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Time-Dependent Variations
in Serum Nitrite, 6-Keto-
Prostaglandin F1α and
Thromboxane B2 Levels Induced
by Lipopolysaccharide in Mice
a a a
Bahar Tunçtan , Sedat Altuğ , Orhan Uludağ &
a
Nurettin Abacioğlu
a
Department of Pharmacology, Faculty of Pharmacy, Gazi
University, Ankara, Turkey
Published online: 09 Aug 2010.

To cite this article: Bahar Tunçtan , Sedat Altuğ , Orhan Uludağ & Nurettin Abacioğlu
(2000) Time-Dependent Variations in Serum Nitrite, 6-Keto-Prostaglandin F1α and
Thromboxane B2 Levels Induced by Lipopolysaccharide in Mice, Biological Rhythm
Research, 31:4, 499-513

To link to this article: http://dx.doi.org/10.1076/0929-1016(200010)31:4;1-2;FT499

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 Biological Rhythm Research, 2000, Vol. 31, No. 4, pp. 499–513 0929-1016/00/3104-0499$15.00
© Swets & Zeitlinger

Time-Dependent Variations in Serum Nitrite,

6-Keto-Prostaglandin F1α and Thromboxane B2 Lev-
els Induced by Lipopolysaccharide in Mice

Bahar Tunçtan, Sedat Altug, Orhan Uludag, Nurettin Abacıoglu

Department of Pharmacology, Faculty of Pharmacy, Gazi University, Ankara, Turkey

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ABSTRACT

Clinical features of certain immuno-inflammatory disorders exhibit time-dependent fluctuations, which

could be related to circadian rhythmicity of proinflammatory mediator production. Many biologically
active substances including nitric oxide (NO) and eicosanoids are released into the circulation in
sepsis. Increased NO and eicosanoid levels have been reported to be responsible from death in septic
shock. The aim of this study was to investigate the variations in the NO and eicosanoid production and
mortality induced by bacterial endotoxin, lipopolysaccharide (LPS) injected either in the morning or
in the evening. Experiments were performed on mice synchronised to 12 h light and 12 h dark (lights
on at 09:00 h). Animals were injected intraperitoneally with LPS (10 mg/kg) at 09:00 (morning) and
21:00 h (evening) alone or in combination with aminoguanidine (NO synthase (NOS) inhibitor) (100
mg/kg) or indomethacin (cyclooxygenase (COX) inhibitor) (100 mg/kg). The serum was separated
from blood samples obtained at nine different time points. Nitrite (stable product of NO), 6-keto-
prostaglandin F1α (6-keto-PGF1α, stable product of prostacyclin) and thromboxane B2 (TxB2, stable
product of thromboxane) concentrations in serum samples were measured. Serum nitrite levels showed
a 24 h circadian rhythmicity depending on LPS injection time. Morning injection caused a peak after
15 h, while evening injection had two peaks after 9 and 18 h. The peak values obtained from morning
and evening injections were significantly decreased by aminoguanidine and indomethacin. When LPS
injected to mice in the morning and in the evening, it gradually increased the mortality rate within 24
h which could be abolished by aminoguanidine, but not indomethacin. Indomethacin-induced inhibi-
tion on LPS-induced nitrite levels was higher in the morning than in the evening. 6-keto-PGF1α and
TxB2 levels were decreased by indomethacin when injected with LPS at both injection times, but not
aminoguanidine. These results showed that there is an interaction between NO and eicosanoids, and
LPS may produce different effects on NOS activity, but not eicosanoid production and mortality,
depending on injection time in the experimental septic shock model in mice. Chronopharmacological
manipulations of NOS and COX pathways and interactions between them could lead to novel thera-
peutic approaches for the treatment of septic shock.

KEYWORDS: Nitric oxide, eicosanoid, lipopolysaccharide, daily variation, mice, septic shock.

Address correspondence to: Nurettin Abacıoglu, Department of Pharmacology, Faculty of Pharmacy,

Gazi University, 06330, Hipodrom, Ankara, Turkey. Phone: (90) (312) 2221225. Fax: (90) (312)
2235018. E-mail: [email protected].
 500 B. TUNÇTAN ET AL.

INTRODUCTION

Septic shock remains a common and devastating problem in critical care units.
The mortality rate from septic shock is still high and ranges from 20 to 75%
(Teplick and Rubin, 1999; Titheradge, 1999). The development of shock causes
in a progressive failure of the circulation to provide blood and oxygen to vital
organs of the body resulting in impaired tissue perfusion. The key symptoms
include a severe fall in blood pressure with hyporeactivity to vasoconstrictor
agents which may lead to the dysfunction or failure of major organs including
lungs, liver, kidneys and brain (multiple organ system failure) and ultimately
death (Georgieff and Tugtekin, 1998; Titheradge, 1999). The underlying hemody-
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namic changes in septic shock consist of a dramatic increase in the production of

eicosanoids (Patrignani et al., 1997; Salvemini et al., 1995), cytokines (Damas et
al., 1997), free radicals (Horn, 1998), and nitric oxide (NO) (de Werra et al.,
1997; Doughty et al., 1998; Grover et al., 1999; Tunçtan et al., 1998). It is current-
ly not known how and when to augment the biologically beneficial changes and
suppress the harmful alterations of the above mediators. Despite of a large number
of new antimicrobic agents, therapies targeted at various aspects of the inflamma-
tory cascade, and significant improvements in supportive therapy for the critically
ill, there has been little progress in the prevention or treatment of sepsis (Horn,
1998; Oh, 1998).
NO and eicosanoids are synthetized enzymatically in numerous tissues and
considered as important mediators of many physiological and pathophysiological
processes (Adams et al., 1999; Dubois et al., 1998; Fukuto and Wink, 1999; Vane
et al., 1998). NO is produced as a reaction product of the conversion of L-arginine
to citrulline through the hydroxylation of an L-arginine guanidino nitrogen by
three isoforms of the enzyme NO synthases (NOS) (Boucher et al., 1999; Stuehr,
1999). Two of these isoforms are expressed in a constitutive manner (constitutive
NOS, cNOS) predominantly in the vascular endothelium (endothelial NOS, eNOS)
and in the nervous system (neuronal NOS, nNOS). Under physiological condi-
tions, these constitutive isoforms of NOS generate low levels of NO in response to
increases in intracellular calcium concentrations. The expression of the third iso-
form (inducible NOS, iNOS) is induced by endotoxin and/or inflammatory cy-
tokines in a variety of cells including macrophages, vascular endothelial and
smooth muscle cells (Beck et al., 1999; Fleming and Busse, 1999; Kirkebøen and
Strand, 1999). Increased production of NO by iNOS has been demonstrated by
many investigators as a major cause for systemic hypotension, vascular hyporeac-
tivity, tissue edema, failure of tissue oxygen use and high mortality rate that are
associated with septic shock (Johnson and Billiar, 1998; Kirkebøen and Strand,
1999; Symenoides and Balk, 1999; Titheradge, 1999; Szabo, 1998). Decomposi-
tion products of NO, nitrite and nitrate, are also increased in the serum or urine of
 TIME-DEPENDENT VARIATIONS 501

animals by exposure to bacterial endotoxin and patients with sepsis (de Werra et
al., 1997; Doughty et al., 1998; Evans et al., 1994; Grover et al., 1999; Rees et al.,
1998; Tunçtan et al., 1998; Vodovotz et al., 1998). The development of selective
iNOS inhibitors has been shown to provide beneficial hemodynamic effects in
septic shock treatment in humans (Grover et al., 1999; Hobbs et al., 1999; Hussein
et al., 1999; Szabo et al., 1999; Wolfe and Dasta, 1995).
COX is the enzyme that catalyzes the conversion of arachidonic acid to pros-
taglandin G2 (PGG2) and PGH2. PGH2 is subsequently converted to a variety of
eicosanoids that include PGI2, PGE2, PGD2, PGF2α, and thromboxane (Tx), de-
pending on the specific enzymes present in the different cells (Smith and Marnett,
1994). Similar to NOS, two major isoforms of COX have been established. COX-
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1 is constitutively expressed in nearly all cell types, but COX-2 is normally absent
in cells. When COX-2 induced, the protein levels increase in a matter of hours in
a variety of cells including macrophages, endothelial and vascular smooth muscle
cells (Dubois et al., 1998; Vane et al., 1998). Like iNOS, the induction of COX-2
by endotoxin and/or cytokines occurs in a variety of inflammatory conditions
such as septic shock resulting in an elevated production of proinflammatory PGs
(Salvemini and Masferrer, 1996). Indeed, as of indexes for COX activity TxA2,
PGE2 and 6-keto-PGF1α (the stable metabolite of PGI2) levels have shown to be
elevated in animals and humans with septic shock (Basu and Eriksson, 1998;
Bernard et al., 1991; Patrignani et al., 1997; Salvemini et al., 1995).
The response of hosts to exogenous stimuli such as lipopolysaccharide (LPS)
or cytokines depends strongly on circadian time of exposure. For instance, some
investigators showed that LPS administration time changes fever response and
mortality rate in pigeons (Nomoto, 1997), ducks (Maloney and Gray, 1998), and
mice (Halberg et al., 1955, 1980). It has also been shown that the levels of COX
products, PGE2, PGF2α and TxB2 (Kunkel et al., 1988; Pandey et al., 1995;
Yosipovitch et al., 1995), and nitrite/nitrate (Globig et al., 1999; Uludag et al.,
1999) show circadian rhythmicity under physiological or pathophysiological con-
ditions in rodents. The cytokines such as tumor necrosis factor-α (TNF-α), inter-
leukins 1, 2, 6, 10 (IL-1, IL-2, IL-6, IL-10), and interferon-γ (IFN-γ) have been
shown to display circadian rhythms under basal or stimulated conditions (Gudewill
et al., 1992; Petrovsky and Harrison, 1997; Young et al., 1995). The cytokines
together with NO and PGs are important mediators of hemodynamic, metabolic,
and immunologic alterations in the host defence. In septic shock, these mediators
are found to be at high concentrations in serum and urine of animals and humans
and the enhanced levels are correlated with decreased survival (Astiz and Rack-
ow, 1998; Damas et al., 1997; Ertel et al., 1991; Johnson and Billiar, 1998;
Ketteler et al., 1998).
In this study, we aimed to investigate the variations in the serum nitrite, 6-
keto-PGF1α and thromboxane B2 (TxB2) levels and mortality depending on injec-
 502 B. TUNÇTAN ET AL.

tion time of LPS, NOS and/or COX enzyme inhibitors in LPS-induced septic
shock model in mice.

MATERIALS AND METHODS

Animals
Local bred male and female albino mice, weighing 20-40 g were used throughout
the experiments according to the proposals of the Declaration of Helsinki and the
U.S. National Institutes of Health Guide for the Care and Use of Laboratory
Animals. Animals were housed in standard transparent cages (20 per cage) in an
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ambient temperature of 24-25°C animal room condition with free access to food
and water. They were synchronised by maintenance of controlled environmental
conditions for at least 2 weeks prior to and throughout the duration of the experi-
ments. The circadian rhythmicity of the animals was entrained by a standardised
12 h light and 12 h dark (lights on at 09:00 h) with a light intensity of approxi-
mately 100 lux. Automatic timer controlled cool fluorescent bulbs were used to
provide lighting. Visualisation and drug administration in the dark were supplied
by photosafe red bulbs. To avoid seasonal variations, all the experiments were
performed from June to August.

Treatments
The following drugs were administered intraperitoneally with control group or
without LPS (10 mg/kg) at 09:00 and 21:00 h: saline, aminoguanidine (100 mg/
kg), indomethacin (100 mg/kg). The blood samples were collected at nine time
points after the drug administration. After blood samples had clotted at room
temperature for 30 min, they were defibrinized and centrifuged at 2000 rpm for 15
minutes. Serum was aspired and frozen at –20°C until analysed.

Nitrite Measurements
Serum nitrite concentrations were measured by using the diazotization method
based on the Griess reaction, which is an indirect assay for NO production (Mar-
letta et al., 1988; Tunçtan et al., 1998). Serum samples (100 µl) were pipetted into
96 well microtiter plates and an equal volume of Griess reagent (1% sulphanyla-
mide (50 µl) and 0.1% naphtylethylenediamine dihydrochloride (50 µl) in 2.5%
ortophosphoric acid) was added to each well. After incubation for 15 minutes at
room temperature, absorbance was measured at 550 nm with a microplate reader
(Diagnostic Pasteur, LP 400) using a reference filter of 620 nm. Standard curves
were also constructed using sodium nitrite concentrations ranging from 0.25-50
µM. Each measurement was performed in triplicate.
 TIME-DEPENDENT VARIATIONS 503

6-keto-PGF1α and TxB2 Measurements

6-keto-PGF1α and TxB2 were measured in the unextracted serum samples by
ELISA according to the manufacturer’s instructions in the 6-keto-PGF1α and
TxB2 assay kits (International Immuno-Diagnostic, 1155 Chess Dr. # 121, Foster
City, CA, 94404-1115). Each experiment was performed in duplicate.

Drugs
LPS (E. coli O11:B4), aminoguanidine and indomethacin were obtained from
Sigma Chemical Co. (St. Louis, USA). NaHCO3 was obtained from Merck (Darm-
stadt, Germany). Indomethacin was dissolved in 5% NaHCO3 solution. All the
other drugs were dissolved in saline.
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Data Analysis
Results were expressed as means ± SEM. The data were analyzed by single
cosinor analysis for 24 h periodicity using a curve-fit programme (2D Table-
Curve, SPSS Inc., San Diego, CA) to elucidate the best curve fitting to the data.
This method describes the rhythm parameters such as mesor (mean of the variable
values throughout the rhythm), acrophase (peak hour of rhythm) and amplitude
(the difference between the peak levels of the rhythm to its mesor). Statistical
comparisons were made by one-way ANOVA followed by Student-Newman-
Keuls multiple comparisons test, and Student’s t test when necessary. Mortality
percentage comparisons were done by Fisher’s exact test. A p value of < .05 was
considered statistically significant.

RESULTS

Serum nitrite levels showed a 24 h circadian rhythmicity regardless of LPS injec-

tion time (Fig. 1). The cosinor analysis parameters are summarized in Table 1.
Basal serum nitrite levels did not show any circadian rhythmcity (p > .05). A
gradual increase in nitrite levels was observed 12 h after LPS injection in the
morning and reached a peak by 15 h (Fig. 1A). Nitrite levels begun to decrease
after 15 h and returned to basal levels within 24 h. LPS-induced nitrite levels were
statistically different from saline at 12, 15, and 18 h (p < .05). This profile of
nitrite levels displayed 24 h circadian rhythm characterised by trough (1.23 ±
0.11, n = 9) and peak (4.20 ± 0.40, n = 17) values after 6 h and 15 h, respectively
(p < .05). There were also found 12, 24 + 12 and 24 + 12 + 8 h rhythms. On the
other hand, the injection of LPS in the evening caused two peaks in nitrite levels
after 9 (5.21 ± 0.73, n = 13) and 18 h (5.03 ± 0.66, n = 9), respectively (Fig. 1B).
The pattern of nitrite levels exhibited 24 h circadian rhythmicity with a statistical
difference from saline at 9, 12, 15, and 18 h (p < .05). In addition, the nitrite level
 504 B. TUNÇTAN ET AL.
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Fig. 1. Circadian changes in serum nitrite levels induced by LPS (10 mg/kg) in mice. Panel A and B
show LPS injection at 09:00 and 21:00 h, respectively. ❍ saline, ● LPS injection at 09:00, ▼
LPS injection at 21:00 h. Data were expressed as the mean ± SEM, n = 4-17. The dark bar
indicates the period of darkness. ★ Statistically significant difference from saline-treated group.
 TIME-DEPENDENT VARIATIONS 505

TABLE 1. Circadian characteristics for serum nitrite levels in mice.

n Amplitude ± SEM Mesor ± SEM Acrophase (95% CI) F p

Saline 22 0.26 ± 0.12 2.06 ± 0.08 04:12 (00:43-07:50) 2.16 > .05
LPS at 09:00 h 17 1.47 ± 0.20* 2.49 ± 0.15* 01.15 (00:46-02:40) 26.81 < .05
LPS at 21:00 h 13 1.40 ± 0.36* 3.43 ± 0.25*+ 03:07 (01:04-05:10) 7.50 < .05

Statistically significant difference from * saline; + LPS at 09:00 h.

rhythmicity displayed 8, 12, 24 + 12 and 24 + 12 + 8 h rhythms. The amplitude

and mesor values for saline rhythm were statistically lower than either morning
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or evening LPS injection. Although there was no statistical difference between

the amplitudes of nitrite levels rhythms depending on injection time of LPS,
mesor values of morning injection were higher than evening injection (Table 1)
(p < .05). Acrophase for morning administration of LPS was significantly shifted
to about 2 h when LPS injected in the evening (Table 1) (p < .05). Morning or
evening injection of LPS to mice gradually increased the mortality rate within
24 h without any significant difference between morning and evening administra-
tions (p > .05) (Fig. 2).

Fig. 2. Time-dependent mortality rate induced by LPS (10 mg/kg) in mice. LPS injected at 09:00 (●)
or at 21:00 h (❍).
 506 B. TUNÇTAN ET AL.
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Fig. 3. Effects of aminoguanidine (AG, 100 mg/kg) and indomethacin (INDO, 100 mg/kg) on LPS
(10 mg/kg)-induced increase in serum nitrite levels in mice. ❑ 09:00 and ■ 21:00 h injections.
Data were expressed as the mean ± SEM, n= 13-17. Statistically significant difference from ★
LPS; + 09:00 h injection.

Fig. 4. Effects of aminoguanidine (AG, 100 mg/kg) and indomethacin (INDO, 100 mg/kg) on LPS
(10 mg/kg)-induced increase in 6-keto-PGF1α levels in mice. ❑ 09:00 and ■ 21:00 h injec-
tions. Data were expressed as the mean ± SEM, n= 6-17. Statistically significant difference
from ★ LPS; + AG.
 TIME-DEPENDENT VARIATIONS 507
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Fig. 5. Effects of aminoguanidine (AG, 100 mg/kg) and indomethacin (INDO, 100 mg/kg) on LPS
(10 mg/kg)-induced increase in TxB2 levels in mice. ❑ 09:00 and ■ 21:00 h injections. Data
were expressed as the mean ± SEM, n= 6-17. Statistically significant difference from ★ LPS; +
AG.

Aminoguanidine (iNOS inhibitor) and indomethacin (COX inhibitor) signifi-

cantly diminished the peak values of nitrite levels induced by either morning or
evening LPS injections (p < .05) (Fig. 3). But, when LPS was administered in the
morning, the elevated nitrite levels were more potently inhibited by indomethacin
than in case of an evening injection (p < .05). Concomitant injections of amino-
guanidine with LPS both in the morning and in the evening, abolished the LPS-
induced mortality after 15 h (25.0%) and 9 h (5.3%), respectively. On the contra-
ry, the effects of indomethacin on the mortality rates induced by morning and
evening LPS injections were not changed significantly (8.3 and 6.7% after 15 and
9 h, respectively).
Coadministration of indomethacin with LPS at morning and evening times of
the day significantly decreased the 6-keto-PGF1α and TxB2 levels (p < .05) while
the morning versus evening administrations exhibited no significant changes on
eicosanoids levels when compared to each other (p > .05). However, concomitant
injection of aminoguanidine with LPS possessed no significant differences on the
LPS-induced eicosanoid levels (p > .05) (Figs. 4, 5).
 508 B. TUNÇTAN ET AL.

DISCUSSION

These results in the LPS-induced septic shock model in mice showed that (1) the
activites of iNOS and COX-2 are increased, (2) there is an interaction between
iNOS and COX-2 pathways, (3) the mediators responsible from mortality in sep-
tic shock may be NO and TxB2 (4) aminoguanidine, but not indomethacin, may be
beneficial in septic shock treatment, and (5) time-dependent variations and inter-
actions between NO and PGs may be occur in septic shock pathogenesis.
The morning or evening injection of LPS to the mice produced different effects
on overproduction of NO, however, caused no significant changes in the in-
creased mortality. In contrast to our findings, the daytime administration of Bru-
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cella or E. coli endotoxin to mice has been shown to decrease the survival rate
when compared to night injection (Halberg et al., 1955; 1980). The diversity of
the results may depend on the source and injection time of LPS and strain of mice.
Serum nitrite levels, as an index for NOS activity, displayed a 24 h rhythm
depending on injection time of LPS. The morning injection caused a peak after 15
h, while the evening injection had two peaks after 9 and 18 h. LPS did not change
the amplitudes, but significantly increased the mesor when injected in the evening
versus morning. The acrophase values of both rhythms were at dark period. The
peak values were significantly decreased, but not differed with the injection time,
and the LPS-induced mortality was abolished with aminoguanidine, a specific
iNOS inhibitor (Corbett and McDaniel, 1996). Our results are in conformity with
previous studies which reported that aminoguanidine and other guanidine deriva-
tives have beneficial effects by inhibition of iNOS and a lesser extent cNOS, and
free radical production (ıskit et al., 1999; Szabo et al., 1999; Takano et al., 1997;
Tunçtan et al., 1998; Wu et al., 1998; Yıldız et al., 1998; Zingarelli et al., 1997).
On the other hand, we couldn’t find any correlation between serum nitrite levels
and mortality in mice in conformity with the results of Evans et al. (1994). Injec-
tion of nonselective COX inhibitor indomethacin (Vane et al., 1998) with LPS
significantly decreased 6-keto-PGF1α and TxB2 levels induced by LPS as previ-
ously shown (Boughton-Smith et al., 1989; Futaki et al., 1997). Indomethacin-
induced inhibition on eicosanoid levels was similar at both injection times. How-
ever, injection of indomethacin either in the morning or in the evening did not
significantly reduce LPS-induced mortality. These results suggest that the over-
production of NO and eicosanoids may be responsible from LPS-induced mortal-
ity and exposure to LPS at different times of the day may change the effects of the
enzyme inhibitors in this experimental septic shock model in mice.
We also investigated the possible effects of iNOS inhibitor aminoguanidine on
eicosanoid production and COX inhibitor indomethacin on NO production. There
is mounting evidence for a direct link between NO production and synthesis of
PGs. Several studies using NOS inhibitors have demonstrated that NO stimulates
 TIME-DEPENDENT VARIATIONS 509

the formation of PGs in in vitro (Salvemini et al., 1993; Watkins et al., 1997) and
in vivo models (Salvemini et al., 1995; Sautebin and Di Rosa, 1994). On the other
hand, NOS inhibitors also act to increase COX-2 expression and PG synthesis in
response to cytokine stimulation and the addition of NO donors reverses the effect
suggesting NO also inhibits COX-2 activity (Amin et al., 1997; Habib et al., 1997;
Swierkosz et al., 1995). Furthermore, some studies suggest that there is no inter-
action between NO and PG synthesis (Boquet et al., 1998; Curtis et al., 1996;
Hamilton and Warner, 1998). However, it is clear that a large group of investiga-
tors have observed NO-stimulated PG biosynthesis, apparently by direct interac-
tion with COX-2. Contrasting data have also been reported concerning the action
of PGs on the L-arginine/NO pathway. It has been shown that PGs inhibit (Marot-
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ta et al., 1992; Raddasi et al., 1993), increase (Gaillard et al., 1992; Milano et al.,
1995) or do not affect (Salvemini et al., 1993) NO production by iNOS in several
in vitro models. This wide variability in the observations may result from the use
of different cell lines and tissues, as well as differences in methods of cell activa-
tion or in inflammatory models (Goodwin et al., 1999). Another explanation for
this diversity may depend on the periodic rhythms in COX and NOS enzyme
activity and/or cytokine production. Although it has been shown that aminoguani-
dine inhibits PG production (Kanematsu et al., 1997; Salvemini et al., 1994;
Sautebin et al., 1998), in our studies, aminoguanidine did not change 6-keto-
PGF1α and TxB2 levels in the sera of septic mice. Similar to previous in vitro
(Illiano et al., 1996) and in vivo (Hamilton and Warner, 1998) findings, our results
indicate that neither morning nor evening administration of aminoguanidine af-
fects eicosanoid production. Indomethacin has been shown to increase (Criado et
al., 1999; Swierkosz et al., 1995), inhibit (Milano et al., 1995; Tunçtan et al.,
1998; Yamashita et al., 1999) or not to affect (Amin et al., 1995; Salvemini et al.,
1993) NOS activity. Our results with indomethacin showed that it is able to
decrease iNOS activity and its effects on NO production may change depending
on injection time. Therefore, PGs appear to exacerbate the overproduction of NO
by iNOS. These results support the observations that NOS activity appears to be
modulated by PGs in this experimental septic shock model in mice.
These results showed that depending on injection time, LPS may produce
different effects on NOS activity, but not eicosanoid production and mortality in
the experimental septic shock model in mice. In addition, it may also suggest that
interactions between NO and the eicosanoids display time-dependent variations.
Chronopharmacological manipulations of NOS and COX pathways and interac-
tions between them could lead to novel therapeutic approaches for the treatment
of septic shock.
 510 B. TUNÇTAN ET AL.

ACKNOWLEDGEMENT

This study was supported by the Research Foundation of Gazi University (Project Code No: EF.02/98-
06).

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