coburn and Mayers 1971).

17.5.2.3 Oxidation
It has been believed that oxidation of CO results in the formation of CO2 involving
cytochrome c oxidase of mitochondria. However, no studies have been reported in
mammalian tissues related to its oxidation and that too under physiological
conditions. The microbes that live in CO oxidize it to form CO2 in the presence of
CO dehydrogenase. Moreover, in chemistry the oxidation or of CO to CO2 is also
well established. Also the atmospheric CO reacts with hydroxyl radicals to yield
HO2 (hydroperoxyl radical) and CO2 (Allen and Root 1957).

17.5.3 Role in Biological Systems

CO performs a diverse role in human physiology, described in the following.

17.5.3.1 CO-Mediated Vasorelaxation

Vasorelaxation by CO can be mediated by three main cellular mechanisms: (1) acti-
vation of sGC, (2) stimulation of big-conductance calcium-activated potassium
channels (BKCa), and (3) NOS induction (Botros and Navar 2006; Fabiani et al.
2008; Li et al. 2008).
It has been reported that the CO releasing molecules (CORMs) dilate renal
afferent arterioles to relax precontracted aortic rings, increase perfusion flow in
cirrhotic liver, and decrease intra-hepatic vascular resistance (Botros and Navar
2006; Van Landeghem et al. 2009).
CO can block potent vasoconstrictors synthesis including endothelin-1 and indi-
rectly reduce vascular resistance. Under the conditions of oxidative stress, CO act
differentially, resulting in vasoconstriction. In 2009, Lamon et al. reported CO
mediated constriction in the renal arteries of rats. They also demonstrated that the
inhibition of pro-oxidant molecules like NADPH oxidase, NOS, and xanthine
oxidase convert CO from constrictor to dilator. Therefore, it is the redox state of
the cell that decides if CO acts as vasodilator or vasoconstrictor (Lamon et al. 2009).
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 599

17.5.3.2 CO-Mediated Cardiac Protection

CO is responsible for cytoprotection after vascular injury. This has been
demonstrated by a number of experiments. During an ischemic injury, a
10–50 μM dose of CORM-3 results in considerable improvement in myocardial
activity, leading to reduced infarct size and less cardiac muscle damage. This was
further confirmed by CORM-3 inhibitor studies. In an isolated heart experiment,
the cardioprotective effects mediated by CORM-3 were eliminated by adding
mitochondrial ATP-dependent potassium channel inhibitor like 5-hydroxydecanoic
acid (Clark et al. 2003; Cepinskas et al. 2008; Nakao et al. 2011). In an experiment
in mice conducted by Clark et al. using a cardiac allograft model, CORM-3 substan-
tially improved the survival rate of transplanted hearts. The CORM-2 can
up-regulate the Bcl-2 (anti-apoptotic protein) expression and down-regulate
caspase-3 activation and protect the liver from ischemia–reperfusion injury
(Cepinskas et al. 2008).

17.5.3.3 Anti-Inflammatory Effects of CO

CO and CORMs have been reported to show anti-inflammatory activity. It has been
reported that CO can inhibit or surmount the initiation of pro-inflammatory enzymes
such as iNOS and cyclooxygenase-2 and inflammatory transcription factor NF-κB
(Freitas et al. 2006; Chin et al. 2007). They also activate the signal transduction
pathways like GC signaling, p38 MAPK, peroxisome proliferator-activated recep-
tor-c (PPARc), HIF-1a, and CCAATT-enhancer-binding protein (C/EBP) -b/d to
exhibit anti-inflammatory effect (Nizamutdinova et al. 2009; Lakkisto et al. 2010;
Nakao et al. 2011). In addition, CO inhibits the production of prostaglandin-2,
pro-inflammatory cytokines, IL-1b, -2, -6, -10, ICAM-1 and macrophage inflamma-
tory protein-1 in macrophages, T cells, and colonic epithelial cells. Needless to say,
the administration of CO or CORMs decreases cell adhesion, neutrophil movement
to the site of inflammation, leukocyte rolling, and LPS (lipopolysaccharide)
activated macrophagic cells (Urquhart et al. 2007; Nizamutdinova et al. 2009).

17.5.3.4 CO and Angiogenesis

It has been well established that angiogenesis process is stimulated by the vascular
endothelial growth factor (VEGF). CO gas or CORMs exposure to VSMCs, human
microvascular endothelial cells, rat primary cardiomyocytes, H9C2 myocytes, and
human umbilical vein endothelial cells (HUVECs) increases the expression of
VEGF. The mechanism of angiogenesis takes place through the involvement of
p38 kinase-dependent pathway or phosphatidylinositol 3-kinase (PI3K)/Akt/mam-
malian target of rapamycin and MEK/ERK-dependent pathways. Additionally,
uptake of CO-donor methylene chloride at single dose 500 mg/kg promotes blood
vessel formation in the infarct heart. This process occurs via VEGF-B and hypoxia
inducible factor (HIF)-1α that accelerates improvement in cardiac muscles and
functions (Li et al. 2005; Lin et al. 2011).
600 R. Deshmukh et al.

17.5.3.5 Anti-Aggregatory Effects of CO

CO can also block platelet aggregation during vascular damage and preserve blood
flow. Wagner et al. reported that monolayer of rat aortic VSMCs culture expresses
increased level of inducible HO-1 when observed under high stress in a time-
dependent manner. This results in increased intracellular cGMP levels in
co-incubated platelets. This is further verified by inhibitor studies. The cells were
treated with HO-1 inhibitor (30 μM snap-IX) that inhibits the stimulatory effect on
the platelet cGMP level induced by sheared VSMCs. Likewise, under hypoxic
conditions CO inhibits the production of platelet-derived growth factor-B in the
endothelial cells (Wagner et al. 1997). Hence, it can be suggested that CO is a
VSMC-generated messenger that can block the platelet aggregation. This aggrega-
tion is facilitated by hemodynamic forces enabling improved blood flow at damaged
cardiac portions. Altogether, more experimentation is required to understand the
basic mechanism of oxidative stress induced by HO-1 up-regulation as well as the
role of CORMs in the inhibition of platelet aggregation under in vivo conditions.

17.5.3.6 Cell Proliferation and Apoptosis of CO

Anti-Apoptotic Effects
CO exhibits apoptosis in various cells and tissues of the body in a very selective and
specific manner. For instance, it shows an anti-apoptotic to hepatocytes,
cardiomyocytes, and endothelial cells, thus protecting the cells and tissues from
injury. The anti-apoptotic activity of CO is dependent on p38 MAPK signaling,
phosphorylation of protein kinase R (e.g., endoplasmic reticulum kinase), and/or
through Akt activation (Choi et al. 2003; Clark et al. 2003; Kim et al. 2008). Besides,
CO also inhibits tumor necrosis factor-(TNF) -α- and endoplasmic reticulum
(ER) stress-induced cellular apoptosis. Interestingly, mitochondrial membrane
permeabilization can be prevented by low CO concentrations (10–100 μM) in
isolated mouse liver cells, thus stopping the formation of pro-apoptotic molecules.
However, high level of CO (250–500 μM) can activate the swelling in mitochondria
(Queiroga et al. 2011).

Pro-Apoptotic Effects
CO exhibits pro-apoptotic effect on fibroblasts and hyper-proliferative smooth
muscle cells (Zheng et al. 2009). It also blocks the smooth muscle cells of the
trachea in humans and propagation of vascular smooth muscle cell in rats both under
hypoxic and normoxic states. Zhou et al. showed an inhibition of fibroblast prolifer-
ation with the decrease degree of fibrosis in mice when exposed to CO. They
discovered that CO blocked the cell cycle in G0/G1 phase via a cGMP-dependent
mechanism. CO at a concentration of 100–200 ppm inhibited the proliferation of
VSMC at G(1)/S transition phase and obstructed the activation and production of
cyclin A. Thus, the HO/CO signaling system offers an important function in the
regulation of cell survival (Zhou et al. 2005).
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 601

17.5.4 Clinical Utility

This section discusses the pathophysiological implications of CO for the following

diseases.

17.5.4.1 Diabetes
Diabetes is a metabolic disease distinguished by high of glucose in the systemic
circulation. It can be classified as insulin-dependent (Type 1) or non-insulin-depen-
dent (Type 2) diabetes mellitus. It has been observed that in diabetic patients there is
an increase exhaled CO levels. The CO concentration in normal healthy persons is
2.9 ppm whereas it is observed to be at 4.0 and 5.0 ppm for Type 1 and Type
2, respectively. Thus, there exists a relationship between glycemia and exhaled CO
levels. This observation was further validated through oral GTT (glucose tolerance
test). In case of healthy humans, there is a connection between blood glucose
concentrations (i.e., 3.9–5.5 mM) and amount of CO expired (i.e., 3.0–6.3 ppm).
This abnormal level is restored to normal within 40 min of glucose intake (Paredi
et al. 1999). This relationship was explained by Lundquist and associate in intact
mouse islets wherein HO activity was initiated by glucose. In an experiment with
Goto-Kakizaki rats model (i.e., defective pancreatic β-cell with HO-2 expression),
low CO was observed with insulin deficiency. This study signified that HO-2 has an
important function in insulin secretion and glucose metabolism (Henningsson et al.
1999).

17.5.4.2 Vascular Proliferative Diseases

VSMC hypertrophy and hyperplasia are the hallmarks of vascular proliferative
diseases. The pathophysiology is due to disturbed redox signaling of cellular prolif-
eration and apoptosis. CO arrests hyper-proliferative VSMCs, increases cell move-
ment, and generation of bone-marrow-derived progenitor cells to shed vessels and
prevent intimal hyperplasia (Raman et al. 2006; Ramlawi et al. 2007).

Hypertension
Hypertension is termed as consistent increased blood pressure distinguished by
increased systolic pressure (i.e., above 150 mm Hg) and high diastolic pressure
(i.e., above 90 mm Hg). It is characterized by vascular structural changes like
atherosclerosis (hardening of blood vessels due to fat deposition) and co-arctation
of aorta (narrowing of the aorta) which results in high resistance and elevated blood
pressure. This hemodynamic stress causes initiation of HO-1 activity, which in turn
stimulates the activity of sGC, and cGMP in VSMCs (Wegiel et al. 2010).
CO at a concentration of 60 ppm can decrease the hypertrophy of left ventricles
and aorta in mouse model which results in the inhibition of angiotensin II-dependent
hypertension. CO can reduce the phosphorylation NOX and Akt resulting in reduc-
ing ROS production, thereby protecting the cells (Ndisang et al. 2004).
Pulmonary arterial hypertension (PAH) is typified as high blood pressure of
lungs. The obstruction in small arteries in the lung causes increased pressure in the
vessels leading to right ventricle failure. CO has a capacity to reduce the PAH.
602 R. Deshmukh et al.

Kobayashi et al. demonstrated that in mice the PAH and right ventricular hypertro-
phy are reversed by inhalation of CO (250 ppm, 1 h/day for 2 or 3 weeks). Moreover,
it also restores the pressure in the pulmonary artery and right ventricule along with
pulmonary vascular structural design (Kobayashi et al. 2007).
The underlying principle in the inhibition of hypertension is the involvement of
the NOS pathway. The HO-1/CO system can be up-regulated by hemin supplemen-
tation, which can be used in the treatment of hypertension. In a study in young
hypertensive rats, hemin therapy was reported to decrease the blood pressure. A 21-
days-based implanted hemin osmotic minipumps releasing 15 mg/kg/day provide
prolonged safety against hypertension in 12-week-old SHRs. It is the accumulation
of residual hemin within VSMCs that results in normalization of blood pressure of
SHR. Thus, this new approach of hemin application at physiologically or therapeutic
concentrations can be a novel alternative approach for the management of
hypertension.

Atherosclerosis
Atherosclerosis is a disease characterized by the hardening of the arteries due to the
deposition of plaque. It slowly and silently blocks arteries, obstructing the blood
flow in the heart. It is one of the primary causes of strokes, heart attacks, and
peripheral vascular disease. It involves inflammation, endothelial dysfunction, and
vascular proliferation. It is evident that during atherosclerosis, HO-1 is up-regulated,
which is important in the management of disease (Johnson et al. 2006).
CO acts as strong anti-atherosclerotic agent owing to VSMCs apoptotic
properties, initiation of endothelial cell proliferation, and anti-inflammatory activity.
Wang et al. reported that in balloon angioplasty-induced vessel injury in rats, CO at a
concentration of 250 ppm blocked the development of atherosclerotic lesions. The
CO blocks leukocyte infiltration and VSMC proliferation thereby resulting in pro-
tection of arteries from hyperplasia (Wang et al. 2001).

17.5.4.3 Myocardial Infarction

MI is also known as heart attack, which is due to necrosis of the myocardium caused
by insufficient blood flow due to embolus, thrombus, or vascular spasm. In MI,
cardiomyopathy occurs quickly, owing to ventricular fibrillation. CO can also give
protection to heart from MI (Shahrbaf et al. 2018).
In hyperoxia-reoxygenation damage, cardiac cells model, CO reduces infarct size
and protects cardiac muscle damage. Stein et al. observed that pre-exposure of CO
gas (1000 ppm) in rat myocardial ischemia–reperfusion injury model decreases the
affected part and also inhibits the movement of monocytes and macrophages in the
infected part. The involvement of p38 MAPK and Akt–eNOS pathways, including
cGMP production is responsible for the cardioprotective effects of CO on cardiac
ischemia–reperfusion injury (Stein et al. 2005). In addition, inhalation of CO
(250 ppm) protects the cardiac system during reperfusion after bypass and improved
cardiac activity (Fujimoto et al. 2004).
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 603

17.5.4.4 Central Nervous System

It has been reported that CO at a low concentration exhibits a protective role in
vascular and neuronal system of CNS. It shows its beneficial effect in cases like
stroke, Alzheimer’s Disease and traumatic brain injury. During various pathological
conditions there is an increase level of ROS resulting in inflammation and injury
to the brain cells. CO/CORMs helps to scavenge the ROS and protect the brain cells
from oxidative injury and apoptosis. The neuroprotective mechanism of CO is
due to either direct binding of heme protein or by indirect inducing of the HO-1.
The increased level of CO up-regulates the synthesis of HO-1 which acts as a
neuroprotective (Choi 2018).
Alzheimer’s Disease is a form of mental deterioration characterized by beta-
amyloid formation resulting in neuron death and brain tissue shrinkage. CORM
also plays an important role in the management of Alzheimer’s Disease. It was
reported that in the human neuroblastoma SH-SY5Y, and in rat hippocampal
neurons, 10 μM CORM-2 protects amyloid-β-induced toxicity (Hettiarachchi et al.
2014).

17.5.4.5 Renal Disorder

In the kidney, CO is generated by the enzymatic action of HO enzymes. The isoform
HO-1 is mainly expressed in the kidney in response to various pathological and
physiological stimuli. It has been demonstrated that CO and CORM inhalation
therapy is beneficial in various renal injuries. It is believed that it protects the renal
cells by limiting the oxidative stress and apoptosis. It increases the viability of cell by
promoting the cell survival pathway. The renal vascular and tubular functions are
regulated through CO in the kidney. It protects renal vessels against excessive
vasoconstriction and limits the sodium reabsorption in the tubule cells, thus playing
a crucial role in the regulation of blood pressure. In addition, during Cisplatin
(CP) therapy of cancer, it accumulates in nephron and results in acute renal injury
and nephrotoxicity. CO and CORM-3 has been reported to have anti-apoptotic
effects and prevents nephrotoxicity (Tayem et al. 2006; Csongradi et al. 2012).

17.5.5 Carbon Monoxide and Various Ion Channels Interaction

Ion channels are the vital constituent of a cell, which is responsible for the activity of
any pharmaceuticals. The activity of CO also depends on its interaction with various
ion channels, such as K+ channels. The K+ channels superfamily is composed of
calcium-activated KCa channels, voltage-dependent Kv, and ATP-sensitive KATP.

17.5.5.1 Carbon Monoxide and KCa Channels

In 1997, Wang et al. conducted a series of experiments in rat tail artery tissues and
demonstrated evidence for a direct interaction of BKCa channels and CO. Their
study claims that CO can relax artery tissues under in vitro conditions. This claim
was further proved by inhibitor studies. They showed that the addition of pharma-
cological inhibition of BKCa channels blocks the CO-induced vasorelaxation. The
604 R. Deshmukh et al.

CO interacts with the histidine residue of BKCa to induce vasorelaxation by the

opening of the channels. CO shows its stimulatory effects by interacting specifically
with BKCa, α subunit (Wang et al. 1997).
In another set of experiments on newborn porcine cerebral arterial smooth muscle
cells, CO at a sub-micromolar level increases the BKCa channel activity and this
activity is independent of the cGMP pathway (Xi et al. 2004). Also, the CO activity
was confirmed using BKCa channel blocking agents, e.g., TEA and iberiotoxin.
They totally eliminate CO-induced vasodilation activity of the pial arterioles of
newborn pigs (Leffler et al. 1999).

17.5.5.2 Carbon Monoxide and KATP Channels

Foresti et al. showed that CO released from CORM-3 (tricarbonylchloro-(glycinato)
ruthenium (II)) induced a relaxation of phenylephrine-precontracted aorta tissues in a
dose-dependent manner. In the above experiment, when Glibenclamide, a KATP
channel blocker, was incubated for 15–30 min, it significantly reduced the vasodila-
tor activity. But till date, no direct electro-physiology experiment has been done to
investigate the direct interaction of KATP channels and CO (Foresti et al. 2004).

17.5.5.3 Carbon Monoxide and Calcium Channels (L-Type)

The regulatory effect of CO on L-type Ca2+ channels is quite controversial. CO
exhibits an inhibitory activity against the cardiac L-type channel in HEK293 cells,
H9c2 cells, and native rat cardiomyocytes. It is believed that CO increases the
mitochondrial ROS formation and inhibits channel activation. As a result, there
occurs a change in the C terminal end of L-type Ca2+ channel at three precise
cysteine residues located at 1789, 1790, and 1810 (Hou et al. 2008). On the contrary,
CO activated L-type Ca2+ channels in recombinant intestinal smooth muscle cells of
human through NO-dependent mechanism. The variation in the cellular redox state
or tissue-specific splice may be the reason behind these contradicting observations
(Dallas et al. 2009).

17.5.6 Therapeutic Applications of Carbon Monoxide and CORMs

In the late 1990s, the concept of delivering low concentrations of CO gas had
begun being investigated for alleviating various pathophysiological conditions
characterized by oxidative stress and inflammatory states. This was a new and
quite provocative proposal concept for management of pathology of disease. How-
ever, it is also evident that CO interferes in oxygen transport and its delivery.
Therefore, its sustained inhalation can cause problems which are related to systemic
effects and thus limiting the use of CO in therapeutics. Later on it was found that this
drawback could be overcome by packing CO in a more stable chemical form called
CORMs. The different types of CO and their properties are given in Table 17.3.
Initially, CO was considered a toxic gas and a “by-product” of heme metabolism
in human physiology. Only recently, the pharmacology of CO has been explored in
human physiology and recognized as a gasotransmitter. CO and CORMs are now
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 605

Table 17.3 Name, chemical structure, and properties of CORMs

Type of Molecular
CORMs Formula Chemical Name Structure
CORM-1 [Mn2(CO)10] Manganese decacarbonyl OO
O O
C C C C
O C Mn Mn C O
C C C
C
O O
OO

CORM-2 ([Ru(CO)3Cl2]2) Tricarbonyldichlororuthenium(II) dimer OC H

Cl
Cl
OC CO
Ru Ru
OC CO
Cl
Cl H CO

CORM-3 (Ru[CO]3Cl Tricarbonylchloro(glycinato)ruthenium H2

OC N
(glycinate)) II OC
Ru
OC O
Cl O

CORM-A1 Na2(H3BCO2) Disodium carboxylato(trihydrido)borate H 2-

H
(1-) H
B
O
HO 2Na

CORM-F3 [C9H5BrFeO5] η-4-(4-bromo-6-methyl-2-pyrone) Br

tricarbonyl iron (0) Fe(CO)3

O O

considered as potential therapeutic agents exhibiting cytoprotective effects. It has

been demonstrated by various preclinical models that CO and CORMs modulate
inflammation, apoptosis, and cell proliferation to restore homeostasis of the body
(Ling et al. 2017). Figure 17.4 illustrates the therapeutic and biological effects of CO
and CORMs in different clinical indications.

17.5.6.1 Inflammation
CO and CORMs can be used in modulating immune-suppression and inflammation.
They act as therapeutic agents in numerous models of inflammatory disease such
as COPD, asthma, and airway hyper-responsiveness, and carrageenan-induced
mesenteric inflammation. In addition, many phase II clinical trials have also
validated the anti-inflammatory activity of these gasotransmitters. CORM-2 can be
employed in the management of allergy as it significantly lowers the release of
histamine and expression of CD203c in the mast cells of guinea pigs and human
basophils (Vannacci et al. 2004).
The lipopolysaccharide (LPS)-activated murine J774 macrophagecells,
stimulates the inflammatory response by the production of NO and TNF- α involving
NF-κB. CORM-2 inhibits the translocation of NF-κB into the nucleus and
completely eliminates the inflammatory response. Also, CORM-2 reduces the
expression of inflammatory molecules like nitrite and iNOS in LPS stimulated
macrophages (Lee et al. 2003). In a Plasmodium infected mouse experimental
cerebral malaria model where the infection stimulated an inflammation, the inhala-
tion of CO for 3 days after the infection considerably reduced the inflammation in
 606
R. Deshmukh et al.

Fig. 17.4 Summary of Therapeutic Applications of CO and CORMs (Adapted from Fig. 2 of Ling et al. 2017)
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 607

mouse brain and improved the survival rate; in contrast there were heavy casualties
in the control group (Pamplona et al. 2007).

17.5.6.2 Cardiovascular Disease

The HO-1/CO/CORMs have been extensively explored for the cure of CVDs.
CORMs are essentially employed in the management of those diseases where the
HO-1 system plays a useful and fundamental function. In HO-1 deficient mice with
an abdominal aortic transplantation model HO-1/ CO/CORMs display a cardio
protective role. In this study, the control mice died within 4 days of transplantation
due to severe arterial thrombosis. In contrast, CORMs in mice considerably
enhanced the survival rate by 62% with reduced platelet aggregation, confirming
the anti-aggregatory properties (Chen et al. 2009).
The exogenous exposure of CO and intravenous infusion of CORM-3 in mouse
coronary artery occlusion model presented a reduced myocardial infarct size and
exhibited a substantial decrease in occurrence of reperfusion-induced tachycardia
and ventricular fibrillation (VF) (Varadi et al. 2007).
In vascular proliferative diseases, CO/CORMs exhibit a notable homeostatic
effect. They block the hyper-proliferation of vascular smooth muscle cells,
suppress atherosclerotic lesions, weaken intimal hyperplasia, and enable
re-endothelialization. In PAH, CO also exhibits its beneficial effect. In mouse
models it restores pulmonary arterial, right ventricular pressure and pulmonary
vascular architecture to near normal. These cardioprotective effects is due to the
involvement of the BK channel, cGMP-dependent pathway, p38 MAPK, ion cardiac
L-type Ca2+channel, and inhibition of CYP450 (Fujimoto et al. 2004).

17.5.6.3 Organ Transplantation and Preservation

In addition to anti-inflammatory and cardiovascular properties mentioned earlier,
CO and CORMs have been utilized in organ transplantations including heart,
kidney, lungs, liver, and pancreatic islet. They are employed throughout the trans-
plantation process and exhibit a beneficiary effect to the donor, organ, and recipient
(Motterlini and Otterbein 2010).
In one of the heart transplantation studies in rodents, CO exposure and CORM-3
administration for 8 days prolonged the cardiac allograft survival. During organ
transplantation CO and CORMs work as organ protectants by reducing ischemia/
reperfusion injury (Fujisaki et al. 2016).
In another experiment, porcine kidney was subjected to 10 min warm ischemia
and 18 h of cold storage. Application of CORM-3 brought back renal blood flow and
enhanced the renal function parameters such as creatinine clearance rate and glo-
merular filtration rate. A similar observation was also obtained in the transplantation
of porcine kidney model where an excised pig kidney graft was transplanted back
into the abdominal cavity after being cold flushed and stored in UW solution
(an organ preservation solution) for 2 days. The CO gas bubbled kidneys had greater
viability and less infiltrates (Bagul et al. 2008).
608 R. Deshmukh et al.

17.5.6.4 Microbial Infection

CO and CORMs exhibited antimicrobial action against varied bacteria, including
gram-negative bacteria, e.g., Escherichia coli or Pseudomonas aeruginosa and
gram-positive like, Staphylococcus aureu. CO inhibits the respiratory chain and
cuts off adenosine triphosphate (ATP) supplies in bacteria, resulting in cell death.
Additionally, CO activates host immune responses and promotes phagocytosis of
bacteria by expression of Toll-like receptor 4. In CORMs, the inner metal core is
responsible for the antibacterial activity. In one study, CORM-2 and tetraethyl
ammonium molybdenum pent carbonyl bromide (ALF062) produced ROS inside
the E. coli, causing DNA lesions and cell death. Apart from antibacterial activity,
they also exhibit anti-leishmanial and anti-trypanosomal effects (Nobre et al. 2007).

17.5.6.5 Cancer
Intriguingly, in cancer therapy HO-1/CO has dichotomous function. At low
concentrations, HO-1/CO, in cancer cells exerts cytoprotective, pro-proliferative
and pro-angiogenic property. However, at high concentration, CO exhibits antitumor
activity. HO-1 overexpression has been established in several tumor cells such as
pancreatic cancer, prostate cancer, melanoma, and Kaposi sarcoma. Generally, CO at
the physiological dose can be formed via tumor cells or tumor-infiltrating
macrophages to produce a pro-tumor effect. Therefore, inhibition of HO-1 can be
utilized for anticancer therapy. For example, in a mouse cancer model small
interfering RNA (siRNA) mediated HO-1 silencing results in lower tumor growth
rates and angiogenesis. Inhaled CO or CORMs at a higher concentration could be
lethal to tumor cells (Simon et al. 2011).
In a CD1 athymic mouse model inhalation of 500 ppm CO for 1 h a day
considerably lowers the cancer cell growth and the resultant angiogenesis. The
anti-tumor activity might be due to mitochondrial ROS generation, acceleration of
oxygen consumption, inhibition of cellular protein synthesis, and reduction of
cellular antioxidants. The high level of CO is toxic to the normal tissue; therefore,
these preclinical data may not be clinically valid (Wegiel et al. 2013).

17.6 Conclusions

The risk-to-benefit ratio of the gaseous molecules as transmitters is quite low if

maintained at lower concentrations. Based on their specificity of molecular mecha-
nism they can be widely utilized for various pathological conditions of the body. The
cellular interactions are pivotal for understanding the signaling of these gaseous
molecules comprising of NO, CO, and H2S. Besides, ammonia is being the latest
addition and methane is under consideration as a likely candidate. The European
Network on Gasotransmitters has set up basic regulations for advanced research in
order to achieve enhanced therapeutic activity. The vital role played, especially, by
NO and CO is at the basis of importance of these gasotransmitters.
NO being the first signaling molecule has provided the underlying concepts in
signal transduction. NO has been recognized as a physiological key element for
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 609

several pharmacological functions of the human body. Diverse kinds of NO (nNOS,

eNOS and iNOS) associated with varied biological activities have been reported
for modulating and transducing the signals within the cells. NO is important for
regulating various disorders in different organs like, brain, kidney, liver, heart, etc.
Besides, NO is also crucial for modulating the tumor activity and immune system.
CO was initially considered an environmental hazard and harmful to human
health. However, its physiological role became evident in the early 1990s in various
pathophysiological conditions. In order to prevent high dose toxicity, a chemically
stable form of CO is produced endogenously termed as CO-releasing molecules
(CORMs). CO and CORMs are now considered as potential therapeutic agents that
exhibit cytoprotective effects.
Further, it has been demonstrated by various preclinical models that CO and
CORMs modulate inflammation, apoptosis, and cell proliferation to restore homeo-
stasis of the body. Lately, it has been reported in the treatment of renal disorder, CNS
regulation, cardiovascular disorder, cancer, and microbial infection. Moreover, the
utilization of exhaled CO as a biomarker in metabolic syndrome like diabetes could
be a newer approach to measure the disease severity and assess the drug therapeutic
efficacy.
Thus, with the advancement of research on various gasotransmitters and explor-
ing their mechanism of action, a new gateway will be opened for the treatment of
various pathological conditions. In future, this will help in drug designing, preven-
tion and treatment of different types of diseases.

References
Abdelall EKA, Abdelhamid AO, Azouz AA (2017) Synthesis and biological evaluations of new
nitric oxide-anti-inflammatory drug hybrids. Bioorg Med Chem Lett 27(18):4358–4369
Abraham NG, Kappas A (2008) Pharmacological and clinical aspects of heme oxygenase.
Pharmacol Rev 60:79–127
Abraham NG, Jiang H, Balazy M, Goodman AI (2003) Methods for measurements of heme
oxygenase (HO) isoforms-mediated synthesis of carbon monoxide and HO-1 and HO-2
proteins. Methods Mol Med 86:399–411
Adela R, Nethi SK, Bagul PK, Barui AK, Mattapally S, Kuncha M, Patra CR, Reddy PN, Banerjee
SK (2015) Hyperglycaemia enhances nitric oxide production in diabetes: a study from south
Indian patients. PLoS One 10(4):e0125270
Akter F, Coghlan G, de Mel A (2016) Nitric oxide in paediatric respiratory disorders: novel
interventions to address associated vascular phenomena? Ther Adv Cardiovasc Dis 10
(4):256–270
Al Omar S, Salama H, Al HM, Al RH, Bunahia M, El Kasem W, Siddiqui FJ, Dilawar M, Yassin H,
Masud F, Mohamed A, Mansour A (2016) Effect of early adjunctive use of oral sildenafil and
inhaled nitric oxide on the outcome of pulmonary hypertension in newborn infants. A feasibility
study. J Neonatal Perinatal Med 9(3):251–259
Alam J, Wicks C, Stewart D, Gong P, Touchard C, Otterbein L, Choi A, Burow M, Tou J.-s. (2000)
Mechanism of Heme Oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial
cells. Role of OF p38 kinase and Nrf2 transcription factor. J Biol Chem 275(36):27694–27702
Allen TA, Root WS (1957) Partition of carbon monoxide and oxygen between air and whole blood
of rats, dogs and men as affected by plasma pH. J Appl Physiol 10(2):186–190
610 R. Deshmukh et al.

Amsel SDJ, Kevin J, Soden KJ, Sielken RL Jr, Valdez-Flora C (2001) Observed versus predicted
carboxyhemoglobin levels in cellulose triacetate workers exposed to methylene chloride. Am J
Ind Med 40(2):180–191
Andreadou I, Iliodromitis EK, Rassaf T, Schulz R, Papapetropoulos A, Ferdinandy P (2015) The
role of gasotransmitters NO, H2S and CO in myocardial ischaemia/reperfusion injury and
cardioprotection by preconditioning, postconditioning and remote conditioning. Br J Pharmacol
172(6):1587–1606
Antosova M, Mokra D, Pepucha L, Plevkova J, Buday T, Sterusky M, Bencova A (2017)
Physiology of nitric oxide in the respiratory system. Physiol Res 66(Supplementum 2):S159–
S172
Archakov AI, Karuzina II, Petushkova N, Lisitsa AV, Zgoda V (2002) Production of carbon
monoxide by cytochrome P450 during iron-dependent lipid peroxidation. Toxicol In Vitro 16
(1):1–10
Arzumanian V, Stankevicius E, Laukeviciene A, Kevelaitis E (2003) Mechanisms of nitric oxide
synthesis and action in cells. Medicina (Kaunas) 39(6):535–541
Austin SA, Katusic ZS (2016) Loss of endothelial nitric oxide synthase promotes p25 generation
and tau phosphorylation in a murine model of Alzheimer’s disease. Circ Res 119
(10):1128–1134
Bagul A, Hosgood S, Kaushik M, Nicholson ML (2008) Carbon monoxide protects against
ischemia-reperfusion injury in an experimental model of controlled nonheartbeating donor
kidney. Transplantation 85(4):576–581
Baran CP, Zeigler MM, Tridandapani S, Marsh CB (2004) The role of ROS and RNS in regulating
life and death of blood monocytes. Curr Pharm Des 10(8):855–866
Basudhar D, Somasundaram V, de Oliveira GA, Kesarwala A, Heinecke JL, Cheng RY, Glynn SA,
Ambs S, Wink DA, Ridnour LA (2017) Nitric oxide Synthase-2-derived nitric oxide drives
multiple pathways of breast Cancer progression. Antioxid Redox Signal 26(18):1044–1058
Bhat KH, Srivastava S, Kotturu SK, Ghosh S, Mukhopadhyay S (2017) The PPE2 protein of
Mycobacterium tuberculosis translocates to host nucleus and inhibits nitric oxide production.
Sci Rep 7:39706
Boehning D, Moon C, Sharma S, Hurt KJ, Hester LD, Ronnett GV, Shugar D, Snyder SH (2003)
Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2.
Neuron 40(1):129–137
Bohlen HG (2015) Nitric oxide and the cardiovascular system. Compr Physiol 5(2):808–823
Botros FT, Navar LG (2006) Interaction between endogenously produced carbon monoxide and
nitric oxide in regulation of renal afferent arterioles. Am J Phys Heart Circ Phys 291(6):H2772–
H2778
Braverman J, Stanley SA (2017a) Nitric oxide modulates macrophage responses to Mycobacterium
tuberculosis infection through activation of HIF-1alpha and repression of NF-kappaB. J
Immunol 199(5):1805–1816
Braverman J, Stanley SA (2017b) Nitric oxide modulates macrophage responses to Mycobacterium
tuberculosis infection through activation of HIF-1alpha and repression of NF-kappaB. J
Immunol 199(5):1805–1816
Burnett AL (2006) The role of nitric oxide in erectile dysfunction: implications for medical therapy.
J Clin Hypertens (Greenwich) 8(12 Suppl 4):53–62
Cartledge J, Minhas S, Eardley I (2001) The role of nitric oxide in penile erection. Expert Opin
Pharmacother 2(1):95–107
Cepinskas G, Katada K, Bihari A, Potter RF (2008) Carbon monoxide liberated from carbon
monoxide-releasing molecule CORM-2 attenuates inflammation in the liver of septic mice.
Am J Physiol Gastrointest Liver Physiol 294(1):G184–G191
Chen K, Maines MD (2000) Nitric oxide induces heme oxygenase-1 via mitogen-activated protein
kinases ERK and p38. Cell Mol Biol (Noisy-le-Grand) 46(3):609–617
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 611

Chen B, Guo L, Fan C, Bolisetty S, Joseph R, Wright MM, Agarwal A, George JF (2009) Carbon
monoxide rescues Heme Oxygenase-1-deficient mice from arterial thrombosis in allogeneic
aortic transplantation. Am J Pathol 175(1):422–429
Cheng Y, Rong J (2017) Therapeutic potential of Heme Oxygenase-1/carbon monoxide system
against ischemia-reperfusion injury. Curr Pharm Des 23(26):3884–3898
Chin BY, Jiang G, Wegiel B, Wang HJ, MacDonald T, Zhang XC, Gallo D, Cszimadia E, Bach FH,
Lee PJ, Otterbein LE (2007) Hypoxia-inducible factor 1α stabilization by carbon monoxide
results in cytoprotective preconditioning. Proc Natl Acad Sci 104(12):5109–5114
Chinta KC, Saini V, Glasgow JN, Mazorodze JH, Rahman MA, Reddy D, Lancaster JR Jr, Steyn AJ
(2016a) The emerging role of gasotransmitters in the pathogenesis of tuberculosis. Nitric Oxide
59:28–41
Chinta KC, Saini V, Glasgow JN, Mazorodze JH, Rahman MA, Reddy D, Lancaster JR Jr, Steyn AJ
(2016b) The emerging role of gasotransmitters in the pathogenesis of tuberculosis. Nitric Oxide
59:28–41
Choi YK (2018) Role of carbon monoxide in neurovascular repair processing. Biomol Ther (Seoul)
26(2):93–100
Choi BM, Pae HO, Kim YM, Chung HT (2003) Nitric oxide-mediated cytoprotection of
hepatocytes from glucose deprivation-induced cytotoxicity: involvement of heme oxygenase-
1. Hepatology 37(4):810–823
Cifuentes D, Poittevin M, Bonnin P, Ngkelo A, Kubis N, Merkulova-Rainon T, Levy BI (2017)
Inactivation of nitric oxide synthesis exacerbates the development of Alzheimer disease pathol-
ogy in APPPS1 mice (amyloid precursor protein/Presenilin-1). Hypertension 70(3):613–623
Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR, Mann BE, Foresti R, Motterlini R (2003)
Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res 93
(2):e2–e8
Coburn RF (1970) The carbon monoxide body stores. Ann N Y Acad Sci U S A 174(1):11–22
Coburn RF, Mayers LB (1971) Myoglobin O2 tension determined from measurement of
carboxymyoglobin in skeletal muscle. Am J Phys 220(1):66–74
Coburn RF, Blakemore WS, Forster RE (1963a) Endogenous carbon monoxide production in man.
J Clin Invest 42:1172–1178
Coburn RF, Blakemore WS, Forster RE (1963b) Endogenous carbon monoxide production in man.
J Clin Invest 42:1172–1178
Csongradi E, Juncos LA, Drummond HA, Vera T, Stec DE (2012) Role of carbon monoxide in
kidney function: is a little carbon monoxide good for the kidney? Curr Pharm Biotechnol 13
(6):819–826
Dallas ML, Scragg JL, Peers C (2009) Inhibition of L-type Ca(2+) channels by carbon monoxide.
Adv Exp Med Biol 648:89–95
Dashwood MR, Crump A, Shi-Wen X, Loesch A (2011) Identification of neuronal nitric oxide
synthase (nNOS) in human penis: a potential role of reduced neuronally-derived nitric oxide in
erectile dysfunction. Curr Pharm Biotechnol 12(9):1316–1321
Divakaran S, Loscalzo J (2017) The role of nitroglycerin and other nitrogen oxides in cardiovascu-
lar therapeutics. J Am Coll Cardiol 70(19):2393–2410
Doroszko A, Dobrowolski P, Radziwon-Balicka A, Skomro R (2018) New insights into the role of
oxidative stress in onset of cardiovascular disease. Oxidative Med Cell Longev 2018:9563831
Durante W, Christodoulides N, Cheng K, Peyton KJ, Sunahara RK, Schafer AI (1997) CAMP
induces heme oxygenase-1 gene expression and carbon monoxide production in vascular
smooth muscle. Am J Physiol 273(1 Pt 2):H317–H323
Fabiani J-N, Achouh PE, Simonet S, Verbeuren TJ (2008) Carbon monoxide induces relaxation of
human internal thoracic and radial arterial grafts. Interact Cardiovasc Thorac Surg 7(6):959–962
Fadel PJ (2017) Nitric oxide and cardiovascular regulation: beyond the endothelium. Hypertension
69(5):778–779
Farrugia G, Szurszewski JH (2014) Carbon monoxide, hydrogen sulfide, and nitric oxide as
signaling molecules in the gastrointestinal tract. Gastroenterology 147(2):303–313
612 R. Deshmukh et al.

Fleissner F, Thum T (2011) Critical role of the nitric oxide/reactive oxygen species balance in
endothelial progenitor dysfunction. Antioxid Redox Signal 15(4):933–948
Fordjour PA, Wang Y, Shi Y, Agyemang K, Akinyi M, Zhang Q, Fan G (2015) Possible
mechanisms of C-reactive protein mediated acute myocardial infarction. Eur J Pharmacol
760:72–80
Foresti R, Hammad J, Clark J, Johnson TR, Mann BE, Friebe A, Green C, Motterlini R (2004)
Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule.
Br J Pharmacol 142(3):453–460
Forstermann U (2010) Nitric oxide and oxidative stress in vascular disease. Pflugers Arch 459
(6):923–939
Forstermann U, Sessa WC (2012) (2012). Nitric oxide synthases: regulation and function. Eur Heart
J 33(7):829–837
Forstermann U, Xia N, Li H (2017) Roles of vascular oxidative stress and nitric oxide in the
pathogenesis of atherosclerosis. Circ Res 120(4):713–735
Freitas A, Alves-Filho JC, Secco DD, Neto AF, Ferreira SH, Barja-Fidalgo C, Cunha FQ (2006)
Heme oxygenase/carbon monoxide-biliverdin pathway down regulates neutrophil rolling, adhe-
sion and migration in acute inflammation. Br J Pharmacol 149(4):345–354
Fujimoto H, Ohno M, Ayabe S, Kobayashi H, Ishizaka N, Kimura H, Yoshida K-I, Nagai R (2004)
Carbon monoxide protects against cardiac ischemia – reperfusion injury in vivo via MAPK and
Akt – eNOS pathways. Arterioscler Thromb Vasc Biol 24(10):1848–1853
Fujisaki N, Kohama K, Nishimura T, Yamashita H, Ishikawa M, Kanematsu A, Yamada T, Lee S,
Yumoto T, Tsukahara K, Kotani J, Nakao A (2016) Donor pretreatment with carbon monoxide
prevents ischemia/reperfusion injury following heart transplantation in rats. Med Gas Res 6
(3):122–129
Garcia-Mata C, Lamattina L (2013) Gasotransmitters are emerging as new guard cell signaling
molecules and regulators of leaf gas exchange. Plant Sci 201-202:66–73
Hayashi S, Omata Y, Sakamoto H, Higashimoto Y, Hara T, Sagara Y, Noguchi M (2004)
Characterization of rat heme oxygenase-3 gene. Implication of processed pseudogenes derived
from heme oxygenase-2 gene. Gene. https://doi.org/10.1016/j.gene.2004.04.002
Heinemann SH, Hoshi T, Westerhausen M, Schiller A (2014) Carbon monoxide--physiology,
detection and controlled release. Chem Commun (Camb) 50(28):3644–3660
Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, Griep A, Axt D,
Remus A, Tzeng TC, Gelpi E, Halle A, Korte M, Latz E, Golenbock DT (2013) NLRP3 is
activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 493
(7434):674–678
Henningsson R, Alm P, Ekström P, Lundquist I (1999) Heme oxygenase and carbon monoxide:
regulatory roles in islet hormone release - A biochemical, immunohistochemical, and confocal
microscopic study. Diabetes 48(1):66–76
Hettiarachchi N, Dallas M, Al-Owais M, Griffiths H, Hooper N, Scragg J, Boyle J, Peers C (2014)
Heme oxygenase-1 protects against Alzheimer’s amyloid-beta(1-42)-induced toxicity via car-
bon monoxide production. Cell Death Dis 5:e1569
Hou S, Xu R, Heinemann SH, Hoshi T (2008) The RCK1 high-affinity Ca2+ sensor confers carbon
monoxide sensitivity to Slo1 BK channels. Proc Natl Acad Sci U S A. https://doi.org/10.1073/
pnas.0800304105
Immenschuh S, Kietzmann T, Hinke V, Wiederhold M, Katz N, Muller-Eberhard U (1998) The rat
heme oxygenase-1 gene is transcriptionally induced via the protein kinase A signaling pathway
in rat hepatocyte cultures. Mol Pharmacol 53:483–491
Johnson TR, Mann BE, Clark J, Foresti R, Green C, Motterlini R (2003) Metal carbonyls: a new
class of pharmaceuticals? Angew Chem Int Ed Engl 42(32):3722–3729
Johnson F, Johnson RA, Durante W, Jackson K, Stevenson BK, Peyton KJ (2006) Metabolic
syndrome increases endogenous carbon monoxide production to promote endothelial dysfunc-
tion in obese Zucker rats. Am J Physiol Regul Integr Comp Physiol 290(3):R601–R608
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 613

Katusic ZS, Austin SA (2014) Endothelial nitric oxide: protector of a healthy mind. Eur Heart J 35
(14):888–894
Keshet R, Erez A (2018) Arginine and the metabolic regulation of nitric oxide synthesis in cancer.
Dis Model Mech 11(8):dmm033332
Kim HS, Loughran PA, Rao J, Billiar TR, Zuckerbraun BS (2008) Carbon monoxide activates
NF-kappaB via ROS generation and Akt pathways to protect against cell death of hepatocytes.
Am J Physiol Gastrointest Liver Physiol 295(1):G146–G152
Kobayashi A, Ishikawa K, Matsumoto H, Kimura S, Kamiyama Y, Maruyama Y (2007) Synergetic
antioxidant and vasodilatory action of carbon monoxide in angiotensin II - induced cardiac
hypertrophy. Hypertension 50(6):1040–1048
Kolluru GK, Shen X, Yuan S, Kevil CG (2017) Gasotransmitter heterocellular signaling. Antioxid
Redox Signal 26(16):936–960
Kovacevic Z, Sahni S, Lok H, Davies MJ, Wink DA, Richardson DR (2017) Regulation and control
of nitric oxide (NO) in macrophages: protecting the “professional killer cell” from its own
cytotoxic arsenal via MRP1 and GSTP1. Biochim Biophys Acta Gen Subj. 1861(5 Pt
A):995–999
Kubic VL, Anders MW (1978) Metabolism of dihalomethanes to carbon monoxide—III: studies on
the mechanism of the reaction. Biochem Pharmacol 27(19):2349–2355
Lakkisto P, Kytö V, Forsten H, Siren J-M, Segersvärd H, Voipio-Pulkki L-M, Laine M, Pulkki K,
Tikkanen I (2010) Heme oxygenase-1 and carbon monoxide promote neovascularization after
myocardial infarction by modulating the expression of HIF-1α, SDF-1α and VEGF-B, vol
635, pp 156–164
Lamon BD, Zhang FF, Puri N, Brodsky SV, Goligorsky MS, Nasjletti A (2009) Dual pathways of
carbon monoxide–mediated vasoregulation. Circ Res 105(8):775–783
Lee T-S, Tsai H-L, Chau L-Y (2003) Induction of heme oxygenase-1 expression in murine
macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Δ12,14-prosta-
glandin J2. J Biol Chem 278:19325–19330
Lee M, Rey K, Besler K, Wang C, Choy J (2017) Immunobiology of nitric oxide and regulation of
inducible nitric oxide synthase. Results Probl Cell Differ 62:181–207
Leffler CW, Nasjletti A, Yu C, Johnson RA, Fedinec AL, Walker N (1999) Carbon monoxide and
cerebral microvascular tone in newborn pigs. Am J Phys Heart Circ Phys 276(5):H1641–H1646
Li VG, Sacerdoti D, Sangras B, Vanella A, Mezentsev A, Scapagnini G, Falck J, Abraham NG
(2005) Carbon monoxide signaling in promoting angiogenesis in human microvessel endothe-
lial cells. Antioxid Redox Signal. https://doi.org/10.1089/ars.2005.7.704
Li A, Xi Q, Umstot ES, Bellner L, Schwartzman ML, Jaggar JH, Leffler CW (2008) Astrocyte-
derived CO is a diffusible messenger that mediates glutamate-induced cerebral arteriolar dilation
by activating smooth muscle cell KCa channels. Circ Res 102(2):234–241
Li L, Hsu A, Moore PK (2009) Actions and interactions of nitric oxide, carbon monoxide and
hydrogen sulphide in the cardiovascular system and in inflammation—a tale of three gases!
Pharmacol Ther. https://doi.org/10.1016/j.pharmthera.2009.05.005
Li H, Horke S, Forstermann U (2014) Vascular oxidative stress, nitric oxide and atherosclerosis.
Atherosclerosis 237(1):208–219
Lin HH, Lai SC, Chau LY (2011) Heme oxygenase-1/carbon monoxide induces vascular endothe-
lial growth factor expression via p38 kinase-dependent activation of Sp1. J Biol Chem 286
(5):3829–3838
Ling K, Men F, Wang W-C, Zhou Y-Q, Zhang H-W, Ye D-W (2017) Carbon monoxide and its
controlled release: therapeutic application, detection, and development of carbon monoxide
releasing molecules (CORMs). J Med Chem. https://doi.org/10.1021/acs.jmedchem.6b01153
Lowenstein CJ, Dinerman JL, Snyder SH (1994) Nitric oxide: a physiologic messenger. Ann Intern
Med 120(3):227–237
Luo S, Lei H, Qin H, Xia Y (2014) Molecular mechanisms of endothelial NO synthase uncoupling.
Curr Pharm Des 20(22):3548–3553
614 R. Deshmukh et al.

MacMicking J, Xie QW, Nathan C (1997a) Nitric oxide and macrophage function. Annu Rev
Immunol 15:323–350
MacMicking J, Xie QW, Nathan C (1997b) Nitric oxide and macrophage function. Annu Rev
Immunol 15:323–350
McCoubrey WK Jr, Huang TJ, Maines MD (1997) Isolation and characterization of a cDNA from
the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 247(2):725–732
Miller MR, Megson IL (2007) Recent developments in nitric oxide donor drugs. Br J Pharmacol
151(3):305–321
Mir JM, Maurya R (2018) A gentle introduction to gasotransmitters with special reference to nitric
oxide: biological and chemical implications. Rev Inorg Chem 38(4):193–220
Moncada S, Higgs A (1993) The L-arginine-nitric oxide pathway. N Engl J Med 329
(27):2002–2012
Motterlini R, Foresti R (2017) Biological signaling by carbon monoxide and carbon monoxide-
releasing molecules. Am J Physiol Cell Physiol 312(3):C302–C313
Motterlini R, Otterbein LE (2010) The therapeutic potential of carbon monoxide. Nat Rev Drug
Discov 9(9):728–743
Munzel T, Daiber A (2018) Inorganic nitrite and nitrate in cardiovascular therapy: A better
alternative to organic nitrates as nitric oxide donors? Vasc Pharmacol 102:1–10
Nagpure BV, Bian JS (2016) Interaction of hydrogen sulfide with nitric oxide in the cardiovascular
system. Oxidative Med Cell Longev 2016:6904327
Nakao A, Huang C-S, Stolz DB, Wang Y, Franks J, Tochigi N, R Billiar T, Toyoda Y, Tzeng E, R
McCurry K (2011) Ex vivo carbon monoxide delivery inhibits intimal hyperplasia in arterialized
vein grafts. Cardiovasc Res. https://doi.org/10.1093/cvr/cvq298
Naseem KM (2005) The role of nitric oxide in cardiovascular diseases. Mol Asp Med 26
(1–2):33–65
Ndisang JF, Tabien HEN, Wang R (2004) Carbon monoxide and hypertension. J Hypertens 22
(6):1057–1074
Neilly PJ, Kirk SJ, Gardiner KR, Rowlands BJ (1994) The L-arginine/nitric oxide pathway--
biological properties and therapeutic applications. Ulster Med J 63(2):193–200
Nizamutdinova IT, Kim YM, Kim HJ, Seo HG, Lee JH, Chang KC (2009) Carbon monoxide (from
CORM-2) inhibits high glucose-induced ICAM-1 expression via AMP-activated protein kinase
and PPAR-γ activations in endothelial cells. Atherosclerosis 207(2):405–411
Nobre LS, Seixas JD, Romão CC, Saraiva LM (2007) Antimicrobial action of carbon monoxide-
releasing compounds. Antimicrob Agents Chemother 51(12):4303–4307
Okuyama H, Yonetani M, Uetani Y, Nakamura H (2001) End-tidal carbon monoxide is predictive
for neonatal non-hemolytic hyperbilirubinemia. Pediatr Int 43(4):329–333
Olah G, Modis K, Toro G, Hellmich MR, Szczesny B, Szabo C (2018) Role of endogenous and
exogenous nitric oxide, carbon monoxide and hydrogen sulfide in HCT116 colon cancer cell
proliferation. Biochem Pharmacol 149:186–204
Omar SA, Webb AJ, Lundberg JO, Weitzberg E (2016) Therapeutic effects of inorganic nitrate and
nitrite in cardiovascular and metabolic diseases. J Intern Med 279(4):315–336
Owens EO (2010) Endogenous carbon monoxide production in disease. Clin Biochem 43
(15):1183–1188
Pamplona A, Ferreira A, Balla J, Jeney V, Balla G, Epiphanio S, Chora Â, Rodrigues CD,
Grégoire I, Cunha-Rodrigues M, Portugal S, P Soares M, Mota M (2007) Heme oxygenase-1
and carbon monoxide suppress the pathogenesis of experimental cerebral malaria. Nat Med. 13
(6):703–710
Papapetropoulos A, Foresti R, Ferdinandy P (2015) Pharmacology of the ‘gasotransmitters’ NO,
CO and H2S: translational opportunities. Br J Pharmacol 172(6):1395–1396
Paredi P, Biernacki W, Invernizzi G, Kharitonov SA, Barnes PJ (1999) Exhaled carbon monoxide
levels elevated in diabetes and correlated with glucose concentration in blood: A new test for
monitoring the disease? Chest 116(4):1007–1011
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 615

Paul BD, Snyder SH (2018a) Gasotransmitter hydrogen sulfide signaling in neuronal health and
disease. Biochem Pharmacol 149:101–109
Paul BD, Snyder SH (2018b) Gasotransmitter hydrogen sulfide signaling in neuronal health and
disease. Biochem Pharmacol 149:101–109
Piazza M, Dieckmann T, Guillemette JG (2018) Investigation of the structure and dynamic of
calmodulin-nitric oxide synthase complexes using NMR spectroscopy. Front Biosci (Landmark
Ed) 23:1902–1922
Pitsikas N (2015) The role of nitric oxide in the object recognition memory. Behav Brain Res
285:200–207
Polhemus DJ, Lefer DJ (2014) Emergence of hydrogen sulfide as an endogenous gaseous signaling
molecule in cardiovascular disease. Circ Res 114(4):730–737
Poulos TL, Li H (2017) Nitric oxide synthase and structure-based inhibitor design. Nitric Oxide
63:68–77
Queiroga C, Almeida A, Alves P, Brenner C, Vieira H (2011) Carbon monoxide prevents hepatic
mitochondrial membrane permeabilization. BMC Cell Biol 12:10
Quillon A, Fromy B, Debret R (2015) Endothelium microenvironment sensing leading to nitric
oxide mediated vasodilation: a review of nervous and biomechanical signals. Nitric Oxide
45:20–26
Raman K, Barbato JE, Ifedigbo E, Ozanich BA, Zenati M, Otterbein L, Tzeng E (2006) Inhaled
carbon monoxide inhibits intimal hyperplasia and provides added benefit with nitric oxide. J
Vasc Surg. 44(1):151–158
Ramlawi B, Scott JR, Feng J, Mieno S, Raman KG, Gallo D, Csizmadia E, Yoke CB, Bach FH,
Otterbein LE, Sellke FW (2007) Inhaled carbon monoxide prevents graft-induced intimal
hyperplasia in swine. J Surg Res 138(1):121–127
Riddell DR, Owen JS (1999) Nitric oxide and platelet aggregation. Vitam Horm 57:25–48
Rochette L, Lorin J, Zeller M, Guilland JC, Lorgis L, Cottin Y, Vergely C (2013) Nitric oxide
synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets?
Pharmacol Ther 140(3):239–257
Ryter SW, Choi AM (2013) Carbon monoxide: present and future indications for a medical gas.
Korean J Intern Med 28(2):123–140
Shahrbaf MA, Mahjoob MP, Khaheshi I, Akbarzadeh MA, Barkhordari E, Naderian M, Tajrishi FZ
(2018) The role of air pollution on ST-elevation myocardial infarction: A narrative mini review.
Futur Cardiol 14(4):301–306
Shefa U, Yeo SG, Kim MS, Song IO, Jung J, Jeong NY, Huh Y (2017) Role of gasotransmitters in
oxidative stresses, neuroinflammation, and neuronal repair. Biomed Res Int. https://doi.org/10.
1155/2017/1689341. Epub 2017 Mar 12
Simon T, Anegon I, Blancou P (2011) Heme oxygenase and carbon monoxide as an immunothera-
peutic approach in transplantation and cancer. Immunotherapy 3(4 Suppl):15–18
Sjostrand T (1952) The formation of carbon monoxide by the decomposition of haemoglobin
in vivo. Acta Physiol Scand 26(4):338–344
Socco S, Bovee RC, Palczewski MB, Hickok JR, Thomas DD (2017) Epigenetics: the third pillar of
nitric oxide signaling. Pharmacol Res 121:52–58
Stein A, Guo Y, Tan W, Wu W-J, Zhu X, Li Q, Luo C, Dawn B, Johnson TR, Motterlini R, Bolli R
(2005) Administration of a CO-releasing molecule induces late preconditioning against
myocardial infarction. J Mol Cell Cardiol 38(1):127–134
Stevenson DK, Vreman HJ, Oh W, Fanaroff AA, Wright LL, Lemons JA, Verter J, Shankaran S,
Tyson JE, Korones SB et al (1994) Bilirubin production in healthy term infants as measured by
carbon monoxide in breath. Clin Chem 40(10):1934–1939
Stewart RD, Fisher TN, Hosko MJ, Peterson JE, Baretta ED, Dodd HC (1972) Carboxyhemoglobin
elevation after exposure to dichloromethane. Science 176(4032):295–296
Sukmansky OI, Reutov VP (2016) Gasotransmitters: physiological role and involvement in the
pathogenesis of the diseases. Usp Fiziol Nauk 47(3):30–58
616 R. Deshmukh et al.

Szabo C (2010) Gaseotransmitters: new frontiers for translational science. Sci Transl Med 2
(59):59ps54. https://doi.org/10.1126/scitranslmed.3000721
Takehito T, Yoshifumi M (1988) Metabolism of dichloromethane and the subsequent binding of its
product, carbon monoxide, to cytochrome P-450 in perfused rat liver. Toxicol Lett 40(1):93–96
Tayem Y, Johnson TR, Mann BE, Green CJ, Motterlini R (2006) Protection against cisplatin-
induced nephrotoxicity by a carbon monoxide-releasing molecule. Am J Physiol Renal Physiol
290(4):F789–F794
Tenhunen R, Marver HS, Schmid R (1968) The enzymatic conversion of heme to bilirubin by
microsomal heme oxygenase. Proc Natl Acad Sci U S A 61(2):748–755
Terry C, Clikeman JA, Hoidal JR, Callahan KS (1999) TNF-alpha and IL-1alpha induce heme
oxygenase-1 via protein kinase C, Ca2+, and phospholipase A2 in endothelial cells. Am J
Physiol 276(5):H1493–H1501
Thom SR (1990) Carbon monoxide-mediated brain lipid peroxidation in the rat. J Appl Physiol 68
(3):997–1003
Traub O, Van Bibber R (1995) Role of nitric oxide in insulin-dependent diabetes mellitus-related
vascular complications. West J Med 162(5):439–445
Truss NJ, Warner T (2011a) Gasotransmitters and platelets. Pharmacol Ther 132(2):196–203
Truss NJ, Warner TD (2011b) Gasotransmitters and platelets. Pharmacol Ther 132(2):196–203
Untereiner A, Wu L, Wang R (2012) The role of carbon monoxide as a gasotransmitter. In:
Hermann A, Sitdikova G, Weiger T (eds) Gasotransmitters: physiology and pathophysiology,
pp 37–70
Urquhart P, Rosignoli G, Cooper D, Motterlini R, Perretti M (2007) Carbon monoxide-releasing
molecules modulate leukocyte-endothelial interactions under flow. J Pharmacol Exp Ther 321
(2):656–662
van den Born JC, Hammes HP, Greffrath W, van Goor H, Hillebrands JL, DFG GRK International
Research Training Group 1874 Diabetic Microvascular Complications (DIAMICOM) (2016)
Gasotransmitters in vascular complications of diabetes. Diabetes 65(2):331–345
Van Landeghem L, Laleman W, Van der Elst I, Zeegers M, Van Pelt J, Cassiman D, Nevens F
(2009) Carbon monoxide produced by intrasinusoidally located haem-oxygenase-1 regulates
the vascular tone in cirrhotic rat liver. Liver Int 29(5):650–660
Vanhoutte PM, Gao Y (2013) Beta blockers, nitric oxide, and cardiovascular disease. Curr Opin
Pharmacol 13(2):265–273
Vannacci A, Di Felice A, Giannini L, Marzocca C, Pierpaolo S, Zagli G, Masini E, Mannaioni PF
(2004) The effect of carbon monoxide releasing molocule on the immunological activation of
guinea-pig mast cells and human basophils. Inflamm Res; 53 Suppl 1:S9–S10
Varadi J, Lekli I, Juhasz B, Bácskay I, Szabo G, Gesztelyi R, Szendrei L, Varga E, Bak I, Foresti R,
Motterlini R, Tosaki A (2007) Beneficial effects of carbon monoxide-releasing molecules on
post-ischemic myocardial recovery, vol 80, pp 1619–1626
Venketaraman V, Talaue MT, Dayaram YK, Peteroy-Kelly MA, Bu W, Connell ND (2003) Nitric
oxide regulation of L-arginine uptake in murine and human macrophages. Tuberculosis (Edinb)
83(5):311–318
Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH (1993) Carbon monoxide: a putative neural
messenger. Science 259(5093):381–384
Von Burg R (1999) Carbon monoxide. J Appl Toxicol 19(5):379–386
Wagner CT, Durante W, Christodoulides N, Hellums JD, Schafer AI (1997) Hemodynamic forces
induce the expression of heme oxygenase in cultured vascular smooth muscle cells. J Clin Invest
100(3):589–596
Wallace JL, Ianaro A, Flannigan KL, Cirino G (2015) Gaseous mediators in resolution of inflam-
mation. Semin Immunol 27(3):227–233
Wang R (1998) Resurgence of carbon monoxide: an endogenous gaseous vasorelaxing factor. Can J
Physiol Pharmacol 76(1):1–15
Wang R (2014) Gasotransmitters: growing pains and joys. Trends Biochem Sci 39(5):227–232
17 Pharmacology of Gasotransmitters (Nitric Oxide and Carbon Monoxide). . . 617

Wang R, Wu L, Wang Z (1997) The direct effect of carbon monoxide on KCa channels in vascular
smooth muscle cells. Pflugers Arch 434(3):285–291
Wang R, Wang Z, Wu L, Hanna ST, Peterson-Wakeman R (2001) Reduced vasorelaxant effect of
carbon monoxide in diabetes and the underlying mechanisms. Diabetes 50(1):166–174
Wang Z, Wu Y, Zhang S, Zhao Y, Yin X, Wang W, Ma X, Liu H (2019) The role of NO-cGMP
pathway inhibition in vascular endothelial-dependent smooth muscle relaxation disorder of
AT1-AA positive rats: protective effects of adiponectin. Nitric Oxide 87:10–22
Wegiel B, Gallo DJ, Raman KG, Karlsson JM, Ozanich B, Chin BY, Tzeng E, Ahmad S, Ahmed A,
Baty CJ, Otterbein LE (2010) Nitric oxide–dependent bone marrow progenitor mobilization by
carbon monoxide enhances endothelial repair after vascular injury. Circulation 121(4):537–548
Wegiel B, Gallo D, Csizmadia E, Harris C, Belcher J, Vercellotti GM, Penacho N, Seth P,
Sukhatme V, Ahmed A, Pandolfi PP, Helczynski L, Bjartell A, Persson JL, Otterbein LE
(2013) Carbon monoxide expedites metabolic exhaustion to inhibit tumor growth. Cancer Res
73(23):7009–7021
Wen L, Feil S, Wolters M, Thunemann M, Regler F, Schmidt K, Friebe A, Olbrich M, Langer H,
Gawaz M, de Wit C, Feil R (2018) A shear-dependent NO-cGMP-cGKI cascade in platelets acts
as an auto-regulatory brake of thrombosis. Nat Commun 9(1):4301
Wobst J, Kessler T, Dang TA, Erdmann J, Schunkert H (2015) Role of sGC-dependent NO
signalling and myocardial infarction risk. J Mol Med (Berl) 93(4):383–394
Wu L, Wang R (2005a) Carbon monoxide: endogenous production, physiological functions, and
pharmacological applications. Pharmacol Rev 57(4):585–630
Wu L, Wang R (2005b) Carbon monoxide: endogenous production, physiological functions, and
pharmacological applications. Pharmacol Rev 57(4):585–630
Wu L, Wang R (2006) Carbon monoxide: endogenous production, physiological functions, and
pharmacological applications. Pharmacol Rev 57(4):585–630
Xi Q, Tcheranova D, Parfenova H, Horowitz B, Leffler CW, Jaggar JH (2004) Carbon monoxide
activates KCa channels in newborn arteriole smooth muscle cells by increasing apparent Ca2+
sensitivity of alpha-subunits. Am J Physiol Heart Circ Physiol 286(2):H610–H618
Yarlagadda K, Hassani J, Foote IP, Markowitz J (2017) The role of nitric oxide in melanoma.
Biochim Biophys Acta Rev Cancer 1868(2):500–509
Zang Y, Popat KC, Reynolds MM (2018) Nitric oxide-mediated fibrinogen deposition prevents
platelet adhesion and activation. Biointerphases 13(6):06E403
Zhao Y, Vanhoutte PM, Leung SW (2015) Vascular nitric oxide: beyond eNOS. J Pharmacol Sci
129(2):83–94
Zheng L, Zhou Z, Lin L, Alber S, Watkins S, Kaminski N, Choi AMK, Morse D (2009) Carbon
monoxide modulates α–smooth muscle actin and small proline rich-1a expression in fibrosis.
Am J Respir Cell Mol Biol 41(1):85–92
Zhou Z, Song R, Fattman C, Greenhill S, Alber S, Oury T, Choi A, Morse D (2005) Carbon
monoxide suppresses bleomycin-induced lung fibrosis, vol 166, pp 27–37
Sodium Channels: As an Eye of the Storm
in Various Clinical Pathologies 18
Vinod Tiwari, Ankit Uniyal, Akhilesh, Anagha Gadepalli,
Vineeta Tiwari, and Somesh Agrawal

Abstract

Voltage-gated sodium ion channels are essential to maintain the excitability and
activity of neurons and neuronal network. Several studies have been done to
explore the basic properties of ion channels, their existence and physiological
characteristics. Till date we know that 11 genes are responsible for encoding of
9 families of sodium channels (Nav 1.1 to Nav 1.9) and are classified according to
varying degrees of sensitivity to Tetrodotoxin (TTX). These are localized in
various sites such as skeletal muscles, central nervous system (CNS), cardiac
muscles, and peripheral sensory neurons. Any aberration in its structure and
function leads to various clinical pathologies such as channelopathies (where
dysregulation in receptors are directly responsible for initiation and progression)
and diseases contributing to dysregulation of expression of sodium channels
cause various neurological disorders. In this chapter, we emphasize the composi-
tion, function, and regulation of sodium channels at the molecular level and the
crucial role of sodium channels in the development and progression of various
disease pathologies such as epilepsy, schizophrenia, familiar hemiplegic migraine
and neuropathic pain.

Keywords

Voltage-gated sodium channels · Composition · Regulation · Channelopathies ·

Neurological disorders · Neuropathic pain

V. Tiwari (*) · A. Uniyal · Akhilesh · A. Gadepalli · V. Tiwari · S. Agrawal

Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology
(B.H.U.), Varanasi, UP, India
e-mail: [email protected]

# Springer Nature Singapore Pte Ltd. 2020 619

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_18
620 V. Tiwari et al.

Abbreviations

AIS Axon initial segment

ARC Activity-regulated cytoskeleton-associated protein
BFNIS Benign familial neonatal-infantile seizures
ChIPs Channel Interacting Proteins
CNS Central nervous system
DRG Dorsal root ganglia
EE Epileptic encephalopathy
FGF Fibroblast growth factor
FHF Fibroblast homologous factor
FHM Familiar hemiplegic migraine
IE Erythromelalgia
IFMT Isoleucine, Phenylalanine, Methionine, and Tryptophan
IHC Immunohistochemistry
PEPD Paroxymal Extreme Pain Disorder
PNS Peripheral Nervous System
Nav Voltage-gated sodium channel
NMDAR N-methyl-D-aspartate receptor
TTX Tetrodotoxin
VGSCs Voltage-Gated Sodium Channels

18.1 Introduction

Voltage-gated sodium ion channels are required for excitation of cells. Their history
can be traced back to 1941, when Kenneth S. Cole and Richard F. Baker confirmed
the existence of voltage-gated membrane pores in giant squid axon (Cole and Curtis
1939). Alan Hodgkin and Andrew Huxley won the Nobel prize for their work on
basic properties of ion channels and their mechanisms of excitation and inhibition in
1952. Later in 1970 and 1981, the existence and physiological characteristics of ion
channels were explored by Bernard Katz, Ricardo, and Miledi et al. and Erwin Neher
and Bert Sakmann et al., using noise analysis and patch clamp techniques, respec-
tively (Cole and Baker 1941; Roger et al. 2015).
So far, 11 genes are responsible for the encoding of 9 families of sodium channels
(Nav 1.1 to Nav 1.9). These genes are classified according to varying degrees of
sensitivity to Tetrodotoxin (TTX) and can be traced to four paralogous locations in
the chromosome segment (Ambrose et al. 1992; Wang et al. 1992; Burgess et al.
1995; Beckers et al. 1996; Kozak and Sangameswaran 1996; Plummer et al. 1998;
Catterall et al. 2005; Bagal et al. 2015) (Table 18.1). More than 20 exons encode for
9 sodium channel α-subunits. Nav channels (1.1, 1.2, 1.3, and 1.7) are situated on the
second chromosome in both humans as well as in the mouse whereas Nav channels
1.5 are situated at 2q24 and in case of mouse, present on chromosome 2. And the
Nav 1.8 and 1.9 channels are present on 3p21–24 in case of humans and chromo-
some 9 in the mouse. Skeletal muscle contains the Nav 1.4 and the CNS contains the
18 Sodium Channels: As an Eye of the Storm in Various Clinical Pathologies 621

Table 18.1 Classification of voltage-gated sodium channels based on location and tetrodotoxin
sensitivity (in human and mouse)
Nav Location
channel Tetrodotoxin
subtype Human Mouse sensitivity References
Nav 1.1 Chromosome Chromosome Sensitive Catterall et al. (2005)
2 2
Nav 1.2 Chromosome Chromosome Sensitive Catterall et al. (2005)
2 2
Nav 1.3 Chromosome Chromosome Sensitive Catterall et al. (2005)
2 2
Nav 1.4 Chromosome Chromosome Sensitive Ambrose et al. (1992); Catterall
17 11 et al. (2005); Wang et al. (1992)
Nav 1.5 2q 24 Chromosome Resistant Catterall et al. (2005)
2
Nav 1.6 Chromosome Chromosome Sensitive Burgess et al. (1995); Catterall
12 15 et al. (2005); Plummer et al.
(1998)
Nav 1.7 Chromosome Chromosome Sensitive Beckers et al. (1996); Catterall
2 2 et al. (2005); Kozak and
Sangameswaran (1996)
Nav 1.8 3p 21–24 Chromosome Resistant Catterall et al. (2005)
9
Nav 1.9 3p 21–24 Chromosome Resistant Catterall et al. (2005)
9

Nav 1.6. The genes of Nav 1.4 are situated on the 17th chromosome (humans) and
11th chromosome (mouse) whereas genes of Nav 1.6 can be traced to the 12th
chromosome (humans) and the 15th chromosome (mouse) (Catterall et al. 2005;
Mantegazza and Catterall 2012). All the sodium channels structures are analogous to
each other, but few amino acid replacements confer this resistance, for example, in
Nav 1.5 which is predominantly localized in cardiac muscles, substitution of phe-
nylalanine to cystine in pore area provides 200 times reduction in sensitivity to TTX
(Yamagishi et al. 2001). A similar replacement is also observed in Nav 1.8 and Nav
1.9, which are localized in peripheral sensory neurons where phenylalanine is
replaced by serine, which provides even greater resistance (Mantegazza and Catterall
2012). Channelopathies are the group of diseases characterized by any aberration in
the structure or function arising from a mutation in the genes encoding for sodium
channels. In this chapter, we will study the composition, function, and regulation at
the molecular level of sodium channels and the crucial role of sodium channels in the
development and progression of various disease pathologies (Roger et al. 2015).
622 V. Tiwari et al.

α subunit
β subunit

DI D II D III D IV

S S S S S S S S S S S S S S S S S S S S S S S S
12345 6 12345 6 12345 6 12345 6

Alanine Asparagine
IFMT Segment

Fig. 18.1 Composition of sodium channel: α subunit (Domains- I, II, III & IV); β subunit and
IFMT segment (Isoleucine, Phenylalanine, Methionine, and Tryptophan)

18.1.1 Composition of Sodium Channels

Sodium channels are heteromeric integral glycoproteins composed of two types of

subunits, α (initiator) and β (regulatory). Each α subunit contains six segments,
which houses four domains (DI-DIV) around the central pore. Arginine-rich seg-
ment 4 (S4) is also known as the “voltage sensor,” and the hairpin segment (P loop)
forms the “ion filter” between segment 5 (S5) and segment 6 (S6) responsible for ion
permeability (King and Vetter 2014). There is a group of amino acids called IFMT,
segment “I” Isoleucine, “F” Phenylalanine, “M” Methionine, “T” Tryptophan,
present between segment 6 of DIII and Segment 1 of DIV (Fig.18.1). This IFMT
segment has an affinity to bind to hydrophobic amino acids (like alanine and
asparagine) between segment 4 and segment 5 of DIII and DIV; this binding is
responsible for the refractive nature of the ion channel (Tata 2013). Pore consists of
tubular vestibule that forms a cavity, ion-selective filter, and intracellular gate
activator. There are two models that explain the organization of voltage-gated
sodium channels: (1) Sliding helix and (2) Helical screw. Both models suggest the
presence of positively charged residues in segment 4 that serves as gating (Frank and
Catterall 2003). These positively charged residues are held together by
corresponding residues having negative charge, of segments 1, 2, and/or 3 in active
state. But in the inactive state, these residues are held inside due to the Coulomb
force of negatively charged resting membrane potential. When depolarization takes
place, changes in membrane polarity occur, which results in relieving of this
electrostatic force. As a result, these positive, rich residues move out opening the
pore, causing an influx of sodium ions, thus initiating the cell activation process. The
outward movement of segment 4 is “voltage dependent.” On the other hand, all the
downstream mechanisms are “voltage independent.”
β subunits are integral regulatory proteins found to be associated with the α
subunit. It consists of three domains: extracellular, transmembrane, and intracellular
domains. β subunits are served as cell adhesion molecules that are responsible for
18 Sodium Channels: As an Eye of the Storm in Various Clinical Pathologies 623

control of the surface expression of voltage-gated sodium channels, cell-to-cell

communication and cellular migration. β1 subunit in association with Neurofascin
186 and Contactin provides increase in surface expression of voltage-gated sodium
channels. β1 subunit knockout leads to disruption of neuronal–glial interaction and
reduction in the number of Nodes of Ranvier in myelinated neurons which results in
disrupted saltatory conduction (Marban et al. 1998).

18.1.2 Molecular Functioning of Sodium Channels

Voltage-gated sodium channels (VGSCs) occur in three forms: (1) active (2) refrac-
tory, (3) closed. From closed state to go on to open state requires depolarization,
changes in membrane polarity to positive, which usually happens during an action
potential, and within a few milli-seconds VGSCs progress to refractory state, also
known as the inactive state, is a mechanism that protects cells from excessive
stimulation. This is followed by the closed state when the cell membrane attains
the normal repolarized potential. During closed state, the “voltage sensor” segment
4 is held inside due to the overall negative resting membrane potential of the cell. The
depolarization relieves the electrostatic force on segment 4 pushing it to move out,
thus opening the intracellular ion gate. This allows sodium ion conductions, leading
to increased sodium concentration inside the cell. When the intracellular gate is
opened, the IFMT segment binds to hydrophobic amino acids (like alanine and
asparagine) between segment 4 and segments 5 of DIII and DIV, as there is no steric
hindrance caused by intracellular ion gate due to conformational change. This state is
called a refractory state/inactive state. In the inactive state, sodium ions cannot pass
through causing prevention of the cell from overstimulation. From a refractory state
to go to closed state again requires a change in membrane potential. Whenever cell
resting membrane potential is restored, segment 4 domain is attracted toward the
cell, augmenting its electrostatic influence on segment 4 resulting in a conforma-
tional change to the closed ion channel (Catterall 1992; Marban et al. 1998)
(Fig. 18.2).

18.2 Regulation of Voltage-Gated Sodium Channels

Channel Interacting Proteins (ChIPs) are endogenous proteins that interact with
voltage-gated Na+ channels. Some examples of ChIPs are connexion 43, Caveolin
3, Calcium-calmodulin kinase 2, ankyrins, telethonin, plakophilin, neuronal precur-
sor cell-expressed developmentally downregulated 4 (nedd4), fibroblast growth
factor (FGF) and its homologous factors (FHFs). These endogenous proteins regu-
late the sodium channel expression and its functioning (Savio-Galimberti et al.
2012).
624 V. Tiwari et al.

Na+
1.Closed Na+ 2.Open
+
+
+
+
S SSSSS Depolarization S
S S SSS
6 54321 4
6 5 321
+
+
+
+
IFMT IFMT Intracellular ion gate
Intracellular ion gate

-ve RMP --------------- ++++++++++++

+ve RMP

3.Inactivated
+
Repolarisation +
S
S S SSS
4
6 5 321
+
+

IFMT Intracellular ion gate

Na+
+ve RMP ++++++++++++

Fig. 18.2 Molecular functioning of sodium channels: Closed, opened and inactivated state; IFMT
segment (Isoleucine, Phenylalanine, Methionine, and Tryptophan), RMP; Resting Membrane
Potential

18.2.1 Cellular Trafficking and Surface Expression of Voltage-Gated

Sodium Channels: Caveolae/Caveolin
Interaction-Kinesin-VGSCs Interaction

Caveolae are membrane folding proteins predominantly found in muscle tissues,

implicated for surface expression of number of receptors and Caveolins are linker
proteins responsible for the rapid surface expression of sodium channels. These
Caveolin proteins act as linker proteins and cause recruitment of voltage-gated ion
channels from the intracellular pool following the cell stimulation. Kinesins are the
molecular motor proteins responsible for the delivery of newly synthesized receptors
(like VGSCs) vesicles from the nucleus, rough endoplasmic reticulum and Golgi
body to the synapse, creating the intracellular pool of vesicles containing receptors.
This intracellular pool of vesicles containing receptors is dynamic in nature. When a
cell is stimulated, these caveolin proteins act as linker proteins causing mobilization
of these vesicles and with the help of caveolae proteins, cause surface expression of
voltage-gated ion channels receptors. Some recent studies show that kif 1B and kif
5A, kif 5B are kinesin members of kinesin 3 and kinesin 1 families respectively
which are implicated in the cellular trafficking of voltage-gated ion channels like
Nav 1.8. Mutation or overexpression of these genes and proteins leads to
18 Sodium Channels: As an Eye of the Storm in Various Clinical Pathologies 625

neuropathic conditions like Charcot and Marie tooth disorder and neuropathic pain.
Another study proved that β cell stimulation leads to increased surface expression of
VGSCs. This is due to intracellular VGSCs pool mobilization by caveolin 3 and
caveolae membrane proteins, VGSCs are surface expressed (Savio-Galimberti et al.
2012; Roger et al. 2015).

18.3 Sodium Channel and Related Clinical Pathologies

Sodium channel as discussed above is crucial for the functioning of any excitable
cells, and any aberration in its structure or functioning leads to various clinical
pathologies. There are two types of disorders that are classified based on the role
played by the sodium channels on pathogenesis of diseases: (1) channelopathies
(where dysfunctional receptors are directly responsible for initiation and progres-
sion) and (2) disease contributing to dysregulated expression of sodium channels
(like cancer, neurological disorders) (Kaplan et al. 2016).

18.3.1 Function of Sodium Channels in the PNS and CNS

Sodium channels are important to maintain the excitable character as well as the
activity of neurons and the neuronal network. They are ubiquitously expressed
throughout the neurocyton and axons in neurons. Denseness of sodium channel is
more at Nodes of Ranvier and axon initial segment (AIS), which is responsible for
speeding nerve impulse conduction. This phenomenon is often referred to as salta-
tory conduction (Kaplan et al. 2016). The action potential is initiated from AIS and
this characteristic is attributed to relatively high sodium channel receptor density at
this site. Various IHC studies have validated this claim of determining distribution of
sodium channel expression along AIS. In such studies Nav1.6 was densely
expressed at distal AIS of retinal ganglion cell of rat, whereas Nav1.1 was
aggregated at proximal AIS. Similar trend in expression was observed in cortical
pyramidal neurons of rat showing increased Nav1.2 and Nav 1.6 at AIS. Hyperpo-
larization of NaV1.6 that depend on NaV 1.2 activity results in differential expres-
sion of sodium channel. When depolarlizing signals enter AIS, activation threshold
of Nav 1.6 crosses more readily as compared to Nav1.2, which consequently
generates action potential. This causes two types of currents: the first one propagates
down the axon and the other results in backpropagating currents, which leads to
activation of Nav1.2. This action potential is propagated from soma to axons and
dendrites. The backpropagating currents are crucial for relating the neuronal output
and regulating neuronal platicity. Dendritic VGSCs also contributes to action poten-
tial by amplifying the depolarlization response. Collectively, this data suggests the
complex relationship of sodium channels distribution and neuronal activity. The data
also hints at the susceptibility of brain networks to any aberrations in sodium
channels function and its number by mutations, disorders, or action of drug (Kaplan
et al. 2016).
626 V. Tiwari et al.

18.3.2 Dysfunctional and Dysregulated Expressions of Sodium

Channels as a Cause for Neurological Disorders

Channelopathies are the group of disorders arising from a mutation in genes

encoding for voltage-sensitive ion channel or dysfunctional receptors arising from
an autoimmune attack. This condition has been indicated for several diseases
including epilepsy, Dravet Syndrome, complications associated with pain like con-
genital pain insensitivity, paroxysmal extreme pain disorder, primary
erythromelalgia and cardiac arrhythmia (characterized by long QT syndrome),
slow ventricular conduction and atrial standstill. Aggressive nature of breast
(Nav1.5) and prostate cancer (Nav1.7) has been related with change in epigenetic
regulation. Dysregulation of sodium channels has also been over-served in diabetes
and chemical-induced neuropathies, neuromuscular disorders (like Eaton Lambert
syndrome, Charcot Marie Tooth disorder) and various neurodegenerative disorders
(like Alzheimer’s, Parkinson’s, and Schizophrenia). Sodium channels have been an
important target due to their major role in these diseases.

18.3.3 Role of Sodium Channels in Epilepsy

Analysis of mutation in VGSCs in epilepsy has shown a complex data that results in
a specific pattern showing connection between channel mutation and epilepsy. In
VGSCs of interneuron, there are alterations in channel biological organization that
are cell dependent in nature; for example, there is a loss of function in Nav1.1 or
Nav1.6 due to depolarization in shifts in the voltage dependence of activation,
hyperpolarization in shifts in the voltage dependence of inactivation, and
haploinsufficiency (Hargus et al. 2013). Contrastingly, alteration in pyramidal cell
channels in NaV1.2 or NaV1.6 leads to depolarization of shifts in the voltage
dependence of inactivation and augmentation in persistent current (Ye et al. 2018).
Haploinsufficiency in SCNIA triggers Dravet Syndrome (Bechi et al. 2012) and
functional dropping in SCN1B (Patino et al. 2009). The function in SCN2A is
associated with BFNIS (Misra et al. 2008). The functional gaining in SCN8A is
related to epileptic encephalopathy and functional loss is related with its nonoccur-
rence (Martin et al. 2007). In patients of epilepsy, additional potential pathological
mechanisms have been suggested, but not yet specifically identified. Mutations in
the voltage sensor in votage-gated sodium channel result in exchange of the amino,
acid, i.e., arginines (charged) with residues (neutral). As a result, voltage sensor
leads to development of a leak current (omega/gating pore current) (Sokolov et al.
2005). In spite of the fact that it is not seen in epilepsy patients till now, in
hypokalemic periodic paralysis, Nav1.4 mutation is associated with an increase in
omega current, proposing involvement in channelopathies (Sokolov et al. 2007).
Resurgence of sodium current is proposed to be involved in epileptical conditions. In
some subtypes of voltage-gated sodium channels, by membrane repolarization
followed by prolonged depolarization pulse, a minor transient ingoing current can
be attained (Raman and Bean 1997). Augmented resurgent current amplitude has
18 Sodium Channels: As an Eye of the Storm in Various Clinical Pathologies 627

been related to augmentation in action potential firing frequency, proposing it as a

possible pathological mechanism in epileptical conditions (Jarecki et al. 2010).
Some important advancement has been made in psychiatric disorders treatment
and diagnosis. From data it has been supported that these disorders occur due to
interplay of environmental and genetic risk factors. These disorders are caused by
structural and functional damage in several brain areas such as the thalamus,
amygdala, midbrain, and the prefrontal cortex. Since the early 1990s, gene mapping
of ion channels have been done on human chromosomes, and identification of
mutations in these genes has been performed. Many neurological disorders like
epilepsy, migraine, and episodic ataxia are caused by dysfunction of brain electrical
circuits, which has been attributed to ion channel mutations. Hence, it has been
suggested that any change in activity of ion channel in the respective brain regions
has been associated with psychiatric disorders etiopathology.

18.3.4 Sodium Channels Involved in Schizophrenia

Schizophrenia is a psychotic disorder with 1% prevalence worldwide. Symptoms

involve negative symptoms (decrease in social interaction, anhedonia), positive
symptoms (hallucination and delusion), and cognitive impairment. Many evidences
have increasingly proposed that schizophrenia is a brain development and plasticity
disorder, which involves strong alteration in activity and excitability of substantia
nigra, hippocampus, ventral tegmental area, and prefrontal cortex. Recently, genetic
linkage and association studies suggested risk genes for schizophrenia, including
some ion channels genes (Imbrici et al. 2013).
By targeting the sequence of 10,198 samples, Rees et al. 2019 confirmed
irregularities of activity of neuron and associated voltage-gated sodium channels
in pathogenesis of schizophrenia. This is a sequence-based study that includes the
schizophrenia of rare coding variants in neuronally expressed genes, including
N-methyl-D-aspartate receptor (NMDAR) complexes and activity-regulated cyto-
skeleton-associated protein (ARC); but, bigger sample number is essential to dis-
close novel genes and particular biological mechanisms. In this study, they have
sequenced 187 genes in a new dataset of 5207 cases and 4991 controls, which are
selected on the basis of previous information related to schizophrenia. These genes
were involved as members of ARC and NMDAR postsynaptic protein complexes,
including voltage-gated sodium and calcium channels. A data for a total of 11,319
cases, 15,854 controls, and 1136 trios has been published sequentially and on the
basis of this data, a rare variant meta-analysis was conducted. From this data, it was
found that there is no single gene significantly involved in schizophrenia but exonic
variants in the ARC (p ¼ 4.0  104) and NMDAR (p ¼ 1.7  105) synaptic
complexes are a significantly involved risk factor for schizophrenia. Other than this,
in this study it was also found that loss-of-function variants and missense variants at
paralog-conserved sites were enhanced in voltage-gated sodium channels, specially
the alpha subunits (p ¼ 8.6  104) involved in schizophrenia. From this study it
was evidenced that multiple voltage-gated sodium channels are involved in
628 V. Tiwari et al.

schizophrenia pathogenesis and verify the involvement of ARC and NMDAR

postsynaptic complexes (Rees et al. 2019).

18.3.5 Sodium Channels Involved in Familial Hemiplegic Migraine

(FHM)

Familial hemiplegic migraine (FHM) can be autosomal-dominant in nature although

its occurrence is rare (Watanabe et al. 1971). Manifestation of illness includes visual
and speech hampering, aura and short duration of motor lethargy to some extent.
Mutations of genes like CACNA1A, ATP1A2, and SCN1A through encoding of
various ion transporting proteins are involved in occurrence of FHM. When com-
pared with migraine arising due to periodic seizures of diseases associated with
nervous system occurrence of FHM is atypical in nature caused due to alteration in a
typical gene. Mutation of gene present in channel generated by calcium ion was the
prime gene to be recognized and termed as CACNA1 α1-subunit and was primarily
responsible for FHM1, which is most affected by the mutation where encoding gene
of catalytic α2 subunit, present in Na+/K+-ATPase and termed as ATP1A2 gets
mutated and around one-fifth of FHM2 families gets affected. Alteration of SCIN1A
gene (Q1489K and L1649Q) has been found to cause FHM3. Formation of pores in
α subunit of neuronal voltage-dependent gated sodium ion channel occurs through
this gene (Dichgans et al. 2005; Carreño et al. 2013). As per current reports, atypical
occurence of gene alteration leading to FHM3 was observed in a Chinese household
on a 62-year-old woman (Shao et al. 2018). Earlier manifestation was transient
ischemic attack which were ultimately identified as FHM having c.4495T>C alter-
ation observed in SCN1A gene. The case study report ultimately deduced about role
of SCN1A gene in FHM pathophysiology. Another study also reported occurrence
of FHM3 through alteration in voltage-dependent sodium channel gates Nav 1.1
which is being encoded by SCNIA gene. This study was conducted to determine
molecular flaws that are inheritable in nature and occur due to alteration related to
FHM3 (L263V, Q1489K, and L1649Q). L1649Q is one of the mutations that failed
to produce measurable current because it significantly reduced cell surface expres-
sion. Production of pronounced currents by two mutations occurs through
co-expression of human beta1 and beta2 accessory subunits by tsA201 cells.
Mutations when compared with WT-Nav1.1 showed principal depletion of func-
tional phenotype that was additionally expressed by depletion of channel accessibil-
ity during consecutive stimulation. Data suggested that Q1489K mutation causes
high current in continuation and decelerates the recovery from fast and slow inacti-
vation as well as increased entry into slow inactivation. L263V, on the other hand,
showed augmentation in functional characteristics which included detained entrance
to slow inactivation, retarded entry but accelerated recovery in case of fast inactiva-
tion as well as high current in continuation. The two alternations (Q1489K and
L1649Q) associated with typical FHM generated either full or partial loss in
producing the outcome. On the other hand, L236V, which is an alteration, leads to
18 Sodium Channels: As an Eye of the Storm in Various Clinical Pathologies 629

attainment of function and was associated with FHM as well as increased occurrence
of generalized epilepsy (Kahlig et al. 2008).

18.3.6 Sodium Channels Involved in Neuropathic Pain

From the generation of action potential to its dissemination, voltage-dependent

sodium ion channels perform a pivotal role. These channels are further identified
as the genes that codes them from Nav1.1 to Nav1.9 with further subclassification
based on function as genes that are sensitive to tetrodotoxin- (TTX-S) or genes that
are resistant to tetrodotoxin (TTX-R). These are present on primary afferent sensory
neurons and recent pharmacological and genetic data has provided evidence of the
involvement of Nav1.3, 1.7, 1.8, and 1.9 in nociceptive transmission. Out of these,
Nav1.8 is a TTX-R sodium channel that is highly localized on primary sensory
afferent neurons.

18.3.6.1 Nav 1.3 Channel

Nav1.3 can be termed as sodium channel sensitive to tetrodotoxin and is found in
nerve cells of embryo but is also present in axotomized sensory nerve cells (Waxman
et al. 2017). Nav1.3 channel produces fast stimulating and nonstimulating kinetics
and quick retrieval from nonstimulating phase. Its presence has also been confirmed
in distal tips of axon in experimental neuromas in rats and in human neuromas.
Hyperresponsiveness and unpremeditated firing of neurons was observed with an
increase in pain and altered electrophysiologal properties as manifestation when Na
1.3 was upregulated, whereas down-regulation of channel showed pain in trigeminal
nerves in trigeminal ganglionic regions in ferret (Nassar et al. 2006). Protein and
mRNA level of Nav1.3 was found to be diminished following administration of
antisense oligodeoxynucleotides through intrathecal route resulting in reduction of
hyperexcitability of DRG neurons and reduced pain in nerves after injury of spinal
cord and sciatic nerve (Lindia et al. 2005).

18.3.6.2 Nav1.7 Channel

Nav1.7 channel is sodium channel sensitive to tetrodotoxin and present largely in
sensory neurons and sympathetic ganglia providing fast stimulating and
nonstimulating kinetics but in turn causes considerably slower recovery period
from fast inactivation making it different from other tetrodotoxin sensitive channels.
Down-regulation of mRNA and protein in Nav1.7 channels was done through tying
of spinal nerve of neuropathic pain. Mutations that cause gain of function of SCN9A
coding for sodium channel Nav1.7 are present in patients suffering from critical pain
syndrome inherited erythromelalgia (IE) (Cregg et al. 2013) and paroxysmal extreme
pain disorder (PEPD) (Dabby et al. 2011), whereas mutations that cause loss of
function of SCN9A gene have been found in patients suffering from inherited
insensitivity to pain and disabled sense of smell. Inherited erythromelalgia is
specified by burning pain and hot skin flashes, which causes change in hyperpolari-
zation of voltage, which in turn effects the activation. Paroxysmal extreme pain
630 V. Tiwari et al.

disorder is manifested by extreme pain and burning sensation in rectal, ocular and
submandibular region, which causes change in depolarizing voltage, which in turn
effects steady-state inactivation.

18.3.6.3 Nav1.8 Channel

Nav1.8 channel is present mainly in sensory neurons of small diameter and trigemi-
nal ganglionic neuron. This channel is tetrodotoxin resistant in nature and produces
lower time period of depolarized stimulation and steady-state voltage-dependent
nonstimulating kinetics but has high rate of retrieval from nonstimulation state.
Depolarized stimulation of Nav1.8 sodium channel leads to upstroke in action
potential by 80–90% of inward channels of sensory neurons from Nav1.8 null
mice (Harty and Waxman 2007). Ongoing nociceptive unit can regulate both
biophysical properties of Nav1.8 as well as its expression. Patients suffering from
chronic pain due to dysfunction of nervous system or with increased sensitivity to
chronic pain in local areas had increased content of Nav1.8 channels in regions
having proximity to peripheral injury site. Manifestation of inflammatory mediators
leads to altered level of tetrodotoxin channel with stimulating threshold shifted
toward more negative potential through stimulation of protein kinase A. Increase
in magnitude of Nav1.8 mediated channel is caused by prostaglandin E2, adenosine,
and serotonin, which results in shift of relation between conductance of voltage
toward negative spectrum and increasing time period of stimulating and
nonstimulating of sodium channels in sensory neurons having small diameter
(Black et al. 2004; Wood et al. 2004).

18.3.6.4 Nav1.9 Channel

Nav1.9 channel is sodium channel resistant to tetrodotoxin and is present mainly in
sensory nerve cells having smaller diameter. Nav1.9 channel provides voltage-
dependent activation close to the potential of membrane present in the resting state
(-70 mV). Steady-state nonstimulation occurs at comparatively positive potential
(-45 mV) (Shah et al. 2010). These kinetic properties suggest that activation of
Nav1.9 may prolong response to subthreshold depolarization and generate a persis-
tence channel. Experiments on electrophysiology on sensory neurons of Nav1.9 null
mice results in lowering of threshold for action potential electrogenesis and produces
persistent channels leading to hyperexcitability. Inflammation leads to increased
expression of Nav1.9 gene along with Nav1.9 mRNA, which ultimately results in
substantial down-regulation of proteins in axotomized afferent neurons and several
in vivo models that are suffering from pain in the somatosensory nervous system
(Lolignier et al. 2011).

18.4 Conclusion

Normal functional and number of sodium channels are crucial for the functioning of
any excitable cells. This, when mutations or disorder arise, which disturbs this
balance, leads to various clinical pathologies. Channelopathies are disarrays arising
18 Sodium Channels: As an Eye of the Storm in Various Clinical Pathologies 631

due to alternations in sodium channels. These disorders include manifestations like

epilepsy, Dravet Syndrome, complications associated with pain, like loss of sensi-
tivity to pain from birth, primary erythromelalgia and manifestation of paroxysmal
extreme pain disorder, irregular heartbeat (signified by extreme QT syndrome), slow
ventricular conduction, and atrial standstill. Aggressive nature of prostate (Nav1.7)
and breast cancer (Nav1.5) has been associated with alteration in epigenetic regula-
tion. Sodium channel upregulation has also been involved in diabetes and chemical
induced neuropathies, presence of neuromuscular anomalies like Eaton Lambert
Syndrome, Charcot Marie tooth disorder along with several disorders that are
neurodegenerative in nature like Alzheimer’s, Schizophrenia, and most importantly
Parkinson’s. Sodium channels are an important target due to their main role in these
diseases. Dravet Syndrome (DS) also leads to haploinsufficiency of SCN1A along
with functional loss of SCN1B. On the other hand, functional gain of SCN2A is
associated with BFNIS. Likewise, functional gain of SCN8A is associated with
epileptic encephalopathy (EE). Substitution of arginine I by uncharged amino acid
residues will lead to alteration in VGSCs voltage sensor and hence lead to outflow in
dispensing of current or alteration in sodium channel function resulting in resurgence
of inward current, leading to increase in neuronal firing frequency. All these changes
are some responsible factors for epilepsy. Sodium channels are also associated with
development of psychotic diseases such as Schizophrenia, alteration in activity of
hippocampus, substantia-nigra, ventral tegmental area, and prefrontal cortex, which
in turn are manifestations associated with the development of Schizophrenia.
Dysregulation in activity of neuron and voltage-gated sodium channels (VGSCs)
was found to be involved in the development of Schizophrenia through targeted
sequencing of 10,198 samples. Development of familial hemiplegic migraine (FHM)
was also found to be related to sodium channels. FHM, which manifests by transient
motor weakness to some extent and difficulty in having conversation along with
optical disturbances is an autosomal dominant type disease although its occurrence is
rare. Alterations in ATP1A2 that encodes for Na+/K + -ATPase dependent alpha2
subunit, which is catalytic in nature, and SCN1A (encodes for pore region of
Nav1.1) are some of the etiological factors involved in the pathogenesis of FHM.
Dysregulation of sodium channels (predominantly Nav1.5, Nav1.7, Nav1.8, and
Nav1.9) leads to hypersensitivity of nervous system, which leads to hyperalgesia and
allodynia observed during neuropathic pain. In another study, alternations that lead
to functional gain of SCN9A gene, which in turn encrypts sodium channel Nav1.7,
was found to be expressed in patients suffering from diseases associated with pain
manifestations such as inherited erythromelalgia (IE) and paroxysmal extreme pain
disorder (PEPD). On the other hand, functional loss of SCN9A is observed in
patients with inborn insensitivity to pain and impaired sense of smell. Sodium
channels targeting is an emerging concept that provides help in mitigation of various
clinical disorders. However, there has been more than 70 years of research in
understanding molecular dynamics and the functioning of sodium channels. The
drugs that effectively target specific Nav channels and that are able to modulate its
expression are yet to be discovered. Kinesins and Caveolae/Caveolin interaction
plays an essential role in maintaining required number of functional VGSCs. Drugs
632 V. Tiwari et al.

that are able to target these markers can be developed or repurposed as an alternative
safe therapy for treatment of channelopathies.

Acknowledgment This work is supported by the Department of Pharmaceutical Engineering and

Technology, Indian Institute of Technology (BHU). Varanasi.

References
Ambrose C et al (1992) The α-subunit of the skeletal muscle sodium channel is encoded proximal to
Tk-1 on mouse chromosome 11. Mamm Genome 3:151–155. https://doi.org/10.1007/
BF00352459
Bagal SK et al (2015) Voltage gated sodium channels as drug discovery targets. Channels
9:360–366. https://doi.org/10.1080/19336950.2015.1079674
Bechi G et al (2012) Pure haploinsufficiency for Dravet syndrome Na V1.1 (SCN1A) sodium
channel truncating mutations. Epilepsia 53:87–100. https://doi.org/10.1111/j.1528-1167.2011.
03346.x
Beckers MC et al (1996) A new sodium channel α-subunit gene (Scn9a) from Schwann cells maps
to the Scn1a, Scn2a, Scn3a cluster of mouse chromosome 2. Genomics 36:202–205. https://doi.
org/10.1006/geno.1996.0447
Black JA et al (2004) Changes in the expression of tetrodotoxin-sensitive sodium channels within
dorsal root ganglia neurons in inflammatory pain. Pain 108:237–247. https://doi.org/10.1016/j.
pain.2003.12.035
Burgess DL et al (1995) Mutation of a new sodium channel gene, Scn8a, in the mouse mutant
“motor endplate disease”. Nat Genet 10:461–465. https://doi.org/10.1038/ng0895-461
Carreño O et al (2013) Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine:
clinical, genetic, and functional studies. Mol Genet Genomic Med 1:206–222. https://doi.org/
10.1002/mgg3.24
Catterall WA (1992) Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev
72:S15–S48. https://doi.org/10.1152/physrev.1992.72.suppl_4.S15
Catterall WA et al (2005) Nomenclature and structure-function relationships of voltage-gated
calcium channels. Pharmacol Rev. https://doi.org/10.1124/pr.57.4.5.units
Cole KS, Baker RF (1941) Longitudinal impedance of the squid giant axon. J Gen Physiol. https://
doi.org/10.1085/jgp.24.6.771
Cole KS, Curtis HJ (1939) Electric impedance of the squid giant axon during activity. J Gen
Physiol. https://doi.org/10.1085/jgp.22.5.649
Cregg R et al (2013) Novel mutations mapping to the fourth sodium channel domain of Nav1.
7 result in variable clinical manifestations of primary erythromelalgia. Neuromol Med 15
(2):265–278. Springer
Dabby R et al (2011) Chronic non-paroxysmal neuropathic pain—novel phenotype of mutation in
the sodium channel SCN9A gene. J Neurol Sci 301(1–2):90–92. Elsevier
Dichgans M et al (2005) Mutation in the neuronal voltage-gated sodium channel SCN1A in familial
hemiplegic migraine. Lancet 366:371–377. https://doi.org/10.1016/S0140-6736(05)66786-4
Frank HY, Catterall WA (2003) Overview of the voltage-gated sodium channel family. Genome
Biol 4(3):207. BioMed Central
Hargus NJ et al (2013) Evidence for a role of Na v 1.6 in facilitating increases in neuronal
hyperexcitability during epileptogenesis. J Neurophysiol 110:1144–1157. https://doi.org/10.
1152/jn.00383.2013
Harty TP, Waxman SG (2007) Inactivation properties of sodium channel Nav1.8 maintain action
potential amplitude in small DRG neurons in the context of depolarization. Mol Pain. https://doi.
org/10.1186/1744-8069-3-12
18 Sodium Channels: As an Eye of the Storm in Various Clinical Pathologies 633

Imbrici P, Camerino DC, Tricarico D (2013) Major channels involved in neuropsychiatric disorders
and therapeutic perspectives. Front Genet 4. https://doi.org/10.3389/fgene.2013.00076
Jarecki BW et al (2010) Human voltage-gated sodium channel mutations that cause inherited
neuronal and muscle channelopathies increase resurgent sodium currents. J Clin Investig
120:369–378. https://doi.org/10.1172/JCI40801
Kahlig KM et al (2008) Divergent sodium channel defects in familial hemiplegic migraine. Proc
Natl Acad Sci 105:9799–9804. https://doi.org/10.1073/pnas.0711717105
Kaplan DI, Isom LL, Petrou S (2016) Role of sodium channels in epilepsy. Cold Spring Harb
Perspect Med 6. https://doi.org/10.1101/cshperspect.a022814
King GF, Vetter I (2014) No gain, no pain: NaV1. 7 as an analgesic target. ACS Chem Neurosci 5
(9):749–751. ACS Publications
Kozak CA, Sangameswaran L (1996) Genetic mapping of the peripheral sodium channel genes,
Scn9a and Scn10a, in the mouse. Mamm Genome 7:787–788. https://doi.org/10.1007/
s003359900235
Lindia JA et al (2005) Relationship between sodium channel NaV1.3 expression and neuropathic
pain behavior in rats. Pain 117:145–153. https://doi.org/10.1016/j.pain.2005.05.027
Lolignier S et al (2011) Nav1.9 channel contributes to mechanical and heat pain hypersensitivity
induced by subacute and chronic inflammation. PLoS One 6:e23083. https://doi.org/10.1371/
journal.pone.0023083
Mantegazza M, Catterall WA (2012) Voltage-gated Na+ channels: structure, function, and patho-
physiology. In: Jasper’s basic mechanisms of the epilepsies. National Center for Biotechnology
Information (US), Bethesda, MD
Marban E, Yamagishi T, Tomaselli GF (1998) Structure and function of voltage-gated sodium
channels. J Physiol 508:647–657. https://doi.org/10.1111/j.1469-7793.1998.647bp.x
Martin MS et al (2007) The voltage-gated sodium channel Scn8a is a genetic modifier of severe
myoclonic epilepsy of infancy. Hum Mol Genet 16:2892–2899. https://doi.org/10.1093/hmg/
ddm248
Misra SN, Kahlig KM, George AL (2008) Impaired NaV1.2 function and reduced cell surface
expression in benign familial neonatal-infantile seizures. Epilepsia 49:1535–1545. https://doi.
org/10.1111/j.1528-1167.2008.01619.x
Nassar MA et al (2006) Nerve injury induces robust allodynia and ectopic discharges in Nav 1.3
null mutant mice. Mol Pain 2:1744-8069-2-33. https://doi.org/10.1186/1744-8069-2-33
Patino GA et al (2009) A functional null mutation of SCN1B in a patient with Dravet syndrome. J
Neurosci 29:10764–10778. https://doi.org/10.1523/JNEUROSCI.2475-09.2009
Plummer NW et al (1998) Exon organization, coding sequence, physical mapping, and polymor-
phic intragenic markers for the human neuronal sodium channel gene SCN8A. Genomics
54:287–296. https://doi.org/10.1006/geno.1998.5550
Raman IM, Bean BP (1997) Resurgent sodium current and action potential formation in dissociated
cerebellar Purkinje neurons. J Neurosci 17(5):4517–4526
Rees E et al (2019) Targeted sequencing of 10,198 samples confirms abnormalities in neuronal
activity and implicates voltage-gated sodium channels in schizophrenia pathogenesis. Biol
Psychiatry 85:554–562. https://doi.org/10.1016/j.biopsych.2018.08.022
Roger S et al (2015) Voltage-gated sodium channels and cancer: is excitability their primary role?
Front Pharmacol 6. https://doi.org/10.3389/fphar.2015.00152
Savio-Galimberti E, Gollob MH, Darbar D (2012) Voltage-gated sodium channels: biophysics,
pharmacology, and related channelopathies. Front Pharmacol 3. https://doi.org/10.3389/fphar.
2012.00124
Shah KU et al (2010) Voltage gated sodium channel blockers: potential treatment for neuropathic
pain. Curr Res Inform Pharm Sci 11:11–16
Shao N et al (2018) Familial hemiplegic migraine type 3 (FHM3) with an SCN1A Mutation in a
Chinese family: a case report. Front Neurol 9. https://doi.org/10.3389/fneur.2018.00976
Sokolov S, Scheuer T, Catterall WA (2005) Ion permeation through a voltage- sensitive gating pore
in brain sodium channels having voltage sensor mutations. Neuron 47:183–189. https://doi.org/
10.1016/j.neuron.2005.06.012
634 V. Tiwari et al.

Sokolov S, Scheuer T, Catterall WA (2007) Gating pore current in an inherited ion channelopathy.
Nature 446:76–78. https://doi.org/10.1038/nature05598
Tata A-G (2013) Effects of cysteine mutations in the S4-S5 linkers from domains D1 to D4 on the
fast inactivation of the voltage-gated sodium channel Nav1. 4. Universität Ulm. Medizinische
Fakultät, Ulm
Wang J et al (1992) Sequence and genomic structure of the human adult skeletal muscle sodium
channel α subunit gene on 17q. Biochem Biophys Res Commun 182:794–801. https://doi.org/
10.1016/0006-291X(92)91802-W
Watanabe K, Sato I, Kosaki T (1971) Familial hemiplegic migraine. No to Hattatsu. https://doi.org/
10.11251/ojjscn1969.3.14
Waxman SG, Kocsis JD, Black JA (2017) Type III sodium channel mRNA is expressed in
embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy. J
Neurophysiol 72:466–470. https://doi.org/10.1152/jn.1994.72.1.466
Wood JN et al (2004) Voltage-gated sodium channels and pain pathways. J Neurobiol 61:55–71.
https://doi.org/10.1002/neu.20094
Yamagishi T et al (2001) Molecular architecture of the voltage-dependent Na channel: functional
evidence for alpha helices in the pore. J Gen Physiol 118:171–182. https://doi.org/10.1085/jgp.
118.2.171
Ye M et al (2018) Differential roles of Na V 1.2 and Na V 1.6 in regulating neuronal excitability at
febrile temperature and distinct contributions to febrile seizures. Sci Rep 8:753. https://doi.org/
10.1038/s41598-017-17344-8
Pharmacology of Potassium Channels
19
Satyendra Deka, Pobitra Borah, Ratnali Bania, Sanjib Das,
and Pran Kishore Deb

Abstract

Potassium channels constitute the largest and most ubiquitous and diverse ion
channel family. They regulate several physiological processes including electrical
signalling, hormone and neurotransmitter release, muscle contraction, cardiac func-
tion, cell proliferation and immune function, which are underscored by the associa-
tion of K+ channel mutations to numerous inherited diseases. Although many
challenges remain unsolved, the availability of diverse expression systems, molecu-
lar cloning and genetic linkage analysis has led to the upgradation of available
topological data, identification of disease-producing loci, and better understanding
of mutation-linked channelopathies. Moreover, in 2016, a new nomenclature
(KNa1.1 for KCa4.1, and KNa1.2 for KCa4.2) has been assigned and implemented
in the IUPHAR database. These advances along with high-throughput screening are
catalysing the discovery process of newer pharmacophores and modulators of K+
channels. This chapter aims to provide a basic understanding of K+ channels and
offers an updated overview on the progress and opportunities of pharmacological
approaches in the exploitation of these channels as therapeutic targets.

Keywords
Potassium channel · Ca2+- and Na+-activated K+ channels · Inwardly rectifying
K+ channels · Two P domain K+ channels · Voltage-gated K+ channels

S. Deka (*) · P. Borah (*) · R. Bania

Pratiksha Institute of Pharmaceutical Sciences, Guwahati, Assam, India
e-mail: [email protected]
S. Das
Department of Pharmaceutical Sciences, Assam University, Silchar, Assam, India
P. K. Deb
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Philadelphia University, Amman,
Jordan

# Springer Nature Singapore Pte Ltd. 2020 635

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_19
636 S. Deka et al.

Abbreviations

1-EBIO 1-Ethyl-2-benzimidazolinone
4-AP 4-Aminopyridine
AF Atrial Fibrillation
AgTx Agitoxins
ALS Amyotropic Lateral Sclerosis
BK Large conductance K+ channels
CHF Congestive Heart Failure
ChTx Charybdotoxin
CLL Chronic Lymphoid Leukaemia
CNS Central Nervous System
DCT Distal Convoluted Tubule
DRG Dorsal Root Ganglia
DTx Dendrotoxin
EA1 Episodic Ataxia type 1
EAG Ether-à-go-go
EGTA Ethylene Glycol Tetraacetic Acid
Elk Eag-Like K+ Channels
Erg Eag-Related Genes
GABA γ-Amino Butyric Acid
GAs General Anaesthetics
GIRK G-protein-activated Inward-Rectifier K+ channels
HERG Human ether-à-go-go
HMs Hypoglossal Motoneurons
HTS High-Throughput Screening
IbTx Iberiotoxin
IK Intermediate Conductance K+ channels
IUPHAR International Union of Basic and Clinical Pharmacology
K2P Two P domain K+ channels
KATP ATP-sensitive K+ channels
KCO Potassium Channel Opener
Kir Inwardly rectifying K+ channels
KT Kaliotoxin
mAb Monoclonal Antibodies
MAC Minimum Alveolar Concentration
MEM Memantine
MgT Margatoxin
MOR μ-Opioid Receptor
mRNA Messenger RNA
MTx Maurotoxin
NBP 3-N-Butylphthalide
NMDAR N-Methyl-D-aspartate receptor
NSAIDs Non-Steroidal Anti-Inflammatory Drugs
19 Pharmacology of Potassium Channels 637

NxTx Noxiustoxin
PHHI Persistent Hyperinsulinaemic Hypoglycaemia of Infancy
PIP2 Phosphatidylinositol-4,5-bisphosphate
PTX Pertussis toxin
RBCs Red Blood Cells
SCLC Small-Cell Lung Cancer cells
SeSAME Seizures, Sensorineural deafness, Ataxia, Mental retardation, Electro-
lyte imbalance
SK Small conductance K+ channels
SSRIs Selective Serotonin Reuptake Inhibitors
SU Sulfonylureas
SUR Sulfonylurea Receptors
T2-DM Type II Diabetes Mellitus
TCAs Tricyclic Antidepressants
TEA Tetraethylammonium
TM Transmembrane domains
TRAM-34 Triarylmethane-34

19.1 Introduction

Potassium channels form the largest and most diverse family among the ion channels
encoded by about 78 genes (D’amico et al. 2013). The first atomic structure of a
prokaryotic K+ channel, KcsA, was determined from bacterium Streptomyces
lividans (Kuang et al. 2015; Garcia et al. 2001; Luzhkov and Åqvist 2005; González
et al. 2012). After this discovery, considerable efforts have been made to interpret the
mechanism of the subtypes of the channel. The genomes of Drosophila and
Caenorhabditis elegans were found to contain 30–100 genes for K+ channels (Miller
2000). The subunit stoichiometry, topology, precise biophysical properties, and
modulation by the ligands and second messengers have been addressed to a great
extent for the K+ channels. These studies have been conducted by using high-
hroughput multiple assays, including T1+ flux assay, ligand binding assay,
voltage-sensitive dye-assay and 86Rb+ flux assay (Yu et al. 2016; Longman and
Hamilton 1992). Potassium channels switch the conformation between an open and
a closed state to conduct K+ ions down the electrochemical gradient of K+ across the
cell membrane (Mackinnon 2003). A typical K+ channel consists of a tetramer with
each monomer possessing one pore-forming (P) domain, which together comprises
the pore for conduction. The potassium channels contain a highly conserved seg-
ment of amino acid sequences known as K+ channel signature sequences (TVGYG)
(Kuang et al. 2015; Miller 2000). These sequences form the selectivity filter for K+
ions, which gives the channels more selectivity for K+ (at least 10,000 times) over
Na+ ions. The specificity is due to the multiple binding sites of the selectivity filter
that mimics a potassium ion hydrated shell. Moreover, the rate of conduction of these
channels is very high (107 ions channel
1 s
1) (Mackinnon 2003). The K+ channels
exist in three states, i.e. the channel is closed in the resting state, open in the activated
638 S. Deka et al.

state and remains non-conductive in the inactivated state. The gating is formed by
the bending of inner helix (intracellular) and the selectivity filter (extracellular)
(Kuang et al. 2015). The wide tissue distribution allows potassium channels to
influence various physiological functions, such as release of neurotransmitters and
hormones, regulation of fluid secretion, controlling of heart rate, smooth and cardiac
muscle contraction and clonal expansion of immune cells (Garcia et al. 1997; Garcia
and Kaczorowski 2005). The activation of K+ channels also regulates cellular
excitability by influencing the action potential waveform. In excitable cells, the
pharmacological activation of these channels reduces the excitability, albeit channel
inhibition leads to excitability of the cell.
Several genetically linked and acquired diseases have been known to be associated
with the alteration of the K+ channel. Disruption of the K+ channel gene underlies
various pathologies involving epilepsy, neurodegeneration, schizophrenia, episodic
ataxia, diabetes, deafness, cardiac arrhythmias, renal diseases, asthma and hyperten-
sion (Bergeron and Bingham 2012). This provides a basis to develop appropriate
pharmacological interventions by targeting these channels. Most interestingly, the
enthusiasm towards this arena was driven by the realization of the fact that the
sulfonylurea class of antidiabetic drugs and class-III antiarrhythmic drugs act via
regulating the K+ channels. Not only this, many K+ channel openers and blockers
also offer therapeutic opportunities in a wide range of areas, including vascular or
non-vascular muscle and immune, neuronal and cardiac systems.
Although continuous progress has been made in developing the underlying
molecular pharmacology of the K+ channels, still some of the subtypes remain
unexplored and in most of the cases existing tools are not sufficient for probing
these subfamilies in higher biological systems (Garcia and Kaczorowski 2016).
Therefore, emphasis has been laid on the discovery of K+ channel modulators in
the last few decades. The basic K+ channel modulators are mainly divided into two
types: peptide venom toxins and small molecules. The peptide toxins exert their
effect either by binding to the outer vestibule (e.g. toxins from snakes, sea anemones,
scorpions and cone snails) or by interacting with the voltage sensor of the channel
(e.g. hanatoxin obtained from spiders). However, the small molecules target the
gating hinge, inner pore or α/β subunit interface. The small molecules either activate
or block the channel. Depending on their selectivity towards target(s), they are also
classified as specific target and multi-target molecules (Tian et al. 2014).
According to the classification of IUPHAR, four structural types of K+ channels
are (a) Ca2+- and Na+-activated K+ channels, (b) inwardly rectifying K+ channels,
(c) two P domain K+ channels and (d) voltage-gated K+ channels (Alexander et al.
2017; Alexander et al. 2013). These individual classes are explained in detail in the
later sections. Moreover, expression of the channel subtypes and their modulators
are summarized in Table 19.1.
Table 19.1 Overview of potassium channels and their modulators
19

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
Ca2+- and Large KCa1.1 KCNMA1 Ubiquitous; brain, NS004, NS1619, Paxilline, TEA, ChTx, Kaczmarek
Na+- conductance hair cells of cochlea, cromakalim, IbTx, slotoxin, EGTA, et al. (2017);
activated (BK) pancreatic β-cells, dehydrosoyasaponin-I, Ni+, aflatrem, Wei et al.
(KCa and skeletal and smooth niflumic acid, MCI-154, verruculogen, penitrem (2005); Coghlan
KNa) muscle, colon, Maxi-K diol, CGS-7181, A et al. (2001);
kidney NS-1608, Kaczorowski
BMS-20435211 et al. (1996);
KCa5.1 KCNU1 Testis, – Ba2+, quinine, quinidine Sanchez and
spermatocytes McManus
KNa1.1 KCNT1 Brain, kidney, testis, Bithionol, niclosamide, Bepridil, quinidine (1996);
hippocampus, loxapine Augustynek
Pharmacology of Potassium Channels

olfactory bulb, et al. (2016);

frontal cortex Ledoux et al.
(2006); Dogan
KNa1.2 KCNT2 Brain, heart Niflumic acid Ba2+, quinidine
et al. (2019);
Small KCa2.1 KCNN1 Brain, aorta, spinal EBIO, NS309, UCL1684, apamin, Faber and Sah
conductance cord, pituitary, GW542573X tubocurarine, (2007); Strøbæk
(SK) oligodendroglioma, pancuroniumatracurium, et al. (2004);
gastric tumour, TEA, lelurotoxin I, Hougaard et al.
glioblastoma dequalinium, UCL2048, (2009);
KCa2.2 KCNN2 Brain, thalamus, EBIO, NS309 and UCL1530 Weatherall et al.
pituitary, (2010); Syme
melanocyte, et al. (2000);
prostate, lung, liver, Castle et al.
heart, skeletal (2003)
muscle,
myometrium,
melanoma,
oligodendroglioma
639

(continued)
Table 19.1 (continued)
640

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
KCa2.3 KCNN3 Brain, lymphocytes, EBIO, NS309
skeletal muscle,
prostate, kidney,
vascular
endothelium, heart,
pancreas, colon,
liver, head, neck,
ovary
Intermediate KCa3.1 KCNN4 RBCs, liver, EBIO, NS309, SK-121, TRAM-34, ChTx, IbTx,
conductance pancreas, benzoxazoles maurotoxin,
(IK) lymphocytes, (zoxazolamine and clotrimazole,
placenta, prostate, chlorzoxazone), isatin nitrendipine, oxime,
colon, lung, colon, derivatives malonate
vascular
endothelium
Inwardly Classical or Kir2.1 KCNJ2 Heart, brain, kidney, PIP2 Spermine, spermidine, González et al.
rectifying strong lung, placenta, putrescine, intracellular (2012); Garcia
(Kir) inward skeletal and smooth Mg2+, Ba2+, Cs+, Rb+, and
rectifier muscle, memantine Kaczorowski
macrophage (2005);
Kir2.2 KCNJ12 Cerebellum, Intracellular Mg2+, Ba2+, Alexander et al.
forebrain, heart, Cs+ (2017); Shieh
kidney, skeletal et al. (2000);
muscle Yamashita et al.
Kir2.3 KCNJ4 Brain, reactive Intracellular alkalization Intracellular Mg2+, Ba2+, (1996); Ishihara
astrocyte, kidney, (pH 6.76), PIP2, Cs+, spermine, et al. (1996);
heart, smooth arachidonic acid, tenidap spermidine, putrescine Alagem et al.
muscle (2001); Hughes
S. Deka et al.
 19

G-protein Kir2.4 KCNJ14 Brain, retina, Extracellular alkalization Intracellular Mg2+, Ba2+, et al. (2000);
activated neuronal cell in Cs+ Camerino et al.
(GIRK) heart (2007)

Kir3.1 KCNJ3 Cerebellum, PIP2, ML297, Tertiapin-Q, Ba2+, Cs+

olfactory bulb,
neocortex,
thalamus,
hippocampus, basal
ganglia, brainstem,
heart
Kir3.2 KCNJ6 Cerebellum, PIP2 Tertiapin, halothane,
pancreatic islets, bupivacaine, verapamil,
Pharmacology of Potassium Channels

brain MK-801, QX-314,

antipsychotics such as
haloperidol, thioridazine,
pimozide, desipramine,
clozapine, fluoxetine
Kir3.3 KCNJ9 Brain PIP2
Kir3.4 KCNJ5 Heart, pancreas, PIP2 Ba2+, Cs+, TEA, 4-AP,
skeletal muscle, tertiapin-Q
lungs, cerebellum,
urinary bladder
ATP- Kir6.1 KCNJ8 Ubiquitous; Cromakalim, diazoxide, Glibenclamide,
sensitive pancreas, neurons, minoxidil, nicorandil, tolbutamide
(KATP) skeletal muscle, iptakalim
vascular smooth
muscle
Kir6.2 KCNJ11 Ubiquitous; Cromakalim, diazoxide, Glibenclamide,
pancreas, neurons, minoxidil, nicorandil tolbutamide
skeletal muscle,
641

brain, heart
(continued)
Table 19.1 (continued)
642

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
K+-transport Kir1.1 KCNJ1 Kidney, pancreatic VU590, VU591 Tertiapin-Q,
islets, skeletal δ-dendrotoxin, Ba2+, Cs+
muscle, pancreas,
spleen, brain, liver,
heart
Kir4.1 KCNJ10 Glia, retina, ear, ATP, PIP2 Ba2+, Cs+, imipramine,
kidney desipramine,
nortryptyline,
amitriptyline
Kir4.2 KCNJ15 Brain, kidney, lung, – Ba2+, Cs+
pancreas, liver,
testis
Kir5.1 KCNJ16 Brain, kidney, – Ba2+, Cs+
thyroid, spleen,
liver, testis, retina
Kir7.1 KCNJ13 Cerebellum, – Ba2+, Cs+
hippocampus,
kidney, thyroid, GI,
stomach, small
intestine, prostate,
testis, lung, retina
Two P TWIK K2P1.1 KCNK1 Brain, heart, kidney, – Garcia et al.
domain or lung, liver, placenta (1997); Gada
(K2P) TWIK-1 and Plant
S. Deka et al.
 19

K2P6.1 KCNK6 Pancreas, heart, Arachidonic acid Ba2+, quinidine, volatile (2019); Enyedi
or placenta, lung, anaesthetics and Czirják
TWIK-2 stomach, eyes, (2010);
embryo Olschewski
K2P7.1 KCNK7 Brain, spinal cord, et al. (2017);
retina Vivier et al.
TREK K2P2.1 KCNK2 Brain, lung, heart Arachidonic acid, Ba2+, PKA, PKC, (2015); Lesage
or Chloroform, halothane, sipatrigine, fluoxetine, (2003); Wright
TREK-1 isoflurane, unsaturated chlorpromazine, et al. (2017);
fatty acid, haloperidol, loxapine, Tian et al.
lysophospholipids, pimozide, fluphenazine, (2019); Hayashi
fenamates, flufenamic mexiletine, propafenone, and Novak
acid, GI-530139 amlodipine, nifedipine (2013);
Staudacher et al.
Pharmacology of Potassium Channels

K2P4.1 KCNK4 Brain, kidney, Arachidonic acid, Gd+, sipatrigine

or placenta, small riluzole, unsaturated fatty (2018a, b); Kim
TRAAK intestine, prostate acid, lysophospholipids (2005)
K2P10.1 KCNK10 Kidney, liver, Arachidonic acid, Norfluoxetine, quinidine,
or pancreas, prostate, halothane, linoleic acid, chlorpromazine,
TREK-2 thymus doecosahexaenoic acid, haloperidol, loxapine,
riluzole, pimozide, fluphenazine
lysophosphatidylcholine,
volatile anaesthetic
halothane, isoflurane,
GI-530139
TASK K2P3.1 KCNK3 Brain, heart, colon, Halothane, isoflurane, Ba2+, external pH (7.3),
or small intestine, volatile anaesthetics arachidonic acid,
TASK-1 lung, pancreas, anandamide, R-(+)-
placenta, prostate, methanandamide
uterus, kidney
(continued)
643
Table 19.1 (continued)
644

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
K2P9.1 KCNK9 Brain Halothane External pH (6.5),
or ruthenium red,
TASK-3 anandamide, R-(+)-
methanandamide
KCNK15 Brain, kidney, heart, – –
lung, pancreas,
liver, placenta,
thyroid, adrenal
gland, salivary
K2P15.1 gland
or
TASK-5
TALK K2P5.1 KCNK5 Brain, kidney, liver, Halothane, volatile External pH (6.5), LAs
or pancreas, placenta, anaesthetics (lidocaine, bupivacaine,
TASK-2 small intestine clofilium)
K2P16.1 KCNK16 Heart, liver, lung, Isoflurane, NO, ROS External pH, Ba2+,
or pancreas, placenta quinidine, chloroform
TALK-1
K2P17.1 KCNK17 Heart, lung, liver, NO, ROS, quinidine, External pH, Ba2+,
or placenta, pancreas propafenone, mexiletine, chloroform, sotalol,
TALK-2 metoprolol, propranolol verapamil, amiodarone,
ranolazine
THIK K2P12.1 KCNK12 Brain, liver, lung, – Propafenone, mexiletine,
or heart, kidney, colon, propranolol, lidocaine
THIK-1 pancreas, spleen,
ovary, placenta,
prostate, thymus,
small intestine
S. Deka et al.
 19

K2P13.1 KCNK13 Brain, lung, heart, Arachidonic acid Ba2+, halothane

or kidney, liver, spleen
THIK-2
TRESK K2P18.1 KCNK18 Cerebellum, Volatile anaesthetics External acidic pH, Ba2+,
cerebrum, (isoflurane, desflurane, quinine, quinidine, free
brainstem, spinal sevoflurane, halothane), fatty acid, lomotrigin,
cord, testis cloxyquin loratidine
Voltage Kv1 Kv1.1 KCNA1 Pons, medulla, – α-Dendrotoxin, Gutman et al.
gated midbrain, margatoxin, TEA, (2005);
(Kv) cerebellum, capsaicin, flecainide, Kaczorowski
hippocampus, nifedipine, diltiazem, and Garcia
auditory nuclei, resiniferatoxin (1999); Garcia
node of Ranvier, et al. (1997);
Pharmacology of Potassium Channels

kidney, retina, heart Felix et al.

Kv1.2 KCNA2 Spinal cord, – α-Dendrotoxin, (1999); Coghlan
cerebral cortex, margatoxin, nifedipine, et al. (2001);
pons, medulla, flecainide, Hill et al.
cerebellum, resiniferatoxin, (1995);
thalamus, anandamide Kuzmenkov
hippocampus, et al. (2015);
retina, Schwann Gutman et al.
cells (2003);
Kv1.3 KCNA3 CNS, T- and – Margatoxin, TEA, Alexander et al.
B-cells, platelets, noxiustoxin, maurotoxin, (2017); Dalby-
microglia, correolide Brown et al.
macrophages, (2006);
osteoclasts, testis, Humphries and
tonsils Dart (2015);

(continued)
645
Table 19.1 (continued)
646

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
Kv1.4 KCNA4 Corpus striatum, – Fampridine, UK78282, Vacher et al.
hippocampus, riluzole, quinidine, (2007);
olfactory bulb, nicardipine Camerino et al.
pancreatic islets, (2007);
heart, skeletal and Alexander et al.
smooth muscle, (2013)
lung carcinoid

Kv1.5 KCNA5 Hippocampus, – Fampridine, verapamil,

cortex, pituitary, nifedipine, quinidine,
macrophages, propafenone,
microglia, cardiac erythromycin, loratidine,
myocytes, vascular terfenadine, ebastine
smooth muscle,
aorta, colon,
stomach
Kv1.6 KCNA6 Spinal cord, – α-Dendrotoxin, TEA,
astrocytes, ChTx, MgTx,
pulmonary artery hongotoxin,
smooth muscle, tumulustoxin
oligodendrocytes,
testis, ovary, heart,
lungs, colon
Kv1.7 KCNA7 Heart, liver, lung, – Fampridine, noxiustoxin,
skeletal muscle, 4-AP, nifedipine,
placenta, CNS amiodarone, quinidine,
flecainide, tedisamil
S. Deka et al.
 19

Kv1.8 KCNA10 CNS, adrenal gland, – Fampridine, Ba2+,

kidney, heart, ChTX, 4-AP, TEA,
skeletal muscle ketoconazole, pimozide,
verapamil
Kv2 Kv2.1 KCNB1 Cerebral cortex, Linoleic acid TEA, hanatoxin,
cerebellum, halothane,
hippocampus, tetrapentylammonium
pancreatic β-cells,
gastric cancer cells,
insulinomas, lung,
retina, cochlea,
heart, skeletal
muscle, germ cell
Pharmacology of Potassium Channels

Kv2.2 KCNB2 Cerebral cortex, – TEA, fampridine,

cerebellum, quinine, phencyclidine
hippocampus,
pancreatic δ-cells,
tongue, GI smooth
muscle, sympathetic
neurons
Kv3 Kv3.1 KCNC1 Cerebellum, – 4-AP, TEA, fampridine,
cochlear and cromakalim, diltiazem,
vestibular nuclei, nifedipine, flecainide,
substantia nigra, resiniferatoxin
skeletal muscle,
lung, testis, germ
cell
Kv3.2 KCNC2 Pancreatic islets, – 4-AP, TEA, fampridine,
Renshaw cells 8-bromo-cGMP,
(spinal verapamil, D-NONOate,
interneurons),
647

(continued)
Table 19.1 (continued)
648

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
fast-GABAergic 3-isobutyl-1-
interneurons, methylxanthine
Schwann cells,
mesenteric artery
Kv3.3 KCNC3 Brainstem, – 4-AP, TEA
forebrain, Purkinje
cells, cerebellum,
motoneurons, lens,
corneal epithelium
Kv3.4 KCNC4 Brainstem, – 4-AP, TEA, sea anemone
hippocampus, toxin BDS-I
skeletal muscle,
parathyroid,
prostate, pancreatic
acinar cells
Kv4 Kv4.1 KCND1 Heart, liver, kidney, – Fampridine
pancreas, thyroid
gland, lung,
stomach, testis,
pulmonary artery
Kv4.2 KCND2 Cerebellum, – Hanatoxin,
thalamus, basal heteropodatoxins
ganglia,
hippocampus,
forebrain, cochlear
nucleus, rodent
heart
S. Deka et al.
 19

Kv4.3 KCND3 Cerebral cortex, – Niflumic acid,

cerebellum, cardiac bupivacaine, DIDS,
myocytes, smooth nicotine
muscle
Kv5 Kv5.1 KCNF1 Brain, liver, heart, –
skeletal muscle,
kidney, pancreas
Kv6 Kv6.1 KCNG1 Brain, uterus, –
skeletal muscle,
testis, ovary,
prostate, germ cell,
kidney, placenta,
pancreas, bone, skin
Pharmacology of Potassium Channels

Kv6.2 KCNG2 Fetal brain, –

myocardium,
germinal centre, B
cells
Kv6.3 KCNG3 Whole brain, spinal –
cord, thymus,
adrenal gland,
pituitary, small
intestine
Kv6.4 KCNG4 Brain, liver, colon, –
small intestine
Kv7 Kv7.1 KCNQ1 Heart, liver, GI – Clofilium, XE991,
tract, kidney, lung, linopirdine, mefloquine,
ear, rectum, azimilide
pancreas, placenta
Kv7.2 KCNQ2 Hippocampus, Retigabine XE991, linopirdine,
thalamus, TEA, L-735821
649

(continued)
Table 19.1 (continued)
650

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
cerebellum,
brainstem, cerebral
cortex, lung, testis,
breast, eye,
placenta, small
intestine, DRG,
sympathetic ganglia
Kv7.3 KCNQ3 Hippocampus, Retigabine Linopirdine, 4-AP,
cerebral cortex, clofilium, CTX, E4031
cerebellum,
brainstem,
thalamus, DRG,
sympathetic
ganglia, colon, eye,
head, neck, retina
Kv7.4 KCNQ4 VSM, outer hair Retigabine XE991, linopirdine,
cells of ear and TEA, bepridil
cochlea, placenta
Kv7.5 KCNQ5 Cerebral cortex, Retigabine, BMS204352 XE991, linopirdine
thalamus,
hippocampus,
skeletal muscle,
VSM, sympathetic
ganglia
Kv8 Kv8.1 KCNV1 Kidney, brain – –
Kv8.2 KCNV2 Lung, kidney, liver, – –
thymus, pancreas,
S. Deka et al.
 19

spleen, testis, ovary,

colon, prostate
Kv9 Kv9.1 KCNS1 Lens, melanocytes, – –
brain,
Kv9.2 KCNS2 Brain, spinal cord, – –
retina, pulmonary
artery
Kv9.3 KCNS3 Brain, breast, eye, – –
colon, heart, kidney,
muscle, skin, testis,
stomach, uterus,
lung
Kv10 Kv10.1 KCNH1 CNS, melanoma – Quinidine, intracellular
Pharmacology of Potassium Channels

cells, tumour cells calcium

Kv10.2 KCNH5 CNS, heart, lung, – Quinidine, intracellular
liver, kidney, calcium
pancreas, placenta,
muscle
Kv11 Kv11.1 KCNH2 Heart, CNS, RPR260243 Astemizole, terfenadine,
lymphocytes, disopyramide, E4031,
endocrine cells, dofetilide, ibutilide,
testis, ovary, MK-499, clofilium,
prostate, tonsil, cisapride, sertindole
uterus, kidney, liver,
lung, pancreas,
microglia, blood
cells, brain
Kv11.2 KCNH6 Brain, – Sipatrigine
hippocampus,
(continued)
651
Table 19.1 (continued)
652

Modulators
Family Subfamily Members Gene Tissue expression Activators Inhibitors/Blockers References
uterus, lactotrophs,
rat pituitary
Kv11.3 KCNH7 Brain, sympathetic – Sertindole, pimozide
ganglia, lactotrophs,
CA pyramidal
neurons, rat
pituitary
Kv12 Kv12.1 KCNH8 Brain, sympathetic – Ba2+
ganglia, lung,
uterus, colon, testis
Kv12.2 KCNH3 Infant brain, – Ba2+
amygdala,
hippocampus, eye
(retinoblastoma),
lung (small-cell
carcinoma)
Kv12.3 KCNH4 Telencephalon, – Ba2+
lung, oesophagus,
pituitary,
cerebellum,
neuroblastoma,
primary B-cell
neoplasia,
oligodendroglioma
S. Deka et al.
19 Pharmacology of Potassium Channels 653

19.2 Ca2+- and Na+-activated K+ Channels

Studies demonstrated that chelation of Ca2+ reduces the K+ efflux in RBCs, while K+
conductance increases after intracellular injection of Ca2+ into neurons (Kaczmarek
et al. 2017). Based on these observations, the first gene from a Drosophila mutant
(slowpoke or slo) encoding a Ca2+-activated K+ channel was cloned and identified
(Kaczmarek et al. 2017; Atkinson et al. 1991). This channel was termed BK or
MaxiK (later named KCa1.1 or Slo1) because of its large conductance capacity and
sensitivity towards both Ca2+ and transmembrane voltage (Contreras et al. 2013;
Greenwood and Leblanc 2007). Screening of cDNA libraries for similar sequences
of a K+-selective pore subsequently led to the discovery of different genes encoding
for other Ca2+-activated K+ channel subtypes. The next two identified classes,
i.e. KCa2 or SK (small conductance) family (KCa2.1 or SK1, KCa2.2 or SK2, and
KCa2.3 or SK3) and KCa3 or IK (intermediate conductance) family (KCa3.1 or SK4),
are insensitive to the membrane voltage (Adelman et al. 2012). Based on structural
resemblance, three other genes were identified and named KCa4.1 (Slack or Slo2.2),
KCa4.2 (Slick or Slo2.1) and KCa5.1 (SLO3).
Later it was observed that KCa4.1 (Slack) and KCa4.2 (Slick) are not only
activated by the intracellular Ca2+ but also regulated by the Na+ and Cl
concentra-
tion in the cytoplasm (Kaczmarek 2013). Based on this, a new nomenclature for the
two channels (KNa1.1 for Slack or former KCa4.1, and KNa1.2 for Slick or former
KCa4.2) was proposed by Kaczmarek et al. (2017), and later it was accepted and
implemented in the IUPHAR database (Alexander et al. 2017; Kaczmarek et al.
2017).
Cryo-electron microscopy and X-ray crystallography revealed that KCa1.1
consists of 7-TM domains distinguishing it from canonical 6-TM K+ channels. It
has an additional S0 domain preceding S1 along with the common S1–S6 domain
sequence with N-terminal lying outside rather than inside of the cell (as shown
in Fig. 19.1) (Meera et al. 1997). The RCK1 and RCK2 domains in the cytoplasmic
C-terminal act as binding sites for Ca2+, and the “gating ring” is formed together by
the eight RCK domains of each tetrameric KCa1.1 channel (Wu et al. 2010; Wei et al.
2005). Unlike the KCa1.1 channel, KCa2 channels contain six α-helical TM (S1–S6)
with a K+-selective pore sequence linking S5 and S6 (Kaczmarek et al. 2017). These
channels are voltage independent, and conduction through the channels occurs only
after Ca2+–calmodulin complexation (Xia et al. 1998). Similarly, KCa3.1 closely
resembles the KCa2 family; however, unitary conductance of this channel is greater
compared to KCa2. Therefore, it is also named intermediate conductance for the K+
or IK channel. The sensitivity of KCa3.1 is also determined by the association of Ca2+ to
calmodulin. KNa1.1 and KNa1.2 differ from KCa1.1 in transmembrane topology as
they lack the S0-TM. Though these are voltage-sensitive channels, they lack the
repeated motif of amino acids in S4 segments like KCa1.1. The two RCK domains
form the gating ring of the pore similar to that of KCa1.1. The KCa5.1 has a similar
topology as KCa1.1 (Kaczmarek et al. 2017). KCa5.1 is expressed mainly in sperm
cells and activated primarily by voltage and internal alkalinization as in the case of
sperm capacitation (Navarro et al. 2007; Zeng et al. 2015).
 654

+
+ +
+
+ +
S0 1 2 3 4 5 6
1 2 3 4 5 6 1 2 3 4 5 6
+
+ +
+
+ +

CaM
RCK2
RCK2
(a) (b) (c)

+++ +++
+
+
1 2 1 2 3 4 1 2 3 4 5 6
+
+

(d) (e) (f)

Fig. 19.1 Schematic diagram of the transmembrane topology of different potassium channels: (a) Large conductance KCa1.1 and KCa5.1 channels with
additional S0 domain in the structure and Ca2+-binding sites RCK1 and RCK2, (b) small and intermediate conductance KCa2.1, KCa2.2, KCa2.3 and KCa3.1
channels with calmodulin as the Ca2+-binding site, (c) large conductance KNa1.1 and KNa1.2 (formerly known as KCa4.1 and KCa4.2) channels with RCK1 and
RCK2 subunits (without S0 domain), (d) inwardly rectifying (Kir) channels or a typical 2-TM domain family, (e) two P domain (K2P) or 4-TM potassium
channels with two P-loops forming a tandem and (f) voltage-gated K+ channels (Kv) with 6-TM domain comprising homomeric and heteromeric tetramers
S. Deka et al.
19 Pharmacology of Potassium Channels 655

19.2.1 Pharmacology of Ca2+- and Na+-activated K+ Channels

Molecular cloning of Ca2+- and Na+-activated K+ channels is anticipated in the

development of the modulators of these channels. These channels are known for
their critical dependency of channel conduction on intracellular Ca2+ and activation
by Na+ (newer concept) and, therefore, implicated in many physiological and
pathological conditions. BK channels are involved mainly in hypertension, coronary
artery spasm, stroke, urinary incontinence and several neurological disorders like
psychoses, schizophrenia and epilepsy (Coghlan et al. 2001; Shieh et al. 2000;
Humphries and Dart 2015). IK channel modulators have long been proposed for
treatment strategies for diarrhoea, asthma, atherosclerosis, sickle cell anaemia and
autoimmune diseases like rheumatoid arthritis (Shieh et al. 2000; Lam et al. 2013;
Grunnet et al. 2011), while SK channel modulators are being investigated as
potential therapy for ataxia, memory disorders, narcolepsy, epilepsy, atrial fibrilla-
tion and alcohol dependence (Kaczmarek et al. 2017; Camerino et al. 2007).
Mutations in the newly classified KNa1.1 result in a variety of early-onset epilepsies
like malignant migrating partial seizures, Ohtahara syndrome and autosomal domi-
nant frontal lobe epilepsy (Kim and Kaczmarek 2014). In contrast, KNa1.2 and
KCa5.1 are least studied as compared to the other subtypes. Because of the wide
range of distribution, the Ca2+- and Na+-activated K+ channels are a good target in
several physiological and pathological conditions (also refer to Table 19.1).

19.2.1.1 Modulators of BK Channels (KCa1.1, KNa1.1, KNa1.2 and KCa5.1)

The association of auxiliary subunits is the key factor for governing pharmacological
properties via KCa1.1 channels. The earlier reported modulators include activators
like glycosylated triterpenes (e.g. dehydrosoyasaponin-I) and blockers such as
indole diterpenes (e.g. paxilline, aflatrem, verruculogen and penitrem A)
(Kaczorowski et al. 1996; Sanchez and McManus 1996; Augustynek et al. 2016).
Paxilline showed enhancement in the binding of ChTx to the BK channels in ligand
binding studies. This effect is determined by the binding of paxilline to the alpha-
subunits of BK channels (Sanchez and McManus 1996). KCa1.1 channels are
activated by benzimidazolones like NS-004 and NS-1619 and induce membrane
hyperpolarization. Other classes of drugs which activate these channels include
benzopyrans (e.g. cromakalim), dihydropyridines (e.g. nitrendipine), biarylamines
(e.g. niflumic acid, MCI-154), terpenoids (e.g. Maxi-K diol), biarylureas
(e.g. CGS-7181, NS-1608) and 3-aryloxyindoles (e.g. BMS-204352) (also refer to
Table 19.1) (Camerino et al. 2007; Calderone 2002; Ledoux et al. 2006).
BMS-20435211 is a vasoactive molecule, being investigated in clinical trials for
its potential as a treatment strategy for migraine (ClinicalTrials.gov Identifier:
NCT03887325). Carbonic anhydrase (CA) inhibitors such as acetazolamide,
bendroflumethiazide, ethoxzolamide and dichlorphenamide also exhibited BK acti-
vation properties. The majority of small-molecule KCa1.1 activators possess struc-
tural homology, i.e. two aromatic rings linked via either a heterocyclic or a urea
spacer (Coghlan et al. 2001). Non-selective activation of BK channels by tamoxifen
656 S. Deka et al.

(oestrogen receptor antagonist) at its therapeutic concentration may explain their role
in tamoxifen-induced QT prolongation and arrhythmia.
The scorpion toxins iberiotoxin (IbTx), charybdotoxin (ChTx) and slotoxin are
specific blockers of KCa1.1 with higher selectivity and potency (Garcia-Valdes et al.
2001). These channels are also blocked by tetraethylammonium (TEA), Ni+ and
ethylene glycol tetraacetic acid (EGTA) (Dogan et al. 2019). Non-selective vascular
BK blockers include gallopamil and verapamil. BK channels are also inhibited by
ketamine (dissociative anaesthetic) and clotrimazole (antifungal) (also refer to
Table 19.1) (Kaczmarek et al. 2017).
No specific pharmacological agents are known to selectively act on KNa1.1,
KNa1.2 and KCa5.1. Drugs such as bithionol, niclosamide and loxapine can
pharmacologically activate these channels. Loxapine is more selective towards
KNa1.1 unlike bithionol, which also activates KCa1.1. Quinidine is known to ame-
liorate the symptoms of malignant migrating partial seizures in infancy by blocking
KNa1.1 currents (Bearden et al. 2014; Yang et al. 2006; De Los Angeles Tejada et al.
2012). The mechanism of quinidine-mediated amelioration of these symptoms is not
very clear as selectivity of quinidine is not limited to KNa1.1 channels. Various
non-specific KNa1.1 blockers include Ba2+, clofilium and bepredil (Bhattacharjee
et al. 2003).
Similarly, KNa1.2 is less explored and reportedly activated by the fenamate class
of NSAIDs like niflumic acid, with low potency. These agents are known to
uncouple the channels from Na+ or transmembrane voltage-regulated modulation
causing greater conductance of current. As they affect other channels including
KCa1.1, this action is non-specific in nature. Other pharmacological blockers of
these channels include clofilium, isoflurane and quinidine (Bhattacharjee et al.
2003; Berg et al. 2007).
The selective pharmacological agents of KCa5.1 are not known yet. However, this
channel is blocked by Ba2+, quinine and quinidine up to some extent (Sánchez-
Carranza et al. 2015; Tang et al. 2010).

19.2.1.2 Modulators of SK Channels (KCa2 Family)

SK channels are smaller conducting channels underlying the afterhyperpolarization
(AHP) currents in the regulation of Ca2+ influx and electrical signalling in neurons.
The functional elucidation of the SK channel has benefited from the discovery of the
SK channel activators. 1-Ethyl-2-benzimidazolinone (1-EBIO) is the prototype of
the activators (Pedarzani et al. 2005; Faber and Sah 2007). 6,7-Dichloro-1H-indole-
2,3-dione-3-oxime (NS309) is also well known as a selective activator of SK
including all the three subtypes (Strøbæk et al. 2004). The first selective KCa2.1
activator, GW542573X, is capable of activating the channel even in the absence of
Ca2+ and is reported to influence the gating process by binding to Ser293 in S5 near
the physical gate (Hougaard et al. 2009). CyPPA is a useful pharmacological tool to
distinguish SK2/SK3 from SK1/IK as these channels have overlapping expression
patterns (Hougaard et al. 2007).
The SK channels are inhibited by (i) natural peptide toxins like bee venom,
apamin and scyllatoxin (lelurotoxin I), (ii) neuromuscular blockers like tubocurarine,
19 Pharmacology of Potassium Channels 657

atracurium and pancuronium, (iii) antiseptics such as dequalinium and

(iv) bis-quinolium analogues such as UCL1684, UCL1848 and UCL1530 (also
refer to Table 19.1) (Wei et al. 2005; Ledoux et al. 2006; Weatherall et al. 2010;
Campos Rosa et al. 2000). UCL1684 is an about 5000 times more selective blocker
of SK compared to IK channels (Fanger et al. 2001; Rosa et al. 1998).

19.2.1.3 Modulators of IK Channels (KCa3.1)

IK channel activators may be of therapeutic interest in hypertension, peripheral
vascular diseases and cystic fibrosis. Although not very specific, 1-EBIO and clinical
benzoxazoles zoxazolamine and chlorzoxazone are described as potential activators
of KCa3 or IK channels. Few isatin derivatives have been extensively studied as IK
channel openers (Syme et al. 2000).
ChTx possesses higher affinity towards the IK channels in T-cells and inhibits
them. IbTx, maurotoxin and their recombinant variants are effective blockers of IK
in thymocytes and erythrocytes (Coghlan et al. 2001; Castle et al. 2003). The
non-selective IK channel blocker clotrimazole reduces T-cell proliferation and
secretion of IFN-γ in T-cells to produce anti-proliferative effects (Jensen et al.
1999). These blockers are believed to inhibit the dehydration of RBCs and, there-
fore, identified as effective therapy for sickle cell anaemia (Goodman et al. 1998).
Clotrimazole is also considered as one of the pharmacological strateies for treatment
of sickle cell anaemia due to its inhibitory effect on the IK channels of RBCs. Newer
analogues of nitrendipine as well as oxime and malonate derivatives are claimed to
possess a selective inhibitory effect on IK (Jensen et al. 2003). TRAM-34
(triarylmethane) is also a potent and selective IK blocker which exhibited in vitro
immunosuppression.

19.3 Inwardly Rectifying K+ Channels (Kir Channels)

Sir Bernard Katz first described the membrane conductance attributed to Kir
channels. He discovered that in frog skeletal muscle fibres, K+ ions move more
readily into the inside compared to the outside (called inward rectification) (Doupnik
2017). These channels were so named because of this characteristic degree of inward
rectification, i.e. prominent asymmetrical conduction of potassium in the inward
direction compared to the outward current (Coghlan et al. 2001). The inward
rectification is observed due to the blockade of depolarized potential (outward
current) by intracellular Mg2+ ions, natural polyamines, putrecine, spermidine and
spermine (Kurata et al. 2004; Köhling and Wolfart 2016; Jiménez-Vargas et al.
2017; Nichols and Lopatin 1997).
X-ray crystallography has detailed the 3D structure of Kir channels to the atomic
level. Structurally, Kir channels are the simplest among the K+ channel families, with
four subunits formed of 2-TM domains separated by a segment of pore-forming
(P) elements (as shown in Fig. 19.1) (Humphries and Dart 2015). The transmem-
brane domain regulates the gating and ion selectivity, whereas the cytoplasmic
domain is involved in the control of gating by G-proteins, Na+ ions and nucleotides.
658 S. Deka et al.

Kir channels conduct K+ ions to a greater extent under hyperpolarization which

decreases under depolarization, thereby maintaining the resting membrane potential
and cellular excitability.
In humans, Kir channels are classified into seven subfamilies encoded by
15 known genes (denoted KCNJx). These seven subfamilies are (1) Kir1.1 or
ROMK1 (KCNJ1), (2) Kir2.1–2.4 (KCNJ2, 4, 12, 14), (3) Kir3.1–3.4 (KCNJ3,
6, 9, 5), (4) Kir4.1–4.2 (KCNJ10, 15), (5) Kir5.1 (KCNJ16), (6) Kir6.1–6.2
(KCNJ8, 11) and (7) Kir7.1 (KCNJ13). These subfamilies differ in their properties
and kinetics producing numerous physiological activities. The Kir channel family
has also been classified into four subsets depending on functional characteristics:
strong inward-rectifier (Kir2.x), G-protein-activated inward-rectifier (Kir3.x),
ATP-sensitive K+ channels (Kir6.x/SURx) and K+-transport channels (Kir1.1, Kir4.
x, Kir5.1 and Kir7.1) (Alexander et al. 2017). The physiological activities of Kir
channels are dependent on localization of channel and regulation of pore opening
and ion flux by ions, lipids, nucleotides, polyamines or other intracellular proteins.
Decrease in intracellular ATP opens up the ATP-sensitive K+ channels (named so),
exhibiting inward rectification. Similarly, G-protein-activated Kir shows inward
rectification via pertussis toxin (PTX)-sensitive G-proteins, orchestrating the cellular
excitability and G-protein signalling. A negatively charged Asp residue in the TM2
helix causes strong inward rectification by increasing the affinity for Mg2+ as in the
case of Kir2.x (Hibino et al. 2010).

19.3.1 Pharmacology of Kir Channels

The Kir channels are widely distributed in neurons, glial cells, cardiac myocytes,
blood cells, osteoclasts, endothelium, epithelium and oocytes exhibiting distinct
roles in both normal and pathophysiological conditions. Alteration in the functions
of these channels may lead to several genetically linked and acquired diseases.
Genetic studies have linked many rare human diseases with Kir channel gene
mutations. For example, recessive loss-of-function mutations in the KCNJ1 gene
or Kir1.1 cause type II Bartter syndrome (Doupnik 2017; Dworakowska and Dolowy
2000), and loss-of-function mutations in Kir2.1 are associated with Andersen syn-
drome (LQT7) (Hibino et al. 2010; Giudicessi and Ackerman 2012). Acquired
pathological conditions such as electric remodelling associated with atrial fibrillation
(AF) can occur due to upregulation of Kir2.1 and Kir3.1/3.4 channel activity. KATP
channels are implicated in glucose metabolism and contractility of heart.
Generalized seizures and SeSAME syndrome are associated with Kir4.1
channelopathy (Bhave et al. 2010; Seifert et al. 2018). Permanent neonatal diabetes
occurs due to gain-of-function mutation in Kir6.2/SUR1. Moreover, new therapeutic
approaches, such as designing of K+ sparing diuretics for CHF and HT by targeting
renal Kir1.1 channels and treating AF by selectively inhibiting atrial Kir3 channels,
have spurred research interest towards this family.
At physiological voltage, generation of PIP2 by ATP-dependent kinases activates
Kir channels (Huang et al. 1998). Ba2+ and Cs+ (classical blockers) are known for
19 Pharmacology of Potassium Channels 659

effective blocking of majority of the Kir channels and are used to explore their
physiological roles (González et al. 2012). This inhibition is more prominent when
the membrane is hyperpolarized. Despite limited number of Kir channel blockers,
physiological and pharmacological assays have explored compounds with selectiv-
ity towards particular types of Kir channels. These compounds are discussed under
specific subtypes.

19.3.1.1 Modulators of Classical or Strong Inward-Rectifier K+ Channels

(Kir2.x)
Recent electrophysiological and pharmacological studies showed that several
reagents possess Kir current blocking properties, but their effects are not specific to
the channels. Specific activators and blockers for classical Kir channels are not well
known. PIP2 can act as an endogenous activator of the Kir2.1 channel. The endoge-
nous Kir2.1 channel blockers include Mg2+, spermine, spermidine and putrescine
(also refer to Table 19.1) (Yamashita et al. 1996; Ishihara et al. 1996). The side effect
of chloroquine, i.e. lethal ventricular arrhythmia, is also linked to the blockade of
Kir2.1 currents. Memantine (MEM), a derivative of amantadine, blocks Kir2.1
currents and regulates the functional activities of microglia or macrophages (Tsai
et al. 2013). The homomeric Kir2.x channels demonstrated a substantial difference in
the sensitivity towards Ba2+ and Cs+ in the heterologous expression system. The
blockage of Kir2.1 by Ba2+ is highly dependent on voltage (Alagem et al. 2001).
Intracellular Mg2+ can act as an endogenous inhibitor of Kir2.2 channels. The gating
mechanism of Kir2.2 can be inhibited by the classical blockers Ba2+ and Cs+
(Takahashi et al. 1994). Intracellular Mg2+, putrescine, spermine and spermidine
are well-known endogenous blockers of Kir2.3 currents (Alexander et al. 2017;
Lopatin et al. 1994). Kir2.3 channels are moderately inhibited by the first-generation
H1-antihistaminics (mepyramine and diphenhydramine) and partially inhibited by a
protein tyrosine kinase inhibitor, genistein. Similarly, intracellular Mg2+ acts as an
endogenous inhibitor of Kir2.4 channels, while Cs+ and Ba2+ act as blockers of the
channels. The blockade of human Kir2.4 channels by Cs+ is more voltage dependent
compared to the Ba2+-mediated blockade (Hughes et al. 2000). Moreover, enhance-
ment in tonic activity and increase in frequency of induced spike discharge in
hypoglossal motoneurons (HMs) caused by Ba2+-mediated blockade indicate that
Kir2.4 channels are the major regulators of excitability of motoneurons in situ
(Töpert et al. 1998).

19.3.1.2 Modulators of G-Protein-Activated Inward-Rectifier K+

Channels (GIRK or Kir3.x)
Gβγ protein-mediated PIP2 activation by lipid kinases causes endogenous activation
of all subtypes of GIRK channels (Huang et al. 1998). ML297 is the first identified
selective and potent molecule which can act as an activator of Kir3 or GIRK. This
molecule possesses good antiepileptic properties (Wydeven et al. 2014; Kaufmann
et al. 2013). Tertiapin, a toxin obtained from honey bee venom, showed Kir3.1/3.4
blocking property. A modified oxidation-resistant toxin, tertiapin Q, blocks IKACh in
isolated cardiac myocytes without affecting other currents. Also, cardiac IKACh
660 S. Deka et al.

channels are blocked by quinine, quinidine and verapamil. Antipsychotics such as

haloperidol, clozapine, thioridazine and pimodine modulate the combination of
subunits, i.e. Kir3.1/3.2 and Kir3.1/3.4, in heterologous expression in Xenopus
oocytes (Kobayashi et al. 2004). Similarly, antipsychotics like imipramine, desipra-
mine, amitriptyline, citalopram, fluoxetine and maprotiline act on Kir3.2, Kir3.1/3.2
and Kir3.1/3.4 channels, which are also targeted by general anaesthetics (GAs) like
halothane, isoflurane, enflurane and F3 (1-chloro-1,2,2-trifluorocyclobutane).
Bupivacaine, a local anaesthetic, acts on Kir3.2, Kir3.1/3.2, Kir3.1/3.4 and Kir3.4
by binding with cytoplasmic regions of the channels without any subunit combina-
tion specificity. Other drugs targeting Kir3.x subunits include MK-801 (NMDAR
antagonist), QX-314 (classical cation channel blocker), ifenprodil (NMDAR antag-
onist) and R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-
benzazepine hydrochloride (dopamine D1R antagonist). Though these compounds
are known to act on Kir3.x subunits, the binding sites of most of them were not
investigated. Montandon et al. (2016) reported that fentanyl regulates GIRK
channel-mediated rhythmic breathing and contributes to the respiratory depression
by μ-opioid receptors (MOR) (Montandon et al. 2016).

19.3.1.3 Modulators of ATP-Sensitive K+ Channels (KATP or Kir6.x/SURx)

KATP channels have the most therapeutic potential among other subtypes and are
modulated by two major classes of drugs, sulfonylureas (SUs) and potassium
channel openers (KCOs). SUs such as glipizide, glimepiride, glibenclamide, tolbu-
tamide and acetohexamide are well known to stimulate insulin secretion in type II
diabetes mellitus (T2-DM) patients (Garcia and Kaczorowski 2005; Sturgess et al.
1985; Challinor-Rogers and McPherson 1994). The resulting effect is due to binding
of the drugs with sulphonylurea receptors (SURx) of KATP channels, causing
membrane depolarization by inhibiting K+ efflux in the pancreatic β-cells (Abraham
et al. 1999). SUs confer more sensitivity towards Kir6.2/SUR1 compared to Kir6.2/
SUR2A (Rubaiy 2016).
KATP channel openers constitute the diverse class and the largest number of small
molecules modulating the K+ channels. Potassium channel openers (KCOs) such as
nicorandil, pinacidil and diazoxide activate the KATP channels (Ackerman and
Clapham 1997; Lawson 2000; Camerino et al. 2007). KCOs are effective in the
therapeutic management of myocardial ischemia, CHF, urinary incontinence, bron-
chial asthma and certain skeletal muscle myopathies (Shieh et al. 2000; Ackerman
and Clapham 1997; Lawson 2000; Camerino et al. 2007). The sensitivity of the
KCOs towards native KATP channels is attributed to the expression of SUR subtypes
in different tissues, i.e. pancreatic β-cell (SUR1), cardiac (SUR2A) and smooth
muscle (SUR2B). The pancreatic β-cell KATP channels are readily opened by
compounds such as diazoxide, which is clinically approved for treatment of
insulinoma-associate hypersecretion of insulin and persistent hyperinsulinaemic
hypoglycaemia of infancy (PHHI) (Akopova 2018). In contrast, these channels are
weakly activated by pinacidil and remain unaffected by nicorandil or cromakalim,
whereas cardiac channels are activated by nicorandil, pinacidil and cromakalim but
not affected by diazoxide, and the smooth muscle KATP channels are activated by all
19 Pharmacology of Potassium Channels 661

the three compounds (Robertson and Steinberg 1990; Edwards and Weston 1995).
The pharmacological properties are known to produce, only when there is
co-expression of Kir6.2 with an appropriate SUR subtype leading to KATP conduc-
tance (Lawson 2000; Quast 1992). The side effects of SUs are also related to their
ability to cross-react with other KATP subtypes. Bimakalin (a selective KCO)
exhibited a dose-dependent vasodilation but failed to show anti-ischemic benefits
in patients with coronary artery disease (CAD) during exercise-induced angina
pectoris (Chan et al. 2008). Levosimendan, a KATP channel opener, showed signifi-
cant reduction in pulmonary capillary wedge pressure in severe low-output HF
patients following cardiac surgery and peripartum cardiomyopathy. Levosimendan
has entered into the clinical studies for heart failure, amyotrophic lateral sclerosis,
ventricular dysfunction, myocardial infarction, hip fracture and cardiorenal syn-
drome (Rubaiy 2016). Another new antihypertensive drug, iptakalim, synthesized
by Thadweik Academy of Medicine, China, also activates KATP channels in the
endothelium of resistance blood vessels (Wang et al. 2015; Duan et al. 2011).
Iptakalim (KCO) as well as fluoxetine (classical antidepressant) showed alleviation
of chronic mild stress depressive behaviour in wild-type mice. However, these
symptoms are partially ameliorated in Kir6.2
/
mice (Fan et al. 2016).
5-Hydroxydecanoic acid (5-HD) is a selective mitochondrial and plasma mem-
brane KATP channel blocker. It is highly used in the study of the physiological role of
mito-KATP channels. Non-sulfonylurea KATP channel blockers such as meglitinide,
nateglinide, repaglinide and mitiglinide are also used to treat T2-DM. These drugs
act by inhibiting pancreatic β-cell KATP channels, i.e. blocking Kir6.2/SUR1
channels. Many derivatives of P1075, a cyanoguanidine K+ opener, have been
known to antagonize the vascular KCOs. These new-generation drugs called PNU
compounds include PNU-37883A, PNU-89692, PNU-97025E and PNU-99963
(Rubaiy 2016; Chowdhury et al. 2017).

19.3.1.4 Modulators of K+-Transport Channels (Kir1.1, Kir4.x, Kir5.1,

Kir7.1)
Unlike other subtypes, the advancement of molecular pharmacology of ROMK or
Kir1.1 is quite limited. Tertiapin (bee venom peptide) is a selective and potent
blocker of the rat Kir1.1 current but exhibits 100 times less potency in human
isoforms. Tertiapin and δ-dendrotoxin bind to the external vestibule of the K+
conduction pore to inhibit Kir1.1 channels (González et al. 2012). Two synthetic
compounds, VU590 and VU591, were identified and screened at Vanderbilt Univer-
sity, USA. These compounds inhibit rat Kir1.1 with IC50 values 290nM and 240nM,
respectively. VU591 contributes to basal K+ secretion by inhibiting ROMK in DCT
without affecting Na+ transport (Bhave et al. 2011).
Tricyclic antidepressants (TCAs) such as imipramine, desipramine, nortryptyline
and amitriptyline block Kir4.1 currents. Voltage-independent inhibition of Kir4.1
currents is observed in the case of selective serotonin reuptake inhibitors (SSRIs)
such as fluoxetine, fluvoxamine and sertraline. The therapeutic activity and adverse
effects of TCAs and SSRIs are thought to be associated with the blockade of
astroglial Kir channels by interacting with T128 and E158 residues of the conduction
662 S. Deka et al.

pore of Kir4.1 channels. No effective blockers of Kir5.1 are reported till date. While
Kir7.1 remains unusually insensitive to the typical blocking property of Ba+ and Cs+,
interestingly, no other activators or blockers are known for this channel.

19.4 Two P Domain K+ Channels (K2P Channels)

The first potassium channel containing two P domain in tandem was the TOK-1
channel discovered from the yeast Saccharomyces cerevisiae (Goldstein et al. 2001;
Ketchum et al. 1995). These channels are referred to as “twin pore” or “tandem pore”
as they possess nonconventional topology, i.e. dimers of dimers: two subunits
forming a mature channel containing two non-identical pore-forming P domains
(P1 and P2) which are arranged in tandem (as shown in Fig. 19.1) (Tian et al. 2014;
Humphries and Dart 2015; Schneider et al. 2014). Most of the K2P channels conduct
voltage-independent outward current as they lack the voltage-sensing S4-TM
domain (Coghlan et al. 2001; Goldstein et al. 2001). These channels also act as the
classical background “leak” K+ channels or open rectifiers and maintain negative
resting membrane potential (Köhling and Wolfart 2016; Piechotta et al. 2011).
Fifteen mammalian KCNK genes encode K2P channels. These channels were
distributed among six clades on the basis of sequence homology and pharmacologi-
cal characteristics. These are as follows: (1) two-pore domain weak inward-
rectifying K+ channels or TWIK (K2P1 or TWIK-1, K2P6 or TWIK-2, and K2P7)
are weak inward rectifiers sensitive to pH; (2) TWIK-related K+ channels or TREK
(K2P2 or TREK-1, K2P4 or TRAAK, and K2P10 or TREK-2) are mechano-gated
channels regulated by several stimuli – pH, stretch, osmolarity, temperature,
neuroprotective agents and volatile anaesthetics; (3) TWIK-related acid-sensitive
K+ channels or TASK (K2P3 or TASK-1, K2P9 or TASK-3, K2P15 or TASK-5) are
inhibited by extracellular acidification and hypoxia; (4) TWIK-related alkaline
pH-activated K+ channels or TALK (K2P5 or TASK-2, K2P16 or TALK-1, and
K2P17 or TALK-2) are sensitive to alkaline pH and oxygen concentration; (5) tandem
pore domain halothane-inhibited K+ channels or THIK (K2P12 or THIK-2, K2P13 or
THIK-1) are sensitive to halothane and arachidonic acid; (6) TWIK-related spinal
cord K+ channels or TRESK (K2P18) are sensitive to free acids, protons, heat,
anaesthetics and increased membrane tension (Jiménez-Vargas et al. 2017;
Feliciangeli et al. 2015; Renigunta et al. 2015; Talley et al. 2003; Lesage and
Lazdunski 2000).

19.4.1 Pharmacology of Two P Domain K+ Channels

Given their critical physiological roles, K2P channels represent a major target for the
modulation of the physiological changes and pathological states. For example, K2P1
and K2P2 channels play an important role in the regulation of atrial size and heart rate
and are, therefore, a putative target for antiarrhythmic drugs. Similarly, K2P18
channels are good candidates for pain management as the pain signalling pathway
19 Pharmacology of Potassium Channels 663

is strongly controlled by the K2P18 channels. Moreover, K2P channels are implicated
in arrhythmia (K2P1), depression (K2P2), atrial fibrillation (K2P3), migraine (K2P18),
cancer (K2P1, K2P5, K2P9) and inflammation (K2P12/13) as well as in the neuro-
pathic pain (K2P18) (also refer to Table 19.1) (Feliciangeli et al. 2015; Gada and
Plant 2019; Li and Toyoda 2015).
Considering the lead optimization and drug discovery process, several practical
limitations were observed. The unique structure of the K2P family restricted the easy
extrapolation from the available data of other K+ channel subfamilies. The lack of a
structural basis of the K2P channels, until the explanation of the crystal structure of
K2P1 and K2P4 in 2012, had stymied the development of pharmacophores of these
channels (Gada and Plant 2019). Moreover, the electrophysiological characterization
for understanding the biological significance of K2P channels has been impeded by
the poor or absent heterologous expression in Xenopus oocytes or COS-7 cells
(Enyedi and CzirjáK 2010). The ubiquitous distribution of many subunits also limits
the therapeutic modulation of specific K2P channels. To develop any pharmacologi-
cal modulator of these subunits, the researchers must overcome these barriers with
available HTS techniques and necessary measures. Nevertheless, the numbers of
small molecules and drugs interacting with the K2P channels are steadily increasing
over time. These channels are weakly sensitive or insensitive to the classical K+
channel blockers including 4-AP, TEA, Cs+ and Ba+ (Dogan et al. 2019). Some
other potent modulators have already been identified and are discussed in the
succeeding section (Tian et al. 2014; Es-Salah-Lamoureux et al. 2010; Kasap and
Dwyer 2018; Gada and Plant 2019).

19.4.1.1 Modulators of the TREK Family

There is evidence that putative inhaled anaesthetics also target K2P channels (Patel
and HonorÉ 2001; Olschewski et al. 2017). The minimum alveolar concentration
(MAC) of general anaesthetics such as chloroform, desflurane and halothane vari-
ably increased in the TREK-1 knockout model. Nitrous oxide (N2O), cyclopropane
and xenon also activate the K2P2 channels (Hudson et al. 2019). Similarly,
halogenated inhaled GAs such as sevoflurane and isoflurane exhibited selective
activation of K2P channels subtypes like TREK-1, TRAAK, TASK-3 and TRESK.
The anaesthetic effect is probably due to the membrane hyperpolarization via the
K2P current in combination with GABAA receptor activation (González et al. 2012).
The fenamate class of NSAIDs (e.g. flufenamic acid, mefenamic acid and niflumic
acid) selectively activates K2P channel by interacting with the N-terminals (Gada and
Plant 2019). Flufenamic acid at 100 μM concentration produced a 250% enhance-
ment of the TREK-1 current, while mefenamic acid and niflumic acid at 100 μM
concentration exhibited 150–180% enhancement of the K+ current (Vivier et al.
2015). Recently, a selective opener of TREK-1 and TREK-2, GI-530139, showed
effectiveness in hyperpolarizing the dorsal root ganglia (DRG) neurons in rats (Gada
and Plant 2019; Loucif et al. 2018). An anti-ischemic and anticonvulsant drug,
riluzole, potentiates TREK-1 as well as TRAAK currents in a dose-dependent
manner. Riluzole is currently used in the treatment of amyotrophic lateral sclerosis
(ALS) (Vivier et al. 2015; Lesage 2003).
664 S. Deka et al.

Sipatrigine, a neuronal Na+ and Ca2+ channel inhibitor, reversibly inhibits both
TREK-1 and TRAAK in a dose-dependent manner. This effect in combination with
glutamate inhibition makes this drug a choice for treatment of depression (Lesage
2003; Meadows et al. 2001; Tsai 2008). 3-N-Butylphthalide (NBP) extracted from
celery seed also reversibly inhibits TREK-1 current. This neuroprotective agent is
used in China for treatment of ischemic stroke (Ji et al. 2011). SSRIs such as
fluoxetine reversibly block TREK-1 and related TASK-3 channels in a voltage-
independent manner. Similarly, norfluoxetine, active metabolite of fluoxetine, is a
more potent blocker of TREK-1 current (Kennard et al. 2005). In addition to SSRIs,
several other antipsychotics such as chlorpromazine, loxapine, haloperidol,
pimozide and fluphenazine also exhibited dose-dependent and reversible inhibition
of both TREK-1 as well as TREK-2 but not TRAAK channels (Thümmler et al.
2007). Other pharmacological classes of drug inhibiting TREK-1 current include
antihypertensive dihydropyridines (e.g. amlodipine and nifedipine), calcium
antagonists (e.g. flunarizine) and antiarrhythmic agents (e.g. mexiletine and
propafenone) (also refer to Table 19.1) (Vivier et al. 2015).

19.4.1.2 Modulators of the TASK Family

TASK-1 and TASK-3 are insensitive to many drugs unless they are used at higher
concentrations. Both the channels are opened by inhalational GAs at clinically
relevant concentrations, targeting a short cytoplasmic region nearer to 4-TM (Patel
et al. 1999). Classical blockers of K+ including Ba2+, TEA, ChTx, aminopyridines
and apamin also produced modest effects on TASK-1 and TASK-3 channels. Drugs
such as quinidine, bupivacaine and ethanol partially inhibit the TASK-1 and TASK-
3 currents, while fluoxetine and norpropoxyphene block only TASK-1 current (also
refer to Table 19.1). Ruthenium red dye is a useful tool to separate TASK-1 and
TASK-3 channels as it nearly causes complete blockade of TASK-3 but has little
effect on TASK-1. However, this cationic dye is not employed to study TASKs in
native systems as it non-specifically blocks many other ion channels (Kim et al.
2000; HajdÚ et al. 2003; CzirjáK and Enyedi 2003).
Anandamide and synthetic cannabinoid receptor agonists such as WIN552122 and
CP55940 are the potent blockers of the human TASK-1 current. Routine use of
anandamide and methanandamide to identify TASK-1 channels in native tissues has
been disproved as they also possess comparable potency for TASK-3 in different
species (Enyedi and CzirjáK 2010). Two TASK channel antagonists, PK-THPP and
A1899, showed effective breathing stimulant property in rats (Cotten 2013). Similarly,
terbinafine was identified as a selective and potent activator of TASK-3 channels in the
whole-cell patch clamp method (Wright et al. 2017). Tian et al. (2019) discovered a
small molecule, NPBA (N-(2-((4-nitro-2-(trifluoromethyl)phenyl)amino)ethyl)
benzamide), which possesses higher selectivity and potency as a novel TASK-3
activator (Tian et al. 2019). Modulators of TASK-5 channels are not yet known.

19.4.1.3 Modulators of TALK, THIK and TRESK Families

Similar to other K2P channels, TASK family members (i.e. TASK-2, TALK-1 and
TALK-2) are also least affected by the classical blockers (e.g. 4-AP, TEA, Cs+).
19 Pharmacology of Potassium Channels 665

Among volatile GAs, chloroform is the potent activator of TASK-2 (Patel and
HonorÉ 2001; Gray et al. 2000; Yost 2000). Clofilium inhibits TASK-2, while
drugs like quinidine, bupivacaine and lidocaine are strong inhibitors of TASK-
2 and weak inhibitors of TALK-1 (Hayashi and Novak 2013; Girard et al. 2001;
Gutman et al. 2005). Whole-cell patch clamp and two-electrode voltage clamp
studies demonstrated the sensitivity of antiarrhythmic drugs to TALK-2 channels
in the Xenopus oocytes expression system. Significant activation was exhibited by
quinidine, propafenone, mexiletine, metoprolol and propranolol, while drugs such as
sotalol, verapamil, amiodarone and ranolazine inhibit the TALK-2 current (also refer
to Table 19.1). Human TALK-2 channels were reported to be sensitive towards
multiple antiarrhythmic drugs (Staudacher et al. 2018a). Activation of the THIK-1
channel by arachidonic acid and inhibition by halothane allow this channel to be
easily distinguished from TREK, which is activated by halothane (Kim 2005).
Therapeutic modulation of THIK-1 channels can make them a suitable target of
antiarrhythmic drugs. Drugs such as propafenone, mexiletine, propranolol and
lidocaine are known to inhibit THIK-1 channels (Staudacher et al. 2018b).
TRESK channels are activated by volatile anaesthetics including isoflurane,
desflurane, sevoflurane and halothane within the clinically used concentrations.
Cloxyquin activates the TRESK channel independent of Ca2+/calcineurin associa-
tion and is hence used for examining TRESK conductions in native cells (Lengyel
et al. 2017). Additionally, TRESK (K2P18) is insensitive to several classical K+
channel blockers, such as 4-AP, apamin and CsCl, and KATP channel blockers, like
tolazamide and glipizide (also refer to Table 19.1). Arachidonic acid, quinine and
quinidine are potent blockers of this channel, while extracellular Ba2+-mediated
inhibition is observed at higher concentrations. Amide local anaesthetics such as
bupivacaine, tetracaine, rupivacaine, chlorprocaine, mepivacaine and lidocaine are
reported to influence mouse and human TRESK. About 240 substances were tested
on mouse TRESK. Among these, zinc, mercuric ions and mibefradil (non-specific to
K2P channels) were found to efficiently inhibit the current. Antidepressants, fluoxe-
tine and sipatrigine also block the TRESK current along with the TREK family
members, while lomotrigin (an anticonvulsant) showed selectivity towards only
TRESK (10-5M). Loratadine (an antihistaminic) also exhibited TRESK inhibition
with a micromolar IC50 (Enyedi and CzirjáK 2015).

19.5 Voltage-Gated K+ Channels (Kv Channels)

The first ever cloned gene from Drosophila was the voltage-gated Shaker channel
followed by the phenotyping of the other potassium channel subtypes in insects, rats
and mammals including humans (Gutman et al. 2005). The Drosophila Shaker
channel acts as a prototype of all voltage-gated channels, consisting of a tetramer
of homologous α-subunits, each comprising 6-TM segments and a circumferentially
arranged membrane re-entering P-loop (Yellen 2002; Jensen et al. 2012). In brief,
the four S1–S4 segments act as voltage sensors, which contain arginine (positively
charged) in the S4 segment, while the K+ conduction pore is formed by the four
666 S. Deka et al.

S5-P-S6 sequences. The gating of the pore is regulated by pulling the S4-S5 linker
(Kuang et al. 2015; Pongs 1999; Sokolova et al. 2001; Kim and Nimigean 2016;
Wulff et al. 2009; Jackson 2017). Both the amino and carboxy terminals are found to
lie on the intracellular side of the membrane (as shown in Fig. 19.1). Membrane
depolarization activates majority of the Kv channels, whereas hyperpolarization
leads to closing of the channel. The Kv channels are encoded by about 40 genes
constituting the largest K+ family, which is categorized under 12 subfamilies. This
wide diversification of the channel arises from several factors, such as
heteromultimerization, modifier subunits, alternate mRNA splicing, post-
translational modification and association of other proteins like calmodulin and
minK (Gutman et al. 2005). The voltage-gated channels are mainly classified as
follows: Kv1 (Kv1.1–1.8), Kv2 (Kv2.1–2.2), Kv3 (3.1–3.4), Kv4 (Kv4.1–4.3), Kv5
(Kv5.1), Kv6 (Kv6.1–6.4), Kv7 (Kv7.1–7.5), Kv8 (Kv8.1–8.2), Kv9 (Kv9.1–9.3),
Kv10 (Kv10.1–10.2), Kv11 (Kv11.1–11.3) and Kv12 (Kv12.1–12.3) subfamilies.

19.5.1 Pharmacology of Voltage-Gated K+ Channels

The Kv channels are often expressed together with voltage-gated sodium or calcium
channels as they regulate the action potential firing. As the Kv current controls the
excitability of the cell, the pharmacological modulation of these channels may
regulate the cellular excitability. Therefore, Kv channels play a significant role in
Ca2+ signalling, proliferation, volume regulation and secretion as well as migration
(Wulff et al. 2009). Not only this, the Kv channel is also found to be involved in the
regulation of cellular activation in lymphocyte and cancer cells; consequently, Kv
blockers have showed effectiveness in inhibition of proliferation and cellular activa-
tion (Chandy et al. 2004; Pardo et al. 1999; Comes et al. 2013). Overall, mutations or
any defects in Kv channel genes are associated with a number of diseases, ranging
from autoimmune diseases to cancer to cardiovascular, neurological and metabolic
disorders. For example, episodic ataxia type 1, or EA1, is associated with loss-of-
function mutations of the KCNA1 gene (Kv1.1) (Maljevic and Lerche 2013;
Lehmann-Horn et al. 2003), whereas loss-of-function mutations of Kv1.1/ Kv1.2
lead to neuromyotonia associated with small-cell lung cancer (SCLC) cells and
limbic encephalitis (Camerino et al. 2007). Kv channels are also implicated in
neuronal apoptosis (Kv1.1, Kv1.3), ischemic cell death (Kv1.5), epilepsy (Kv1.1,
Kv1.2, Kv1.4, Kv3.2, Kv4.2, Kv4.3), Alzheimer’s disease (Kv3.4, Kv4.2), tinnitus
(Kv7.1– Kv7.5) pain (Kv7.1–Kv7.5) and neuropsychiatric disorders (Kv7.1–Kv7.5)
(also refer to Table 19.1) (Tsantoulas 2015; Shah and Aizenman 2014; Langguth
et al. 2016).
The pharmacological modulation of Kv channels can be effective in the therapeu-
tic management of various human channelopathies, which is further underlined by
the transgenic mice models. Metal ions, peptide toxins and small molecules are well
known to regulate Kv current conduction by either external or internal binding to the
conducting pore or by altering the channel gating mechanism (Wulff and Zhorov
19 Pharmacology of Potassium Channels 667

2008). However, the lack of well-defined crystal structures of physiologically

important channels (e.g. Kv1.5, Kv7.2 and Kv11.1) is one of the major hurdles in
the rational design of modulators. Importantly, impressive progress has been made in
the structural study in the last decade. Beside this, antibodies and toxins have been
continuously engineered to obtain better delivery of the drug to the channel-
expressing cells (Wulff et al. 2009; Zhou et al. 1998). The early-stage development
of Kv modulators presents many challenges, but the fact cannot be denied that there
are considerable opportunities for forthcoming success. Tremendous work has been
devoted to identify the agents that interact with the voltage-gated K+ channels with
higher affinity.

19.5.1.1 Modulators of the Kv1 Family

The Kv1 family is sensitive to the classical non-peptidyl blockers (TEA, 4-AP), but
dramatic variation is observed between members of the families (Mathie et al. 1998).
Kv1.1 channels are selectively blocked by the dendrotoxins (DTx) isolated from
green and black mamba snakes. Among the different DTx, δ-DTx and dendrotoxin
K are highly selective and currently used as a diagnostic tool to determine the
presence of Kv1.1 channels in real cells (Vacher et al. 2007). Other well-known
peptide toxins such as ChTx, maurotoxin (MTx), noxiustoxin (NxTx) and kaliotoxin
(KTx) also block Kv1.1 channels (Kaczorowski and Garcia 1999). Many newer
toxins have been isolated and identified as modulators of these channels, but the
selectivity can only be achieved by protein engineering. Small molecules inhibiting
this channel also include capsaicin, flecainide, nifedipine, diltiazem and
resiniferatoxin (also refer to Table 19.1) (Gutman et al. 2005).
The Kv1.2 channel is the most abundant Kv1 subtype in the mammalian CNS.
Due to the lack of a specific tyrosine residue for TEA binding, the Kv1.2 channel
offers resistance to the higher concentration of external TEA. However, 4-AP shows
sensitivity at the normal concentration range required to block the Kv1.1 channel.
Tityustoxin-Kα from Tityus serrulatus (a Brazilian scorpion) blocks Kv1.2 subunits.
These subunits are also inhibited by δ-DTx and dendrotoxin K. The elucidation of
the functional roles of Kv1.2 channels in CNS can be done by using a 28-amino-acid
peptide toxin, OsK2, isolated from Orthochirus scrobiculosus (Central Asian scor-
pion) (Dudina et al. 2001). Nifedipine, flecainide, resiniferatoxin and anandamide
have also been reported to block Kv1.2 channel conduction.
Scorpion venom NxTx was the first identified peptidyl blocker of potassium
channels. This venom inhibited the Kv1.3 channel obtained from heterologous
expression in Xenopus and in Jurkat cells (Garcia et al. 1997; Swanson et al. 1990;
Sands et al. 1989). ChTx has been known to inhibit Kv1.3 channels in human T-cells
and neuronal tissue (Swanson et al. 1990). In search of selective toxins, margatoxin
(MgTx) from scorpion Centruroides margaritatus was characterized as a high-
affinity blocker against Kv1.3 channels (Augustynek et al. 2016). Importantly,
MgTx does not show any effect on ChTx-sensitive K+ channels (Garcia-Calvo
et al. 1993). Other toxins from scorpion are KTx, agitoxins (AgTx), Pandinus
imperator venom (Pi1, Pi2 and Pi3) and Centruroides limbatus venom
(hongotoxin-1) which also inhibit Kv1.3 channels (Panyi 2005). Peptidyl toxin
668 S. Deka et al.

from sea anemone (e.g. BgK, ShK and AsKS) and spider venom (e.g. hanatoxin
1 and hanatoxin 2) are also well-known blockers of the Kv channels (Garcia et al.
1997). A natural triterpene, correolide, is a potent blocker of Kv1.3 but showed
affinity for other members of the Kv1 family (Felix et al. 1999). Pharmacological
agents such as naltrexone, sulfamidbenzamidoindane, UK-78282, WIN-17317-3
and CP 339818 have been extensively studied for their inhibitory effect on Kv1.3
channels (Coghlan et al. 2001; Hill et al. 1995).
Fairly mild acidosis (pH ¼ 6.5) can inhibit the Kv1.4 current due to the slow
recovery of N-type inactivation, which is a useful diagnostic characteristic of this
subtype (Claydon et al. 2000). Kv1.4 channels are blocked by 4-AP (0.7–13 mM) but
exhibit resistance to external TEA, DTx or similar toxins. Few small molecules are
known to inhibit the Kv1.4 current, including UK78282, riluzole, quinidine and
nicardipine (also refer to Table 19.1) (Gutman et al. 2005; Kuzmenkov et al. 2015).
Abundant distribution of the Kv1.5 channel makes it undoubtedly one of the most
important channels. They are insensitive to TEA while sensitive to 4-AP even at
relatively lower concentrations. Channel inhibition is exhibited by several therapeu-
tic classes including calcium channel blockers (e.g. verapamil and nifedipine),
antiarrythmic drugs (e.g. quinidine, clofilium and propafenone), antibiotics
(e.g. erythromycin) and antihistaminics (e.g. loratidine, terfenadine and ebastine)
(also refer to Table 19.1) (Zhang et al. 1997; Grissmer et al. 1994; Malayev et al.
1995; Gutman et al. 2005; Gutman et al. 2003).
Kv1.6 is sensitive to both TEA and 4-AP. Venoms like ChTx, MgTx, hongotoxin
and tumulustoxin were identified as the potent blockers of these channels. Kv1.7
channels are insensitive towards external TEA but blocked by 4-AP, nifedipine,
amiodarone, quinidine, flecainide and tedisamil, whereas reported blockers of Kv1.8
include Ba2+, ChTX, 4-AP, TEA, ketoconazole, pimozide and verapamil (Gutman
et al. 2005; Kuzmenkov et al. 2015; Gutman et al. 2003).

19.5.1.2 Modulators of the Kv2 Family

The Kv2 family (Kv2.1 and Kv2.2) is activated at low thresholds, so-called delayed
rectifier channels. Linoleic acid acts as an activator of the Kv2.1 channel, while no
activator is reported for Kv2.2. The classical blockers 4-AP and TEA are insensitive
to these channels. The isolated venom, hanatoxin, from Chilean tarantulas binds to
the S3/S4 linker to inhibit channel opening (Swartz and Mackinnon 1997a, b).
Hanatoxin is known to act as the gating inhibitor of the Kv2.1 channel. Several
blockers of Kv2.1 and Kv2.2 channels are well known. Both the channels are
sensitive to blockers like TEA and 4-AP. Specifically, the conduction of Kv2.1 is
inhibited by internal and external Ba2+, internal Mg2+, halothane and
tetrapentylammonium, whereas the Kv2.2 current is inhibited by quinine and phen-
cyclidine (Gutman et al. 2005).

19.5.1.3 Modulators of the Kv3 Family

The Kv3 family plays many important physiological and pharmacological roles,
especially in mammalian CNS. The unique feature of Kv3 includes channel activa-
tion at a depolarized potential which is more positive than
20 mV. Kv3.1 and Kv3.2
have showed little inactivation unlike Kv3.4, which exhibited rapid N-type
19 Pharmacology of Potassium Channels 669

inactivation (Rudy and Mcbain 2001). Kv3 manifests extraordinarily high sensitivity
towards 4-AP and TEA because of the presence of the tyrosine residue at the
carboxyl terminal of P-loop. Drugs like cromakalim, diltiazem, nifedipine, flecainide
and resiniferatoxin also inhibit Kv3.1 channels. The Kv3.2 current is blocked by
8-bromo-cGMP, verapamil, D-NONOate and 3-isobutyl-1-methylxanthine. Kv3.3 is
reported to show variable inactivation kinetics in contrast to other Kv3 family
members. The subunits of Kv3.3 are sensitive to hypoxia and hence implicated in
oxygen sensing in pulmonary vasculature (Guo et al. 2008). Blood-depressing
substance (BDS) from sea anemone, Anemonia sulcata, has two isoforms, BDS I
and BDS II, which are selective and effective blockers of Kv3.4 (also refer
to Table 19.1). Although the specificity of BDS I towards Kv3.4 has been
questioned, these toxins at higher concentrations were earlier employed as selective
tools for Kv3.4 (Riazanski et al. 2001; Yeung et al. 2005).

19.5.1.4 Modulators of the Kv4 Family

Kv4 (Kv4.1, Kv4.2 and Kv4.3) channels are strongly influenced by the accessory
proteins like Kv4 channel-interacting proteins (KChIPs), DP66 and DPP10 to alter
the channel gating (An et al. 2000). Absence of KChIPs in knockout mice shows
increase in susceptibility of ventricular tachycardia and seizures (Gutman et al.
2005). These channels are relatively resistant to mM concentrations of TEA and
provide moderate sensitivity to 4-AP. Fampridine is known to inhibit the Kv4.1
channel (Alexander et al. 2017). No other blocking agents are reported to block the
Kv4.1 channel. The slower voltage-dependent activation and inactivation rates of
Kv4.2 were blocked by hanatoxin and heteropodatoxins (HpTx1, HpTx2 and
HpTx3) in Xenopus oocytes. Quinidine and flecainide are also potent blockers of
the Kv4.2 current. Also, arachidonic acid modulates Kv4.2 by interacting with
KChIPs (Holmqvist et al. 2001), whereas niflumic acid, bupivacaine, DIDS (Cl-
channel blocker) and nicotine (at concentrations seen during smoking) block the
Kv4.3 conduction in oocytes (Gutman et al. 2005). It is reported that the level of
Kv4.3 mRNA decreases in patients with paroxysmal AF (Gutman et al. 2005).

19.5.1.5 Modulators of the Kv7 Family

The Kv7.1 channel encoded by the KCNQ1 gene exhibits delayed rectifier current
with varying activation kinetics between expression systems. Several pharmacolog-
ical agents are already in consideration with regard to blocking of the Kv7.1 channel
and its complexes. Clofilium and XE911 are potent inhibitors of the Kv7.1 channel.
The novel benzodiazepine derivatives, L-735821 and L-364373, showed a distinct
effect on the Kv7.1 channel. The former compound increases the ventricular action
potential duration by inhibiting the Kv7.1 current, while the latter apparently
activates the Kv7.1 conduction (Salata et al. 1996; Salata et al. 1998). Chromanol
293B has demonstrated inhibition of the KCNQ1/minK complex. XE991, meflo-
quine and azimilide are also reported as selective blockers of the Kv7.1 channel.
Kv7.2 and Kv7.3 constitute heteromultimers to produce larger currents (Langguth
et al. 2016; Haick and Byron 2016). However, both the channels have slightly
different gating mechanisms and sensitivities towards inhibitors (Robbins 2001).
670 S. Deka et al.

Kv7.2 channels are resistant to 4-AP (2 mM) but are effectively blocked by TEA as
they possess the tyrosine residue in P-loop. Kv7.2 channels are also blocked by
XE991 and L-735821. In contrast, Kv7.3 channels are insensitive to external TEA
(5 mM) and are inhibited by 4-AP, clofilium, ChTx and E4031 (Dalby-Brown et al.
2006). At present, little pharmacological data is available for Kv7.4 channels. This
channel shows intermediate sensitivity to TEA. Retigabine or ezogabine, a Kv7.4
activator, was approved by FDA in 2011 for adjuvant therapy of partial-onset
seizures in adults (Maljevic and Lerche 2013; Gribkoff 2003; Humphries and Dart
2015; Jenkinson 2006). The other compounds inhibiting this channel include
bepridil and XE991 (Dalby-Brown et al. 2006; Augustynek et al. 2016). Kv7.5
channels are activated by BMS204352 and retigabine, and further promote conduc-
tion of the channels. TEA exerts little sensitivity towards Kv7.5 channels. XE991
also demonstrated selective inhibitory effect on Kv7.5 channels. Linopirdine is
known to block all the members of the Kv7 family (Camerino et al. 2007;
Kuzmenkov et al. 2015).

19.5.1.6 Modulators of Kv5, Kv6, Kv8 and Kv9 Families

The Kv5 family (Kv5.1), the Kv6 family (Kv6.1, Kv6.2, Kv6.3 and Kv6.4) and the
Kv8 family (Kv8.1 and Kv8.2) have no function of their own but modulate the Kv2
channels’ function. These channels along with Kv9 channels are referred to as
modifiers.

19.5.1.7 Modulators of Kv10, Kv11 and Kv12 Families (EAG Family)

The EAG (ether-à-go-go) comprises the eag, eag-related genes (erg) and eag-like K+
channels (elk) subfamilies. The eag 1 and 2 (Kv10.1, Kv10.2); erg 1, 2 and 3 (Kv11.1,
Kv11.2, Kv11.3); and elk1 and 2 (Kv12.1, Kv12.2, Kv12.3) have been identified so
far in mammalian brain (Alexander et al. 2017; Alexander et al. 2013; Dai and
Zagotta 2017). Among the EAG family, Kv11.1 or HERG (human ether-à-go-go) is
one of the notable members, as defect in the channel underlies LQT2 syndrome
(Giudicessi and Ackerman 2012).
Quinidine and intracellular calcium are known to block both Kv10.1 and Kv10.2
channels. The monoclonal antibody, mAb62, an anti-Kv10.1, was studied in vitro on
the human breast cell line (MDA-MB-435S) and in vivo using tumour model nude
mice. It showed accumulation for at least 1 week in the tumour cells in vivo due to
Cy5.5 fluorophore labelling (Napp et al. 2016). A monoclonal antibody, mAb56,
antagonizes the Kv10.1 (hEag1) current and exhibits anti-proliferative and in vivo
tumour cell growth inhibition in breast, ovarian, colon, melanoma, fibrosarcoma and
pancreatic carcinoma (Gómez-Varela et al. 2007).
The HERG (Kv11.1) channels are effectively blocked by class III antiarrhythmic
methanesulphonamides like dofetilide, MK-499 and E-4031. Several other
compounds including antihistaminics (e.g. terfenadine), antibiotics
(e.g. erythromycin), prokinetic agents (e.g. cisapride) and antipsychotics
(e.g. sertindole) are known to block HERG channels (also refer to Table 19.1)
(D’amico et al. 2013). Combination therapy of ketoconazole and terfenadine also
prolonged the cardiac action potential synergistically by targeting HERG.
19 Pharmacology of Potassium Channels 671

CD160130 preferentially blocks the KV11.1 IB isoform expressed in leukaemic cells

and induces apoptosis of primary cells of chronic lymphoid leukaemia (CLL)
(Pillozzi et al. 2002; Pillozzi et al. 2007). Halofantrine and mefloquine block
HERG channels, where the severe side effect of the former can be associated with
HERG blockade. Moreover, ergotoxin specifically blocks HERG but showed no
affinity to eag or elk channels. A drug like sipatrigine is reported to block Kv11.2,
whereas Kv11.3 is blocked by pimozide and sertindole (Gutman et al. 2005). On the
other hand, the pharmacology of the elk subfamilies is very limited. These channels
are resistant to TEA (100 mM) but can be effectively blocked by Ba2+ (1 mM)
(Trudeau et al. 1999).

19.6 Conclusion

Potassium channels have been extensively investigated for molecular diversity,

subunit stoichiometry, channel assembly, precise biophysical properties and modu-
lation by different pharmacological agents. The ubiquitous distribution and diversi-
fication of these channels allow them to play a pivotal role in the regulation of
various cellular signalling processes that governs the action potential waveform,
stimulus-secretion coupling, electrolyte transport and regulation of cellular volume.
Concurrent with better insight into the channel topology and functions, the under-
standing of the pathophysiological regulation and genetic mutation of K+ channels
could lead to the identification and validation of novel modulators as a therapeutic
strategy in disease conditions. Apart from peptide and non-peptide toxins, K+
channels are the well-known targets of several pharmacological classes of drugs,
including GAs; antiarrhythmic, antihypertensive, anti-ischemic, antidiabetic and
antipsychotic drugs; and NSAIDs. Unlike the other subtypes, remarkable progress
has been observed in the case of KATP channels. Compounds like diazoxide (KCO)
and retigabine (Kv7.4 activator) are already approved for clinical use in PHHI and
adjuvant therapy of partial-onset seizures in adults, respectively. Many other
compounds such as levosimendan and BMS-204352 are under active clinical devel-
opment. In addition to this, the recent introduction of new insight into KNa1.1
(formerly KCa4.1) and KNa1.2 (formerly KCa4.2) will definitely catalyse a transfor-
mation in the research and drug discovery process. Together with these progresses,
the possibility of in-depth exploitation of other K+ channel subtypes as potential
pharmacological targets has also spurred the research thrive. In addition to the well-
known venom toxins and small molecules, unravelling precise tissue-specific and
targeted drug delivery has led to the introduction of gene therapy as a novel means
for delivery of mAbs to target specific channel subtypes. Monoclonal antibodies
such as mAb56 and mAb62, specific for inhibition of Kv10.1, have already been
reported to possess higher therapeutic potential in targeting tumour cells in vitro.
However, many challenges and hurdles in targeting these channel proteins remain to
be resolved, including lack of proper structural data as well as defined molecular
pharmacology of many K+ channel subtypes. The promising high-throughput
screening methods are expected to accelerate the identification and development of
672 S. Deka et al.

novel pharmacological agents targeting these channels. It is to be anticipated that the

improvised technology and newer delivery approaches could collectively enhance
the in-depth knowledge of channelopathies and will disclose newer
pharmacotherapeutic avenues in the upcoming years.

References
Abraham MR, Jahangir A, Alekseev AE, Terzic A (1999) Channelopathies of inwardly rectifying
potassium channels. FASEB J 13:1901–1910
Ackerman MJ, Clapham DE (1997) Ion channels—basic science and clinical disease. N Engl J Med
336:1575–1586
Adelman JP, Maylie J, Sah P (2012) Small-conductance Ca2+-activated K+ channels: form and
function. Annu Rev Physiol 74:245–269
Akopova OV (2018) Direct and off-target effects of ATP-sensitive potassium channels opener
diazoxide. J Drug Metab Toxicol 8:227
Alagem N, Dvir M, Reuveny E (2001) Mechanism of Ba2+ block of a mouse inwardly rectifying K
+ channel: differential contribution by two discrete residues. J Physiol 534:381–393
Alexander SP, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Catterall WA, Spedding M,
Peters JA, Harmar AJ (2013) The concise guide to pharmacology 2013/14: ion channels. Br J
Pharmacol 170:1607–1651
Alexander SP, Striessnig J, Kelly E, Marrion NV, Peters JA, Faccenda E, Harding SD, Pawson AJ,
Sharman JL, Southan C (2017) The concise guide to pharmacology 2017/18: Voltage-gated ion
channels. Br J Pharmacol 174:S160–S194
An WF, Bowlby MR, Betty M, Cao J, Ling H-P, Mendoza G, Hinson JW, Mattsson KI, Strassle
BW, Trimmer JS (2000) Modulation of A-type potassium channels by a family of calcium
sensors. Nature 403:553
Atkinson NS, Robertson GA, Ganetzky B (1991) A component of calcium-activated potassium
channels encoded by the Drosophila slo locus. Science 253:551–555
Augustynek B, Kunz WS, Szewczyk A (2016) Guide to the pharmacology of mitochondrial
potassium channels. Pharmacol Mitochondria 240:103–127. Springer
Bearden D, Strong A, Ehnot J, Digiovine M, Dlugos D, Goldberg EM (2014) Targeted treatment of
migrating partial seizures of infancy with quinidine. Ann Neurol 76:457–461
Berg AP, Sen N, Bayliss DA (2007) TrpC3/C7 and Slo2. 1 are molecular targets for metabotropic
glutamate receptor signaling in rat striatal cholinergic interneurons. J Neurosci 27:8845–8856
Bergeron Z, Bingham J-P (2012) Scorpion toxins specific for potassium (K+) channels: a historical
overview of peptide bioengineering. Toxins 4:1082–1119
Bhattacharjee A, Joiner WJ, Wu M, Yang Y, Sigworth FJ, Kaczmarek LK (2003) Slick (Slo2. 1), a
rapidly-gating sodium-activated potassium channel inhibited by ATP. J Neurosci
23:11681–11691
Bhave G, Lonergan D, Chauder BA, Denton JS (2010) Small-molecule modulators of inward
rectifier K+ channels: recent advances and future possibilities. Future Med Chem 2:757–774
Bhave G, Chauder BA, Liu W, Dawson ES, Kadakia R, Nguyen TT, Lewis LM, Meiler J, Weaver
CD, Satlin LM (2011) Development of a selective small-molecule inhibitor of Kir1. 1, the renal
outer medullary potassium channel. Mol Pharmacol 79:42–50
Calderone V (2002) Large-conductance, Ca2+-activated K+ channels: function, pharmacology and
drugs. Curr Med Chem 9:1385–1395
Camerino DC, Tricarico D, Desaphy J-F (2007) Ion channel pharmacology. Neurotherapeutics
4:184–198
Campos Rosa J, Galanakis D, Piergentili A, Bhandari K, Ganellin CR, Dunn PM, Jenkinson DH
(2000) Synthesis, molecular modeling, and pharmacological testing of bis-quinolinium
19 Pharmacology of Potassium Channels 673

cyclophanes: potent, non-peptidic blockers of the apamin-sensitive Ca2+-activated K+ channel.

J Med Chem 43:420–431
Castle N, London D, Creech C, Fajloun Z, Stocker J, Sabatier J-M (2003) Maurotoxin: a potent
inhibitor of intermediate conductance Ca2+-activated potassium channels. Mol Pharmacol
63:409–418
Challinor-Rogers JL, McPherson GA (1994) Potassium channel openers and other regulators of
KATP channels. Clin Exp Pharmacol Physiol 21:583–597
Chan KW, Wheeler A, Csanády L (2008) Sulfonylurea receptors type 1 and 2A randomly assemble
to form heteromeric KATP channels of mixed subunit composition. J Gen Physiol 131:43–58
Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD (2004) K+ channels as
targets for specific immunomodulation. Trends Pharmacol Sci 25:280–289
Chowdhury UR, Dosa PI, Fautsch MP (2017) ATP sensitive potassium channel openers: a new
class of ocular hypotensive agents. Exp Eye Res 158:85–93
Claydon T, Boyett M, Sivaprasadarao A, Ishii K, Owen J, O’beirne H, Leach R, Komukai K,
Orchard C (2000) Inhibition of the K+ channel Kv1. 4 by acidosis: protonation of an extracel-
lular histidine slows the recovery from N-type inactivation. J Physiol 526:253–264
Coghlan MJ, Carroll WA, Gopalakrishnan M (2001) Recent developments in the biology and
medicinal chemistry of potassium channel modulators: update from a decade of progress. J Med
Chem 44:1627–1653
Comes N, Bielanska J, Vallejo-Gracia A, Serrano-AlbarráS A, Marruecos L, Gómez D, Soler C,
Condom E, RamóN Y, Cajal S, Hernández-Losa J (2013) The voltage-dependent K+ channels
Kv1. 3 and Kv1. 5 in human cancer. Front Physiol 4:283
Contreras GF, Castillo K, Enrique N, Carrasquel-Ursulaez W, Castillo JP, Milesi V, Neely A,
Alvarez O, Ferreira G, Gonzalez C (2013) A BK (Slo1) channel journey from molecule to
physiology. Channels 7:442–458
Cotten JF (2013) TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore potassium channel
antagonists stimulate breathing in isoflurane anesthetized rats. Anesth Analg 4:116
CzirjáK G, Enyedi P (2003) Ruthenium red inhibits TASK-3 potassium channel by interconnecting
glutamate 70 of the two subunits. Mol Pharmacol 63:646–652
D’amico M, Gasparoli L, Arcangeli A (2013) Potassium channels: novel emerging biomarkers and
targets for therapy in cancer. Recent Pat Anticancer Drug Discov 8:53–65
Dai G, Zagotta WN (2017) Molecular mechanism of voltage-dependent potentiation of KCNH
potassium channels. Elife 6:e26355
Dalby-Brown W, Hansen HH, Korsgaard MP, Mirza N, Olesen S-P (2006) Kv7 channels: function,
pharmacology and channel modulators. Curr Top Med Chem 6:999–1023
De Los Angeles Tejada M, Stolpe K, Meinild A-K, Klaerke DA (2012) Clofilium inhibits Slick and
Slack potassium channels. Biologics 6:465
Dogan MF, Yildiz O, Arslan SO, Ulusoy KG (2019) Potassium channels in vascular smooth
muscle: a pathophysiological and pharmacological perspective. Fundam Clin Pharmacol
Doupnik CA (2017) Venom-derived peptides inhibiting Kir channels: past, present, and future.
Neuropharmacology 127:161–172
Duan R, Cui W, Wang H (2011) Mutational analysis of the Kir6. 1 gene in Chinese hypertensive
patients treated with the novel ATP-sensitive potassium channel opener iptakalim. Exp Ther
Med 2:757–760
Dudina EE, Korolkova YV, Bocharova NE, Koshelev SG, Egorov TA, Huys I, Tytgat J, Grishin EV
(2001) OsK2, a new selective inhibitor of Kv1. 2 potassium channels purified from the venom of
the scorpion Orthochirus scrobiculosus. Biochem Biophys Res Commun 286:841–847
Dworakowska B, Dolowy K (2000) Ion channels-related diseases. Acta Biochim Pol 47:685–703
Edwards G, Weston AH (1995) Pharmacology of the potassium channel openers. Cardiovasc Drugs
Ther 9:185–193
Enyedi P, CzirjáK G (2010) Molecular background of leak K+ currents: two-pore domain potas-
sium channels. Physiol Rev 90:559–605
674 S. Deka et al.

Enyedi P, CzirjáK G (2015) Properties, regulation, pharmacology, and functions of the K 2P

channel, TRESK. Pflügers Arch 467:945–958
Es-Salah-Lamoureux Z, Steele DF, Fedida D (2010) Research into the therapeutic roles of two-
pore-domain potassium channels. Trends Pharmacol Sci 31:587–595
Faber EL, Sah P (2007) Functions of SK channels in central neurons. Clin Exp Pharmacol Physiol
34:1077–1083
Fan Y, Kong H, Ye X, Ding J, Hu G (2016) ATP-sensitive potassium channels: uncovering novel
targets for treating depression. Brain Struct Funct 221:3111–3122
Fanger CM, Rauer H, Neben AL, Miller MJ, Rauer H, Wulff H, Rosa JC, Ganellin CR, Chandy
KG, Cahalan MD (2001) Calcium-activated potassium channels sustain calcium signaling in T
lymphocytes SELECTIVE blockers and manipulated channel expression levels. J Biol Chem
276:12249–12256
Feliciangeli S, Chatelain FC, Bichet D, Lesage F (2015) The family of K2P channels: salient
structural and functional properties. J Physiol 593:2587–2603
Felix JP, Bugianesi RM, Schmalhofer WA, Borris R, Goetz MA, Hensens OD, Bao J-M, Kayser F,
Parsons WH, Rupprecht K (1999) Identification and biochemical characterization of a novel
nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1. 3. Bio-
chemistry 38:4922–4930
Gada K, Plant LD (2019) Two-pore domain potassium channels: emerging targets for novel
analgesic drugs: IUPHAR Review 26. Br J Pharmacol 176:256–266
Garcia ML, Kaczorowski GJ (2005) Potassium channels as targets for therapeutic intervention. Sci
Signal 302:pe46
Garcia ML, Kaczorowski GJ (2016) Ion channels find a pathway for therapeutic success. Proc Natl
Acad Sci 113:5472–5474
Garcia ML, Hanner M, Knaus H-G, Koch R, Schmalhofer W, Slaughter RS, Kaczorowski GJ
(1997) Pharmacology of potassium channels. In: Advances in pharmacology. Elsevier,
Amsterdam
Garcia M, Gao Y-D, McManus O, Kaczorowski G (2001) Potassium channels: from scorpion
venoms to high-resolution structure. Toxicon 39:739–748
Garcia-Calvo M, Leonard R, Novick J, Stevens S, Schmalhofer W, Kaczorowski G, Garcia M
(1993) Purification, characterization, and biosynthesis of margatoxin, a component of
Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium
channels. J Biol Chem 268:18866–18874
Garcia-Valdes J, Zamudio FZ, Toro L, Possan LD (2001) Slotoxin, αKTx1. 11, a new scorpion
peptide blocker of MaxiK channels that differentiates between α and α+ β (β1 or β4) complexes.
FEBS Lett 505:369–373
Girard C, Duprat F, Terrenoire C, Tinel N, Fosset M, Romey G, Lazdunski M, Lesage F (2001)
Genomic and functional characteristics of novel human pancreatic 2P domain K+ channels.
Biochem Biophys Res Commun 282:249–256
Giudicessi JR, Ackerman MJ (2012) Potassium-channel mutations and cardiac arrhythmias—
diagnosis and therapy. Nat Rev Cardiol 9:319
Goldstein SA, Bockenhauer D, O’kelly I, Zilberberg N (2001) Potassium leak channels and the
KCNK family of two-P-domain subunits. Nat Rev Neurosci 2:175
Gómez-Varela D, Zwick-Wallasch E, Knötgen H, Sánchez A, Hettmann T, Ossipov D, Weseloh R,
Contreras-Jurado C, Rothe M, Stühmer W (2007) Monoclonal antibody blockade of the human
Eag1 potassium channel function exerts antitumor activity. Cancer Res 67:7343–7349
González C, Baez-Nieto D, Valencia I, OyarzúN I, Rojas P, Naranjo D, Latorre R (2012) K+
channels: function-structural overview. Compr Physiol 2:2087–2149
Goodman S, Pace BS, Shartava A (1998) New therapeutic approaches to sickle cell disease:
Targeting RBC membrane oxidative damage. Cell Mol Biol Lett 4(03)
Gray AT, Zhao BB, Kindler CH, Winegar BD, Mazurek MJ, Xu J, Chavez RA, Forsayeth JR, Yost
CS (2000) Volatile anesthetics activate the human tandem pore domain baseline K+ channel
KCNK5. Anesthesiology 92:1722–1730
19 Pharmacology of Potassium Channels 675

Greenwood IA, Leblanc N (2007) Overlapping pharmacology of Ca2+-activated Cl
and K+

channels. Trends Pharmacol Sci 28:1–5
Gribkoff VK (2003) The therapeutic potential of neuronal KCNQ channel modulators. Expert Opin
Ther Targets 7:737–748
Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz MJ, Auperin
DD, Chandy KG (1994) Pharmacological characterization of five cloned voltage-gated K+
channels, types Kv1. 1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol
Pharmacol 45:1227–1234
Grunnet M, Bentzen BH, SØrensen US, Diness JG (2011) Cardiac ion channels and mechanisms
for protection against atrial fibrillation. Rev Physiol Biochem Pharmacol 162:1–58. Springer
Guo L, Tang X, Tian H, Liu Y, Wang Z, Wu H, Wang J, Guo S, Zhu D (2008) Subacute hypoxia
suppresses Kv3. 4 channel expression and whole-cell K+ currents through endogenous
15-hydroxyeicosatetraenoic acid in pulmonary arterial smooth muscle cells. Eur J Pharmacol
587:187–195
Gutman GA, Chandy KG, Adelman JP, Aiyar J, Bayliss DA, Clapham DE, Covarriubias M, Desir
GV, Furuichi K, Ganetzky B (2003) International Union of Pharmacology. XLI Compendium of
voltage-gated ion channels: potassium channels. Pharmacol Rev 55:583–586
Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D, Pardo LA, Robertson GA,
Rudy B, Sanguinetti MC, Stühmer W (2005) International Union of Pharmacology. LIII
Nomenclature and molecular relationships of voltage-gated potassium channels. Pharmacol
Rev 57:473–508
Haick JM, Byron KL (2016) Novel treatment strategies for smooth muscle disorders: targeting Kv7
potassium channels. Pharmacol Ther 165:14–25
HajdÚ P, Ulens C, Panyı G, Tytgat J (2003) Drug-and mutagenesis-induced changes in the
selectivity filter of a cardiac two-pore background K+ channel. Cardiovasc Res 58:46–54
Hayashi M, Novak I (2013) Molecular basis of potassium channels in pancreatic duct epithelial
cells. Channels 7:432–441
Hibino H, Inanobe A, Furutani K, Murakami S, Findlay I, Kurachi Y (2010) Inwardly rectifying
potassium channels: their structure, function, and physiological roles. Physiol Rev 90:291–366
Hill RJ, Grant AM, Volberg W, Rapp L, Faltynek C, Miller D, Pagani K, Baizman E, Wang S,
Guiles JW (1995) WIN 17317-3: novel nonpeptide antagonist of voltage-activated K+ channels
in human T lymphocytes. Mol Pharmacol 48:98–104
Holmqvist MH, Cao J, Knoppers MH, Jurman ME, Distefano PS, Rhodes KJ, Xie Y, An WF (2001)
Kinetic modulation of Kv4-mediated A-current by arachidonic acid is dependent on potassium
channel interacting proteins. J Neurosci 21:4154–4161
Hougaard C, Eriksen B, Jørgensen S, Johansen T, Dyhring T, Madsen L, Strøbaek D,
Christophersen P (2007) Selective positive modulation of the SK3 and SK2 subtypes of small
conductance Ca2+-activated K+ channels. Br J Pharmacol 151:655–665
Hougaard C, Jensen ML, Dale TJ, Miller DD, Davies DJ, Eriksen BL, Strøbæk D, Trezise DJ,
Christophersen P (2009) Selective activation of the SK1 subtype of human small-conductance
Ca2+-activated K+ channels by 4-(2-methoxyphenylcarbamoyloxymethyl)-piperidine-1-car-
boxylic acid tert-butyl ester (GW542573X) is dependent on serine 293 in the S5 segment.
Mol Pharmacol 76:569–578
Huang C-L, Feng S, Hilgemann DW (1998) Direct activation of inward rectifier potassium channels
by PIP 2 and its stabilization by Gβγ. Nature 391:803
Hudson AE, Herold KF, Hemmings HC Jr (2019) Pharmacology of inhaled anesthetics Pharmacol
Physiol Anesth vol 2E. 217–240. Elsevier, Philadelphia, PA
Hughes BA, Kumar G, Yuan Y, Swaminathan A, Yan D, Sharma A, Plumley L, Yang-Feng TL,
Swaroop A (2000) Cloning and functional expression of human retinal Kir2. 4, a pH-sensitive
inwardly rectifying K+ channel. Am J Physiol Cell Physiol 279:C771–C784
Humphries ES, Dart C (2015) Neuronal and cardiovascular potassium channels as therapeutic drug
targets: promise and pitfalls. J Biomol Screen 20:1055–1073
676 S. Deka et al.

Ishihara K, Hiraoka M, Ochi R (1996) The tetravalent organic cation spermine causes the gating of
the IRK1 channel expressed in murine fibroblast cells. J Physiol 491:367–381
Jackson WF (2017) Potassium channels in regulation of vascular smooth muscle contraction and
growth. Adv Pharmacol 78:89–144. Elsevier
Jenkinson DH (2006) Potassium channels–multiplicity and challenges. Br J Pharmacol 147:S63–
S71
Jensen BS, Ødum N, JØrgensen NK, Christophersen P, Olesen S-P (1999) Inhibition of T cell
proliferation by selective block of Ca2+-activated K+ channels. Proc Natl Acad Sci
96:10917–10921
Jensen BS, Olsen S-P, Jørgensen TD, Strøbæk D, Christophersen P, Ødum N (2003) Chemical
compounds having ion channel blocking activity for the treatment of immune dysfunction.
Google Patents
Jensen MØ, Jogini V, Borhani DW, Leffler AE, Dror RO, Shaw DE (2012) Mechanism of voltage
gating in potassium channels. Science 336:229–233
Ji X-C, Zhao W-H, Cao D-X, Shi Q-Q, Wang X-L (2011) Novel neuroprotectant chiral 3-n-
butylphthalide inhibits tandem-pore-domain potassium channel TREK-1. Acta Pharmacol Sin
32:182
Jiménez-Vargas JM, Possani LD, Luna-Ramirez K (2017) Arthropod toxins acting on neuronal
potassium channels. Neuropharmacology 127:139–160
Kaczmarek LK (2013) Slack, slick, and sodium-activated potassium channels. ISRN Neurosci
2013:354262
Kaczmarek LK, Aldrich RW, Chandy KG, Grissmer S, Wei AD, Wulff H (2017) International
Union of Basic and Clinical Pharmacology. C Nomenclature and properties of calcium-activated
and sodium-activated potassium channels. Pharmacol Rev 69:1–11
Kaczorowski GJ, Garcia ML (1999) Pharmacology of voltage-gated and calcium-activated potas-
sium channels. Curr Opin Chem Biol 3:448–458
Kaczorowski GJ, Knaus H-G, Leonard RJ, McManus OB, Garcia ML (1996) High-conductance
calcium-activated potassium channels; structure, pharmacology, and function. J Bioenerg
Biomembr 28:255–267
Kasap M, Dwyer DS (2018) Two-pore domain potassium channels (K2Ps) as drug targets in
neuroinflammation. Neuroinflammation:413–427. https://doi.org/10.1016/B978-0-12-811709-
5.00022-3. Elsevier
Kaufmann K, Romaine I, Days E, Pascual C, Malik A, Yang L, Zou B, Du Y, Sliwoski G, Morrison
RD (2013) Ml297 (VU0456810), the first potent and selective activator of the GIRK potassium
channel, displays antiepileptic properties in mice. ACS Chem Nerosci 4:1278–1286
Kennard LE, Chumbley JR, Ranatunga KM, Armstrong SJ, Veale EL, Mathie A (2005) Inhibition
of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite
norfluoxetine. Br J Pharmacol 144:821–829
Ketchum KA, Joiner WJ, Sellers AJ, Kaczmarek LK, Goldstein SA (1995) A new family of
outwardly rectifying potassium channel proteins with two pore domains in tandem. Nature
376:690
Kim D (2005) Physiology and pharmacology of two-pore domain potassium channels. Curr Pharm
Des 11:2717–2736
Kim GE, Kaczmarek LK (2014) Emerging role of the KCNT1 Slack channel in intellectual
disability. Front Cell Neurosci 8:209
Kim DM, Nimigean CM (2016) Voltage-gated potassium channels: a structural examination of
selectivity and gating. Cold Spring Harb Perspect Biol 8:a029231
Kim Y, Bang H, Kim D (2000) TASK-3, a new member of the tandem pore K+ channel family. J
Biol Chem 275:9340–9347
Kobayashi T, Washiyama K, Ikeda K (2004) Inhibition of G protein-activated inwardly rectifying K
+ channels by various antidepressant drugs. Neuropsychopharmacology 29:1841
Köhling R, Wolfart J (2016) Potassium channels in epilepsy. Cold Spring Harb Perspect Med 6:
a022871
19 Pharmacology of Potassium Channels 677

Kuang Q, Purhonen P, Hebert H (2015) Structure of potassium channels. Cell Mol Life Sci
72:3677–3693
Kurata HT, Phillips LR, Rose T, Loussouarn G, Herlitze S, Fritzenschaft H, Enkvetchakul D,
Nichols CG, Baukrowitz T (2004) Molecular basis of inward rectification: polyamine interac-
tion sites located by combined channel and ligand mutagenesis. J Gen Physiol 124:541–554
Kuzmenkov A, Grishin E, Vassilevski A (2015) Diversity of potassium channel ligands: focus on
scorpion toxins. Biochemistry (Mosc) 80:1764–1799
Lam J, Coleman N, Garing ALA, Wulff H (2013) The therapeutic potential of small-conductance
KCa2 channels in neurodegenerative and psychiatric diseases. Expert Opin Ther Targets
17:1203–1220
Langguth B, Elgoyhen AB, Schlee W (2016) Potassium channels as promising new targets for
pharmacologic treatment of tinnitus: can Internet-based ‘crowd sensing’initiated by patients
speed up the transition from bench to bedside? Expert Opin Ther Targets 20:251–254. Taylor &
Francis
Lawson K (2000) Potassium channel openers as potential therapeutic weapons in ion channel
disease. Kidney Int 57:838–845
Ledoux J, Werner ME, Brayden JE, Nelson MT (2006) Calcium-activated potassium channels and
the regulation of vascular tone. Physiology 21:69–78
Lehmann-Horn F, Lerche H, Jurkat-Rott K (2003) Skeletal muscle channelopathies: myotonias,
periodic paralyses and malignant hyperthermia. In: Handbook of clinical neurophysiology, vol
2. Elsevier, Amsterdam, pp 457–483
Lengyel M, Dobolyi A, CzirjáK G, Enyedi P (2017) Selective and state-dependent activation of
TRESK (K2P18. 1) background potassium channel by cloxyquin. Br J Pharmacol
174:2102–2113
Lesage F (2003) Pharmacology of neuronal background potassium channels. Neuropharmacology
44:1–7
Lesage F, Lazdunski M (2000) Molecular and functional properties of two-pore-domain potassium
channels. Am J Physiol Renal Physiol 279:F793–F801
Li X-Y, Toyoda H (2015) Role of leak potassium channels in pain signaling. Brain Res Bull
119:73–79
Longman SD, Hamilton TC (1992) Potassium channel activator drugs: mechanism of action,
pharmacological properties, and therapeutic potential. Med Res Rev 12:73–148
Lopatin AN, Makhina EN, Nichols CG (1994) Potassium channel block by cytoplasmic polyamines
as the mechanism of intrinsic rectification. Nature 372:366
Loucif AJ, Saintot PP, Liu J, Antonio BM, Zellmer SG, Yoger K, Veale EL, Wilbrey A, Omoto K,
Cao L (2018) GI-530159, a novel, selective, mechanosensitive two-pore-domain potassium
(K2P) channel opener, reduces rat dorsal root ganglion neuron excitability. Br J Pharmacol
175:2272–2283
Luzhkov V, Åqvist J (2005) Ions and blockers in potassium channels: insights from free energy
simulations. Biochim Biophys Acta 1747:109–120
Mackinnon R (2003) Potassium channels. FEBS Lett 555:62–65
Malayev A, Nelson D, Philipson L (1995) Mechanism of clofilium block of the human Kv1.
5 delayed rectifier potassium channel. Mol Pharmacol 47:198–205
Maljevic S, Lerche H (2013) Potassium channels: a review of broadening therapeutic possibilities
for neurological diseases. J Neurol 260:2201–2211
Mathie A, Wooltorton JR, Watkins CS (1998) Voltage-activated potassium channels in mammalian
neurons and their block by novel pharmacological agents. Gen Pharmacol Vasc S 30:13–24
Meadows H, Chapman C, Duckworth D, Kelsell R, Murdock P, Nasir S, Rennie G, Randall A
(2001) The neuroprotective agent sipatrigine (BW619C89) potently inhibits the human tandem
pore-domain K+ channels TREK-1 and TRAAK. Brain Res 892:94–101
Meera P, Wallner M, Song M, Toro L (1997) Large conductance voltage-and calcium-dependent K
+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal
678 S. Deka et al.

transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C

terminus. Proc Natl Acad Sci 94:14066–14071
Miller C (2000) An overview of the potassium channel family. Genome Biol 1:reviews0004. 1
Montandon G, Ren J, Victoria NC, Liu H, Wickman K, Greer JJ, Horner RL (2016) G-protein–
gated inwardly rectifying potassium channels modulate respiratory depression by opioids.
Anesthesiology 124:641–650
Napp J, Pardo LA, Hartung F, Tietze LF, StÜhmer W, Alves F (2016) In vivo imaging of tumour
xenografts with an antibody targeting the potassium channel K v 10.1. Eur Biophys J
45:721–733
Navarro B, Kirichok Y, Clapham DE (2007) KSper, a pH-sensitive K+ current that controls sperm
membrane potential. Proc Natl Acad Sci 104:7688–7692
Nichols C, Lopatin A (1997) Inward rectifier potassium channels. Annu Rev Physiol 59:171–191
Olschewski A, Veale EL, Nagy BM, Nagaraj C, Kwapiszewska G, Antigny F, Lambert M,
Humbert M, CzirjáK G, Enyedi P (2017) TASK-1 (KCNK3) channels in the lung: from cell
biology to clinical implications. Eur Respir J 50:1700754
Panyi G (2005) Biophysical and pharmacological aspects of K+ channels in T lymphocytes. Eur
Biophys J 34:515–529
Pardo LA, Del Camino D, Sánchez A, Alves F, Brüggemann A, Beckh S, StÜhmer W (1999)
Oncogenic potential of EAG K+ channels. EMBO J 18:5540–5547
Patel AJ, HonorÉ E (2001) Properties and modulation of mammalian 2P domain K+ channels.
Trends Neurosci 24:339–346
Patel AJ, Honoré E, Lesage F, Fink M, Romey G, Lazdunski M (1999) Inhalational anesthetics
activate two-pore-domain background K+ channels. Nat Neurosci 2:422
Pedarzani P, McCutcheon JE, Rogge G, Jensen BS, Christophersen P, Hougaard C, Strøbæk D,
Stocker M (2005) Specific enhancement of SK channel activity selectively potentiates the
afterhyperpolarizing current IAHP and modulates the firing properties of hippocampal pyrami-
dal neurons. J Biol Chem 280:41404–41411
Piechotta PL, Rapedius M, Stansfeld PJ, Bollepalli MK, Erhlich G, Andres-Enguix I,
Fritzenschaft H, Decher N, Sansom MS, Tucker SJ (2011) The pore structure and gating
mechanism of K2P channels. EMBO J 30:3607–3619
Pillozzi S, Brizzi MF, Balzi M, Crociani O, Cherubini A, Guasti L, Bartolozzi B, Becchetti A,
Wanke E, Bernabei P (2002) HERG potassium channels are constitutively expressed in primary
human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemo-
poietic progenitors. Leukemia 16:1791
Pillozzi S, Brizzi MF, Bernabei PA, Bartolozzi B, Caporale R, Basile V, Boddi V, Pegoraro L,
Becchetti A, Arcangeli A (2007) VEGFR-1 (FLT-1), β1 integrin, and hERG K+ channel for a
macromolecular signaling complex in acute myeloid leukemia: role in cell migration and
clinical outcome. Blood 110:1238–1250
Pongs O (1999) Voltage-gated potassium channels: from hyperexcitability to excitement. FEBS
Lett 452:31–35
Quast U (1992) Potassium channel openers: pharmacological and clinical aspects. Fundam Clin
Pharmacol 6:279–293
Renigunta V, SchlichthÖrl G, Daut J (2015) Much more than a leak: structure and function of K
2P-channels. Pflügers Arch 467:867–894
Riazanski V, Becker A, Chen J, Sochivko D, Lie A, Wiestler OD, Elger CE, Beck H (2001)
Functional and molecular analysis of transient voltage-dependent K+ currents in rat hippocam-
pal granule cells. J Physiol 537:391–406
Robbins J (2001) KCNQ potassium channels: physiology, pathophysiology, and pharmacology.
Pharmacol Ther 90:1–19
Robertson DW, Steinberg MI (1990) Potassium channel modulators: scientific applications and
therapeutic promise. J Med Chem 33:1529–1541
Rosa JC, Galanakis D, Ganellin CR, Dunn PM, Jenkinson DH (1998) Bis-quinolinium
cyclophanes: 6, 10-diaza-3 (1, 3), 8 (1, 4)-dibenzena-1, 5 (1, 4)-diquinolinacyclodecaphane
19 Pharmacology of Potassium Channels 679

(UCL 1684), the first nanomolar, non-peptidic blocker of the apamin-sensitive Ca2+-activated
K+ channel. J Med Chem 41:2–5
Rubaiy HN (2016) The therapeutic agents that target ATP-sensitive potassium channels. Acta
Pharm 66:23–34
Rudy B, Mcbain CJ (2001) Kv3 channels: voltage-gated K+ channels designed for high-frequency
repetitive firing. Trends Neurosci 24:517–526
Salata J, Jurkiewicz N, Sanguinetti M, Siegl P, Claremon D, Remy D, Elliot J, Libby B (1996) The
novel class III antiarrhythmic agent, L-735,821 is a potent and selective blocker of IKs in guinea
pig ventricular myocytes. Circulation 94:1–529
Salata JJ, Jurkiewicz NK, Wang J, Evans BE, Orme HT, Sanguinetti MC (1998) A novel
benzodiazepine that activates cardiac slow delayed rectifier K+ currents. Mol Pharmacol
54:220–230
Sanchez M, McManus O (1996) Paxilline inhibition of the alpha-subunit of the high-conductance
calcium-activated potassium channel. Neuropharmacology 35:963–968
Sánchez-Carranza O, Torres-Rodríguez P, Darszon A, Treviño CL, López-González I (2015)
Pharmacology of hSlo3 channels and their contribution in the capacitation-associated hyperpo-
larization of human sperm. Biochem Biophys Res Commun 466:554–559
Sands S, Lewis R, Cahalan M (1989) Charybdotoxin blocks voltage-gated K+ channels in human
and murine T lymphocytes. J Gen Physiol 93:1061–1074
Schneider ER, Anderson EO, Gracheva EO, Bagriantsev SN (2014) Temperature sensitivity of
two-pore (K2P) potassium channels. Curr Top Membr 74:113–133. Elsevier
Seifert G, Henneberger C, Steinhaeuser C (2018) Diversity of astrocyte potassium channels: An
update. Brain Res Bull 136:26–36
Shah NH, Aizenman E (2014) Voltage-gated potassium channels at the crossroads of neuronal
function, ischemic tolerance, and neurodegeneration. Transl Stroke Res 5:38–58
Shieh C-C, Coghlan M, Sullivan JP, Gopalakrishnan M (2000) Potassium channels: molecular
defects, diseases, and therapeutic opportunities. Pharmacol Rev 52:557–594
Sokolova O, Kolmakova-Partensky L, Grigorieff N (2001) Three-dimensional structure of a
voltage-gated potassium channel at 2.5 nm resolution. Structure 9:215–220
Staudacher I, Illg C, Chai S, Deschenes I, Seehausen S, Gramlich D, Müller ME, Wieder T, Rahm
A-K, Mayer C (2018a) Cardiovascular pharmacology of K 2P 17.1 (TASK-4, TALK-2) two-
pore-domain K+ channels. Naunyn Schmiedebergs Arch Pharmacol 391:1119–1131
Staudacher I, Seehausen S, Illg C, Lugenbiel P, Schweizer PA, Katus HA, Thomas D (2018b)
Cardiac K2P13. 1 (THIK-1) two-pore-domain K+ channels: pharmacological regulation and
remodeling in atrial fibrillation. Prog Biophys Mol Biol 144:128–138
Strøbæk D, Teuber L, JØrgensen TD, Ahring PK, Kjær K, Hansen RS, Olesen SP,
Christophersen P, Skaaning-Jensen B (2004) Activation of human IK and SK Ca2+-activated
K+ channels by NS309 (6, 7-dichloro-1H-indole-2, 3-dione 3-oxime). Biochim Biophys Acta
1665:1–5
Sturgess N, Cook D, Ashford MJ, Hales CN (1985) The sulphonylurea receptor may be an
ATP-sensitive potassium channel. Lancet 326:474–475
Swanson R, Marshall J, Smith JS, Williams JB, Boyle MB, Folander K, Luneau CJ, Antanavage J,
Oliva C, Buhrow SA (1990) Cloning and expression of cDNA and genomic clones encoding
three delayed rectifier potassium channels in rat brain. Neuron 4:929–939
Swartz KJ, Mackinnon R (1997a) Hanatoxin modifies the gating of a voltage-dependent K+
channel through multiple binding sites. Neuron 18:665–673
Swartz KJ, Mackinnon R (1997b) Mapping the receptor site for hanatoxin, a gating modifier of
voltage-dependent K+ channels. Neuron 18:675–682
Syme CA, Gerlach AC, Singh AK, Devor DC (2000) Pharmacological activation of cloned
intermediate-and small-conductance Ca2+-activated K+ channels. Am J Physiol Cell Physiol
278:C570–C581
680 S. Deka et al.

Takahashi N, Morishige K, Jahangir A, Yamada M, Findlay I, Koyama H, Kurachi Y (1994)

Molecular cloning and functional expression of cDNA encoding a second class of inward
rectifier potassium channels in the mouse brain. J Biol Chem 269:23274–23279
Talley EM, Sirois JE, Lei Q, Bayliss DA (2003) Two-pore-Domain (KCNK) potassium channels:
dynamic roles in neuronal function. Neuroscientist 9:46–56
Tang Q-Y, Zhang Z, Xia X-M, Lingle CJ (2010) Block of mouse Slo1 and Slo3 K+ channels by
CTX, IbTX, TEA, 4-AP and quinidine. Channels 4:22–41
Thümmler S, Duprat F, Lazdunski M (2007) Antipsychotics inhibit TREK but not TRAAK
channels. Biochem Biophys Res Commun 354:284–289
Tian C, Zhu R, Zhu L, Qiu T, Cao Z, Kang T (2014) Potassium channels: structures, diseases, and
modulators. Chem Biol Drug Des 83:1–26
Tian F, Qiu Y, Lan X, Li M, Yang H, Gao Z (2019) A Small-molecule compound selectively
activates K2P channel TASK-3 by acting at two distant clusters of residues. Mol Pharmacol
96:26–35
Töpert C, Döring F, Wischmeyer E, Karschin C, Brockhaus J, Ballanyi K, Derst C, Karschin A
(1998) Kir2. 4: a novel K+ inward rectifier channel associated with motoneurons of cranial
nerve nuclei. J Neurosci 18:4096–4105
Trudeau MC, Titus SA, Branchaw JL, Ganetzky B, Robertson GA (1999) Functional analysis of a
mouse brain Elk-type K+ channel. J Neurosci 19:2906–2918
Tsai S-J (2008) Sipatrigine could have therapeutic potential for major depression and bipolar
depression through antagonism of the two-pore-domain K+ channel TREK-1. Med Hypotheses
70:548–550
Tsai K-L, Chang H-F, Wu S-N (2013) The inhibition of inwardly rectifying K+ channels by
memantine in macrophages and microglial cells. Cell Physiol Biochem 31:938–951
Tsantoulas C (2015) Emerging potassium channel targets for the treatment of pain. Curr Opin
Support Palliat Care 9:147–154
Vacher H, Mohapatra DP, Misonou H, Trimmer JS (2007) Regulation of Kv1 channel trafficking by
the mamba snake neurotoxin dendrotoxin K. FASEB J 21:906–914
Vivier D, Bennis K, Lesage F, Ducki S (2015) Perspectives on the two-pore domain potassium
channel TREK-1 (TWIK-related K+ channel 1). A novel therapeutic target? miniperspective. J
Med Chem 59:5149–5157
Wang S-Y, Cui W-Y, Wang H (2015) The new antihypertensive drug iptakalim activates
ATP-sensitive potassium channels in the endothelium of resistance blood vessels. Acta
Pharmacol Sin 36:1444
Weatherall KL, Goodchild SJ, Jane DE, Marrion NV (2010) Small conductance calcium-activated
potassium channels: from structure to function. Prog Neurobiol 91:242–255
Wei AD, Gutman GA, Aldrich R, Chandy KG, Grissmer S, Wulff H (2005) International Union of
Pharmacology. LII Nomenclature and molecular relationships of calcium-activated potassium
channels. Pharmacol Rev 57:463–472
Wright PD, Veale EL, Mccoull D, Tickle DC, Large JM, Ococks E, Gothard G, Kettleborough C,
Mathie A, Jerman J (2017) Terbinafine is a novel and selective activator of the two-pore domain
potassium channel TASK3. Biochem Biophys Res Commun 493:444–450
Wu Y, Yang Y, Ye S, Jiang Y (2010) Structure of the gating ring from the human large-conductance
Ca 2+-gated K+ channel. Nature 466:393
Wulff H, Zhorov BS (2008) K+ channel modulators for the treatment of neurological disorders and
autoimmune diseases. Chem Rev 108:1744–1773
Wulff H, Castle NA, Pardo LA (2009) Voltage-gated potassium channels as therapeutic targets. Nat
Rev Drug Discov 8:982
Wydeven N, De Velasco EMF, Du Y, Benneyworth MA, Hearing MC, Fischer RA, Thomas MJ,
Weaver CD, Wickman K (2014) Mechanisms underlying the activation of G-protein–gated
inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297. Proc Natl Acad
Sci 111:10755–10760
19 Pharmacology of Potassium Channels 681

Xia X-M, Fakler B, Rivard A, Wayman G, Johnson-Pais T, Keen J, Ishii T, Hirschberg B, Bond C,
Lutsenko S (1998) Mechanism of calcium gating in small-conductance calcium-activated
potassium channels. Nature 395:503
Yamashita T, Horio Y, Yamada M, Takahashi N, Kondo C, Kurachi Y (1996) Competition between
Mg2+ and spermine for a cloned IRK2 channel expressed in a human cell line. J Physiol
493:143–156
Yang B, Gribkoff VK, Pan J, Damagnez V, Dworetzky SI, Boissard CG, Bhattacharjee A, Yan Y,
Sigworth FJ, Kaczmarek LK (2006) Pharmacological activation and inhibition of Slack (Slo2.
2) channels. Neuropharmacology 51:896–906
Yellen G (2002) The voltage-gated potassium channels and their relatives. Nature 419:35
Yeung SYM, Thompson D, Wang Z, Fedida D, Robertson B (2005) Modulation of Kv3 subfamily
potassium currents by the sea anemone toxin BDS: significance for CNS and biophysical
studies. J Neurosci 25:8735–8745
Yost CS (2000) Tandem pore domain K channels an important site of volatile anesthetic action.
Curr Drug Targets 1:207–217
Yu H-B, Li M, Wang W-P, Wang X-L (2016) High throughput screening technologies for ion
channels. Acta Pharmacol Sin 37:34
Zeng X-H, Yang C, Xia X-M, Liu M, Lingle CJ (2015) SLO3 auxiliary subunit LRRC52 controls
gating of sperm KSPER currents and is critical for normal fertility. Proc Natl Acad Sci
112:2599–2604
Zhang X, Anderson JW, Fedida D (1997) Characterization of nifedipine block of the human heart
delayed rectifier, hKv1. 5. J Pharmacol Exp Ther 281:1247–1256
Zhou B-Y, Ma W, Huang X-Y (1998) Specific antibodies to the external vestibule of voltage-gated
potassium channels block current. J Gen Physiol 111:555–563
Pharmacology of Calcium Channel
20
Santanu Mallik and Pratap Chandra Acharya

Abstract

Calcium channel plays a very crucial role in the regulation of various

vital functions of the body. The effects and modes of action of various drugs on
calcium channels have been outlined with every detail. The pharmacology of
various subunits and subtypes of this channel has been discussed in view of drug
development in the near future. The role of Cav1 channel in health problems such
as Parkinson’s disease, cardiovascular diseases, subtypes of Cav2 channels in
terms of G-protein inhibition and synaptic vesicle release, Cav3 channels for
peptide toxins, and α2δ ligands in amino acid transportation and synaptic trans-
mission has been highlighted in the literature. Diseases like obesity, epilepsy, and
anxiety can be treated easily by targeting the T-type calcium channel which is one
of the best potential therapeutic targets for the aforesaid diseases. In epilepsy and
neuropathic pain, α2δ subunit is the most prominent area of therapeutic target by
the gabapentinoid drugs. Thus, scientific research on calcium channel pharma-
cology may become revolutionary in the management of various chronic
diseases. In this chapter, all possible attention has been given to describe the
super selectivity of different subtypes of calcium channels by focusing on
isoforms of channels and biophysical properties in various target tissues.

S. Mallik
Department of Pharmacy, Tripura University (A Central University), Suryamaninagar, Tripura (W),
India
Bharat Pharmaceutical Technology, Amtali, Agartala, Tripura (W), India
P. C. Acharya (*)
Department of Pharmacy, Tripura University (A Central University), Suryamaninagar, Tripura (W),
India
e-mail: [email protected]

# Springer Nature Singapore Pte Ltd. 2020 683

P. Kumar, P. K. Deb (eds.), Frontiers in Pharmacology of Neurotransmitters,
https://doi.org/10.1007/978-981-15-3556-7_20
684 S. Mallik and P. C. Acharya

Keywords

Calcium channel · Voltage-gated calcium channel Cav1.2 · Cav1.3 · Cav2.3 ·

Cavβ · L-type calcium channel · Drug selectivity · α2δ ligand · Splice variants

Abbreviations

AID α-Interaction domain

CRMP-2 Collapsin response mediator protein-2
CYP450 3A4 Cytochrome P450 3A4
DUB Deubiquitinating enzyme
GABA γ-Aminobutyric acid
GPCR G-protein-coupled receptors
IP3 Inositol 1,4,5-trisphosphate
NAGly N-Arachidonylglycine
pS Picosiemens
TAT Transactivator of transcription
US-FDA United States Food and Drug Administration
USP5 Ubiquitin-specific peptidase 5
ω-TRTX-Hg1a ω-Theraphotoxin-Hg1a

20.1 Introduction

Erwin Neher and Bert Sakmann, Nobel laureates (1970), invented the technique
“patch clamp” (De la Peña and Gomis 2019) which confirmed the practical existence
of ion channels in a physiological environment. Ion channels are transmembrane
glycoprotein pores having the open and close properties like gates. These channels
allow the movement of ions in a regulated manner. Electrochemical gradient governs
the direction and rate of ion movement. The transportation is always down the
gradient and the speed of ion transportation through the channels is very high
which is approximately about 107 ions/s (Sanchez-Sandoval and Gomora 2019;
Uzieliene et al. 2018). The types of calcium channels are shown in Tables 20.1
and 20.2 (Zamponi et al. 2015; Spedding and Paoletti 1992).
In the recent medical science, diseases like migraine, epilepsy, hypertension, and
cerebral ischemia and rarer diseases like night blindness have been treated by
targeting calcium channels. The channels allow calcium ions to flow out of the
endoplasmic reticulum or to flow into the cell (Zamponi et al. 2010; Marchetti 2013).
The opening of such ion channels is initiated due to the changes in voltage across the
membrane, for example, signal propagation of nerve cells. One more factor which
helps open the channel is the ligand molecule like inositol 1,4,5-triphosphate (IP3)
(Prakriya and Lewis 2015; Simms and Zamponi 2014). The pancreatic beta cells,
neurons, myocytes, etc., have calcium channels. Such kind of channels forms most of
the systematic molecular connection between biochemical signaling intracellularly
20 Pharmacology of Calcium Channel 685

Table 20.1 Types and subtypes of calcium channels

1. Voltage-dependent calcium ion-selective channels
L-type
T-type
N-type
P-type
2. Calcium ion-selective channels
Calcium ion release channels in the sarcoplasmic reticulum
Receptor-operated calcium ion channels
3. Voltage-dependent ion channels (no calcium ion selectivity)
(Na+, K+, etc.)

Table 20.2 Calcium Ca2+ currents Family α1 subunit

channel currents and α1
L-type Cav1.1 Cav1
subunits (Dolphin 2016;
Cav1.2
Hirano et al. 2017)
Cav1.3
Cav1.4
P-type Cav2.1 Cav2
N-type Cav2.2
R-type Cav2.3
T-type Cav3.1 Cav3
Cav3.2
Cav3.3

and is responsible for the depolarization of membranes. Calcium channels are

responsible for the contraction of muscles, release of hormones, regulation of
membrane excitements, regulation of gene expressions, etc. (Badou et al. 2013;
Nanou and Catterall 2018; Nieto-Rostro et al. 2018).
Functioning of contraction–excitation coupling (Santulli et al. 2017), endocrine
and exocrine gland secretion (Gambardella et al. 2017), secretion of hormones
(DiMeglio and Imel 2019), platelet aggregation (Aggarwal et al. 2016), excitation–
transcription coupling (Kim and Kim 2018), release of neurotransmitters (Bruckner
et al. 2017), and chemotaxis (Larsch et al. 2015) are initiated by the calcium
channels. Thus, calcium plays a critical role as an initiator and modulator of cellular
functioning (Scott et al. 2016). This is the reason why calcium is considered as an
important regulatory component in the human body for various cellular activities. It
is important to focus on the biochemical and pharmacological depiction of calcium
channels to understand its whole contribution and process mechanism (Raffaello
et al. 2016).
Millions of calcium ions enter to the cells through calcium channels and help in
contraction–excitation coupling and release of neurotransmitters in muscles and
nerves. These channels are also available in neutrophils, lymphocytes, plant cells,
and sperms which are also considered as non-excitable cells. Calcium channels are
also found in intracellular membranes, but the type may vary with those present in
the plasma membrane (Medrihan et al. 2013; Südhof 2013).
686 S. Mallik and P. C. Acharya

Fig. 20.1 Pictorial representation of α1, β, α-2δ and γ subunits of calcium channel. Four homolo-
gous domains of α1 subunit (1–4) with helical structures have been presented

The opening and closing of the calcium channels are the fundamental properties.
Depending on the mechanism of these properties, calcium channels are categorized
(Fig. 20.1) under two broad headings: (1) voltage dependent and (2) receptor
operated. Voltage-dependent calcium channels are further subclassified depending
on the pharmacological sensitivities, kinetic properties, and voltage sensitivities
(Zamponi 2016).

1. Voltage-dependent calcium channels (Zamponi et al. 2015; Catterall and

Swanson 2015):
(a) L-type: Generally, this type of channel is activated by high voltages and has
large conductance of ~25 pS (picosiemens).
(b) T-type: This type of channel is activated by minimum voltages and is a
transient current with comparably low conductance of ~9 pS.
(c) N-type: It is an intermediate channel in between L- and T-types. This type of
channel conducts an intermediate size of ~15 pS transient current and is
usually activated with high voltages.

It has been reported that L- and T-type channels are present in skeletal and cardiac
muscles where N-type channel is absent. In sympathetic neurons of ganglia, T-type
is not present but both L- and N-types are available. The T-, L-, and N-types of
receptors coexist in dorsal root ganglia sensory neurons. Furthermore, differences
have been observed in the same type of channel located at different organs. For
instance, L-type calcium channel present in the skeletal muscle and heart shows
differences in electrophysiological and pharmacological properties (Kappel et al.
2013; Rose et al. 2013; Li et al. 2017a, b).
Receptor-operated calcium channels or calcium channels operated by receptor-
dependent mechanism are usually opened after receiving signals from an activated
associate receptor (Fig. 20.2). Chloride and ion channels are two major types
20 Pharmacology of Calcium Channel 687

Fig. 20.2 Speculative representation of voltage-dependent and receptor-operated calcium

channels and their mechanism of action

which come under receptor-dependent calcium channel. Chloride channels are

generally associated with glycine and γ-aminobutyric acid (GABA) receptor, and
nonspecific ion channels are found associated with nicotinic acetylcholine receptor
(Szabó et al. 2014; Schwab et al. 2012; Yocum et al. 2018).

20.1.1 Pharmacology of Calcium Channels and Its State Dependency

Drug affinity entirely depends on the specific gating status of the channels for the
drugs that target ion channels. State dependency is the reason behind the clinical
utility of drug molecules. On the other hand, experimental use of a drug is an
evidential proof of successful state-dependent inhibition. It is very important to
contemplate hypothetical considerations of state-dependent inhibition, which
includes drug-inactivation synergism, use-dependent block (the rate of nerve stimu-
lation is proportional to the degree of block), guarded receptors (acyclic), and cyclic
or allosteric receptor (Yoder et al. 2018).
The idea of drug binding was introduced by Armstrong in 1966 and Strichartz in
1973. Different strengths to resting closed (R), open (O), and closed inactivated
(I) ion channels have been reported. Later on, the idea was accepted as the theoretical
concept of a modulated drug receptor (Zaydman et al. 2014; Lenaeus et al. 2017).
688 S. Mallik and P. C. Acharya

Depending on this hypothesis, study on the biological phenomena with the help of
physics and drug or medication action experiments on ion channels helped the
medical science understand in a better way about the repression of signals of local
anesthetics and sodium channel inhibitors such as the antiarrhythmic drugs,
antiepileptics, and actions of other drugs (Borowicz and Banach 2014; Tikhonov
and Zhorov 2017).

20.1.2 Drug Action and State Dependency

The three conformational states of calcium channels have been widely accepted: R
state or resting, nonconducting; O state or open, conducting, or excited; and I state or
inactivated, nonconducting. During the depolarization process, channels move to I
through O from R and again back to the R state during the change in cell’s membrane
potential (hyperpolarization) (Alexander et al. 2015/16).

20.1.2.1 Drug Inhibition of the R State

The scientific findings are insufficient to understand the mechanism of R state-
dependent inhibition. This interaction is realistic but suggested the transformation
to O or I states which usually initiate drug dissociation from resting channels.
Factually, for Ca2+ channels, no single evidence has been reported for a “use-
dependent release” from the same block.
The difficulties with the interpretation of R state are represented in Fig. l. Peak
current inhibition or the initial block by the drug mibefradil during the first test pulse
may be elucidated as inhibition of resting channels (RB) with selectivity (Tang et al.
2016; Yang et al. 2014a, b).

20.1.2.2 State-Dependent Channel Inhibition – “O” and “I”

The binding of drug occurs only in the O state, and at this stage, the flow of the
current gets interrupted which is depicted in Fig. 20.3b. Cumulative suppression of
the current can be predicted from this model representation as shown in Fig. 20.3c.
There are two exponential time courses which have been associated with these
channels: recovery from inactivation (“fast phase”) and dissociation of the drug
channel complexes (“slow phase”) (Fig. 20.3) (Striessnig et al. 2014; Laurent et al.
2016).
The last phase is almost undetectable as the concentration of drug is nearly similar
to the saturation. The slow inactivation model as shown in Fig. 20.4a, b produces a
very similar attribute of use-dependent current inhibition (Fig. 20.4c). However,
among the two exponential time courses of the recovery processes, one depicts the
recovery from slow inactivation and the other indicates recovery from the fast
inactivated state.

20.1.2.3 Calcium Channel Inhibition – Molecular Studies

Recently, new important insights have been explored into the molecular events
underlying calcium channel block by conducting pharmacological studies on
20 Pharmacology of Calcium Channel 689

Fig. 20.3 Hypothetical open channel block model. (a) Open state model depicting kinetic scheme
of calcium channel, (b) molecular mechanism involved in an open state, (c) changes in kinetics of
use-dependent and open channel inhibition in reference to control, and (d) model graphical
representation of normalized current versus membrane potential in open state kinetics

recombinant and mutant calcium channels of different subunit compositions. The

subunit compositions strongly regulate the properties of high-voltage-activated
calcium channels. Particularly, the four subunits strongly modulate the inactivation
of high-voltage-activated channels (Prakriya and Lewis 2015; Zhang et al. 2017).

20.1.2.4 Mechanisms of Drug Action – At Molecular Level

The molecular level drug action can be easily understood by the actions of diltiazem,
phenylalkylamines, and mibefradil.
The pore-forming transmembrane segments IIIS5, IIIS6, and IVS6 contain very
important determinative of the putative receptor sites for phenylalkylamines, diltia-
zem, and dihydropyridines (Vega-Vela et al. 2017). Various studies reveal that a
change in inactivation kinetics helps many of the mutation processes within the
putative drug-receptor region (Laurent 2017). Moreover, it is evident that the affinity
of phenylalkylamines, diltiazem, and mibefradil has been impaired by the mutation
mechanism because it can also influence the channel activation. Various studies
suggest that the process of channel inactivation is closely linked to state-dependent
inhibition of Ca2+ channels (Zheng 2013; Santos et al. 2017). It is difficult to explain
690 S. Mallik and P. C. Acharya

Fig. 20.4 Hypothetical model of inactivated channel block. (a) Inactivated state model depicting
selective state transition kinetic scheme of calcium channel, (b) selective inhibition mechanism
involved in an inactivated state, (c) current kinetics of use-dependent and inactivated channel
inhibition in reference to control where D1 represents the initial impact of current on drugs in
comparison to control, and (d) model graphical representation of normalized current versus
membrane potential in inactivated state channel block kinetics

in depth about all recent findings on recombinant channels in terms of state-

dependent pharmacology. For example, mibefradil represents a benchmark of an
open channel blocker, showing insignificant changes in channel availability. How-
ever, these channels are expected to be more efficiently inhibited by “open channel
blockers” (Krouse et al. 2015).
Mibefradil-sensitive co-expression of the 0.12.1 subunit does not increase but
usually decelerating by nature. This result is inconsistent with respect to channel
block model opening. The same observation was found for Cav1.2 inhibition by
phenylalkylamines (Matsunami et al. 2012; Woo et al. 2019).

20.2 Cav1 Channels – Pharmacology

20.2.1 Clinical Pharmacology

For decades, treatment of hypertension and myocardial ischemia has been based on
the L-type calcium channel blockers. They are the first-line choice for the above
health issues and first choice for the physicians. The arterial vasodilators
20 Pharmacology of Calcium Channel 691

dihydropyridines generally reduce arterial muscle tone, resistance of peripheral

vascular tissues, and coronary and peripheral artery vasospasm. Dihydropyridines
also reduce cardiac oxygen demand by lowering the arterial blood pressure. With the
properties of spasmolytic effect, antianginal actions of dihydropyridines can easily
be established. Dihydropyridines show negative inotropic actions and remain inef-
fective to sinoatrial node and atrioventricular node function at its precise therapeutic
doses. Furthermore, verapamil and diltiazem are also negative dromotropic,
chronotropic, and inotropic to their antihypertensive, vasodilating, and spasmolytic
properties and thus inhibit myocardial oxygen consumption and increased heart rate
due to exercise. In the treatment of hypertensive patients with angina pectoris, these
drugs are suitable due to their direct cardiodepressant effects (Katzung 2017; Jaisser
and Farman 2016).
The vasodilating effects of Ca2+ channel blockers result in unwanted effects like
flushing, headache, dizziness, and hypotension at any therapeutic dose. Long-term use
of dihydropyridines has therapy-limiting side effects of peripheral edema associated
with ankle swelling. One of the major side effects of verapamil is it causes constipation
and can be explained by L-type calcium channel inhibition in intestinal smooth
muscles. Life-threatening health issues like reduced left ventricular function, cardiac
arrhythmia, or atrioventricular block (AV block) can also be caused by verapamil. This
problem becomes more prominent for the patients who administered β-adrenoceptor
blockers or have previous medical history of heart-related diseases (Katzung 2017;
Fardal and Lygre 2015).
No evidence has been reported for muscle weakness in skeletal muscles from the
unit of Cav1.1 channels or increased hearing thresholds from inhibition of Cav1.3 in
inner hair cells of the cochlea. At the same time, loss of vision from the unit of
Cav1.4 in photoreceptor of the retina or disturbances in the central nervous system
from the block of Cav1.2 and/or Cav1.3 in the brain have not yet been reported.
L-type calcium channels always serve important functions where Ca2+ channel
blockers cause no significant side effects at therapeutic doses in other tissues. Ca2+
channel blocker overdose may suppress insulin secretion, and as a result, hypergly-
cemia may occur only when plasma attains toxic level (Vallejo-Illarramendi et al.
2014).

20.2.2 Molecular Pharmacology

Ca2+ channel blockers can be categorized under different chemical classes

depending on their clinical use. Nifedipine, felodipine, or amlodipine which falls
under dihydropyridine group is most widely used. Benzothiazepine (e.g., diltiazem)
and phenylalkylamine (eg., verapamil) usually interact with drug binding domains
with high-affinity and activation gate of L-type calcium channel α1 subunits near to
the pore (Fig. 20.4) (Bladen et al. 2014a, b; Chaugai et al. 2018; Ataei et al. 2019).
Binding is stereoselective and reversible in nature. The binding takes place with
dissociation constants in nanomolar range (0.1–50 nM) (Glossmann and Striessnig
1990). Cycling of the channel with normal voltage dependency through its open,
692 S. Mallik and P. C. Acharya

resting, and inactivated conditions which is also known as modulated receptor model
usually interferes with bound drugs (Bain 2019).
Dihydropyridines, uncharged, stabilize primarily and prevail upon inactivated
channel states. Inactivated channel conformation shows higher affinity and thus
decreases with increased availability of inactivated channel states at voltage-
dependent block or more depolarized membrane potentials (Zhao et al. 2019)
which is favored by the open channel state and always favors the access of
benzothiazepines and phenylalkylamines. Blocking of pore directly together with
slowed recovery from inactivation with stabilization of inactivated channel state
results in pronounced frequency or use-dependent inhibition.
Dihydropyridines which are Ca2+ channel activators also exist. For different
reasons, the sensitivity of L-type calcium channels for dihydropyridine Ca2+ channel
blockers varies in different tissues (Surmeier et al. 2019). Variable contribution of
these L-type calcium channels to total L-type current is one of the prominent
explanations. Cav1.4 and Cav1.3 exhibit about 5- to 10-fold down to
dihydropyridines than Cav1.2, which is demonstrated as negative membrane
potentials in heterologous expression systems (Ortner et al. 2014; Kang et al.
2012). Sinoatrial node is usually dominated by Cav1.3 which can explain the
relatively weak inhibition of L-type pacemaker currents (Mesirca et al. 2015).
L-type current in substitute splicing of α1 subunits is another important factor
affecting dihydropyridine sensitivity. It has been expositional that dihydropyridines,
at lower concentrations, inhibit currents in arterial smooth muscle than in the
functional myocardium for Cav1.2. The presence of dihydropyridine-sensitive splice
variants predominantly expressed in arterial smooth muscle has been established by
rigorous analysis of Cav1.2 α1 splice variants present in heart and smooth muscles.
Moderate negative voltage activates some of these splice variants and brings out a
steady-state Ca2+ window or inward current nearer to the resting potential of cardiac
muscles (Li et al. 2017a, b; Fan et al. 2005). More amount of depolarized resting
membrane potential available in smooth muscle usually favors inactivated channels
obstructed by dihydropyridines in comparison to cardiomyocytes (Liao and Soong
2010).
Another way to enhance dihydropyridine sensitivity is that few of such splice
variants are liable to more promised inactivation of steady state. There is also
evidence that the drug binding domain 5s is affected by alternative splicing of
Cav1.2 α1 and therefore the dihydropyridines can access the inactivated channels.
Dihydropyridine sensitivity of Cav1.3 is slightly affected by alternative splicing in
the C-terminus (Jang et al. 2013).

20.2.3 L-Type Calcium Channel as Potential Targets for Various

Diseases

The pharmacotherapeutic potential of L-type calcium channel blocking in other

tissues is a serious point of consideration. The physiological and pathophysiological
role of L-type calcium channels other than the cardiovascular system has raised
20 Pharmacology of Calcium Channel 693

importance to draw the attention of researchers. Efficient inhibition of L-type

calcium channels in the brain is particularly the prime matter of concern.
Therapeutically relevant pharmacological effects can be theorized from findings
in human mutations and mutant mice to consider Parkinson’s disease and its
neuroprotection as well as treatment of neuropsychiatric disorders like autism
spectrum disorders and febrile seizures. Although clinical use of calcium channel
blockers in cardiovascular diseases are well established, they may be used for other
neurodegenerative indications (Bidaud et al. 2006; Matta et al. 2015).

20.2.3.1 Parkinson’s Disease

A clinical trial of phase III has been started off to study the neuroprotective potential of
the dihydropyridine isradipine in the early stage of Parkinson’s disease (Lotia and
Jankovic 2016). This phase III study has been initiated depending on the
robust preclinical outcomes regarding the key role of L-type calcium channel arbitrated
Ca2+ load in substantia nigra pars compacta neurons. For instance, isradipine is currently
licensed to treat high blood pressure. Presently, the findings from preclinical in vivo
neurotoxin-influenced Parkinson’s disease models are not sufficient to predict
whether Cav1.2, Cav1.3, or both isoforms are responsible for the proposed toxicity of
Ca2+ ion. Calcium-mediated side effects like peripheral edema and/or hypotension, in
clinical trials, limit long-term treatment of Parkinson’s disease with large doses of
dihydropyridines providing a strong support for efforts to discover calcium chan-
nel selective inhibitors (Surmeier et al. 2017; Yang et al. 2014a, b; Caricati-Neto and
Bergantin 2016).

20.2.3.2 Neuropsychiatric Disease

Calcium voltage-gated channel α1 gene belongs to the family of genes which
provides instructions for generating calcium channels. Genome-wide calcium
channels have revealed a strong group of intronic single nucleotide polymorphisms
in calcium voltage-gated channel α1 and the responsiveness for psychiatric
disorders, including schizophrenia, manic-depressive illness, and severe depression
in individuals. As reported in psychiatric genetics, this is one of the most consistent
associations (Nanou and Catterall 2018; Quach et al. 2015).
The recent scientific findings indicate that the single nucleotide polymorphisms
can increase mutations in voltage-gated α1 channel leading to autism in Timothy
syndrome. These facts strongly advocate the re-evaluation of calcium channel
blockers for the treatment of schizophrenia, bipolar disease, and major depression
in certain individuals (Gershon et al. 2014; Lee et al. 2016).

20.2.3.3 Cardiovascular Disease

Recently discovered Cav1.3 plays a key role in aldosterone secretion. It may be one
of the reasons why therapeutic doses of the dihydropyridine class of calcium channel
blockers show no robust inhibitory effects on aldosterone secretion in humans. In the
near future, potent Cav1.3 selective inhibitors might be discovered (Striessnig et al.
2014). Such inhibitors may not affect cardiac inotropy due to the unavailability
of Cav1.3 channel in ventricular myocardium. The merged mechanism of action
694 S. Mallik and P. C. Acharya

seems to be clinically fruitful in patients with heart failure, in which elevated heart
rate has been noticed may be due to increased sympathetic drive along with
aldosterone which may be caused by secondary aldosteronism (Gordan et al.
2015). Heart rate at high level is always a risk factor in heart failure. But lowering
of heart rate with specificity may be improved by cardiovascular outcomes with the
hyperpolarization-activated cyclic nucleotide-gated channel blocking bradycardia
agent ivabradine (Tse et al. 2017).
Physiological functions controlled by different L-type calcium channel isoforms
identify these types of channels as new drug targets. It is a matter of high con-
cern because the nonselective channel blockers are in clinical use for a long time.
Furthermore, Cav1.3 selective Ca2+ channel blockers have high therapeutic potential
for many indications including neuropsychiatric disorders (Lu et al. 2015).

20.3 Cav2 Channels – Pharmacology

20.3.1 Cav2.1 and Cav2.3 Channels and Their Prospective Roles

Peptide toxins that are collected from the venoms of organisms like fish-hunting
molluscs, spiders, and scorpions can inhibit voltage-gated calcium channels. For
example, polypeptide ω -agatoxin, subtype IVA, having a molecular weight of 5210
kDa, collected from the venom of the North American funnel web spider
Agelenopsis aperta, inhibits Cav2.1 channels (Lian et al. 2014). If they are carefully
regulated with compounds that normalize deviant gain of function, Cav2.1 channels
are not considered as good pharmacological targets in large extent where Cav2.3
channel inhibitors could potentially have an influential effect in pain (Duda et al.
2016) and seizure disorders (Kim et al. 2015).
These channels lack specific small organic inhibitors. The inhibitor of Cav2.3
channels, SNX-482 spider toxin, isolated from the venom of the spider
Hysterocrates gigas also targets Cav1.2 L-type calcium channels with A-type K+
currents. SNX-482 is also known as ω-theraphotoxin-Hg1a, or ω-TRTX-Hg1a
(Zamponi 2016; Moutal et al. 2017; Rousset et al. 2015).

20.3.2 Cav2.2 Channels and Their Roles

In the context of Cav2.1 and Cav2.3 channels, there is extensive information

available to the researchers related to N-type calcium channel inhibitors. The
Cav2.2 channel-mediated cellular activities can be easily controlled for therapeutic
purposes by four significant principles (Chai et al. 2017).
20 Pharmacology of Calcium Channel 695

20.3.2.1 Direct Block of Cav2.2 Channel by Small Organic Molecules

and Peptides
The peptides present in the venoms of a variety of raptorial organisms significantly
retard Cav2.2 channels. Peptide toxin, ω-conotoxin GVIA, extracted from the fish-
hunting cone snail Conus geographus selectively and potently inhibits Cav2.2
channels (Lewis et al. 2000; Zhang et al. 2015) (Fig. 20.6, pathway 2). To distin-
guish between P/Q-type and N-type currents in varying neurons, ω-conotoxin GVIA
and ω-agatoxin IVA have been widely used. On the other hand, the above toxins also
typify significant modes of action, namely, gating modification and pore block.
Small 27-amino-acid peptide ω -conotoxin GVIA is made up of a backbone that is
contrived by three disulfide bonds. The ω -conotoxin GVIA physically lodges into
the permeation pathway and blocks the channels (Ramírez et al. 2017; Thapa et al.
2014).
The virtually irreversible blocking occurs if the unblocking rate constant remains
low. The ω-agatoxin IVA is a much larger peptide (83 amino acids) than the
ω
-conotoxin GVIA and acts by blocking voltage sensor movement. If any mem-
brane does not repetitively depolarize, it can produce poorly reversible inhibition.
Repetitive depolarization allows the voltage sensors of the channel to remove the
bound toxin (Kuwahara and Kimura 2015; Mir et al. 2016). Spider toxins like
α-grammotoxin SIA which is a peptide by nature and isolated from the venom of
the tarantula Grammostola spatulata act as gating inhibitors and inhibit both Cav2.1
and Cav2.2 channels. Another example is SNX-482 which is obtained from the
tarantula Hysterocrates gigas and is a Cav2.3 channel blocker (Xu et al. 2018;
Osteen et al. 2016; Bourinet et al. 2001).
It has been reported that fish-hunting molluscs produce enriched palette of
calcium channel blockers having significant selectivity for Cav2.2 channels. The
25-amino-acid Cav2.2 channel pore-blocking toxin called ω-conotoxin MVIIA has
been isolated from the venom of the snail Conus magus and can mediate potent
analgesia in cancer patients with pain of cancer (Thompson et al. 2006) and
rodents (Gardezi et al. 2016). The above context fits with the significant role of
Cav2.2 channels in neurotransmitter released from afferent terminals. The Cav2.2
channel inhibitors such as ω-conotoxins MVIIB, MVIIC, and MVIID have been
obtained from C. magus. In the same way, snails like Conus striatus, Conus fulmen,
and Conus catus produce various Ca2+ channel blocking peptides. Such peptides
usually have similar disulfide bridge arrangements and act as pore blockers. Among
them, most of the peptides act selectively on Cav2.2 channels, for example, ω-
conotoxins SIA, FVIA, CVID, etc. (Motin et al. 2007). Few other peptides like ω-
conotoxins SIB and MVIIC have the role to block Cav2.1 channel. It has been
reported that ω-conotoxin CVID has undergone analgesic tests in clinical trials (Bajaj
and Han 2019; Carstens et al. 2011; Bourinet and Zamponi 2017).
The construction of chimeric channel and site-directed mutagenesis helps in
investigating the blocking site for ω-conotoxins GVIA and MIIVA in the Cav2.2
subunit (Page et al. 2016). The above study indicates that the large extracellular
domain IIIS5–S6 region is a key factor to determine ω-conotoxin GVIA block.
Moreover, the dramatic reversibility of ω-conotoxins GVIA and MVIIA block
696 S. Mallik and P. C. Acharya

occurs due to proline and mutagens of a single glycine residue in the 1326-amino-
acid position of the channel. An ensuing study reveals that ω-conotoxins MVIIA and
CVID cause changes in both the extent of inhibition and kinetics of the
co-expression of Cav2.2 δ subunit channels (Wang et al. 2016; Mollica et al. 2015).
Various peptide toxins are highly selective and are high-affinity blockers to
several Ca2+ channel subtypes, but they cannot cross the blood–brain barrier. A lot
of pore-blocking conotoxins do not perform effectively as state-dependent blockers,
namely, local anesthetics and anticonvulsants (Norton 2017; Bourinet and Zamponi
2017; Duggan and Tuck 2015). But the above issue can be overcome by increasing
affinity and selectivity of developing small organic blocking agents. The
peptidylamines are designed to mimic the pore-blocking activity of the larger ω-
conotoxin molecules. This is executed by linking acid group of N,N-disubstituted
leucine to amine group of tyrosine. The high-affinity block of Cav2.2 channels has
been evidenced in various literatures. Moreover, phenylalanine and
benzyloxyaniline derivatives have strong affinity toward Cav2.2 channel blockers,
and management of pain has been reported with scientific evidences (Jin et al. 2013;
Robinson et al. 2017; Brady et al. 2013).
Antipsychotics with D2 dopamine receptor-blocking activity are examples of few
important Cav2.2 channel blockers (Siafis et al. 2018) (Fig. 20.6, pathway 2), which
contain a core morpholine, piperidine, and/or piperazine structure and usually linked
to 1 or 2 diphenyl moieties through alkyl chains. Such agents are renowned for their
blocking property of N-type channels (Brimblecombe et al. 2015). Furthermore,
several major compounds falling under this section have been confirmed for the
management of pain in animal models. The dependency and intoxicating narcotic
properties of ethanol are also abated, and the derivatives like pyrazole piperidines
and amino-piperidine sulfonamide showed mixed action on Cav2.2 and Cav2.3
channels (François et al. 2014; Striessnig et al. 2015; Striessnig et al. 2014).
Cilnidipine is a drug which comes under dihydropyridines and acts as a blocker of
L-type calcium channel. At the same time, it is also reported that some other drugs of
the same class can also block N-type calcium channels with high accord. These
drugs have shown analgesic properties in rats and kidney protective as well as
antihypertensive properties in human volunteers. Such favorable effects of drugs
may be imputable to their action on the sympathetic nervous system of N-type
channels (Shetty et al. 2013; Adake et al. 2015; Manthri et al. 2015).
Moreover, other examples of Cav2.2 channel blockers found in the literature
include (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-
(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1) which is an oxindole
compound. This compound has analgesic properties and is a state-dependent inhibi-
tor. Further, long-chain aliphatic monoamines (Beedle and Zamponi 2000) and
farnesol also block (Roullet et al. 1999) Cav2.2 channels with stronger accord and
reveal favored blockade of inactivated channels (Nimmrich and Eckert 2013; Page
et al. 2016; Ripsch et al. 2012).
The inhibitions of Cav2.2 channels are also done by other classes of
pharmacophores indicating that these channels have promising pharmacotherapeutic
implications. Left side shift has been observed in the steady-state inactivation curve
20 Pharmacology of Calcium Channel 697

of state-dependent blockage of Cav2.2 channels along with frequency-dependent

inhibition of activity. However, small molecule Cav2.2 channel blockers are
unknown in comparison to the peptide toxins. The physical interaction site of
Cavβ subunit and mutation affecting only few nucleotides in the domain I–II region
of Cav2.1 has no evidence for the effect of piperidine block (Adams and Berecki
2013; Schroeder et al. 2006; M’Dahoma et al. 2016).

20.3.2.2 Cav2.2 Channel and G-Protein Inhibition

Various G-protein-coupled receptors are practically connected to Cav2.2 channels
(Fig. 20.5, pathway 1). Nucleotide exchange in the associated Gα subunit initiates
the activation of these receptors and produces active signaling molecules like Gα-
guanosine triphosphate and Gβγ subunit. The physical combination of Cav2.2
channel and Gβγ subunit regulates the potent voltage-dependent inhibition of chan-
nel response (Adams et al. 2012; Jurkovicova-Tarabova and Lacinova 2019).
The regulation of Cav2.1 channel occurs in parallel manner and goes through in a
much smaller degree of inhibition. A large majority of drugs act through several G-
protein-coupled receptors, and such receptors are linked with other downstream
responder systems. The objective activity of a receptor agonist is niggardly linked
to Cav2.2 in the case of opioid receptors. Morphine, a μ-opioid receptor agonist, is a
potent analgesic which interacts with opioid receptors (Wright et al. 2015; Taylor
2009). The drug impedes Cav2.2 channels in dorsal horn synapses and activates G-
protein-coupled potassium channels. It is believed that the receptor-induced inhibi-
tion of Cav2.2 channel reduces presynaptic calcium levels. As a result, it reduces
synaptic communication between afferent nerve terminals. Morphine acts at μ-
opioid receptors in the central nervous system, but it is very difficult to establish a
clear relation between physiological outcome and moderation of Cav2.2 channels
(Montandon et al. 2016; Baloh 2019).
Morphine is considered to be very selective for the μ-opioid receptors and
discriminating agonists of the rest three other receptors, namely, delta-, kappa-,
and nociceptin receptors of the extended opioid receptor family. The selec-
tive blocking of the μ-opioid receptors occurs through inhibition of Cav2.2 channels.
The activation of μ-opioid receptors induces analgesia in numerous animal models.
But clinically, there are no evidences of targeting analgesia by δ-opioid and
nociceptin receptor. Only pentazocine, which is a k-opioid receptor agonist, is
used as an analgesic (Turnaturi et al. 2016; Jokinen 2017; Winters et al. 2019).
Gamma-aminobutyric acid B (GABAB) receptors or metabolic receptors are
another type of receptors that interfere with Cav2.2 calcium channels in dorsal
horn synapses. Although systemic GABA agonists such as baclofen is associated
with CNS side effects, it is used intrathecally to treat spasticity and central pain
arising due to spinal cord or in brain injury. The most interesting part is that the
α-conotoxin Vc1.1, a disulfide-bonded peptide, and the structurally related peptide
Rg1A have been evidenced to trigger outlying GABAB and resulting in Cav2.2
channel inhibition along with analgesia when delivered through intramuscular or
intrathecal route. Improved oral bioavailability of acyclic version of the Vc1.1 has
698 S. Mallik and P. C. Acharya

Fig. 20.5 Pathway 1 represents the modulation of drugs, toxins, and signals. L-type calcium
channel active drugs are shown here. The potency of L-type channel blockers depends on mem-
brane potential as shown in pathway 2. Different signaling pathways like G-protein (pathway 2a) or
enzymes activated by G-protein-coupled receptor (GPCR) (pathway 2b) or receptor tyrosine
kinases (RTKs) (pathway 2c)

been designed for better therapeutic response (Cai et al. 2018; Ren et al. 2019; Liu
et al. 2018; Bowery 2016).
Overall, Cav2.2 channels are very important effectors of 7-transmembrane helix
receptors. The physiological importance of this regulation can be clearly exemplified
in the primary afferent pain pathway. The GPCR agonists can also regulate their
downstream effects in many other physiological processes via Ca2+ channels.
This process is connected to the entry of calcium that plays crucial role in cellular
20 Pharmacology of Calcium Channel 699

physiology – the modulation and triggering of neurotransmitter release (Morrill et al.

2015; Patel et al. 2018).

20.3.2.3 Cav2.2 Channel Trafficking and Inhibition

CRMP-2, or collapsin response mediator protein-2, and Cav2.2-type calcium
channels are associated with each other (Fig. 20.6, pathway 3), and the interactivity
supports the channels in the plasma membrane. It is assumed that the rate of channel
internalization has been slowed down and reversibly facilitates Cav2.2 channel-
mediated release of, for example, calcitonin and peptide types of neurotransmitters
(François-Moutal et al. 2015; Buchta et al. 2019).
In an opposite way, unsettling of Cav2.2 channel reciprocity with collapsin
response mediator protein-2 can be attained by using peptides. TAT (transactivator
of transcription) peptides or cell-penetrating peptides reduce the density of Cav2.2
channel in the plasma membrane and arbitrate analgesic effects in various pain
managements. Thus, without blocking the function of a channel, calcium entry can
be controlled by targeting the mechanism which controls channel density in the
plasma membrane as arbitrated by Cav2.2. The other association mechanism of these
channels with the ancillary Cav2.2 δ subunit is very censorious for Cav2.2 channel
trafficking (Chew and Khanna 2018; Cassidy et al. 2014).

20.3.2.4 Synaptic Vesicle Release Machinery and Cav2.2 Coupling

The proteins which are involved in fast synaptic transmission are physically
associated with Cav2.2 channels. Synthetic synprint peptides block Cav2.2
channel-mediated synaptic transmission and initiate the competitive interference of
Cav2.2 interaction with syntaxin 1A. From the above illustration, it can be concluded
that without changing the function or density of Cav2.2 channel, the pharmacologi-
cal manipulation of Cav2.2 channel-mediated physiological processes can be
executed easily (Wong et al. 2014; Ferron et al. 2014).
It is possible to identify small organic factitious of these synprint peptides with
the support of Cav2.2 channel trafficking regulators. However, for management and
treatment of conditions like pain, such facilities may be utilized to target Cav2.2
channel-mediated synaptic transmission as a better futuristic consideration.

20.4 Cav3 Channels – Pharmacology

20.4.1 Peptide Toxins

As the peptide toxins are unsuccessful in crossing the blood–brain barrier, they are
not administered orally as therapeutic agents. Kurtoxin has high affinity to inhibit
Cav3.1 calcium channels. This peptide is extracted from the venom of the scorpion
species Parabuthus transvaalicus. The above compound functions as a gating
modifier in a manner comparable to that described for the ω -agatoxin IVA, the
P-type channel blocker. Again, kurtoxin targets both N- and L-type calcium channel
700 S. Mallik and P. C. Acharya

Fig. 20.6 N-type channels regulated by toxins, drugs, and various signals. Pathway 2 indicates the
most pertinent group of active drugs. A different type of signaling pathway has been shown in
pathway 3 representing the CRMP-2 (scaffolding of proteins)
20 Pharmacology of Calcium Channel 701

isoforms and also shows its effects on sodium channel (Bourinet and Zamponi 2017;
King et al. 2008; McDonough 2007; Mary et al. 2018).
The structure of kurtoxin shows similarities to other scorpion toxins. However,
the unique surface properties of kurtoxin are the main reason for its action on T-type
channels. Two additional Parabuthus transvaalicus scorpion toxins (KLI and KLII)
also exert blocking effects on T-type calcium channels. Both toxins have weak
impact on Cav3.3 channels but can easily block sodium channels and T-type
channels (Antal and Martin-Caraballo 2019; Nanclares et al. 2018; Housley et al.
2017).
The most prominent sodium channel inhibitors are protoxins I and II which are
peptides collected from the Thrixopelma pruriens tarantula. Subtype-dependent
Cav3 channels are blocked by both peptides. Protoxin I advantageously blocks
Cav3.1 channels over Cav3.3 and Cav3.2 channels. Having the best accord for
Cav3.2 channels, protoxin II favors to act as a gating modifier. PsPTx3 is another
peptide spider toxin, extracted from Theraphosidae tarantula, which has properties to
block T-type calcium channels and perceptible selectivity for Cav3.2 channels (Kyle
et al. 2017; Bladen et al. 2014a, b; Klint et al. 2012).
In overall comparison with N-type calcium channels, peptide toxin impedes Cav3
channels which remain relatively limited and derived mostly from arachnids. Most
importantly, peptide blockers of T-type calcium channels should not be restricted to
those derived from venomous species. For instance, an intrinsic agonist of the
chemokine receptor CCR-2 (C-C motif chemokine receptor 2), monocyte
chemoattractant protein-1, can directly and actively inhibit Cav3.2 T-type calcium
channels (Ellinor et al. 1994; Silva et al. 2018; Bellampalli et al. 2019).

20.4.2 Inorganic Ions

The responsiveness to extracellularly applied nickel ions is a crucial feature of

differentiating features of T-type calcium channels. With a minimum difference of
one order of magnitude, Cav3.2 channels display a greater accord for nickel ions
when compared to channels of Cav3.1 and Cav3.3. Generally, Cav3.2 channels show
a distinctive histidine remnant inside the domain S3–S4 loop at position 191 which is
the key reason behind such affinity (Antal and Martin-Caraballo 2019; Kang et al.
2006).
In the channels, the same remnant acts as a prime redox injunction site. This
proceeds to hindrance of channel functionality by ascorbate, and in the presence of
L-cysteine, the upregulation of channel function has been observed. During the
presence of metal zinc ion, the distinctive regulation of Cav3 isoforms also takes
place. The zinc ion strongly inhibits Cav3.2 channels in comparison to two other
Cav3 isoforms. But under certain conditions, zinc ions also act as agonists of Cav3.3
channels by reducing the rate of deactivation (Voisin et al. 2016; Wagner et al. 2013;
Bogeski et al. 2011).
On the other hand, magnesium ions also influence to moderate the activity of
T-type channel. It has been reported that blocking affinity of distinctive magnesium
blocking in solutions containing external calcium and barium underlies the
702 S. Mallik and P. C. Acharya

perceptible differentiations in the barium and calcium magnitude of Cav3.1 (Srebro

et al. 2017; Barua et al. 2019).
Like the divalent metal ions like Mg2+, Ca2+, etc., T-type channels are
also blocked by trivalent metal ions like Al3+, Fe3+, etc. Specially, one of the most
potent lanthanides, yttrium, has been used for cloned human Cav3.1 channels,
having an affinity of around 30 nM. Upon increasing the concentration of
permeations, the block was greatly attenuated which indicates that the trivalent
ions usually act by physically obstructing the pore of the channels (Himeno and
Fujishiro 2019; Wang et al. 2018).
In a nutshell, for the selectivity and activity of T-type calcium channel, the metal
ions are considered as very potent inhibitors. However, such ions are not
recommended for therapeutic approaches but can be used as research tools for
further discovery of various alternatives.

20.4.3 Organic Molecules

There are no shortfalls of T-type organic calcium channel blockers when compared
with peptide toxins. Various categories of T-type calcium channel blockers were
brought to light in different time frame (Fig. 20.7). The drug amiloride is one of the
very first accepted T-type calcium channel blockers which acts as diuretics, blocking
the Cav3.2 channels of about a single degree of magnitude with higher accord
compared to Cav3.3 and Cav3.1 channels. But it is a fact that amiloride is not a
distinct T-type calcium channel inhibitor. The antiepileptic agent ethosuximide
comes under the succinimide group. This compound displays state-dependent inhi-
bition and is a low-affinity blocker of all three categories of Cav3 channel isoforms
(Zamponi and Diaz 2017; Striessnig et al. 2015; Seo et al. 2007).
Due to the pretended selective inhibition of mibefradil, a T-type calcium channel,
initially it generated a significant excitement in this field. The United States Food
and Drug Administration (US-FDA) had approved the drug earlier for the treatment
of hypertension. But later on, it was reported across the world that the drug is
metabolized by cytochrome P450 and had severe drug–drug interactions. Hence,
mibefradil was withdrawn from the market. Few years later, a derivative of
mibefradil (NNC-55-0396) was developed which has very less interaction with
cytochrome P450 3A4 (CYP3A4) (Oshima et al. 2005; Wang et al. 2017; Okuyama
et al. 2018).
T-type calcium channels react with synthetic cannabinoid and endocannabinoid
receptor ligands. Anandamide, a neurotransmitter, and its derivative NAGly
(N-arachidonylglycine) arbitrate mighty inhibition of Cav3 calcium channels. A
synthetic carbazole derivative, NMP-7, acts as an agonist of cannabinoid receptors.
The aforesaid compound and its derivatives usually involve in blocking of T-type Ca
channels (Nam 2018; Qian et al. 2017).
Neuroleptic drugs like diphenylbutylpiperidines are familiar for their actions as
D2 dopamine receptor antagonists. Numerous members of this class of compounds
like penfluridol and pimozide significantly inhibit Cav3 channels. Major drug
20 Pharmacology of Calcium Channel 703

Fig. 20.7 Pictorial representation of different modulatory inhibitions of T-type calcium channels
like kurtoxin. The figure shows that inhibitors may block the passage physically or by binding the
gating mechanism (pathway 1). On the other hand, pathway 2a represents the direct regulation of
calcium channel by activation of GPCRs or indirect regulation by CaMKII protein kinase (pathway
2b). The channel degradation may occur due to interference of isopeptidase T or also known
as USP5 (ubiquitin-specific peptidase 5) (pathway 3) (Zamponi et al. 2015; Watanabe et al. 2015)
704 S. Mallik and P. C. Acharya

discovery initiatives confined to the piperidine core pharmacophore have helped in

the synthesis of a number of T-type channel inhibitors with great selectivity and
potency. For example, it includes a compound which is termed as Z944 and usually
mediates potent Cav3 channel inhibition in several in vivo models of pain (Yang
et al. 2016; Roebuck et al. 2018). Moreover both Z944 and TTA-A2, another Cav3
inhibitor, mediates state-dependent preferential inhibition of Cav3.2 T-type currents.
It is also reported that increasing the dose of TTA-A2 leads to the sleep and high-fat
diet-induced weight gain (Tomita et al. 2019; Snutch and Zamponi 2018; Santi et al.
2002).
It is reported that the T-type calcium channels have the aptness to interrelate with
certain categories of dihydropyridines. Nimodipine and niguldipine, the L-type
calcium channel blocking agents, also potently block T-type channels. Several
scientific evidences depict that few types of dihydropyridine show better blocking
activity on T-type channels rather than L-type channels. On the other hand, ST101
compound evinces a channel-activating response which is useful to further disentan-
gle the role of T-type channels (Gadotti et al. 2015; Kuo et al. 2014; Zamponi et al.
2015).
Various classes of compounds as mentioned above have been tested for inflam-
matory and neuropathic pain in rodent models to arbitrate analgesia. It has been
reported that two T-type channel blockers, namely, Z944 and ABT-639, have been
tested in humans for efficacy and safety in pain management (Jarvis et al. 2014).
Thus, the importance of Cav3.2 T-type calcium channels has been revealed in the
foremost dorsal root pain pathway. Contrarily, ethosuximide is one of the prime
T-type channel blocking antiepileptic drugs used in the absence seizures by targeting
T-type calcium channels. In the same way, T-type calcium channels include
zonisamide and valproic acid that are used clinically for the treatment of epilepsy
(Powell et al. 2014; Snutch and Zamponi 2018).

20.4.4 Intervention with Cav3 Channel Regulation

An extracellular signaling molecule controls T-type calcium channels. This can

be potent to be utilized for therapeutic uses. It has been reported that the T-type
Cav3.2 channels are regulated by oxidation-reduction modulation. The inhibition of
Cav3.2 channel activity is initiated by ascorbates via catalyzed oxidization of metals.
The oxidation-reduction activity regulates the increase of Cav3.2 current amplitudes
by L-cysteine (Guse 2015; Loperena and Harrison 2017). Such oxidation-reduction
regulation takes place at a definite remnant, which is also involved in blocking Ni2+
of such channels, resulting in hyperalgesia. Hydrogen sulfide induces abnormally
heightened sensitivity to pain or hyperalgesia through the actions of Cav3.2 channels
in the same way. Again, administration of polaprezinc can arbitrate analgesia
through its antioxidant activity in a model for interstitial cystitis (Latham et al.
2009; Watanabe et al. 2015).
By regulating intracellular messenger, Cav3 channel activity can be altered in a
different way too. Such activity includes effects of direct binding of G-proteins,
20 Pharmacology of Calcium Channel 705

protein kinases, and phosphatases. Thus, the “kiss of death” process for a protein,
that is, ubiquitinating and deubiquitinating enzyme (DUB), namely, isopeptidases,
deubiquitinases, and ubiquitin proteases, controls Cav3.2 channels, and the T-type
channel regulation can possibly be used as a therapeutic approach for the manage-
ment of pain (François et al. 2015; Joksimovic et al. 2018).

20.5 a2d Ligands – Pharmacology

20.5.1 Mechanism of Action – a2d Ligands

The α2δ subunit’s molecular mechanism of action studies indicate that pregabalin
and gabapentin produce very little or zero acute retardation of calcium currents in
neuronal cell bodies transfected cells (Offord and Isom 2016). But there are few
evidences of small acute inhibitory effects. The research shows that gabapentin is
unable to inhibit calcium currents in model mouse dorsal root ganglion neurons. The
current in dorsal root ganglions from α2δ-1 is overexpressed in mice and inhibited
by gabapentin (Tano et al. 2019).
It is also found that persistent application of the above drug markedly reduces
calcium currents generated by several different α1/β/α2δ subunit combinations.
During the use of α2δ-1 or α2δ-2, the effect has been observed (Kazim 2017).
However, in the mutant α2δ, α2δ, and α2δ-3 subunits the effect does not occur
and also equally true for the subunits which bind very poorly with gabapentin. This
type of evidences strongly indicates that effects of gabapentin are in fact occurring
through binding to α2δ subunits (Freynhagen et al. 2016; Ikeda et al. 2018; Heyes
et al. 2015).

20.5.2 a2d-1 Splice Variants and Binding of Gabapentin

The alternative splice variant of α2δ-1 (ΔA+BΔC) shows a reduced accord for
gabapentin, whose ion is increased after nerve injury in dorsal root ganglion neurons.
This indicates the possibility of variation in the expression extent of splice variants in
neuropathic pain of individual patients (Patel and Dickenson 2016; Brockhaus et al.
2018).

20.5.3 a2d Subunits and Ligand Binding Sites

The binding site for the antiepileptic drugs like gabapentin and pregabalin is
considered to be at α2δ-1 and α2δ-2 subunits. These compounds are intended to
increase the activation of GABA 2 receptor and synthesized to be immutable
lipophilic analogs. Factually gabapentin does not affect GABA levels or GABA
receptors. However, gabapentin is effective in few cases of epilepsy that are
characterized by loss of consciousness and convulsions in human and other
706 S. Mallik and P. C. Acharya

experimental animal models (Donaldson and Beazley-Long 2016). A key step to

untangle the function of gabapentin is to refine and point out the binding sites in the
cerebrum by utilizing proteomic approaches and radiolabeled gabapentin. Investiga-
tional results indicate about the primary binding site which is α2δ-1. The
3
H-gabapentin has been observed to refuse to bind with α2δ-3, but easily accepts
bonding with α2δ-2. Accessory proteins of voltage-gated calcium channels, the
α2δ subunits, are not considered as drug targets (Celli et al. 2017; Kadurin et al.
2016).
It is considered that the α2δ protein does not possess any binding site to ligands
without having δ-1 in gabapentin. The Hill coefficient data near to 1 indicates lack of
binding cooperativity for single affinity binding site. Initially, it was considered α2δ-
1 subunit as the binding site for gabapentin. The perceptible accord for gabapentin
binding indicated a certain sequential elevation at various steps of decontamination
of α2δ from model pig cerebrum (Zvejniece et al. 2015, Faria et al. 2017).
The affinity of 3H-gabapentin towards α2δ-1 is found to be increased threefold
times during dialysis of brain membranes. Such results indicate the presence of
endogenic ligand that occupies such sites. But its activity remains dubious and the
nature is unknown. Howbeit, there are many endogenic amino acids like L-leucine
which binds with α2δ subunits. The structure and function studies indicate that
C-terminal lop of α2δ-1 repudiated binding to 3H-gabapentin (Arsene et al. 2018).
Eventually, residues like the third arginine (R) in RRR motif are essential for
gabapentin binding, as identified in α2δ-1. The evolution of RRR to RRA in forms
of calcium current enhancement and calcium channel trafficking in both α2δ-1 and
α2δ-2 has typically reduced the activity of α2δ-1 and α2δ subunits (Weiss and
Zamponi 2017).
It has been observed that various endogenous small molecules bind to α2δ-1 and
α2δ-2. The RRR motif is situated at the upstream of the von Willebrand factor A
domain, and it is however anticipated to fit constructionally at the base of the von
Willebrand factor A. The transformations of RRR to RRA lessen the accord of
gabapentin binding for both α2δ-2 and α2δ-1. Anxiolytic influences of pregabalin
has been observed in mice model by binding to α2δ-1 rather than α2δ-2. Both
gabapentin and pregabalin show similar interests for α2δ-1 and α2δ-2 subunits,
and therefore improved side effect profile of the α2δ-1 selective ligands might be
observed (Tano et al. 2019; Song et al. 2015).
By using ligand binding assays, it is easy to identify the compounds that displace
3
H-pregabalin or 3H-gabapentin. Such displacement occurs with improved pharma-
cokinetics and impactful affinity for α2δ-1 or with selectivity toward α2δ-1. The
major side effect of gabapentin, ataxia, is mediated via binding to α2δ-2. There are
other similar compounds like gabapentin which has also been found to bind to α2δ -1
(Chen et al. 2019; Dolphin 2016).
20 Pharmacology of Calcium Channel 707

20.5.4 a2d Ligand Drugs, Synaptic Transmission, and Transmitter

Release

The impact of α2δ ligand drugs on transmission of synapse and release of transmitter
is a very important subject of discussion in the context of pharmacology. The
presynaptic terminals are strongly expressed by α2δ-1 subunits. However, pain
pathways can be explained by the observation of chronic effects of gabapentinoid
drugs, and the drastic effects of gabapentinoid drugs to impede release of transmitter
and synaptic transmission have been noticed in few in vitro systems (Uchitel et al.
2010; Patel et al. 2000). Deficiency of gabapentinoid drugs has acute effect on
somatic calcium currents in various systems. But such drugs are believed to have
differential effects on presynaptic terminal calcium currents in comparison to cell
bodies. As a result, more rapid inhibition of ion occurs at that stage.
Therefore, calcium channel trafficking has been noticed prominently due to the
higher turnover of calcium channel in presynaptic terminals than in somata.
Depending on the reciprocity between these different processes and presynaptic
sensitization, gabapentinoid drugs might act rapidly or more slowly. Activation of
protein kinase C is an accurate example of this phenomenon (Rogawski and Bazil
2008; Gong et al. 2018).

20.5.5 Role of Amino Acid Transporters

The transport of gamma-aminobutyric acid in vitro is not facilitated with the

presence of pregabalin and gabapentin. However, at neutral pH, both the drugs
act as zwitterions. They use neutral amino acid transporter system L for uptaking
cell membranes (Akanuma et al. 2018; Takahashi et al. 2018).

20.6 Cab Subunits – Pharmacological Roles

20.6.1 Cavb Subunit Biochemistry

The very first source of Caβ subunits was identified in the purified skeletal muscle
voltage-gated calcium channel complex, and the gene for Cavβ1 was cloned later
on. As the time proceeded, Cavβ2, Caβ3, and Caβ4, three more calcium subunit
genes, were identified by similar studies. Cytoplasmic proteins or the Cavβ subunits
participate in the binding process with high accord to the intracellular loop in
between domains I and II of the Cav1 and Cav2 α1 subunits. The above binding
motif is termed as the α-interaction territory which is actually an 18-amino-acid
region in the proximal part of the I–II linker (Miriyala et al. 2008; Jangsangthong
et al. 2011; Findeisen et al. 2017).
Cavβ subunits having a protected src homology-3 domain (or SH3 domain) were
found by homology modeling processes. This subunit is also found in guanylate
kinase like domain which is interconnected by a flexible loop. Due to the mutations
708 S. Mallik and P. C. Acharya

in the active site, the kinase activity is zero in the domain of guanylate kinase. There
are three studies which have resolved the crystal structure of the conserved domains
in various β subunits (Newman and Prehoda 2009; Stölting et al. 2015). The study
showed that the AID (α-interaction domain) peptide interaction site is in a groove in
the domain like guanylate kinase. The α-helical structure of the α-interaction domain
in the intact I–II loop is inflicted by binding to the Caβ subunit. This is predicted to
continue till the end of S6 in transmembrane domain I. In this way, β subunits are
considered as safeguards to induce folding of the α1 subunit with accuracy
(Hofmann et al. 2015; Kazim 2017; Hidalgo et al. 2019).

20.6.2 Pathophysiology and Potential Pharmacology Involving

Cavb Subunits

Most common and rare diseases like epilepsy, cardiac dysfunction, and others are
connected to Cavβ subunit. Hypothetical consideration about designing of a drug
targeting the indentation within Cavβ into which the α-interaction domain peptide is
incorporated could impede the interaction between the β subunits and Cavα1. In this
way, it reduces the function of calcium channel, which on the other hand is beneficial
in certain medical conditions such as hypertension and chronic pain (Dolphin 2016;
Felix and Weiss 2017).
However, the interactivity between Cavβ and the alpha-interacting domain region
is of very high accord and includes a number of remnants. It is difficult to extract out
the process through which small molecules compete for conjugation and the selec-
tivity of different Cavα1 and β subunits. The use of cell-penetrating peptides
describes the interference between the interaction between Cav2.2 and collapsin
response mediator protein (Angelini 2015; Butcher et al. 2006; Korkosh et al. 2019).
To study the specific roles for different calcium channel isoforms, a number of
splice variants and supplementary subunits have been identified, aided by existence
of human mutations and knockout mouse models of these channels and their
supplementary subunits. Physiological activities that are controlled by Cav1.2 and
Cav1.3 are identified through different procedures. Both channels act as potentially
novel drug targets if selectivity can be achieved (Dolphin 2016; Thalhammer et al.
2017). For the treatment of hypertension, nonselective blockers of these channels
have been used and their profiles of adverse effect have been studied very closely.
Depolarized potentials have been found in vascular Cav1.2, and due to this, the
selectivity of dihydropyridines is nevertheless attained in vivo for aiming the tissue.
It is intended to bind with greater accord to inactivated channels. Few specific drugs
have promising therapeutic efficacy for selective Cav1.3 blockers with various
indications like neuroprotection, neuropsychiatric diseases, Parkinson’s disease,
and resistant hypertension associated with hyperaldosteronism (Ross 2018; Torres
et al. 2015).
Ensuring novel classes of Cav2.2 channel blockers with enormous selectivity,
accord, and use dependence is a challenge for the researchers. Ziconotide (peptide ω -
20 Pharmacology of Calcium Channel 709

conotoxin MVIIA) has been administered intrathecally for use in intractable pain.
The Cav2.2 channel blockers which act as analgesics might also be effective in
conditions such as drug anxiety and dependence. The Cav3 (T-type) channels are
important regulators of pacemaker activity and neuronal firing and play crucial roles
in the cardiovascular system (Elies et al. 2016; Woon and Balijepalli 2015). Their
dysfunction contributes to a number of chronic complications such as epilepsy and
pain. In this way, they are both potential and actual drug targets for such medical
conditions, namely, ethosuximide. But due to the protection of absence seizures by
ethosuximide, the present-day clinical use of many types of such promising T-type
channel blocking small organic molecules has been largely restricted (Bourinet et al.
2016; Lee et al. 2019).
The Cav3 auxiliary subunit α2δ1 is a well promising drug target for pregabalin
and gabapentin and has been used in chronic neuropathic pain. When combined with
other drugs in several forms, it is useful in the treatment of epilepsy. The drug also
shows binding interest with α2δ2, but not α2δ3. A selective or more potent α2δ1
ligand would have less side effects, and whether a similar ligand targeting α2δ3
might be of therapeutic use is still under investigation (Choi et al. 2016; Rui et al.
2016; Chew and Khanna 2018).
But few specific calcium channel blockers are likely to hold eminent promises
and hope for therapeutic involvement in coming future.

20.7 Conclusions

Different types of calcium ion channels like L-type, T-type, and N-type found in
muscle cells, neurons, or endocrine cells can be easily distinguished by their
pharmacological responses. All the three types of ion channels are available in
dorsal root ganglion with different activation threshold and identical selectivity.
Animal studies reveal that these ion channels can regulate the use of targeted drug
delivery effectively. The Cav1.2 and Cav1.3 subunits have strong indications of
enhancing selectivity of novel drugs, whereas nonselective dihydropyridines and
other types of calcium channel blockers have been used for the treatment of elevated
blood pressure for decades. The drug ziconotide, a peptide ω  -conotoxin MVIIA, is
popularly used in unmanageable pain of individuals through intrathecal route and
acts by blocking of Cav2.2 channel.
In modern times, the management of epilepsy and chronic neuropathic pain has
been widely controlled by targeting α2δ-1 subunit with minimal side effects. Drugs
like pregabalin and gabapentin usually bind to the ligand binding site to exert
therapeutic activity.
The Cav3 or T-type channels are connected with the cardiovascular system by
regulating the action of pacemaker. Furthermore, it is essential to understand their
interaction with anandamide, arachidonic acid, redox agents, and G-proteins. The
randomized clinical investigation further indicates the promising use of Cav3 ligands
in various disease management related to pain, heart, hypertension. In a nutshell, cal-
cium channel blocking agents, depending on the selectivity and mechanism of their
710 S. Mallik and P. C. Acharya

pharmacological action, are going to hold great commitment for therapeutic arbitra-
tion in the coming days.

References
Adake P, Somashekar HS, Rafeeq PM, Umar D, Basheer B, Baroudi K (2015) Comparison of
amlodipine with cilnidipine on antihypertensive efficacy and incidence of pedal edema in mild
to moderate hypertensive individuals: a prospective study. J Adv Pharm Technol Res 6(2):81
Adams DJ, Berecki G (2013) Mechanisms of conotoxin inhibition of N-type (Cav2. 2) calcium
channels. Biochim Biophys Acta 1828(7):1619–1628
Adams DJ, Callaghan B, Berecki G (2012) Analgesic conotoxins: block and G protein-coupled
receptor modulation of N-type (CaV2. 2) calcium channels. Br J Pharmacol 166(2):486–500
Aggarwal S, Loomba RS, Arora RR (2016) Effects of concurrent calcium channel blocker on
antiplatelet efficacy of clopidogrel therapy: a systematic review. Am J Ther 23(1):e29–e36
Akanuma SI, Yamakoshi A, Sugouchi T, Kubo Y, Hartz AM, Bauer B, Hosoya KI (2018) Role of
L-type amino acid transporter 1 at the inner blood-retinal barrier in the blood-to-retina transport
of gabapentin. Mol Pharm 15(6):2327–2337
Alexander SP, Peters JA, Kelly E, Marrion N, Benson HE, Faccenda E, Pawson AJ, Sharman JL,
Southan C, Davies JA (2015) The Concise Guide to pharmacology 2015/16: ligand-gated ion
channels. Br J Pharmacol 172(24):5870–5903
Angelini M (2015) Role of CLIC1 and L-type calcium channels in the pathophysiology of
glioblastoma and ventricular arrhythmias. PhD Thesis, University of California Los Angeles,
The United States of America
Antal L, Martin-Caraballo M (2019) T-type calcium channels in cancer. Cancer 11(2):134
Arsene CG, Kaiser P, Paleari R, Henrion A, Spannagl M, Mosca A (2018) Determination of HbA2
by quantitative bottom-up proteomics and isotope dilution mass spectrometry. Clin Chim Acta
487:318–324
Ataei S, Abaspanah S, Haddadi R, Mohammadi M, Nili-Ahmadabadi A (2019) Therapeutic
potential of dihydropyridine calcium channel blockers on oxidative injury caused by
organophosphates in cortex and cerebellum: an in vivo study. Indian J Clin Biochem 1–8.
https://doi.org/10.1007/s12291-019-00830-3 (Article in press)
Badou A, Jha MK, Matza D, Flavell RA (2013) Emerging roles of L-type voltage-gated and other
calcium channels in T lymphocytes. Front Immunol 4:243
Bain A (2019) Use of calcium channel blockers in cardiovascular disease. Br J of Cardiac Nurs 14
(2):64–70
Bajaj S, Han J (2019) Venom-derived peptide modulators of cation-selective channels: friend, foe
or frenemy. Front Pharmacol 10:58
Baloh RW (2019) Promising new treatments for chronic pain. In: Sciatica and chronic pain.
Springer, Cham, pp 109–120
Barua S, Kim JY, Kim JY, Kim JH, Lee JE (2019) Therapeutic effect of agmatine on neurological
disease: focus on ion channels and receptors. Neurochem Res 44(4):735–750
Beedle AM, Zamponi GW (2000) Block of voltage-dependent calcium channels by aliphatic
monoamines. Biophys J 79(1):260–270
Bellampalli SS, Ji Y, Moutal A, Cai S, Wijeratne EK, Gandini MA, Yu J, Chefdeville A, Dorame A,
Chew LA, Madura CL (2019) Betulinic acid, derived from the desert lavender Hyptis emoryi,
attenuates paclitaxel-, HIV-, and nerve injury–associated peripheral sensory neuropathy via
block of N-and T-type calcium channels. Pain 160(1):117–135
Bidaud I, Mezghrani A, Swayne LA, Monteil A, Lory P (2006) Voltage-gated calcium channels in
genetic diseases. Biochim Biophys Acta 1763(11):1169–1174
20 Pharmacology of Calcium Channel 711

Bladen C, Gündüz MG, Şimşek R, Şafak C, Zamponi GW (2014a) Synthesis and evaluation of
1, 4-dihydropyridine derivatives with calcium channel blocking activity. Pflügers Arch 466
(7):1355–1363
Bladen C, Hamid J, Souza IA, Zamponi GW (2014b) Block of T-type calcium channels by
protoxins I and II. Mol Brain 7(1):36
Bogeski I, Kappl R, Kummerow C, Gulaboski R, Hoth M, Niemeyer BA (2011) Redox regulation
of calcium ion channels: chemical and physiological aspects. Cell Calcium 50(5):407–423
Borowicz KK, Banach M (2014) Antiarrhythmic drugs and epilepsy. Pharmacol Rep 66
(4):545–551
Bourinet E, Zamponi GW (2017) Block of voltage-gated calcium channels by peptide toxins.
Neuropharmacology 127:109–115
Bourinet E, Stotz SC, Spaetgens RL, Dayanithi G, Lemos J, Nargeot J, Zamponi GW (2001)
Interaction of SNX482 with domains III and IV inhibits activation gating of alpha
(1E) (Ca(V)2.3) calcium channels. Biophys J 81(1):79–88
Bourinet E, Francois A, Laffray S (2016) T-type calcium channels in neuropathic pain. Pain 157:
S15–S22
Bowery NG (2016) Baclofen: therapeutic use and potential of the prototypic GABA B receptor
agonist. In: GABAB receptor. Humana Press, Cham, pp 337–356
Brady R, Baell J, Norton R (2013) Strategies for the development of conotoxins as new therapeutic
leads. Mar Drugs 11(7):2293–2313
Brimblecombe KR, Gracie CJ, Platt NJ, Cragg SJ (2015) Gating of dopamine transmission by
calcium and axonal N-, Q-, T- and L-type voltage-gated calcium channels differs between
striatal domains. J Physiol 593:929–946
Brockhaus J, Schreitmüller M, Repetto D, Klatt O, Reissner C, Elmslie K, Heine M, Missler M
(2018) α-Neurexins together with α2δ-1 auxiliary subunits regulate Ca2+ influx through CaV2.
1 channels. J Neurosci 38(38):8277–8294
Bruckner JJ, Zhan H, Gratz SJ, Rao M, Ukken F, Zilberg G, O’Connor-Giles KM (2017) Fife
organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release. J
Cell Biol 216(1):231–246
Buchta WC, Moutal A, Hines B, Garcia-Keller C, Smith AC, Kalivas P, Khanna R, Riegel A
(2019) Dynamic CRMP2 regulation of CaV2. 2 in the prefrontal cortex contributes to the
reinstatement of cocaine seeking. bioRxiv, 533083
Butcher AJ, Leroy J, Richards MW, Pratt WS, Dolphin AC (2006) The importance of occupancy
rather than affinity of Cavβ subunits for the calcium channel I–II linker in relation to calcium
channel function. J Physiol 574(2):387–398
Cai F, Xu N, Liu Z, Ding R, Yu S, Dong M, Wang S, Shen J, Tae HS, Adams DJ, Zhang X (2018)
Targeting of N-Type calcium channels via GABAB-receptor activation by α-Conotoxin Vc1.
1 variants displaying improved analgesic activity. J Med Chem 61(22):10198–10205
Caricati-Neto A, Bergantin LB (2016) New therapeutic strategy of Alzheimer’s and Parkinson’s
diseases: pharmacological modulation of neural Ca/cAMP intracellular signaling interaction
2. Asian J Pharm Pharmacol 2(6):136–143
Carstens BB, Clark RJ, Daly NL, Harvey PJ, Kaas Q, Craik DJ (2011) Engineering of conotoxins
for the treatment of pain. Curr Pharm Des 17(38):4242–4253
Cassidy JS, Ferron L, Kadurin I, Pratt WS, Dolphin AC (2014) Functional exofacially tagged
N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits. Proc Natl Acad
Sci 111(24):8979–8984
Catterall WA, Swanson TM (2015) Structural basis for pharmacology of voltage-gated sodium and
calcium channels. Mol Pharmacol 88(1):141–150
Celli R, Santolini I, Guiducci M, Van Luijtelaar G, Parisi P, Striano P, Gradini R, Battaglia G,
Ngomba TR, Nicoletti F (2017) The α2δ subunit and absence epilepsy: beyond calcium
channels? Curr Neuropharmacol 15(6):918–925
712 S. Mallik and P. C. Acharya

Chai Z, Wang C, Huang R, Wang Y, Zhang X, Wu Q, Wang Y, Wu X, Zheng L, Zhang C, Guo W

(2017) CaV2. 2 gates calcium-independent but voltage-dependent secretion in mammalian
sensory neurons. Neuron 96(6):1317–1326
Chaugai S, Sherpa LY, Sepehry AA, Kerman SRJ, Arima H (2018) Effects of long-and intermedi-
ate-acting dihydropyridine calcium channel blockers in hypertension: a systematic review and
meta-analysis of 18 prospective, randomized, actively controlled trials. J Cardiovasc Pharmacol
Ther 23(5):433–445
Chen Y, Chen SR, Chen H, Zhang J, Pan HL (2019) Increased α2δ-1–NMDA receptor coupling
potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuro-
pathic pain. J Neurochem 148(2):252–274
Chew LA, Khanna R (2018) CRMP2 and voltage-gated ion channels: potential roles in neuropathic
pain. Neuronal Signal 2(1). https://doi.org/10.1042/NS20170220
Choi S, Yu E, Hwang E, Llinás RR (2016) Pathophysiological implication of CaV3. 1 T-type Ca2+
channels in trigeminal neuropathic pain. Proc Natl Acad Sci 113(8):2270–2275
De la Peña E, Gomis A (2019) Characterization of TRPC channels in a heterologous system using
calcium imaging and the patch-clamp technique. In: TRP channels. Humana, New York, NY, pp
83–97
DiMeglio LA, Imel EA (2019) Calcium and phosphate: hormonal regulation and metabolism. In:
Basic and applied bone biology. Academic Press, Cambridge, pp 257–282
Dolphin AC (2016) Voltage-gated calcium channels and their auxiliary subunits: physiology and
pathophysiology and pharmacology. J Physiol 594(19):5369–5390
Donaldson LF, Beazley-Long N (2016) Alternative RNA splicing: contribution to pain and
potential therapeutic strategy. Drug Discov Today 21(11):1787–1798
Duda J, Pötschke C, Liss B (2016) Converging roles of ion channels, calcium, metabolic stress, and
activity pattern of Substantia nigra dopaminergic neurons in health and Parkinson’s disease. J
Neurochem 139:156–178
Duggan P, Tuck K (2015) Bioactive mimetics of conotoxins and other venom peptides. Toxins 7
(10):4175–4198
Elies J, Scragg JL, Boyle JP, Gamper N, Peers C (2016) Regulation of the T-type Ca2+ channel
Cav3.2 by hydrogen sulfide: emerging controversies concerning the role of H2S in nociception.
J Physiol 594(15):4119–4129
Ellinor PT, Zhang JF, Horne WA, Tsien RW (1994) Structural determinants of the blockade of
N-type calcium channels by a peptide neurotoxin. Nature 372(6503):272
Fan QI, Vanderpool KM, Chung HS, Marsh JD (2005) The L-type calcium channel alpha 1C
subunit gene undergoes extensive, uncoordinated alternative splicing. Mol Cell Biochem 269
(1):153–163
Fardal Ø, Lygre H (2015) Management of periodontal disease in patients using calcium channel
blockers–gingival overgrowth, prescribed medications, treatment responses and added treat-
ment costs. J Clin Periodontol 42(7):640–646
Faria LC, Gu F, Parada I, Barres B, Luo ZD, Prince DA (2017) Epileptiform activity and behavioral
arrests in mice overexpressing the calcium channel subunit α2δ-1. Neurobiol Dis 102:70–80
Felix R, Weiss N (2017) Ubiquitination and proteasome-mediated degradation of voltage-gated Ca
2+ channels and potential pathophysiological implications. Gen Physiol Biophys 36:1–5
Ferron L, Nieto-Rostro M, Cassidy JS, Dolphin AC (2014) Fragile X mental retardation protein
controls synaptic vesicle exocytosis by modulating N-type calcium channel density. Nat
Commun 5:3628
Findeisen F, Campiglio M, Jo H, Abderemane-Ali F, Rumpf CH, Pope L, Rossen ND, Flucher BE,
DeGrado WF, Minor DL Jr (2017) Stapled voltage-gated calcium channel (CaV) α-interaction
domain (AID) peptides act As selective protein–protein interaction inhibitors of CaV function.
ACS Chem Nerosci 8(6):1313–1326
François A, Laffray S, Pizzoccaro A, Eschalier A, Bourinet E (2014) T-type calcium channels in
chronic pain: mouse models and specific blockers. Pflügers Arch 466(4):707–717
20 Pharmacology of Calcium Channel 713

François A, Schüetter N, Laffray S, Sanguesa J, Pizzoccaro A, Dubel S, Mantilleri A, Nargeot J,

Noël J, Wood JN, Moqrich A (2015) The low-threshold calcium channel Cav3. 2 determines
low-threshold mechanoreceptor function. Cell Rep 10(3):370–382
François-Moutal L, Wang Y, Moutal A, Cottier KE, Melemedjian OK, Yang X, Wang Y, Ju W,
Largent-Milnes TM, Khanna M, Vanderah TW (2015) A membrane-delimited N-myristoylated
CRMP2 peptide aptamer inhibits CaV2. 2 trafficking and reverses inflammatory and postopera-
tive pain behaviors. Pain 156(7):1247
Freynhagen R, Backonja M, Schug S, Lyndon G, Parsons B, Watt S, Behar R (2016) Pregabalin
for the treatment of drug and alcohol withdrawal symptoms: a comprehensive review. CNS
Drugs 30(12):1191–1200
Gadotti VM, Bladen C, Zhang FX, Chen L, Gündüz MG, Şimşek R, Şafak C, Zamponi GW (2015)
Analgesic effect of a broad-spectrum dihydropyridine inhibitor of voltage-gated calcium
channels. Pflügers Arch 467(12):2485–2493
Gambardella J, Trimarco B, Iaccarino G, Santulli G (2017) New insights in cardiac calcium
handling and excitation-contraction coupling. In: Heart failure: from research to clinical prac-
tice. Springer, Cham, pp 373–385
Gardezi SR, Nath AR, Li Q, Stanley EF (2016) Characterization of a synaptic vesicle binding motif
on the distal CaV2.2 channel C-terminal. Front Cell Neurosci 10:145
Gershon ES, Grennan K, Busnello J, Badner JA, Ovsiew F, Memon S, Alliey-Rodriguez N,
Cooper J, Romanos B, Liu C (2014) A rare mutation of CACNA1C in a patient with bipolar
disorder, and decreased gene expression associated with a bipolar-associated common SNP of
CACNA1C in brain. Mol Psychiatry 19(8):890
Glossmann H, Striessnig J (1990) Molecular properties of calcium channels. Rev Physiol Biochem
Pharmacol 114:1–105
Gong N, Park J, Luo ZD (2018) Injury-induced maladaptation and dysregulation of calcium
channel α2δ subunit proteins and its contribution to neuropathic pain development. Br J
Pharmacol 175(12):2231–2243
Gordan R, Gwathmey JK, Xie LH (2015) Autonomic and endocrine control of cardiovascular
function. World J Cardiol 7(4):204
Guse AH (2015) Calcium mobilizing second messengers derived from NAD. Biochim Biophys
Acta 1854(9):1132–1137
Heyes S, Pratt WS, Rees E, Dahimene S, Ferron L, Owen MJ, Dolphin AC (2015) Genetic
disruption of voltage-gated calcium channels in psychiatric and neurological disorders. Prog
Neurobiol 134:36–54
Hidalgo P, Guzman G, Guzman RE, Jordan N (2019) A tripartite interaction among the calcium
channel α1-and β-subunits and F-actin increases the readily releasable pool of vesicles and its
recovery after depletion. Front Cell Neurosci 13:125
Himeno S, Fujishiro H (2019) Roles of metal transporters in cellular cadmium transport in
mammals. In: Cadmium toxicity. Springer, Singapore, pp 163–178
Hirano M, Takada Y, Wong CF, Yamaguchi K, Kotani H, Kurokawa T, Mori MX, Snutch TP,
Ronjat M, De Waard M, Mori Y (2017) C-terminal splice variants of P/Q-type Ca2+ channel
CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins. J Biol
Chem 292(22):9365–9381
Hofmann F, Belkacemi A, Flockerzi V (2015) Emerging alternative functions for the auxiliary
subunits of the voltage-gated calcium channels. Curr Mol Pharmacol 8(2):162–168
Housley DM, Housley GD, Liddell MJ, Jennings EA (2017) Scorpion toxin peptide action at the
ion channel subunit level. Neuropharmacology 127:46–78
Ikeda H, Yonemochi N, Ardianto C, Yang L, Kamei J (2018) Pregabalin increases food intake
through dopaminergic systems in the hypothalamus. Brain Res 1701:219–226
Jaisser F, Farman N (2016) Emerging roles of the mineralocorticoid receptor in pathology: toward
new paradigms in clinical pharmacology. Pharmacol Rev 68(1):49–75
714 S. Mallik and P. C. Acharya

Jang SJ, Choi HW, Choi DL, Cho S, Rim HK, Choi HE, Kim KS, Huang M, Rhim H, Lee KT, Lee
JY (2013) In vitro cytotoxicity on human ovarian cancer cells by T-type calcium channel
blockers. Bioorg Med Chem Lett 23(24):6656–6662
Jangsangthong W, Kuzmenkina E, Böhnke AK, Herzig S (2011) Single-channel monitoring of
reversible L-type Ca2+ channel CaV α1-CaVβ subunit interaction. Biophys J 101
(11):2661–2670
Jarvis MF, Scott VE, McGaraughty S, Chu KL, Xu J, Niforatos W, Milicic I, Joshi S, Zhang Q, Xia
Z (2014) A peripherally acting, selective T-type calcium channel blocker, ABT-639, effectively
reduces nociceptive and neuropathic pain in rats. Biochem Pharmacol 89(4):536–544
Jin AH, Dutertre S, Kaas Q, Lavergne V, Kubala P, Lewis RJ, Alewood PF (2013) Transcriptomic
messiness in the venom duct of Conus miles contributes to conotoxin diversity. Mol Cell
Proteomics 12(12):3824–3833
Jokinen V (2017) Opioid analgesia: modulation by drug interactions and glial activation. PhD
thesis, University of Helsinki, Finland
Joksimovic SL, Joksimovic SM, Tesic V, García-Caballero A, Feseha S, Zamponi GW, Jevtovic-
Todorovic V, Todorovic SM (2018) Selective inhibition of CaV3. 2 channels reverses hyperex-
citability of peripheral nociceptors and alleviates postsurgical pain. Sci Signal 11(545):
eaao4425
Jurkovicova-Tarabova B, Lacinova L (2019) Structure, function and regulation of CaV 2.2 N-type
calcium channels. Gen Physiol Biophys. https://doi.org/10.4149/gpb_2019004
Kadurin I, Ferron L, Rothwell SW, Meyer JO, Douglas LR, Bauer CS, Lana B, Margas W,
Alexopoulos O, Nieto-Rostro M, Pratt WS (2016) Proteolytic maturation of α2δ represents a
checkpoint for activation and neuronal trafficking of latent calcium channels. Elife 5:e21143
Kang HW, Park JY, Jeong SW, Kim JA, Moon HJ, Perez-Reyes E, Lee JH (2006) A molecular
determinant of nickel inhibition in Cav3. 2 T-type calcium channels. J Biol Chem 281
(8):4823–4830
Kang S, Cooper G, Dunne SF, Dusel B, Luan CH, Surmeier DJ, Silverman RB (2012) CaV1.3-
selective L-type calcium channel antagonists as potential new therapeutics for Parkinson’s
disease. Nature Commun 3:1146
Kappel VD, Zanatta L, Postal BG, Silva FRMB (2013) Rutin potentiates calcium uptake via
voltage-dependent calcium channel associated with stimulation of glucose uptake in skeletal
muscle. Arch Biochem Biophys 532(2):55–60
Katzung BG (2017) Basic and clinical pharmacology. McGraw-Hill Education, New York, NY
Kazim AS (2017) Roles of voltage-gated Ca2+ channel subunits in pancreatic β cells. PhD thesis,
Lund University, Sweden
Kim SK, Kim TK (2018) New molecular insights into the excitation-transcription coupling. Neuron
98(3):453–456
Kim TY, Maki T, Zhou Y, Sakai K, Mizuno Y, Ishikawa A, Tanaka R, Niimi K, Li W, Nagano N,
Takahashi E (2015) Absence-like seizures and their pharmacological profile in tottering-6j mice.
Biochem Biophys Res Commun 463(1-2):148–153
King GF, Escoubas P, Nicholson GM (2008) Peptide toxins that selectively target insect NaV and
CaV channels. Channels 2(2):100–116
Klint JK, Senff S, Rupasinghe DB, Er SY, Herzig V, Nicholson GM, King GF (2012) Spider-
venom peptides that target voltage-gated sodium channels: pharmacological tools and potential
therapeutic leads. Toxicon 60(4):478–491
Korkosh VS, Kisilev AM, Mikhaylov EN, Kostareva AA, Zhorov BS (2019) Atomic mechanisms
of timothy syndrome-associated mutations in calcium channel Cav1. 2. Front Physiol 10:335
Krouse AJ, Gray L, Macdonald T, McCray J (2015) Repurposing and rescuing of mibefradil, an
antihypertensive, for cancer: a case study. Drug Repurposing Rescue Repositioning 1(1):36–39
Kuo IYT, Howitt L, Sandow SL, McFarlane A, Hansen PB, Hill CE (2014) Role of T-type channels
in vasomotor function: team player or chameleon? Pflügers Arch 466(4):767–779
Kuwahara K, Kimura T (2015) The organ-protective effect of N-type Ca2+ channel blockade.
Pharmacol Ther 151:1–7
20 Pharmacology of Calcium Channel 715

Kyle DJ, Park JH, Purdue Pharma LP (2017) Sodium channel blocking peptides and the use thereof.
US Patent 9,718,865
Larsch J, Flavell SW, Liu Q, Gordus A, Albrecht DR, Bargmann CI (2015) A circuit for gradient
climbing in C. elegans chemotaxis. Cell Rep 12(11):1748–1760
Latham JR, Pathirathna S, Jagodic MM, Choe WJ, Levin ME, Nelson MT, Lee WY, Krishnan K,
Covey DF, Todorovic SM, Jevtovic-Todorovic V (2009) Selective T-type calcium channel
blockade alleviates hyperalgesia in ob/ob mice. Diabetes 58(11):2656–2665
Laurent S (2017) Antihypertensive drugs. Pharmacol Res 124:116–125
Laurent B, Murail S, Shahsavar A, Sauguet L, Delarue M, Baaden M (2016) Sites of anesthetic
inhibitory action on a cationic ligand-gated ion channel. Structure 24(4):595–605
Lee AS, De Jesús-Cortés H, Kabir ZD, Knobbe W, Orr M, Burgdorf C, Huntington P, McDaniel L,
Britt JK, Hoffmann F, Brat DJ (2016) The neuropsychiatric disease-associated gene cacna1c
mediates survival of young hippocampal neurons. Eneuro 3(2). https://doi.org/10.1523/
ENEURO.0006-16.2016
Lee MS, Newbold EJ, Papapetropoulos S (2019) Selective T-type calcium channel modulator
CX-8998 fully suppresses seizures in the GAERS genetic model of epilepsy at human achiev-
able concentrations (P1. 5-026)
Lenaeus MJ, El-Din TMG, Ing C, Ramanadane K, Pomès R, Zheng N, Catterall WA (2017)
Structures of closed and open states of a voltage-gated sodium channel. Proc Natl Acad Sci
114(15):E3051–E3060
Lewis RJ, Nielsen KJ, Craik DJ, Loughnan ML, Adams DA, Sharpe IA, Luchian T, Adams DJ,
Bond T, Thomas L, Jones A (2000) Novel ω-conotoxins from Conus catus discriminate among
neuronal calcium channel subtypes. J Biol Chem 275(45):35335–35344
Li Y, Tatsui CE, Rhines LD, North RY, Harrison DS, Cassidy RM, Johansson CA, Kosturakis AK,
Edwards DD, Zhang H, Dougherty PM (2017a) Dorsal root ganglion neurons become hyperex-
citable and increase expression of voltage-gated T-type calcium channels (Cav3. 2) in
paclitaxel-induced peripheral neuropathy. Pain 158(3):417
Li G, Wang J, Liao P, Bartels P, Zhang H, Yu D, Liang MC, Poh KK, Yu CY, Jiang F, Yong TF
(2017b) Exclusion of alternative exon 33 of CaV1. 2 calcium channels in heart is
proarrhythmogenic. Proc Natl Acad Sci 114(21):E4288–E4295
Lian LY, Pandalaneni SR, Todd PA, Martin VM, Burgoyne RD, Haynes LP (2014) Demonstration
of binding of neuronal calcium sensor-1 to the cav2. 1 p/q-type calcium channel. Biochemistry
53(38):6052–6062
Liao P, Soong TW (2010) Ca V 1.2 channelopathies: from arrhythmias to autism, bipolar disorder,
and immunodeficiency. Pflügers Arch 460(2):353–359
Liu Z, Bartels P, Sadeghi M, Du T, Dai Q, Zhu C, Yu S, Wang S, Dong M, Sun T, Guo J (2018) A
novel α-conopeptide Eu1. 6 inhibits N-type (Ca V 2.2) calcium channels and exhibits potent
analgesic activity. Sci Rep 8(1):1004
Loperena R, Harrison DG (2017) Oxidative stress and hypertensive diseases. Med Clin 101
(1):169–193
Lotia M, Jankovic J (2016) New and emerging medical therapies in Parkinson’s disease. Expert
Opin Pharmacother 17(7):895–909
Lu L, Sirish P, Zhang Z, Woltz RL, Li N, Timofeyev V, Knowlton AA, Zhang XD, Yamoah EN,
Chiamvimonvat N (2015) Regulation of gene transcription by voltage-gated L-type calcium
channel, Cav1. 3. J Biol Chem 290(8):4663–4676
M’Dahoma S, Gadotti VM, Zhang FX, Park B, Nam JH, Onnis V, Balboni G, Lee JY, Zamponi
GW (2016) Effect of the T-type channel blocker KYS-05090S in mouse models of acute and
neuropathic pain. Pflügers Arch 468(2):193–199
Manthri S, Veena D, Ambari RB, Puchchakayala G (2015) A prospective interventional study on
clinical effects of cilnidipine in hypertensive patients. IJPRIF 8(10):70–76
Marchetti C (2013) Role of calcium channels in heavy metal toxicity. ISRN Toxicol 2013:184360
716 S. Mallik and P. C. Acharya

Mary R, Giribaldi J, Lesport P, Bourinet E, Dutertre S (2018) Discovery, synthesis and characteri-
zation of PmuTx1–A new spider toxin that blocks T-type calcium channels Cav3. 2. Toxicon
149:101
Matsunami M, Miki T, Nishiura K, Hayashi Y, Okawa Y, Nishikawa H, Sekiguchi F, Kubo L,
Ozaki T, Tsujiuchi T, Kawabata A (2012) Involvement of the endogenous hydrogen sulfide/
Cav3. 2 T-type Ca2+ channel pathway in cystitis-related bladder pain in mice. Br J Pharmacol
167(4):917–928
Matta C, Zákány R, Mobasheri A (2015) Voltage-dependent calcium channels in chondrocytes:
roles in health and disease. Curr Rheumatol Rep 17(7):43
McDonough SI (2007) Gating modifier toxins of voltage-gated calcium channels. Toxicon 49
(2):202–212
Medrihan L, Cesca F, Raimondi A, Lignani G, Baldelli P, Benfenati F (2013) Synapsin II
desynchronizes neurotransmitter release at inhibitory synapses by interacting with presynaptic
calcium channels. Nat Commun 4:1512
Mesirca P, Torrente AG, Mangoni ME (2015) Functional role of voltage gated Ca2+ channels in
heart automaticity. Front Physiol 6:19
Mir R, Karim S, Amjad Kamal M, Wilson MC, Mirza Z (2016) Conotoxins: structure, therapeutic
potential and pharmacological applications. Curr Pharm Des 22(5):582–589
Miriyala J, Nguyen T, Yue DT, Colecraft HM (2008) Role of CaVβ subunits, and lack of functional
reserve, in protein kinase A modulation of cardiac CaV1. 2 channels. Circ Res 102(7):e54–e64
Mollica A, Costante R, Novellino E, Stefanucci A, Pieretti S, Zador F, Samavati R, Borsodi A,
Benyhe S, Vetter I, Lewis RJ (2015) Design, synthesis and biological evaluation of two opioid
agonist and Cav2. 2 blocker multitarget ligands. Chem Biol Drug Des 86(2):156–162
Montandon G, Ren J, Victoria NC, Liu H, Wickman K, Greer JJ, Horner RL (2016) G-protein–
gated Inwardly Rectifying Potassium Channels Modulate Respiratory Depression by Opioids.
Anesthesiology 124(3):641–650
Morrill GA, Kostellow AB, Gupta RK (2015) Computational analysis of the extracellular domain of
the Ca2+-sensing receptor: an alternate model for the Ca2+ sensing region. Biochem Biophys
Res Commun 459(1):36–41
Motin L, Yasuda T, Schroeder CI, Lewis RJ, Adams DJ (2007) ω-Conotoxin CVIB differentially
inhibits native and recombinant N-and P/Q-type calcium channels. Eur J Neurosci 25
(2):435–444
Moutal A, Dustrude ET, Khanna R (2017) Sensitization of ion channels contributes to central and
peripheral dysfunction in neurofibromatosis type 1. Mol Neurobiol 54(5):3342–3349
Nam G (2018) T-type calcium channel blockers: a patent review (2012–2018). Expert Opin Ther
Pat 28(12):883–901
Nanclares C, Baraibar AM, Gandía L (2018) L-type calcium channels in exocytosis and endocytosis
of chromaffin cells. Pflügers Arch 470(1):53–60
Nanou E, Catterall WA (2018) Calcium channels, synaptic plasticity, and neuropsychiatric disease.
Neuron 98(3):466–481
Newman RA, Prehoda KE (2009) Intramolecular interactions between the Src homology
3 guanylate kinase domains of discs large regulate its function in asymmetric cell division. J
Biol Chem 284(19):12924–12932
Nieto-Rostro M, Ramgoolam K, Pratt WS, Kulik A, Dolphin AC (2018) Ablation of α2δ-1 inhibits
cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo.
Proc Natl Acad Sci 115(51):E12043–E12052
Nimmrich V, Eckert A (2013) Calcium channel blockers and dementia. Br J Pharmacol 169
(6):1203–1210
Norton RS (2017) Enhancing the therapeutic potential of peptide toxins. Expert Opin Drug
Discovery 12(6):611–623
Offord J, Isom LL (2016) Drugging the undruggable: gabapentin, pregabalin and the calcium
channel α2δ subunit. Crit Rev Biochem Mol Biol 51(4):246–256
20 Pharmacology of Calcium Channel 717

Okuyama Y, Hirawa N, Fujita M, Fujiwara A, Ehara Y, Yatsu K, Sumida K, Kagimoto M,

Katsumata M, Kobayashi Y, Saka S (2018) The effects of anti-hypertensive drugs and the
mechanism of hypertension in vascular smooth muscle cell-specific ATP2B1 knockout mice.
Hypertens Res 41(2):80
Ortner NJ, Bock G, Vandael DH, Mauersberger R, Draheim HJ, Gust R, Carbone E, Tuluc P,
Striessnig J (2014) Pyrimidine-2, 4, 6-triones are a new class of voltage-gated L-type Ca 2+
channel activators. Nat Commun 5:3897
Oshima T, Ozono R, Yano Y, Higashi Y, Teragawa H, Miho N, Ishida T, Ishida M, Yoshizumi M,
Kambe M (2005) Beneficial effect of T-type calcium channel blockers on endothelial function in
patients with essential hypertension. Hypertens Res 28(11):889
Osteen JD, Herzig V, Gilchrist J, Emrick JJ, Zhang C, Wang X, Castro J, Garcia-Caraballo S,
Grundy L, Rychkov GY, Weyer AD (2016) Selective spider toxins reveal a role for the Na V
1.1 channel in mechanical pain. Nature 534(7608):494
Page KM, Rothwell SW, Dolphin AC (2016) The CaVβ subunit protects the I-II loop of the voltage-
gated calcium channel CaV2.2 from proteasomal degradation but not oligoubiquitination. J Biol
Chem 291(39):20402–20416
Patel R, Dickenson AH (2016) Mechanisms of the gabapentinoids and α2δ-1 calcium channel
subunit in neuropathic pain. Pharmacol Res Perspect 4(2):e00205
Patel MK, Gonzalez MI, Bramwell S, Pinnock RD, Lee K (2000) Gabapentin inhibits excitatory
synaptic transmission in the hyperalgesic spinal cord. Br J Pharmacol 130(8):1731–1734
Patel R, Montagut-Bordas C, Dickenson AH (2018) Calcium channel modulation as a target in
chronic pain control. Br J Pharmacol 175(12):2173–2184
Powell KL, Cain SM, Snutch TP, O’Brien TJ (2014) Low threshold T-type calcium channels as
targets for novel epilepsy treatments. Br J Clin Pharmacol 77(5):729–739
Prakriya M, Lewis RS (2015) Store-operated calcium channels. Physiol Rev 95(4):1383–1436
Qian WJ, Yin N, Gao F, Miao Y, Li Q, Li F, Sun XH, Yang XL, Wang Z (2017) Cannabinoid CB1
and CB2 receptors differentially modulate L-and T-type Ca2+ channels in rat retinal ganglion
cells. Neuropharmacology 124:143–156
Quach TT, Honnorat J, Kolattukudy PE, Khanna R, Duchemin AM (2015) CRMPs: critical
molecules for neurite morphogenesis and neuropsychiatric diseases. Mol Psychiatry 20(9):1037
Raffaello A, Mammucari C, Gherardi G, Rizzuto R (2016) Calcium at the center of cell signaling:
interplay between endoplasmic reticulum, mitochondria, and lysosomes. Trends Biochem Sci
41(12):1035–1049
Ramírez D, Gonzalez W, Fissore R, Carvacho I (2017) Conotoxins as tools to understand the
physiological function of voltage-gated calcium (CaV) channels. Marine Drugs 15(10):313
Ren J, Zhu X, Xu P, Li R, Fu Y, Dong S, Zhangsun D, Wu Y, Luo S (2019) d-amino acid
substitution of α-conotoxin RgIA identifies its critical residues and improves the enzymatic
stability. Marine Drugs 17(3):142
Ripsch MS, Ballard CJ, Khanna M, Hurley JH, White FA, Khanna R (2012) A peptide uncoupling
CRMP-2 from the presynaptic Ca 2+ channel complex demonstrates efficacy in animal models
of migraine and AIDS therapy-induced neuropathy. Transl Neurosci 3(1):1–8
Robinson SD, Li Q, Bandyopadhyay PK, Gajewiak J, Yandell M, Papenfuss AT, Purcell AW,
Norton RS, Safavi-Hemami H (2017) Hormone-like peptides in the venoms of marine cone
snails. Gen Comp Endocrinol 244:11–18
Roebuck AJ, Marks WN, Liu MC, Tahir NB, Zabder NK, Snutch TP, Howland JG (2018)
Effects of the T-type calcium channel antagonist Z944 on paired associates learning and
locomotor activity in rats treated with the NMDA receptor antagonist MK-801. Psychophar-
macology (Berl) 235(11):3339–3350
Rogawski MA, Bazil CW (2008) New molecular targets for antiepileptic drugs: α 2 δ, SV2A, and K
v 7/KCNQ/M potassium channels. Curr Neurol Neurosci Rep 8(4):345–352
Rose KE, Lunardi N, Boscolo A, Dong X, Erisir A, Jevtovic-Todorovic V, Todorovic SM (2013)
Immunohistological demonstration of CaV3. 2 T-type voltage-gated calcium channel
718 S. Mallik and P. C. Acharya

expression in soma of dorsal root ganglion neurons and peripheral axons of rat and mouse.
Neuroscience 250:263–274
Ross JA (2018) The role of norepinephrine in modulating amyloid beta peptides: implications for
neurodegenerative and psychiatric disease. Drexel University
Roullet JB, Spaetgens RL, Burlingame T, Feng ZP, Zamponi GW (1999) Modulation of neuronal
voltage-gated calcium channels by farnesol. J Biol Chem 274:25439–25446
Rousset M, Cens T, Menard C, Bowerman M, Bellis M, Brusés J, Raoul C, Scamps F, Charnet P
(2015) Regulation of neuronal high-voltage activated CaV2 Ca2+ channels by the small GTPase
RhoA. Neuropharmacology 97:201–209
Rui LI, Shen L, Huang Y (2016) Effect of gabapentin on Cav3. 2 channels in dorsal root ganglia of
rats with neuropathic pain. Chin J Anesthesiol 36(11):1371–1374
Sanchez-Sandoval AL, Gomora JC (2019) Contribution of voltage-gated sodium channel
β-subunits to cervical cancer cells metastatic behavior. Cancer Cell Int 19(1):35
Santi CM, Cayabyab FS, Sutton KG, McRory JE, Mezeyova J, Hamming KS, Parker D, Stea A,
Snutch TP (2002) Differential inhibition of T-type calcium channels by neuroleptics. J Neurosci
22(2):396–403
Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, Karlsson A, Al-Lazikani B,
Hersey A, Oprea TI, Overington JP (2017) A comprehensive map of molecular drug targets. Nat
Rev Drug Discov 16(1):19
Santulli G, Lewis DR, Marks AR (2017) Physiology and pathophysiology of excitation–contraction
coupling: the functional role of ryanodine receptor. J Muscle Res Cell Motil 38(1):37–45
Schroeder CI, Doering CJ, Zamponi GW, Lewis RJ (2006) N-type calcium channel blockers: novel
therapeutics for the treatment of pain. Med Chem 2(5):535–543
Schwab A, Fabian A, Hanley PJ, Stock C (2012) Role of ion channels and transporters in cell
migration. Physiol Rev 92(4):1865–1913
Scott I, Ramirez-Reveco A, Parodi J (2016) The restraint of bovine sperm cell motility increases
survival: role of extracellular calcium in the phenomena. J Vet Sci Technol 7(359):2
Seo HN, Choi JY, Choe YJ, Kim Y, Rhim H, Lee SH, Kim J, Joo DJ, Lee JY (2007) Discovery of
potent T-type calcium channel blocker. Bioorg Med Chem Lett 17(21):5740–5743
Shetty R, Vivek G, Naha K, Tumkur A, Raj A, Bairy KL (2013) Excellent tolerance to cilnidipine in
hypertensives with amlodipine-induced edema. N Am J Med Sci 5(1):47
Siafis S, Tzachanis D, Samara M, Papazisis G (2018) Antipsychotic drugs: from receptor-binding
profiles to metabolic side effects. Curr Neuropharmacol 16(8):1210–1223
Silva RB, Greggio S, Venturin GT, da Costa JC, Gomez MV, Campos MM (2018) Beneficial
effects of the calcium channel blocker ctk 01512-2 in a mouse model of multiple sclerosis. Mol
Neurobiol 55(12):9307–9327
Simms BA, Zamponi GW (2014) Neuronal voltage-gated calcium channels: structure, function, and
dysfunction. Neuron 82(1):24–45
Snutch TP, Zamponi GW (2018) Recent advances in the development of T-type calcium channel
blockers for pain intervention. Br J Pharmacol 175(12):2375–2383
Song L, Espinoza-Fuenzalida IA, Etheridge S, Jones OT, Fitzgerald EM (2015) The R-domain:
identification of an N-terminal region of the α2δ-1 subunit which is necessary and sufficient for
its effects on Cav2. 2 calcium currents. Curr Mol Pharmacol 8(2):169–179
Spedding M, Paoletti R (1992) Classification of calcium channels and the sites of action of drugs
modifying channel function. Pharmacol Rev 44(3):363–376
Srebro D, Vuckovic S, Milovanovic A, Kosutic J, Savic Vujovic K, Prostran M (2017) Magnesium
in pain research: state of the art. Curr Med Chem 24(4):424–434
Stölting G, de Oliveira RC, Guzman RE, Miranda-Laferte E, Conrad R, Jordan N, Schmidt S,
Hendriks J, Gensch T, Hidalgo P (2015) Direct interaction of CaVβ with actin up-regulates
L-type calcium currents in HL-1 cardiomyocytes. J Biol Chem 290(8):4561–4572
Striessnig J, Pinggera A, Kaur G, Bock G, Tuluc P (2014) L-type Ca2+ channels in heart and brain.
Wiley Interdiscip Rev Membr Transp Signal 3(2):15–38
20 Pharmacology of Calcium Channel 719

Striessnig J, Ortner NJ, Pinggera A (2015) Pharmacology of L-type calcium channels: novel drugs
for old targets? Curr Mol Pharmacol 8(2):110–122
Südhof TC (2013) A molecular machine for neurotransmitter release: synaptotagmin and beyond.
Nat Med 19(10):1227
Surmeier DJ Jr, Silverman RB, Kang SS, Cooper G (2019) Northwestern University, 2019.
SELECTIVE CALCIUM CHANNEL ANTAGONISTS. U.S. Patent Application 16/167,055
Surmeier DJ, Schumacker PT, Guzman JD, Ilijic E, Yang B, Zampese E (2017) Calcium and
Parkinson’s disease. Biochem Biophys Res Commun 483(4):1013–1019
Szabó GG, Lenkey N, Holderith N, Andrási T, Nusser Z, Hájos N (2014) Presynaptic calcium
channel inhibition underlies CB1 cannabinoid receptor-mediated suppression of GABA release.
J Neurosci 34(23):7958–7963
Takahashi Y, Nishimura T, Higuchi K, Noguchi S, Tega Y, Kurosawa T, Deguchi Y, Tomi M
(2018) Transport of pregabalin via L-type amino acid transporter 1 (SLC7A5) in human brain
capillary endothelial cell line. Pharm Res 35(12):246
Tang L, El-Din TMG, Swanson TM, Pryde DC, Scheuer T, Zheng N, Catterall WA (2016)
Structural basis for inhibition of a voltage-gated Ca 2+ channel by Ca2+ antagonist drugs.
Nature 537(7618):117
Tano YK, Wakimoto S, Tamura S, Kubota K, Domon Y, Arakawa N, Saito M, Sava B, Buisson B
(2019) Effects of mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium
channels, on N-type calcium channel currents of rat dorsal root ganglion culture neurons.
Pharmazie 74(3):147–149
Taylor CP (2009) Mechanisms of analgesia by gabapentin and pregabalin–Calcium channel α2-δ
[Cavα2-δ] ligands. Pain 142(1):13–16
Thalhammer A, Contestabile A, Ermolyuk YS, Ng T, Volynski KE, Soong TW, Goda Y, Cingolani
LA (2017) Alternative splicing of P/Q-Type Ca2+ channels shapes presynaptic plasticity. Cell
Rep 20(2):333–343
Thapa P, Espiritu MJ, Cabalteja CC, Bingham JP (2014) Conotoxins and their regulatory
considerations. Regul Toxicol Pharmacol 70(1):197–202
Thompson JC, Dunbar E, Laye RR (2006) Treatment challenges and complications with ziconotide
monotherapy in established pump patients. Pain Physician 9:147–152
Tikhonov DB, Zhorov BS (2017) Mechanism of sodium channel block by local anesthetics,
antiarrhythmics, and anticonvulsants. J Gen Physiol 149(4):465–481
Tomita S, Sekiguchi F, Deguchi T, Miyazaki T, Ikeda Y, Tsubota M, Yoshida S, Du Nguyen H,
Okada T, Toyooka N, Kawabata A (2019) Critical role of Cav3. 2 T-type calcium channels in
the peripheral neuropathy induced by bortezomib, a proteasome-inhibiting chemotherapeutic
agent, in mice. Toxicology 413:33–39
Torres M, Marcilla-Etxenike A, Fiol-deRoque MA, Escribá PV, Busquets X (2015) The unfolded
protein response in the therapeutic effect of hydroxy-DHA against Alzheimer’s disease. Apo-
ptosis 20(5):712–724
Tse G, Liu T, Li KHC, Laxton V, Wong AOT, Chan YWF, Keung W, Chan CW, Li RA (2017)
Tachycardia-bradycardia syndrome: electrophysiological mechanisms and future therapeutic
approaches. Int J Mol Med 39(3):519–526
Turnaturi R, Arico G, Ronsisvalle G, Pasquinucci L, Parenti C (2016) Multitarget opioid/non-
opioid ligands: a potential approach in pain management. Curr Med Chem 23(40):4506–4528
Uchitel OD, Di Guilmi MN, Urbano FJ, Gonzalez-Inchauspe C (2010) Acute modulation of
calcium currents and synaptic transmission by gabapentinoids. Channels 4(6):490–496
Uzieliene I, Bernotas P, Mobasheri A, Bernotiene E (2018) The role of physical stimuli on calcium
channels in chondrogenic differentiation of mesenchymal stem cells. Int J Mol Sci 19(10):2998
Vallejo-Illarramendi A, Toral-Ojeda I, Aldanondo G, de Munain AL (2014) Dysregulation of
calcium homeostasis in muscular dystrophies. Expert Rev Mol Med 16:e16
Vega-Vela NE, Osorio D, Avila-Rodriguez M, Gonzalez J, García-Segura LM, Echeverria V,
Barreto GE (2017) L-type calcium channels modulation by estradiol. Mol Neurobiol 54
(7):4996–5007
720 S. Mallik and P. C. Acharya

Voisin T, Bourinet E, Lory P (2016) Genetic alteration of the metal/redox modulation of Cav3.
2 T-type calcium channel reveals its role in neuronal excitability. J Physiol 594(13):3561–3574
Wagner S, Rokita AG, Anderson ME, Maier LS (2013) Redox regulation of sodium and calcium
handling. Antioxid Redox Signal 18(9):1063–1077
Wang F, Yan Z, Liu Z, Wang S, Wu Q, Yu S, Ding J, Dai Q (2016) Molecular basis of toxicity of
N-type calcium channel inhibitor MVIIA. Neuropharmacology 101:137–145
Wang AL, Iadecola C, Wang G (2017) New generations of dihydropyridines for treatment of
hypertension. J Geriatr Cardiol 14(1):67
Wang X, Chen Y, Meng Z, Zhang Q, Zhai J (2018) Effect of Trivalent “Calcium-like” Cations on
Ionic Transport Behaviors of Artificial Calcium-Responsive Nanochannels. J Phys Chem C 122
(43):24863–24870
Watanabe M, Ueda T, Shibata Y, Kumamoto N, Shimada S, Ugawa S (2015) Expression and
regulation of Cav3. 2 T-type calcium channels during inflammatory hyperalgesia in mouse
dorsal root ganglion neurons. PLoS One 10(5):e0127572
Weiss N, Zamponi GW (2017) Trafficking of neuronal calcium channels. Neuronal Signal
1(1):1–16
Winters BL, Christie MJ, Vaughan CW (2019) Electrophysiological actions of N/OFQ. Springer,
New York, NY
Wong FK, Nath AR, Chen RH, Gardezi SR, Li Q, Stanley EF (2014) Synaptic vesicle tethering and
the CaV2. 2 distal C-terminal. Front Cell Neurosci 8:71
Woo LA, Tkachenko S, Ding M, Plowright AT, Engkvist O, Andersson H, Drowley L, Barrett I,
Firth M, Akerblad P, Wolf MJ (2019) High-content phenotypic assay for proliferation of human
iPSC-derived cardiomyocytes identifies L-type calcium channels as targets. J Mol Cell Cardiol
127:204–214
Woon MT, Balijepalli RC (2015) L-type Ca2+ channel Cavb subunits associate with and differen-
tially regulate the cardiac Cav3. 2 T-type Ca2+ channel currents. Biophys J 108(2):578a–579a
Wright AB, Norimatsu Y, McIntosh JM, Elmslie KS (2015) Limited efficacy of α-conopeptides,
Vc1. 1 and RgIA, to inhibit sensory neuron CaV current. eNeuro 2(1). https://doi.org/10.1523/
ENEURO.0057-14.2015
Xu Y, Sun J, Liu H, Sun J, Yu Y, Su Y, Cui Y, Zhao M, Zhang J (2018) Scorpion toxins targeting
voltage-gated sodium channels associated with pain. Curr Pharm Biotechnol 19(11):848–855
Yang R, Xiong Z, Liu C, Liu L (2014a) Inhibitory effects of capsaicin on voltage-gated potassium
channels by TRPV1-independent pathway. Cell Mol Neurobiol 34(4):565–576
Yang YC, Tai CH, Pan MK, Kuo CC (2014b) The T-type calcium channel as a new therapeutic
target for Parkinson’s disease. Pflügers Arch 466(4):747–755
Yang S, Ben-Shalom R, Ahn M, Liptak AT, van Rijn RM, Whistler JL, Bender KJ (2016)
β-arrestin-dependent dopaminergic regulation of calcium channel activity in the axon initial
segment. Cell Rep 16(6):1518–1526
Yocum GT, Perez-Zoghbi JF, Danielsson J, Kuforiji AS, Zhang Y, Li G, Rashid Roni MS,
Kodali R, Stafford DC, Arnold LA, Cook JM (2018) A novel GABAA receptor ligand
MIDD0301 with limited blood-brain barrier penetration relaxes airway smooth muscle
ex vivo and in vivo. Am J Physiol Lung Cell Mol Physiol 316(2):L385–L390
Yoder N, Yoshioka C, Gouaux E (2018) Gating mechanisms of acid-sensing ion channels. Nature
555(7696):397
Zamponi GW (2016) Targeting voltage-gated calcium channels in neurological and psychiatric
diseases. Nat Rev Drug Discov 15(1):19
Zamponi GW, Diaz P (2017) UTI LP and University of Montana, 2017. T-type calcium channel
modulator and uses thereof. U.S. Patent Application 15/518,473
Zamponi GW, Lory P, Perez-Reyes E (2010) Role of voltage-gated calcium channels in epilepsy.
Pflügers Arch 460(2):395–403
Zamponi GW, Striessnig J, Koschak A, Dolphin AC (2015) The physiology, pathology, and
pharmacology of voltage-gated calcium channels and their future therapeutic potential.
Pharmacol Rev 67(4):821–870
20 Pharmacology of Calcium Channel 721

Zaydman MA, Kasimova MA, McFarland K, Beller Z, Hou P, Kinser HE, Liang H, Zhang G, Shi J,
Tarek M, Cui J (2014) Domain–domain interactions determine the gating, permeation, pharma-
cology, and subunit modulation of the IKs ion channel. Elife 3:e03606
Zhang S, Yang L, Zhang K, Liu X, Dai W, Zhang C, Yong Z, Li J, Zheng J (2015) ZC88, a novel
N-type calcium channel blocker from 4-amino-piperidine derivatives state-dependent inhibits
Cav2. 2 calcium channels. Brain Res 1605:12–21
Zhang Y, Cruickshanks N, Yuan F, Wang B, Pahuski M, Wulfkuhle J, Gallagher I, Koeppel AF,
Hatef S, Papanicolas C, Lee J (2017) Targetable T-type calcium channels drive glioblastoma.
Cancer Res 77(13):3479–3490
Zhao Y, Huang G, Wu J, Wu Q, Gao S, Yan Z, Lei J, Yan N (2019) Molecular basis for ligand
modulation of a mammalian voltage-gated Ca2+ channel. Cell 177(6):1495–1506
Zheng J (2013) Molecular mechanism of TRP channels. Compr Physiol 3(1):221–242
Zvejniece L, Vavers E, Svalbe B, Veinberg G, Rizhanova K, Liepins V, Kalvinsh I, Dambrova M
(2015) R-phenibut binds to the α2–δ subunit of voltage-dependent calcium channels and exerts
gabapentin-like anti-nociceptive effects. Pharmacol Biochem Behav 137:23–29