Selective Inducible Nitric Oxide Synthase Research Articles

In Vivo Nitric Oxide Synthase Inhibitors Can Be Deprived of ThisActivity: Unexpected Influence of the Tetrachloroplatinate(II) Counteranion.Crystal Structures of Bis(S‐Methyl‐Isothiouronium)‐N,N′‐Bis(3‐Guanidinopropyl)Piperazinium and HexamidiniumTetrachloroplatinates(II) Salts

The synthesis and crystal structures of bis(S-methylisothiouronium) (MSTUH)+, N,N′-bis((3- guanidinopropyl)piperazinium (PipeC3GuaH4)4+ and hexamidinium (HexaH2)2+ tetrachloro platinate(ll) salts ( called hereafter PtMSTU, PtPipeC3Gua and PtHexa respectively ) were investigated. These compounds contain the “amidine” function ( - C(=NH)NH2 ) in which the H atoms supplied by the acid have become attached to the imino group of each terminal amidino function. Moreover, in PtPipeC3Gua, the nitrogen atoms of the chair-piperazine moiety are also protonated. The influence of tetrachloroplatinate(ll) counteranion ( versus sulfate, nitrate and diisethionate ) in the in vivo nitrite inhibition by the (MSTUH)+, (PipeC3GuaH4)4+ and (HexaH2)2+ cations was investigated. The three tetrachloroplatinate(ll) salts, unexpectedly, do not inhibit significantly the in vivo nitrite production in comparison with the other salts (sulfate, nitrate and diisethionate and their corresponding previous countercations) which exhibit NO synthase inhibition, especially bis(S-methylisothiouronium) sulfate, a selective and potent inducible NO synthase (iNOS) inhibitor commonly used as standard.

A blinded randomised, controlled trial comparing dopamine, noradrenaline, S-methylisothiourea sulphate and volume in a porcine model of endotoxin shock

Objective: To investigate the association between levels of nitric oxide metabolites and the cardiovascular response to endotoxin shock; and to compare the effects of dopamine, noradrenaline and S-methylisothiourea sulphate (SMT), a relatively selective inducible nitric oxide synthase inhibitor, on haemodynamics, oxygen transport, markers of global and regional perfusion and survival in an immature pig model of endotoxin shock. Design: Randomised, blinded laboratory study. Setting: Animal research laboratory in a university hospital. Subjects: Three- to four-week-old pigs. There were eight animals in each of four groups. Interventions: The animals were anaesthetised and ventilated. Peripheral arterial, pulmonary artery and central venous cannulae and a gastric tonometer were inserted. Endotoxin shock was induced by a continuous infusion of E. coli lipopolysaccharide. Animals were randomly allocated to one of four groups: i) dopamine at 5-20 μg/kg/min, ii) noradrenaline at 0.1-0.5 μg/kg/min, iii) SMT at 20-80 μg/kg/min, or iv) placebo, to which the investigators were blinded. The study drug was commenced 1 hour after initiation of endotoxaemia, and then infused continuously for 6 hours. Volume resuscitation was given to all groups throughout the study to maintain a pulmonary artery wedge pressure at 2-4 mm Hg above pre-endotoxin baseline levels. Measurements: At baseline after cannulation, 1 hour after the initiation of endotoxaemia, and every 90 minutes after the commencement of the study drug infusion, the following data were recorded: heart rate, systemic blood pressure, mean pulmonary artery pressure (PAP) and pulmonary artery wedge pressure (PAWP), central venous pressure (CVP), gastric intramucosal pH (pHi), base deficit, blood lactate, mixed venous oxygen saturation (SvO2), cardiac index, systemic and pulmonary vascular resistance indices (SVRI and PVRI) and oxygen delivery (DO2). Blood was collected for assay of combined serum nitrate and nitrite (nitrogen oxides). Endpoints: The major endpoints were: the relationship between background nitric oxide (NO) activity and initial haemodynamic response to endotoxin; and the relative effect of the study drugs over time on: DO2, SVR, PVR, blood lactate, pHi, SvO2 and base deficit. Results: There was a significant, albeit weak, relationship between baseline serum nitrogen oxide concentrations and the initial cardiovascular response to endotoxin: levels of serum nitrogen oxides were positively associated with change in cardiac index; and negatively associated with the change in SVRI and PVRI. SMT produced greater increases in SVRI than the other three treatment arms, but caused a decrease in DO2. Dopamine increased DO2 in the early stages, but not later in the course of the study. SMT produced a greater increase in PVR than dopamine. Conclusions: Endogenous NO activity may be one determinant of the cardiovascular response to infection. Use of SMT in endotoxin shock resulted in vasoconstriction and reduced DO2, compared with catecholamines or volume alone.

A blinded randomised, controlled trial comparing dopamine, noradrenaline, S-methylisothiourea sulphate and volume in a porcine model of endotoxin shock

Objective: To investigate the association between levels of nitric oxide metabolites and the cardiovascular response to endotoxin shock; and to compare the effects of dopamine, noradrenaline and S-methylisothiourea sulphate (SMT), a relatively selective inducible nitric oxide synthase inhibitor, on haemodynamics, oxygen transport, markers of global and regional perfusion and survival in an immature pig model of endotoxin shock. Design: Randomised, blinded laboratory study. Setting: Animal research laboratory in a university hospital. Subjects: Three- to four-week-old pigs. There were eight animals in each of four groups. Interventions: The animals were anaesthetised and ventilated. Peripheral arterial, pulmonary artery and central venous cannulae and a gastric tonometer were inserted. Endotoxin shock was induced by a continuous infusion of E. coli lipopolysaccharide. Animals were randomly allocated to one of four groups: i) dopamine at 5-20 μg/kg/min, ii) noradrenaline at 0.1-0.5 μg/kg/min, iii) SMT at 20-80 μg/kg/min, or iv) placebo, to which the investigators were blinded. The study drug was commenced 1 hour after initiation of endotoxaemia, and then infused continuously for 6 hours. Volume resuscitation was given to all groups throughout the study to maintain a pulmonary artery wedge pressure at 2-4 mm Hg above pre-endotoxin baseline levels. Measurements: At baseline after cannulation, 1 hour after the initiation of endotoxaemia, and every 90 minutes after the commencement of the study drug infusion, the following data were recorded: heart rate, systemic blood pressure, mean pulmonary artery pressure (PAP) and pulmonary artery wedge pressure (PAWP), central venous pressure (CVP), gastric intramucosal pH (pHi), base deficit, blood lactate, mixed venous oxygen saturation (SvO2), cardiac index, systemic and pulmonary vascular resistance indices (SVRI and PVRI) and oxygen delivery (DO2). Blood was collected for assay of combined serum nitrate and nitrite (nitrogen oxides). Endpoints: The major endpoints were: the relationship between background nitric oxide (NO) activity and initial haemodynamic response to endotoxin; and the relative effect of the study drugs over time on: DO2, SVR, PVR, blood lactate, pHi, SvO2 and base deficit. Results: There was a significant, albeit weak, relationship between baseline serum nitrogen oxide concentrations and the initial cardiovascular response to endotoxin: levels of serum nitrogen oxides were positively associated with change in cardiac index; and negatively associated with the change in SVRI and PVRI. SMT produced greater increases in SVRI than the other three treatment arms, but caused a decrease in DO2. Dopamine increased DO2 in the early stages, but not later in the course of the study. SMT produced a greater increase in PVR than dopamine. Conclusions: Endogenous NO activity may be one determinant of the cardiovascular response to infection. Use of SMT in endotoxin shock resulted in vasoconstriction and reduced DO2, compared with catecholamines or volume alone.

ROLE OF NITRIC OXIDE IN PATHOGENESIS OF SEROTONINE-INDUCED GASTRIC LESIONS IN RATS

We investigated the role of nitric oxide (NO) in the development of gastric mucosal lesions induced by serotonine (5-HT) in rats. Repeated subcutaneous administration of 5-HT (20 mg kg−1) produced damage in the stomach with severe edema in the submucosa. Gastric lesions induced by 5-HT were prevented by simultaneous administration of aminoguanidine, a selective inducible NO synthase (iNOS) inhibitor, as well as by methysergide, a 5-HT antagonist. In addition, the lesions were inhibited by pretreatment with the antioxidative drugs, such as allopurinol (a xanthine oxidase inhibitor) and hydroxyurea (a neutrophil reducing agent). Following 5-HT treatment, the Ca2+-independent NOS activity in the gastric mucosa was significantly increased within 6 h and remained elevated for 2 days thereafter. The serum NOx levels increased 12 h after the administration of 5-HT, reaching a peak 24 h later. Gastric mucosal thiobarbituric acid (TBA) reactants and myeloperoxidase (MPO) activity were also significantly increased after 2 days treatment with 5-HT. Our results suggest that: (1) the repeated administration of 5-HT induced inflammatory gastric lesions in the rat stomach; (2) iNOS is upreguated during 5-HT treatment, and NO produced by iNOS contributes to development of gastric lesions in response to 5-HT, in addition to the oxyradical formation; and (3) the deleterious role of NO in this model may be accounted for by a cytotoxic action of peroxynitrite that is formed in the presence of NO and superoxide radicals.

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin.

We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5-2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by N(G)-nitro-L-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5-20 mg/kg, i.v.), but not by an inactive enantiomer, N(G)-nitro-D-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1-100 microg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE2 (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 microg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 microg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE2, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin.

Actions of isoform-selective and non-selective nitric oxide synthase inhibitors on endotoxin-induced vascular leakage in rat colon

The effects of the nitric oxide (NO) synthase inhibitor, N-(3-(aminomethyl)benzyl)-acetamidine (1400W) which is selective for the inducible isoform of NO synthase, on rat colonic microvascular injury provoked by Escherichia coli endotoxin (3 mg/kg i.v.) has been compared to those of aminoguanidine (25–50 mg/kg, s.c.), N G-iminoethyl- l-ornithine ( l-NIO, 15–30 mg/kg, s.c.) and N G-nitro- l-arginine methyl ester ( l-NAME, 2–5 mg/kg, s.c.). Administration of aminoguanidine, l-NIO or l-NAME concurrently with endotoxin provoked microvascular albumin leakage 1 h later, presumably by inhibiting constitutive NO synthase, whereas 1400W (0.1–10 mg/kg, s.c.) had no such effect. Administration of all these agents during the expression of inducible NO synthase (i.e. 3 h after endotoxin challenge) attenuated the subsequent endotoxin-provoked albumin leakage 1 h later. Moreover, concurrent administration of 1400W (0.2–5 mg/kg, s.c.; doses that did not affect systemic arterial blood pressure) with endotoxin suppressed the subsequent rise in albumin leakage after 5 h. These findings indicate that 1400W is a potent inhibitor of colonic microvascular injury associated with induction of NO synthase in vivo. 1400W will thus be useful to investigate in vivo the therapeutic potential of a selective inducible NO synthase inhibitor in inflammation.

Aminoguanidine corrects hyperdynamic circulation without ameliorating portal hypertension and portal hypertensive gastropathy in anesthetized portal hypertensive rats

Background/Aims: Portal hypertension and hyperdynamic circulation (i.e. generalized vasolidation and increased cardiac output and regional organ blood flows) may play an important role in the development of portal hypertensive gastropathy. This study investigated the effect of chronic administration of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to portal hypertensive rats on hemodynamics and the development of portal hypertensive gastropathy. Methods: Partial portal vein-ligated or sham-operated rats were randomly assigned to receive either placebo (distilled water) or aminoguanidine (∼100 mg/kg per day subcutaneously) for 2 days prior to and 14 days. Hemodynamic studies with a thermodilution technique and gastric morphometric analysis were performed at 14 days after the operation. Results: In rats given placebo, portal vein-ligated rats had a significantly lower mean arterial pressure and systemic vascular resistance associated with a significantly higher cardiac index and portal pressure than sham-operated rats ( p<0.05). In portal vein-ligated rats aminoguanidine induced a significant increase in mean arterial pressure and systemic vascular resistance accompanied by a significant decrease in cardiac index ( p<0.05) without changes in portal pressure ( p<0.05). Despite persistence of portal hypertension, the aminoguanidine-treated portal vein-ligated rats had similar mean arterial pressure, cardiac index, and systemic vascular resistance as seen in placebo-treated sham-operated rats. The mean cross-sectional area of gastric mucosal vessels was significantly higher in placebo-treated portal vein-ligated than in placebo-treated sham-operated rats ( p<0.05). Treatment with aminoguadinidine did not induce changes in the mean cross-sectional area of gastric mucosal vessels in either portal vein-ligated or sham-operated rats ( p<0.05). Conclusion: The results show that in portal hypertensive rats long-term aminoguanidine therapy corrects the hyperdynamic circulation without inducing changes in portal pressure and ameliorating the development of portal hypertensive gastropathy. This study suggests that, instead of correcting hyperdynamic circulation, treatment of portal hypertensive gastropathy should be aimed at reducing portal pressure.

Experimental treatments for asthma.

There has been increased recognition of the importance of inflammatory cells and their products in the pathogenesis of asthma. From this recognition has evolved a number of new approaches to treat the various components of the asthmatic inflammatory response. Nonselective anti-inflammatory agents such as cyclosporine and gold appear to decrease symptoms and allow a steroid-sparing effect in many cases, though side effects from cyclosporine often necessitate dose reduction. Novel oral compounds as the 5-lipoxygenase inhibitors have been effective in controlling asthma symptoms triggered by various stimuli, and the cysteinyl leukotriene receptor antagonists have shown promise in this regard as well. Neurokinin antagonists, inhaled loop diuretics, and lidocaine may play significant roles in asthma therapy through inhibition of neurogenic inflammation and possibly mast cell function. Inhibition of mast cell products by existing drugs such as heparin or the development of specific inhibitors of mast cell tryptase may also be effective agents, as are selective phosphodiesterase inhibitors, which appear to have anti-inflammatory properties. Finally, specific cytokine antagonists, agonists, inhibitors of T-cell function, selective inducible nitric oxide synthase inhibitors, and even gene-directed strategies may provide not only insights into the pathogenesis of asthma but also novel therapeutic approaches to treat the inflammation in this disease.

U-19451A: A selective inducible nitric oxide synthase inhibitor

Drugs with high selectivity for iNOS inhibition may be useful for treatment of neurodegenerative disorders, chronic inflammatory diseases, and septic shock. Therefore, U-19451A (2-benzyl-2-thio-pseudourea hydrochloride), a potential NOS inhibitor, has been investigated for its selectivity for iNOS using tissues, primary cerebellar granule cell cultures and glial cell cultures. Lungs isolated from rats treated with intravenous injection of E coli lipopolysaccharide and glial cell cultures treated with the same bacterial toxin plus γ-interferon were used for iNOS activity. Rat cerebellum and primary cerebellar granule cell cultures were utilized for neuronal NOS (nNOS) activity. S-methylthiourea (SMT) and L-nitroarginine methyl ester (L-NAME), selective iNOS and nNOS inhibitors, respectively, were chosen as standards. Both U-19451A and SMT were 4-times more selective for iNOS as compared to nNOS in tissues. U-19451A was more selective than SMT for iNOS inhibition using cultures. L-NAME was 16–31 times more selective for inhibiting nNOS activity. Based on the selectivity of U-19451A for iNOS inhibition, this drug would be expected to be effective in the treatment of diseases with inflammatory pathology without producing side effects associated with nNOS inhibition.

Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase

Adjuvant-induced arthritis is a model of chronic inflammation that exhibits several pathological changes similar to those occurring in rheumatoid arthritis, an autoimmune disease in humans characterized by chronic inflammation of the joints. We have examined the role of inducible nitric oxide synthase in producing the pathological changes associated with adjuvant-induced arthritis. Plasma nitrite concentrations were maximally elevated 14 days following adjuvant administration compared to untreated control animals. Arthritic changes in the paw were first observed between days 10–12 and were maximally elevated 21 days following adjuvant administration. Inducible nitric oxide synthase immunoreactivity was found localized in the synovial tissue from adjuvant-treated rats, while untreated controls exhibited no inducible nitric oxide synthase staining. Two selective inducible nitric oxide synthase inhibitors, aminoguanidine and N-iminoethyl- l-lysine, suppressed the increase in plasma nitrite levels and joint inflammation associated with adjuvant-induced arthritis in a dose-dependent manner. N-Iminoethyl- l-lysine attenuated the inducible nitric oxide synthase immunoreactivity in adjuvant-treated rats. Blood pressure was not affected by the highest dose of N-iminoethyl- l-lysine administered in the drinking water, indicating a lack of inhibition of constitutive nitric oxide synthase.

Nitric oxide synthase inhibition can initiate or prevent gut inflammation: Role of enzyme source

The role of nitric oxide in gut inflammation was evaluated by comparing the effects of selective or nonselective inhibitors of nitric oxide synthase (NOS). Aminoguanidine, a selective inducible NOS (iNOS) inhibitor, or NG-nitro-l-arginine methyl ester (l-NAME) were administered via the drinking water to normal guinea pigs or following induction of ileitis with trinitrobenzene sulfonic acid (TNBS 30 mg/kg). Aminoguanidine had no detectable effect in normal animals. In contrast,l-NAME caused a time and dose-dependent increase in ileal myeloperoxidase activity and circulating leukocyte numbers. Only the ileum was inflamed withl-NAME treatment. In TNBS ileitis, both NOS inhibitors were protective, inhibiting in a dose-dependent manner granulocyte infiltration and submucosal fibrosis, with concurrent reductions in substance P immunoreactivity, epithelial protein leak and bowel wall thickening. Aminoguanidine was remarkably potent with an EC50 value of 100 ng/ml (drinking water concentration).l-NAME was approximately 100-fold less potent than aminoguanidine. We conclude from this pharmacological profile, that a lack of cNOS activity or an excess of iNOS activity can lead to gut inflammation. Aminoguanidine is the most potent inhibitor of experimental inflammatory bowel disease yet reported.