Endothelin receptor antagonist bosentan improves survival in a murine caecal ligation and puncture model of septic shock

The role of endothelin peptides was evaluated on survival and organ injury in a model of polymicrobial sepsis, induced by caecal ligation and puncture with particular emphasis on the timing of the administration of its blocker bosentan in Swiss albino mice (20-40 g). The cardiovascular response pattern in this experimental model was characterized by an early, "hyperdynamic" phase starting at 5 h, followed by a late but "hypodynamic" phase that commence after 20 h, provided that the animals are "resuscitated" by injecting 1 ml of saline i.p. at the end of the surgery. However, if saline resuscitation is omitted, then only hypodynamic pattern is observed starting at 5 h without any hyperdynamic phase. Thus, mice were first allocated into saline-resuscitated or unresuscitated groups and endothelin receptor antagonist bosentan (30 mg kg(-1), i.p., either 5 or 20 h after caecal ligation and puncture) was then administered. The control animals received the solvent of bosentan (i.e., saline: %0.9 NaCl, w/v). The survival rates in each group (n=14) were recorded over the following 144 h. In unresuscitated mice, the overall survival at 144 h was 14.3% in controls while bosentan treatment at 5 h (78.6%, P=0.0018) or 20 h (64.3%, P=0.0183) have both significantly improved the survival. However, in saline-resuscitated mice, bosentan administered at 20 h has significantly improved the survival (71.4%, P=0.0213) while its administration at 5 h has yielded exactly the same percent of survival (i.e., 21.4%) as observed in control animals. The beneficial effects of bosentan in preventing the tissue injury due to caecal ligation and puncture were also observed histopathologically in liver, spleen and kidney. Therefore, we concluded that the blockade of endothelin receptors by using bosentan during the later (hypodynamic) stages of septic shock is a promising therapeutic manoeuvre.     

Increase in gastric intramucosal hydrogen ion concentration following endotoxin challenge in the rat and the actions of nitric oxide synthase inhibitors

1. Cardiovascular events and outcome in septic shock may be predicted by monitoring the fall in intramural pH (pHi), as an index of splanchnic perfusion and mucosal ischaemia. In the present study, a small animal model for monitoring the changes of gastric pHi or intramucosal [H+] following challenge with the endotoxin lipopolysaccharide (LPS) was developed in the rat. The role of nitric oxide (NO) in these events in this model was evaluated using the non-selective NO synthase (NOS) inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA). 2. The pHi and intramucosal [H+] were evaluated in omeprazole-pretreated rats (30 mg/kg, i.p.) using the Henderson equation after estimating the PCO2 and the bicarbonate concentration in gastric wall. To measure gastric wall PCO2, the oesophagus was intubated and the pylorus ligated. The PCO2 was measured by a blood gas analyser in 2 mL saline instilled for 30 min in the gastric lumen to equilibrate with the gastric wall. The pHi was measured under basal conditions and 3 and 5 h after LPS (3 mg/kg) administration. Separate groups received treatment with L-NMMA (25-50 mg/kg) or L-NAME concomitantly or 2.5 h after administration of LPS. 3. Intravenous administration of Escherichia coli LPS provoked a significant fall in gastric pHi from 7.37 to 7.18 (median values; n =10-19) determined after 5 h. In groups treated concurrently with LPS and L-NAME (5 mg/kg; n = 19), there was a similar increase in intramucosal [H+] as that induced by LPS alone (n = 15) in those animals that survived. In contrast, L-NAME (5 mg/kg; n = 12), given 2.5 h after LPS challenge, at a time at which inducible NOS is known to be significantly expressed, prevented the increase in intramucosal [H+] at 3 and 5 h after LPS challenge. Similarly, L-NMMA (25-50 mg/kg; n = 23), given 2.5 h after LPS challenge, dose-dependently inhibited the increase in intramucosal [H+] at 3 and 5 h. 4. In conclusion, these findings indicate that this rat model could be useful in exploring the pathophysiology of acute endotoxin shock. Delayed administration of L-NAME and L-NMMA abolished the increase in gastric intramucosal [H+], supporting the involvement of excess NO in the tissue dysfunction associated with endotoxin shock. This suggests the potential value of this small animal model in evaluating the therapeutic activity of novel agents for use in septic shock.     

Endotoxin stimulates fecal pellet output in rats through a neural mechanism

The effects of endotoxin on fecal pellet output and the neural mechanisms involved in this response were investigated in conscious rats. E. coli endotoxin (40 micro g/kg i.p.) significantly increased fecal excretion for 3 h after the injection. Water content in feces was not modified by endotoxin. Ablation of primary afferent neurons by systemic administration of high doses of capsaicin (20+30+50 mg/kg s.c.) to adult rats prevented the stimulatory effect of endotoxin and so did abdominal vagotomy. Adrenoceptor blockade with phentolamine (5 mg/kg i.p.) + propranolol (3 mg/kg i.p.) did not modify pellet output in endotoxin-treated rats while muscarinic receptor blockade with atropine (1 mg/kg i.p.) abolished the stimulatory effect of endotoxin. Finally, the increase in pellet output induced by endotoxin was prevented in animals receiving the substance P receptor antagonist D-Pro2, D-Trp7,9-substance P (2 mg/kg i.p.) or the NO-synthase inhibitor L-NAME (10 mg/kg i.p.). None of the above treatments modified pellet output in saline-treated rats.These observations indicate that endotoxin increases fecal pellet output through a nervous reflex in which capsaicin-sensitive afferent neurons and the release of excitatory (acetylcholine and substance P) and inhibitory (NO) neurotransmitters in the colonic wall are involved.     

Role for endogenous endothelin in the regulation of plasma volume and albumin escape during endotoxin shock in conscious rats

To explore the role of endogenous endothelin (ET) in the regulation of vascular functions, we studied the effects endothelin receptor blockade on blood pressure, plasma volume and albumin escape during endotoxin shock in conscious, chronically catheterized rats. Red blood cell volume and plasma volume were determined by using chromium-51-tagged erythrocytes and iodine-125-labelled albumin, respectively. Intravenous injection of lipopolysaccharide (LPS, 10 mg kg(-1)) resulted in hypotension, haemoconcentration, and increased total-body albumin escape, which is reflected by a 30% reduction in plasma volume. Plasma ET-1 concentrations increased 2.1 fold and 5.4 fold at 30 and 120 min post-LPS, respectively. LPS-induced losses in plasma volume and albumin escape were significantly attenuated by pretreatment of animals with the dual ET(A)/ET(B) receptor antagonist bosentan (17.4 micromol kg(-1), i.v. 15 min prior to LPS) or the ET(A) receptor antagonist FR 139317 (3.8 micromol kg(-1), i.v.) during both the immediate and delayed phases of endotoxin shock. The inhibitory actions of bosentan and FR 139317 were similar. Both antagonists augmented the hypotensive action of LPS. Administration of bosentan or FR 139317 70 min after injection of LPS also attenuated further losses in plasma volume and increases in total body and organ albumin escape rates with the exception of the lung and kidney. These results indicate a role for endogenous endothelin in mediating losses in plasma volume and albumin escape elicited by LPS predominantly through activation of ET(A) receptors, and suggest that by attenuating these events, ET(A) or dual ET(A)/ET(B) receptor blockers may be useful agents in the therapy of septic shock.     

Preventive effects of a biscoclaurine alkaloid, cepharanthine, on endotoxin or tumor necrosis factor-alpha-induced septic shock symptoms: involvement of from cell death in L929 cells and nitric oxide production in raw 264.7 cells

The preventive effects of cepharanthine, a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, on the lethality and cell death caused by endotoxin or tumor necrosis factor (TNF)-alpha-induced syndrome in septic shock were investigated. In these experiments, we estimated the survival of mice treated with a lethal dose of endotoxin (50 mg/kg, i.p.) or recombinant human (rh) TNF-alpha (10,000 units/mouse, i.v.) together with a sublethal dose (1 mg/kg, i.p.) of endotoxin. Cepharanthine clearly protected mice from endotoxin-induced and endotoxin/rhTNF-alpha-induced lethal shock. In in vitro experiments, cepharanthine (3 micro g/ml) definitely inhibited cell death in mouse L929 fibroblast cells incubated with rhTNF-alpha (100 units/ml) at 37 degrees C for 24 h. On the other hand, non-apoptotic programmed death of cells was observed by fluorescence microscopy in rhTNF-alpha (100 units/ml)-treated L929 cells. In the 3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay after 48-h drug exposure, the cell proliferation of L929 cells was significantly increased by the addition of cepharanthine (1 and 3 micro g/ml). It seems that the preventive effect of cepharanthine on rhTNF-alpha-induced cytotoxicity in fibroblast cells occurs through an increase of cell proliferation by the drug. In addition, cepharanthine suppressed nitric oxide (NO) production by endotoxin-stimulated Raw 264.7 mouse macrophage cells. These findings suggest that cepharanthine prevents lethality or cytotoxicity through suppression of endotoxin-induced NO in macrophages and that its effects are possibly mediated by the enhancement of the proliferation of fibroblast cells. Cepharanthine may therefore protect against some of the various disturbances caused by endotoxin through its ability to inhibit NO production in septic shock.     

Quercetin, coenzyme Q10, and L-canavanine as protective agents against lipid peroxidation and nitric oxide generation in endotoxin-induced shock in rat brain.

The present study was designed to evaluate the possible protective effect of quercetin, coenzyme Q10 (CoQ10), or L-canavanine treatments against endotoxin-induced shock in rat brain. Shock was induced by i.p. injection of 10 mg x kg(-1)of lipopolysaccharide (LPS) and was biochemically manifested 2 h after injection as an increase in brain malondialdehyde (MDA), total nitrite/nitrate (NO(x)), glutathione peroxidase (GSHPx), and blood lactate level/activity. On the other hand, endotoxemia resulted in reduced brain glutathione (GSH) and phospholipids' content as well as the serum sulfhydryl groups' (SH-group) value. Pretreatment with quercetin (200 mg x kg(-1)per os) 2 h before LPS injection diminished the shock-induced increases in brain MDA, and NO(x)levels while elevating the reduced brain phospholipids' and serum SH groups' content. CoQ10 administered at a dose of 200 mg x kg(-1)per os for 7 days prior to shock induction, reduced the elevated levels of brain MDA, NO(x), and GSHPx level/activity due to redundancy. The same treatment caused a 3-fold increase in the reduced brain GSH level and normalized the depressed phospholipids' content. Treatment of animals with L-canavanine (50 mg x kg(-1)i.p.) simultaneously with LPS injection, reduced the elevated level of blood lactate. Brain superoxide dismutase (SOD) level was neither affected by endotoxin nor by different treatments. In conclusion, this study indicates that SOD may not reflect the level of peroxidation and points to the value of quercetin, CoQ10, and L-canavanine in ameliorating the oxidative status of brain during the early phase of endotoxic shock.     

The lazaroid, U-74389G, inhibits inducible nitric oxide synthase activity, reverses vascular failure and protects against endotoxin shock

The aim of our study was to investigate the effect of the 21-aminosteroid U-74389G [21- < 4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl-pregna-1,4,9,(11) triene-3,20-dione(z)-2-butenedionate] on the l-arginine-nitric oxide (NO) pathway in a rat model of endotoxin shock. Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg/kg of Salmonella Enteritidis lipopolysaccharide (LPS). Rats were treated with U-74389G (7.5, 15 and 30 mg/kg i.v.) or vehicle (1 ml/kg i.v.) 5 min after endotoxin challenge. Lipopolysaccharide administration reduced survival rate (0%, 72 h after endotoxin administration) decreased mean arterial blood pressure, enhanced plasma concentration of bilirubin and alanine aminotransferase and increased plasma nitrite concentrations. Lipopolysaccharide injection also increased the activity of inducible NO synthase in the liver and in the aorta. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (1 nM-10 microM). In addition lipopolysaccharide (50 microg/ml for 4 h) in vitro stimulation significantly increased nitrite production in peritoneal macrophages harvested from normal rats. Treatment with U-74389G (15 and 30 mg/kg i.v., 5 min after endotoxin challenge) significantly protected against lipopolysaccharide-induced lethality (90% survival rate 24 h and 80% 72 h after lipopolysaccharide injection, respectively, following the highest dose of the drug), reduced hypotension, ameliorated liver function, decreased plasma nitrite levels, restored the hyporeactivity of aortic rings to their control values and inhibited the activity of inducible NO synthase in the liver and in the aorta. Finally, U-74389G in vitro (12.5, 25 and 50 microM) significantly inhibited nitrite production in endotoxin stimulated peritoneal macrophages. The data suggest that U-74389G may exert beneficial effects in an experimental model of septic shock by inhibiting the activity of the inducible NO synthase.     

Nitric oxide and sensory afferent neurones modulate the protective effects of low-dose endotoxin on rat gastric mucosal damage

Pretreatment (1 h) with low doses (5–40 μg/kg i.p.) of Escherichia coli endotoxin dose dependently reduced the gastric mucosal damage induced by a 10 min challenge with 1 ml ethanol (50% and 100%) in conscious rats. Treatment with the nitric oxide synthesis inhibitor, oxide synthesis inhibitor, N^G-nitro- -arginine methyl ester (L-NAME, 5 and 10 mg/kg i.p.), significantly inhibited the protective effects of endotoxin (40 μg/kg i.p.). The actions of L-NAME were reversed by the prior administration of -arginine (100 mg/kg i.p.). The protective effects of endotoxin were not influenced by pretreatment with dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg s.c.). However, ablation of sensory afferent neurones by capsaicin pretreatment (20, 30 and 50 mg/kg s.c.) abolished the mucosa protective effects of endotoxin (40 μg/kg). These findings suggest that the protection elicited by low doses of endotoxin against ethanol-induced mucosal damage involves synthesis of nitric oxide and activation of sensory neurones.     

Role of glutathione in stabilization of nitric oxide during hypertension developed by inhibition of nitric oxide synthase in the rat

The present study examined the role of glutathione in the development of hypertension induced by long-term inhibition of nitric oxide (NO)-synthase. Three groups of rats were investigated: control group, L-NAME group: group with NO-synthase inhibition by N(G)-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg per day) for 2 weeks, and BSO group: group with glutathione synthesis inhibitor L-buthionine sulfoximine (BSO, 1.4 mmol/kg per 12 h) for 3 days. All the groups were subjected to an acute i.v. experiment in which the given substances were exchanged between groups. There was no change in systolic blood pressure (SBP) in the control group after 1 and 2 h of acute BSO (1.4 mmol/kg, i.v.) treatment. In the L-NAME group, SBP increased significantly by 10% after 2 h of acute BSO treatment. In the BSO group, SBP did not change vs control; however, after 2 h of acute L-NAME (10 mg/kg, i.v.) treatment, the increase in SBP exceeded by 12% (P<0.05) that of the control group. Along with the increase in SBP, acute BSO treatment significantly potentiated the decrease in plasma nitrite/nitrate concentration in the L-NAME group. The acute BSO-induced glutathione decrease was significantly greater in the L-NAME group than in the control group. In NO-deficient hypertensive rats, the results are indicative of a decrease in glutathione synthesis and a stabilizing role of glutathione.     

Dual effect of nitric oxide in articular inflammatory pain in zymosan-induced arthritis in rats

The contribution of nitric oxide (NO) to articular pain in arthritis induced by zymosan (1 mg, intra articular) in rats was assessed by measuring articular incapacitation (AI). Systemic treatment with the non-selective NO synthase (NOS) inhibitor L-NAME (10 - 100 mg kg(-1) i.p.) or with the selective iNOS inhibitors aminoguanidine (AG; 10 - 100 mg kg(-1) i.p.) or 1400W (0.5 - 1 mg kg(-1) s.c.) inhibited the AI induced by injection of zymosan 30 min later. Local (intra articular) treatment with the NOS inhibitors (L-NAME or AG, 0.1 - 1 micromol; 1400W, 0.01 (micromol) 30 min before zymosan also inhibited the AI. Systemic or local treatment with the NOS inhibitors (L-NAME; AG, 100 mg kg(-1) i.p. or 0.1 micromol joint(-1); 1400W, 1 mg kg(-1) s.c. or 0.01 micromol joint(-1)), 2 h after zymosan did not affect the subsequent AI. Local treatment with the NO donors SNP or SIN-1, 2 h after zymosan did inhibit AI. L-NAME and AG, given i.p. inhibited nitrite but not prostaglandin E(2) (PGE(2)) levels in the joints. L-NAME (100 mg kg(-1)) but not AG (100 mg kg(-1)) increased mean arterial blood pressure. Neither L-NAME, AG nor the NO donor SIN-1 altered articular oedema induced by zymosan. In conclusion, inhibitors of iNOS decrease pain in zymosan arthritis only when given before the zymosan. This was not due to inhibition of articular PGE(2) release or oedema. NO donors also promoted antinociception in zymosan arthritis without affecting oedema.     

The role of nitrergic system in lidocaine-induced convulsion in the mouse

The effects of N-nitro-L-arginine-methyl ester (L-NAME) a nitric oxide (NO) synthase inhibitor and L-arginine, a NO precursor, were investigated on lidocaine-induced convulsions. In the first experiment, four groups of mice received physiological saline (0.9%), L-arginine (300 mg/kg, i.p.), L-NAME (100 mg/kg, i.p.) and diazepam (2 mg/kg), respectively. Thirty minutes after these injections, all mice received lidocaine (50 mg/kg, i.p.). In the second experiment, four groups of mice received similar treatment in the first experiment, and 30 min after these injections, all mice received a higher dose of lidocaine (80 mg/kg). L-NAME (100 mg/kg, i.p.) and diazepam (2 mg/kg) significantly decreased the incidence of lidocaine (50 mg/kg)-induced convulsions. In contrast, the L-arginine treatment increased the incidence of lidocaine (80 mg/kg, i.p.)-induced convulsions significantly. These results may suggest that NO is a proconvulsant mediator in lidocaine-induced convulsions.     

EFFECT OF DIPYRIDAMOLE ON BLOOD GLUCOSE AND LIVER CYCLIC AMP LEVELS AND PLATELET COUNT DURING ENDOTOXAEMIA IN MICE

1. Blood glucose, liver cyclic AMP (cAMP) levels, and platelet count were studied at different times during a 12 h interval after intravenous (i.v.) endotoxin injection (40 mg/kg) in mice. 2. Blood glucose and liver cAMP levels showed a progressive decrease in endotoxic mice and the decrease was significant after 8 and 12 h. 3. Dipyridamole (50 mg/kg) administered intraperitoneally (i.p.) 7 h post-endotoxin improved blood glucose and liver cAMP levels. 4. Platelet counts were decreased after 1 h in endotoxic mice and remained decreased up to 8 h. Dipyridamole administered soon after endotoxin improved platelet counts in endotoxic mice and increased survival rate to 100%.     

ADRENOCORTICOTROPHIC HORMONE-INDUCED HYPERTENSION IN THE RAT: EFFECTS OF THE ENDOTHELIN ANTAGONIST BOSENTAN

1. The effects of the endothelin antagonist bosentan on adrenocorticotrophic hormone (ACTH)-induced hypertension were examined in the conscious male Sprague-Dawley rat. 2. In order to confirm endothelin antagonism, 18 rats were randomly divided into two groups: receiving either (i) endothelin-1 (0.125, 0.25, 0.5 and 1 nmol/kg, i.v.); or (ii) endothelin-1 at these doses following bosentan (100 mg/kg gavage) and mean arterial pressure recorded (study A). Subsequently, 40 male rats (320 +/- 5 g) were randomly divided into four groups (n = 10): (i) Sham (0.9% saline, s.c.) + 5% acacia gum gavage; (ii) ACTH (500 micrograms/kg per day, s.c.) + 5% acacia gum gavage; (iii) Sham injection + bosentan (100 mg/kg per day) gavage; or (iv) ACTH + bosentan. Six control days (C1-C6) were followed by 11 treatment days (T0-T10). Systolic blood pressure, water intake, urine volume, food intake and bodyweight were measured every second day (study B). 3. Bosentan significantly attenuated the endothelin-1-induced blood pressure rise at 0.125 nmol/kg (P < 0.05), but not at higher doses. 4. Bosentan at a dose which attenuated endothelin-1-induced blood pressure increase had no effect on either blood pressure or metabolic parameters in ACTH-treated rats. 5. These results suggest that endothelin does not play a major role in ACTH-induced hypertension.     

Anxiolytic effects of acute morphine can be modulated by nitric oxide systems

This study was performed to investigate whether nitric oxide (NO) precursor (L-arginine), NO donor (S-nitroso-N-acetylpenicillamine, SNAP) and NO synthase inhibitors [N(G)-nitro-L-arginine-methylester (L-NAME) and N(G)-nitro-L-arginine (L-NOARG)] modulate morphine-induced anxiolytic effects in the plus-maze. L-Arginine (100, 200 and 300 mg kg(-1), i.p.) and SNAP (4, 8 and 10 mg kg(-1), i.p.) reduced the anxiolytic effect of morphine (20 mg kg(-1), s.c.). L-NAME (10, 20 and 40 mg/kg, i.p.) and L-NOARG (10, 15 and 20 mg kg(-1), i.p.) enhanced the anxiolytic effects of morphine (20 mg kg(-1), s.c.). On the other hand, L-arginine and SNAP increased the morphine-induced locomotor activity. L-NAME decreased the morphine-induced locomotor activity, but L-NOARG did not modify the morphine-induced locomotor activity. Therefore, these results suggest that the anxiolytic effects of morphine can be modulated by NO systems.     

Impaired vascular sensitivity to nitric oxide in the coronary microvasculature after endotoxaemia

1. The effects of endotoxaemia on coronary vasodilator responses to bradykinin (BK), sodium nitroprusside (SNP) and nicardipine were investigated in the rat isolated heart perfused at constant flow ex vivo. 2. Dose-dependent reductions in coronary perfusion pressure reaching a maximum of 56+/-3 and 57+/-5 mmHg were observed for BK and SNP respectively. The BK response was biphasic, consisting of a rapid dilator response that was insensitive to N(G)nitro-L-arginine methyl ester (L-NAME, 0.1 mM) and a second slower component whose duration was attenuated by L-NAME. 3. Hearts obtained from rats treated with endotoxin (2.5 mg kg(-1), i.p.) for 2 or 6 h had increased basal coronary perfusion pressure and reduced vasodilator responses to BK or SNP. Dilator responses to nicardipine were not affected by endotoxin treatment. In vitro perfusion of hearts from endotoxin-treated rats with L-NAME (0.1 mM) restored SNP responses to control values. 4. Treatment with dexamethasone (1 mg kg(-1)), 1 h before endotoxin did not alter the endotoxin-induced impairment of dilator responses to BK or SNP. 5. These results show that coronary microvascular responses are altered following endotoxin exposure. Endotoxin results in increased coronary microvascular tone despite induction of NO synthase and inhibits the dilator response to BK and SNP, vasodilators that act via the release of NO. Responses to SNP in endotoxin-treated hearts were restored to control values in the presence of L-NAME suggesting that enhanced endogenous NO synthesis might saturate guanylate cyclase resulting in reduced response to NO donors. The reduced response to vasodilators and increased coronary resistance might be important in determining the response of the coronary circulation to systemic inflammation and infection.     

The role of nitric oxide and platelet-activating factor in the inhibition by endotoxin of pentagastrin-stimulated gastric acid secretion.

Administration of E. coli endotoxin (1 mg/kg i.v.) abolished the acid secretory response induced by a bolus injection of pentagastrin (100 micrograms/kg i.v.) in the continuously perfused stomach of the anaesthetized rat. Endotoxin administration did not modify mean systemic arterial blood pressure. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 5-20 mg/kg i.v.), but not dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg i.m.), substantially restored the secretory responses to pentagastrin. The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.v.), but not by its enantiomer D-arginine (100 mg/kg i.v.). L-NAME (10 mg/kg i.v.) increased blood pressure but this does not seem to be the mechanism by which endotoxin-induced acid inhibition was prevented, since similar systemic pressor responses induced by noradrenaline (15 micrograms/kg per min i.v.) had no such effect. The platelet-activating factor (PAF) receptor antagonist, WEB 2086 (2 mg/kg), induced a partial reversal of the inhibition by endotoxin of acid responses to pentagastrin. In endotoxin-treated rats, the combined administration of L-NAME (10 mg/kg) and WEB 2086 (2 mg/kg) completely restored the degree of H+ output induced by pentagastrin to levels similar to those of control, vehicle-treated animals. These findings suggest that endotoxin-induced acute inhibition of acid responses to pentagastrin involves NO synthesis and the release of PAF.     

The effects of nitric oxide on the acquisition and expression of nicotine-induced conditioned place preference in mice

In the present study, the possible role of nitric oxide on the conditioned place preference (CPP) induced by nicotine in mice was investigated. Intraperitoneal (i.p.) injections of nicotine (1 mg/kg) and the nitric oxide (NO) precursor, L-arginine (200 and 500 mg/kg), produced significant place preference. However, injection of mecamylamine (0.05 and 0.1 mg/kg; i.p.) or the NO synthase (NOS) inhibitor, L-Nitro-amino-methyl-ester, L-NAME (5-20 mg/kg; i.p.), had no effect. Ineffective doses of nicotine in combination with ineffective doses of L-arginine produced significant place preference. Administration of L-arginine (50, 100 and 150 mg/kg; i.p.) on the test day reduced the expression of nicotine-induced place preference. Nicotine injection (0.25, 0.5 and 0.75 mg/kg) on the test day reduced the expression of place preference induced by L-arginine, while both mecamylamine (0.05 and 0.1 mg/kg) and L-NAME (5, 10 and 20 mg/kg) inhibited the acquisition of place preference induced by nicotine (1 mg/kg) and L-arginine (200 mg/kg). Moreover, neither of the antagonists reduced the expression of nicotine- or L-arginine-induced place preference. It is suggested that nitric oxide may play an important role in nicotine-induced place preference.     

Airway function,oedema, cell infiltration and nitric oxide generation in conscious ozone-exposed guinea-pigs: effects of dexamethasone and rolipram

1. The effects of ozone inhalation (90 min, 2.15+/-0.05 p.p.m.) and their modification by dexamethasone (20 mg kg(-1)) or the phosphodiesterase-4 inhibitor, rolipram (1 mg kg(-1)), administered (i.p.) 24 and 0.5 h before and 24 h after ozone exposure were examined in conscious guinea-pigs. 2. Ozone caused an early-phase bronchoconstriction (EPB) as a fall in specific airways conductance (sG(aw)) measured by whole body plethysmography, followed at 5 h by a late-phase bronchoconstriction (LPB) and increased respiratory rate. Rolipram did not alter this profile but dexamethasone inhibited the EPB. 3. Airway hyperreactivity to inhaled histamine (1 mM, 20 s) occurred at 0.5, 2, 12, 24 and 48 h after ozone inhalation, the 2 h change being abolished by rolipram and dexamethasone. 4. Bronchoalveolar lavage fluid (BALF) macrophages, eosinophils and neutrophils were significantly (P<0.05) elevated at 12, 24 and 48 h after ozone exposure, the 48 h influx being significantly attenuated (P<0.05) by rolipram and dexamethasone. 5. BALF nitric oxide (NO) metabolites decreased 0.5 h after ozone exposure by 52%, recovered at 2 h and significantly increased at 12 (101%) and 24 h (127%). The elevated NO was unaffected by rolipram or dexamethasone. 6. Lung oedema, measured from wet/dry weight differences, was significant 12, 24 and 48 h after ozone exposure, the latter being significantly attenuated (P<0.05) by rolipram and dexamethasone. 7. Ozone exposure of guinea-pigs produced features common to COPD. Although rolipram and dexamethasone did not affect the airway function changes, they inhibited the inflammation, airway hyperreactivity and oedema.     

The role of nitric oxide in aloe-induced diarrhoea in the rat

The role of nitric oxide (NO) on aloe-induced diarrhoea was studied in the rat. Nine hours after oral administration, aloe produced diarrhoea at doses of 5 g kg(-1)(20% rats with diarrhoea) and 20 g kg(-1) (100% of rats with diarrhoea). Lower doses of aloe (0.1 and 1 g kg(-1) did not produce a diarrhoeal response. Pre-treatment (i.p.) of rats with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME 2.5-25 mg kg(-1) reduced the diarrhoea induced by aloe (20 g kg(-1) 9 h after its oral administration. L-NAME (25 mg kg(-1)) also reduced the increase in faecal water excretion produced by aloe (20 g kg(-1). L-arginine (1500 mg kg(-1), i.p.), administered to rats pre-treated with L-NAME (25 mg kg(-1), drastically reduced the effect of L-NAME on diarrhoea and increase in faecal water excretion induced by aloe (20 g kg(-1). Given alone, L-arginine did not modify aloe-induced diarrhoea. Basal Ca2+ -dependent NO synthase activity in the rat colon was dose-dependently inhibited by aloe (0.1-20 g kg(-1)) and by aloin (0.1-1 g kg(-1)), the active ingredient of aloe. These results suggest that endogenous NO modulates the diarrhoeal effect of aloe.     

Regional blood flow responses to acute ANG II infusion: effects of nitric oxide synthase inhibition.

We hypothesized that nitric oxide (NO) opposes regional vasoconstriction caused by acute angiotensin II (ANG II) infusion in conscious rats. Mean arterial pressure (MAP), blood flow, and vascular conductance (regional blood flow/ MAP; ml/min/100 g/mm Hg) were measured and/or calculated before and at 2 min of ANG II infusion (0.05 or 1 microg/kg/min, i.a.) in the absence and presence of NO synthase (NOS) inhibition [N(G)-nitro-L-arginine methyl ester (L-NAME), 0.25 or 1 mg/kg, i.a.]. ANG II reduced stomach and hindlimb conductance only after NOS inhibition. For example, whereas 0.05 microg/kg/min ANG II did not attenuate conductance in the stomach (i.e., 1.04+/-0.08 to 0.93+/-0.12 ml/min/100 g/mm Hg), this variable was reduced (i.e., 0.57+/-0.14 to 0.34-/+0.05 ml/min/100 g/mm Hg; p < 0.05) when ANG II was infused after 0.25 mg/kg L-NAME. In addition, whereas hindlimb conductance was similar before and after administering 1 microg/kg/min ANG II (i.e., 0.13+/-0.01 and 0.09+/-0.02, respectively), this variable was reduced (i.e., 0.07+/-0.01 and 0.02+/-0.00, respectively; p < 0.05) when ANG II was infused after 1 mg/kg L-NAME. These findings indicate that NO opposes ANG II-induced vasoconstriction in the stomach and hindlimb. In contrast, whereas both doses of ANG II decreased (p < 0.05) vascular conductance in the kidneys and small and large intestine regardless of whether NOS inhibition was present, absolute vascular conductance was lower (p < 0.05) after L-NAME. For example, 1 microg/kg ANG II reduced renal conductance from 3.34+/-0.31 to 1.22+/-0.14 (p < 0.05). After 1 mg/kg L-NAME, renal conductance decreased from 1.39+/-0.18 to 0.72+/-0.16 (p < 0.05) during ANG II administration. Therefore the constrictor effects of NOS inhibition and ANG II are additive in these circulations. Taken together, our results indicate that the ability of NO to oppose ANG II-induced constriction is not homogeneous among regional circulations.