Attenuation by prolonged nitric oxide synthase inhibition of the enhancement of fibrinolysis caused by environmental stress in the rat.

1. Nitric oxide (NO) suppresses platelet aggregation and plasminogen activator inhibitor (PAI) release from platelets, playing physiological and/or pathological roles in the haemostatic system. We investigated the effect of NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, on the disseminated intravascular coagulation (DIC)-like phenomena in rats under environmental stress, induced by prolonged fluctuation in air temperature, known as SART (specific alternation of rhythm in temperature) stress. 2. Exposure of rats to SART stress for 7 days caused mild DIC-like symptoms such as thrombocytopenia, hypofibrinogenemia, decreased factor VIII: coagulant activity and shortened euglobulin clot lysis time (ECLT). The enhanced fibrinolysis was accompanied by a marked decrease in the activity of plasma PAI. 3. L-NAME, but not its D-enantiomer, when administered orally at 0.3-10 mg kg-1, twice a day for 7-day exposure to stress, inhibited the stress-induced decrease in fibrinogen levels in a dose-dependent manner, whereas it failed to alter platelet count, factor VIII:coagulant activity and plasma protein levels in stressed rats. All these parameters in unstressed rats were resistant to L-NAME at 10 mg kg-1. 4. Repeated treatment with 10 mg kg-1 of L-NAME blocked the shortening of ECLT and the decrease in PAI activity following stress exposure, although it was without effect in unstressed rats. 5. The inhibitory effects of L-NAME at 10 mg kg-1 on the stress-induced alterations in fibrinogen levels and in ECLT were significantly reduced by coadministered L-arginine at 1000 mg kg-1. 6. These findings demonstrate that repeated administration of L-NAME attenuates the enhanced fibrinolysis, without aggravating thrombocytopenia, in SART-stressed rats. Endogenous NO appears to contribute to the stress-induced development of fibrinolysis by suppressing, plasma PAI activity, most probably as a result of inhibition of the PAI release from platelets.     

Evidence that endogenous nitric oxide modulates plasma fibrinogen levels in the rat.

This study investigated the effect of prolonged inhibition of nitric oxide (NO) synthase on plasma fibrinogen levels and platelet count in the rats. NG-nitro-L-arginine methyl ester (L-NAME), when administered orally twice a day, at 10-100 mg kg-1, for 7 consecutive days, 14 times in all, significantly elevated fibrinogen levels and systolic blood pressure in a dose-dependent manner. The same dose range of L-NAME failed to alter platelet count and plasma protein concentrations. The increase in fibrinogen levels produced by chronic treatment with L-NAME at 30 mg kg-1 was reversed by L-arginine at 500-1500 mg kg-1 in a dose-dependent manner. These findings suggest that endogenous NO tonically acts to reduce plasma fibrinogen levels in rats under physiological conditions.     

Interactions of constitutive nitric oxide with PAF and thromboxane on rat intestinal vascular integrity in acute endotoxaemia.

1. The involvement of endogenous platelet activating factor (PAF) and thromboxane A2 in the acute microvascular damage in the ileum and colon induced by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) following endotoxin administration was investigated in the rat over a 1 h period. 2. Administration of L-NAME (1-10 mg kg-1, s.c.) concurrently with E. coli lipopolysaccharide (LPS; 3 mg kg-1, i.v.) dose-dependently increased vascular permeability in the ileum and colon, as determined by the leakage of radiolabelled albumin, and caused macroscopic mucosal damage in the ileum determined 1 h later. Neither LPS administration nor L-NAME (5 mg kg-1) alone affected resting vascular permeability. 3. Infusion of phenylephrine (10 micrograms kg-1 min-1, i.v. for 1 h) caused an elevation in blood pressure similar to that found following L-NAME administration (5 mg kg-1, i.v. or s.c.), but did not increase intestinal vascular permeability, when administered with LPS (3 mg kg-1, i.v.). 4. The increased vascular permeability in the ileum and colon and macroscopic damage in the ileum, induced by L-NAME (5 mg kg-1, s.c.) and LPS (3 mg kg-1, i.v.) was dose-dependently inhibited following s.c. pretreatment (15 min before challenge) with the thromboxane synthase inhibitors, OKY 1581 (5-25 mg kg-1) or 1-benzyl-imidazole (1-50 mg kg-1), or with the thromboxane receptor antagonist, BM 13177 (0.2-2 mg kg-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock.

1. The role of an enhanced formation of nitric oxide (NO) and the relative importance of the constitutive and inducible NO synthase (NOS) for the development of immediate (within 60 min) and delayed (at 180 min) vascular hyporeactivity to noradrenaline was investigated in a model of circulatory shock induced by endotoxin (lipopolysaccharide; LPS) in the rat. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate. In addition, the calcium-dependent and calcium-independent NOS activity was measured ex vivo by the conversion of [3H]-arginine to [3H]-citrulline in homogenates from several organs obtained from vehicle- and LPS-treated rats. 3. E. coli LPS (10 mg kg-1, i.v. bolus) caused a rapid (within 5 min) and sustained fall in MAP. At 30 and 60 min after LPS, pressor responses to noradrenaline (0.3, 1 or 3 micrograms kg-1, i.v.) were significantly reduced. The pressor responses were restored by NG-nitro-L-arginine methyl ester (L-NAME, 1 mg kg-1, i.v. at 60 min), a potent inhibitor of NO synthesis. In contrast, L-NAME did not potentiate the noradrenaline-induced pressor responses in control animals. 4. Dexamethasone (3 mg kg-1, i.v., 60 min prior to LPS), a potent inhibitor of the induction of NOS, did not alter initial MAP or pressor responses to noradrenaline in control rats, but significantly attenuated the LPS-induced fall in MAP at 15 to 60 min after LPS. Dexamethasone did not influence the development of the LPS-induced immediate (within 60 min) hyporeactivity to noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective effect of S-nitroso-N-acetyl-penicillamine in endotoxin-induced acute intestinal damage in the rat

Macroscopic jejunal damage and plasma leakage induced within 15 min by E. coli lipopolysaccharide (LPS 50 mg kg-1 i.v.) in the rat was enhanced by the inhibitor of nitric oxide (NO) formation, NG-monomethyl-L-arginine (L-NMMA 50 mg kg-1 i.v.). The nitro-vasodilator, S-nitroso-N-acetyl-penicillamine (SNAP; 10 micrograms kg-1 min-1 i.v.), which generates NO, attenuated both LPS-induced intestinal damage and the enhancement of such damage and plasma leakage produced by L-NMMA. Endogenous NO may thus have a protective role in the intestinal vasculature that can be mimicked by generators of NO.     

Effect of L-NAME on the synthesis of plasma fibrinogen in mice

The effect of nitric oxide (NO) on plasma fibrinogen was investigated by treating mice with oral N(G)-nitro-L-arginine-methyl ester (L-NAME), a non-selective NO synthase (NOS) inhibitor. Treatment with L-NAME significantly increased the concentration of plasma fibrinogen and hepatic expression of the alpha- and beta-chains of fibrinogen, whereas plasma NO(2)(-) level and hepatic expression of endothelial NOS (eNOS) mRNA were significantly decreased. These results suggest that L-NAME treatment can increase plasma fibrinogen levels via an increase in expression of fibrinogen mRNA in the liver, and NO may be involved in plasma fibrinogen increase.     

Polygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages

Polygonum cuspidatum water extract (PCWE) was shown to be a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). PCWE was compared to baicalin isolated from Scutellaria baicalensis Georgi and berberine of Coptidis rhizoma and Phellodendri cortex, for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. Both PCWE and the compounds inhibited LPS-induced NO production in a concentration-dependent manner without a cytotoxicity. The decrease in NO production was in parallel with the inhibition of LPS-induced iNOS gene expression by PCWE and the compounds. In contrast, iNOS enzyme activity was not inhibited by PCWE and two agents. In addition, only PCWE inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity, while baicalin or berberine did not. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS protein expression, which was inhibited by these PCWE and two agents, although LPS-induced COX-2 protein expression was not affected by NLA and L-NAME. PCWE inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cell, however, baicalin or berberine did not. From the results, it was concluded that co-treatment with NOS inhibitors and PCWE effectively blocks acute production of NO and inhibits expression of iNOS and COX-2 genes.     

PF-04886847 (an inhibitor of plasma kallikrein) attenuates inflammatory mediators and activation of blood coagulation in rat model of lipopolysaccharide (LPS)-induced sepsis

The plasma kallikrein-mediated proteolysis regulates both thrombosis and inflammation. Previous study has shown that PF-04886847 is a potent and competitive inhibitor of kallikrein, suggesting that it might be useful for the treatment of kallikrein-kinin mediated inflammatory and thrombotic disorders. In the rat model of lipopolysaccharide (LPS) -induced sepsis used in this study, pretreatment of rats with PF-04886847 (1 mg/kg) prior to LPS (10 mg/kg) prevented endotoxin-induced increase in granulocyte count in the systemic circulation. PF-04886847 significantly reduced the elevated plasma 6-keto PGF1α levels in LPS treated rats, suggesting that PF-04886847 could be useful in preventing hypotensive shock during sepsis. PF-04886847 did not inhibit LPS-induced increase in plasma TNF-α level. Pretreatment of rats with PF-04886847 prior to LPS did not attenuate endotoxin-induced decrease in platelet count and plasma fibrinogen levels as well as increase in plasma D-dimer levels. PF-04886847 did not protect the animals against LPS-mediated acute hepatic and renal injury and disseminated intravascular coagulation (DIC). Since prekallikrein (the zymogen form of plasma kallikrein) deficient patients have prolonged activated partial thromboplastin time (aPTT) without having any bleeding disorder, the anti-thrombotic property and mechanism of action of PF-04886847 was assessed. In a rabbit balloon injury model designed to mimic clinical conditions of acute thrombotic events, PF-04886847 reduced thrombus mass dose-dependently. PF-04886847 (1 mg/kg) prolonged both aPTT and prothrombin time (PT) in a dose-dependent manner. Although the findings of this study indicate that PF-04886847 possesses limited anti-thrombotic and anti-inflammatory effects, PF-04886847 may have therapeutic potential in other kallikrein-kinin mediated diseases.     

Changes in nitric oxide release in vivo in response to vasoactive substances.

1. Changes in the release of nitric oxide (NO) in vivo were studied in rats following the administration of endothelium-dependent and -independent vasodilators as well as the NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). NO production was assessed by measuring variations of nitrate in plasma by capillary ion analysis. 2. Intravenous administration of the endothelium-dependent vasodilators, bradykinin (2 and 10 micrograms kg-1 min-1) or substance P (0.3-3 micrograms kg-1 min-1) caused a transient dose-dependent hypotension followed by an increase in plasma nitrate concentration (maximal increments: 33 +/- 5% and 38 +/- 6%, for bradykinin and substance P, respectively). Prior administration of L-NAME (10 mg kg-1 min-1) inhibited the hypotension and increase in plasma nitrate caused by these substances. Intravenous administration of sodium nitrate (200 micrograms kg-1) also produced a transitory elevation in plasma nitrate which was similar in magnitude as that caused by the vasodilators. A rapid and transitory increment in plasma nitrate was observed after i.v. administration of authentic NO (400 micrograms kg-1). 3. Rats receiving the endothelium-dependent vasodilators, prostacyclin (0.6 micrograms kg-1 min-1) or adenosine (3 mg kg-1 min-1) intravenously showed a drop in blood pressure paralleled by a decrease in plasma nitrate (maximal decreases: 34 +/- 5% and 24 +/- 4%, for prostacyclin and adenosine, respectively). A similar effect on the plasmatic concentration of nitrate was observed when L-NAME (10 mg kg-1 min-1, i.v.) was administered to the animals. 4. This study demonstrates that (i) changes in plasma nitrate can be detected in vivo after stimulation or inhibition of NO synthase, (ii) an increased production of NO, measured as plasma nitrate, is related to the hypotension caused by bradykinin and substance P and (iii) a diminished concentration of plasmatic nitrate is associated to the hypotension induced by adenosine or prostacyclin (endothelium-independent vasodilators), suggesting that the L-arginine: NO pathway is capable of rapid down-regulation in response to a fall in blood pressure.     

Antagonism by salvianolic acid B of lipopolysaccharide‐induced disseminated intravascular coagulation in rabbits

The aim of the present study was to investigate the effects of salvianolic acid B on lipopolysaccharide (LPS)‐induced disseminated intravascular coagulation (DIC) in rabbits. Continuous infusion of LPS was used to induce a DIC model in rabbits. Treatment with salvianolic acid B (1, 3 or 6 mg/kg) was started simultaneously with LPS infusion (0.5 mg/kg LPS in 60 mL saline; 10 mL/h over a period of 6 h) through the contralateral marginal ear vein. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were determined, as were plasma levels of fibrin–fibrinogen degradation products (FDP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), protein C activity, antithrombin III (ATIII) and tumour necrosis factor (TNF)‐α concentration. The gradual impairment of haemostatic parameters was induced by continuous infusion of LPS. There were marked increases in APTT, PT, BUN, ALT and plasma TNF‐α and marked decreases in the platelet count, fibrinogen, FDP, protein C and ATIII. The intravenous administration of 1, 3 or 6 mg/kg salvianolic acid B attenuated the increases in APTT, PT, BUN, ALT and plasma TNF‐α and the decreases in fibrinogen, platelet, FDP, protein C and ATIII induced by LPS infusion. These observations indicate that salvianolic acid B has an effect against LPS‐induced DIC in rabbits.     

The role of nitric oxide as an endogenous regulator of platelet and neutrophil activation within the pulmonary circulation of the rabbit.

1. Intravenous (i.v.) administration of adenosine diphosphate (ADP), platelet activating factor (PAF) and thrombin induced a dose-related accumulation of 111indium-labelled platelets within the thoracic region of anaesthetized rabbits. 2. I.v. administration of the inhibitor of NO biosynthesis, L-NG-nitro arginine methyl ester (L-NAME; 10 mg kg-1) significantly potentiated the peak platelet accumulation induced by ADP, PAF and thrombin. Additionally L-NAME prolonged the disaggregation of platelets in comparison to D-NAME (10 mg kg-1). Such changes were reversible by the administration of L-arginine (900 mg kg-1). 3. I.v. administration of PAF induced a small accumulation of 111indium-labelled neutrophils within the pulmonary circulation which could be greatly potentiated by pretreatment of the animals with L-NAME. In contrast, thrombin administration did not cause significant accumulation of 11indium-labelled erythrocytes in the pulmonary circulation of anaesthetized rabbits. 4. Intracarotid (i.c.) administration of thrombin induced a marked accumulation of radiolabelled platelets within the cranial vasculature which was not potentiated by the prior administration of L-NAME (at either 10 mg kg-1 or 100 mg kg-1). 5. These results suggest that endogenous NO may regulate platelet and polymorphonuclear leukocyte activation within the pulmonary but not the cerebral circulation of rabbits.     

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.     

Effects of UR-12633, a new antagonist of platelet-activating factor, in rodent models of endotoxic shock.

1. The effects of the selective and potent novel platelet-activating factor (PAF) antagonist, UR-12633 (1-(3,3-diphenylpropionyl)-4-(3-pyridylcyanomethyl)piperidin e) on several markers of endotoxic shock syndrome were evaluated in rats and mice. 2. UR-12633, administered 60 min after E. coli lipopolysaccharide (LPS), reversed the LPS-induced sustained hypotension in rats at doses of 0.01 to 1 mg kg-1, i.v. The reference compound WEB-2086 (1 mg kg-1) also reversed the LPS-induced hypotension. UR-12633 (1 mg kg-1), administered 10 min before LPS, almost fully inhibited sustained hypotension. The immediate hypotension (within 1 min) caused by LPS was not prevented by either UR-12633 or WEB-2086. 3. Pretreatment with 10 mg kg-1, i.v. of either UR-12633 or WEB-2086 inhibited the increase in disseminated intravascular coagulation markers, such as activated partial thromboplastin time (55 and 74% inhibition, respectively), and prothrombin time (22 and 72% inhibition) and prevented the decrease in plasma fibrinogen content (100 and 29% inhibition). 4. Increases in acid phosphatase (ACP) plasma activity, a marker of lysosomal activation, and in lactate dehydrogenase (LDH), a marker of tissue damage, were inhibited by pretreatment with 10 mg kg-1, i.v. of either UR-12633 or WEB-2086 (100% and 69% inhibition, ACP; 62 and 48% inhibition, LDH). Hyperglycaemia (71 and 46%) and hyperlactacidaemia (92 and 56%) were also inhibited. 5. UR-12633, but not WEB-2086, inhibited the LPS-induced increase in vascular permeability in rats, as shown by prevention of haemoconcentration and, to a lesser degree, the increase in Evans blue dye extravasation. 6. In a series of nine reference compounds and UR-12633, we found a high correlation (P < 0.001) between PAF antagonist activity, measured as the inhibition of PAF-induced rabbit platelet aggregation or PAF-induced mortality in mice and the inhibition of LPS-induced mortality. 7. In spite of the multifactorial nature of endotoxic shock, in which many mediators may be involved, the new potent PAF antagonist, UR-12633, proved effective in protecting against changes in most shock markers. These data strongly suggest a key role for PAF in the pathogenesis of endotoxic shock in rodents.     

Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide

1. Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS-induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS-induced plasma leakage using iNOS deficient mice. 2. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100 - 400 microg site(-1)) produced a dose-related increase in dye leakage in both iNOS deficient and wild-type mice with about 40% less dye leakage in iNOS deficient mice. 3. Indomethacin (5 mg kg(-1)), N-[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide (NS-398) (1 mg kg(-1)), diphenhydramine (10 mg kg(-1)) and anti-TNF-alpha antibody (dilution 1 : 400, 10 ml kg(-1)) inhibited the LPS-induced dye leakage in both iNOS deficient and wild-type mice, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg kg(-1)) or aminoguanidine (10 mg kg(-1)) inhibited that in wild-type but not in iNOS deficient mice. 4. Pretreatment with LPS (0.15 mg kg(-1) i.p.) 4 h before decreased the LPS-induced dye leakage in wild-type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild-type but not in iNOS deficient mice. 5. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF-alpha. The tolerance against LPS-induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.     

Time-dependent enhancement or inhibition of endotoxin-induced vascular injury in rat intestine by nitric oxide synthase inhibitors.

1. The effects of the nitric oxide (NO) synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), on the vascular damage induced by the endotoxin, E. coli lipopolysaccharide (LPS), in the ileum and colon were investigated in the conscious rat over a 5 h period. 2. Administration of LPS (3 mg kg-1, i.v.) increased ileal and colonic vascular injury after a lag period of 2 h, as determined by the leakage of radiolabelled albumin. 3. Administration of L-NAME (1-5 mg kg-1, s.c.) concurrently with LPS, produced a dose-dependent increase in vascular albumin leakage in the intestinal tissues, when determined over a 5 h period. Vascular albumin leakage with LPS and L-NAME (5 mg kg-1) was substantially increased after 1 h, reached maximal levels 3 h after administration, and then slowly declined. 4. L-NMMA (50 mg kg-1, s.c.), likewise elevated intestinal albumin leakage when administered concurrently with LPS, but this reached maximal levels after 1 h and rapidly declined over the subsequent 2 h. 5. In control rats, in the absence of LPS challenge, neither L-NAME (5 mg kg-1, s.c.) nor L-NMMA (50 mg kg-1, s.c.) increased intestinal vascular leakage of albumin over a 5 h period. 6. By contrast, when L-NAME (1-5 mg kg-1, s.c.) or L-NMMA (12.5-50 mg kg-1, s.c.) was injected 3 h after LPS, a dose-dependent reduction in the LPS-provoked vascular albumin leakage was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide as a mediator of the laxative action of magnesium sulphate.

1. Magnesium sulphate was studied for its effects on diarrhoea, fluid secretion, gastrointestinal transit and nitric oxide (NO) synthase activity in rats. 2. At a dose of 2 g kg-1 orally magnesium sulphate produced diarrhoea that was delayed in onset and intensity in a dose-related manner by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). This was prevented by the NO precursor, L-arginine and the NO donating compound, isosorbide-5-mononitrate (IMN). 3. Nitric oxide synthase activity was stimulated in gut tissue from rats given magnesium sulphate and this was inhibited by L-NAME. Dexamethasone (1 mg kg-1, i.p.), an inhibitor of inducible NO synthase, had no effect on magnesium sulphate-induced diarrhoea. 4. Magnesium sulphate stimulated fluid and electrolyte accumulation in the intestinal lumen; these effects were prevented by L-NAME but not D-NAME. 5. Gastrointestinal transit of a non-absorbable marker (charcoal suspension) was increased by oral magnesium sulphate from a mean value of 54.1% to 72.9% (P < 0.01), and this was prevented by pretreatment with L-NAME. 6. The results demonstrate that oral magnesium sulphate produces diarrhoea in rats by increasing the accumulation of fluid in the intestinal lumen and enhancing flow from the proximal to distal intestine. The mechanism involves release of NO, probably through stimulation of the constitutive form of NO synthase. Whether or not the effects of magnesium sulphate are due to an osmotic action or an intrinsic effect of the magnesium or sulphate ions cannot be determined from these experiments.     

Attenuation by nitrosothiol NO donors of acute intestinal microvascular dysfunction in the rat.

1. The effects of the nitric oxide (NO) donors, S-nitroso-glutathione (SNOG) and S-nitroso-N-acetyl-penicillamine (SNAP), on the acute intestinal microvascular dysfunction induced by NG-nitro-L-arginine methyl ester (L-NAME) in combination with low doses of endotoxin were investigated in the anaesthetized rat. 2. Administration of L-NAME (5 mg kg-1, s.c.) concurrently with E. coli lipopolysaccharide (LPS, 3 mg kg-1, i.v.) provoked the leakage of radiolabelled albumin in the ileum and colon, as a measure of microvascular damage, determined 1 h after challenge. 3. Intravenous infusion of SNOG or SNAP (1-10 micrograms kg-1 min-1) dose-dependently attenuated the microvascular leakage induced by L-NAME and LPS. 4. Infusion of the lowest doses of SNOG or SNAP (1 microgram kg-1 min-1, i.v.) that significantly reduced the albumin leakage, did not affect the increase in blood pressure in response to L-NAME in LPS-treated rats. Higher doses of SNOG or SNAP (5-10 micrograms kg-1 min-1, i.v.) dose-dependently reduced this increase in blood pressure. 5. In control studies, intravenous infusion of glutathione (10 micrograms kg-1 min-1) or N-acetyl-penicillamine (10 micrograms kg-1 min-1) had no effect on microvascular leakage in the ileum and colon induced by LPS and L-NAME. 6. Pretreatment with rabbit anti-rat neutrophil serum (0.4 ml kg-1, i.p., 4 h before challenge), which reduced the neutrophil count in peripheral arterial blood, also inhibited the microvascular leakage in the ileum and colon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of nitric oxide modulators on pylorus-ligation-induced ulcers in the rat.

In the present investigation, the effect of nitric oxide (NO) modulators on pylorus-ligation-induced gastric ulcers in rats was studied. Sodium nitroprusside (SNP, 1 mg kg-1), a NO donor, l-arginine (l-Arg, 300 mg kg-1), the NO precursor, nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase (NOS) inhibitor and lipopolysaccharide (LPS, 3 mg kg-1), a NOS inducer have been administered prior to pylorus ligation. The effects of these interventions on the gastric mucosal nitrite content, the incidence of ulcers, the ulcer index, the volume of gastric secretions and the free and total acidity 4 h after pylorus ligation were investigated. SNP, l-Arg and LPS pretreatment increased the mucosal nitrite contents and protected the animals against pyloric-ligation-induced increase in acidity and ulcer index. However, inhibition of NOS activity by l-NAME (10 mg kg-1) decreased the nitrite content and augmented the ulcer-induced increase in the gastric acid contents. Coadministration of l-Arg with l-NAME prevented the l-NAME-induced changes. Interventions which increased the mucosal nitrite content were found to be protective against ulcers. However, the NOS inhibitor l-NAME decreased mucosal nitrite levels and was ulcerogenic. Results obtained thus indicate the protective effect of NO on the pyloric-ligation-induced ulcers in the rat.     

Induction of nitric oxide synthase and microvascular injury in the rat jejunum provoked by indomethacin.

1. The role of nitric oxide (NO) formed by the inducible isoform of NO synthase (NOS) in the generation of indomethacin-induced intestinal microvascular leakage was investigated in the rat. 2. Indomethacin (10 mg kg-1, s.c.) provoked an elevation of vascular leakage of radiolabelled human serum albumin in the jejunum over 48 h, commencing 18 h after its administration. This was associated with the induction of a calcium-independent NOS, as assessed by the conversion of radiolabelled L-arginine to citrulline. 3. Pretreatment with the glucocorticoid, dexamethasone (1 mg kg-1 day-1, s.c.) inhibited the induction of NOS and reduced jejunal microvascular leakage, determined 24 and 48 h after indomethacin. 4. Administration of the broad-spectrum antibiotic, ampicillin (800 mg kg-1 day-1, p.o.) likewise inhibited both the induction of NOS and the plasma leakage observed 24 and 48 h after indomethacin. 5. Ampicillin pretreatment did not, however, inhibit the induction of NOS, determined 5 h following endotoxin (3 mg kg-1 i.v.) challenge. Furthermore, incubation with ampicillin (1 mM, 10 min) did not inhibit the activity of the calcium-independent isoform in vitro. 6. Administration of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 2-10 mg kg-1, s.c.), at the time of the detectable expression of the inducible NOS (18 h after indomethacin), dose-dependently attenuated the plasma leakage, determined 6 later. This effect was reversed by pretreatment with L-arginine (300 mg kg-1, s.c.) 15 min before L-NAME. 7. These findings suggest that induction of a calcium-independent NOS following indomethacin administration involves gut bacteria and leads to microvascular injury in the rat jejunum.