The L-arginine: nitric oxide pathway, cellular transduction and immunological roles.
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Arginine↗
Biomedical subjects
S Moncada
Publications and source records attributed to S Moncada.
Induction of macrophage parasiticidal activity by Staphylococcus aureus and exotoxins through the nitric oxide synthesis pathway.
Murine peritoneal macrophages stimulated in vitro with killed Gram-positive bacteria Staphylococcus aureus or its membrane components in the presence of interferon-gamma (IFN-gamma) expressed high levels of nitric oxide (NO) synthase and produced large amounts of NO in a dose-dependent manner. This is not due to the contamination by Gram-negative endotoxin because the stimulatory activity was not affected by the addition of polymyxin B. The expression of the NO synthase and the synthesis of NO by macrophages stimulated with toxic shock syndrome toxin-1 (TSST), lipoteichoic acid (LTA) or killed whole S. aureus together with IFN-gamma was inhibited by the glucocorticoid, dexamethasone or by the specific inhibitor of NO synthesis, L-N-iminoethyl-ornithine (L-NIO). The exotoxins together with IFN-gamma also activated macrophages to kill the intracellular parasite Leishmania major. The leishmanicidal activity was completely inhibited by L-NIO.
Animals↗
Effect of local intra-arterial asymmetric dimethylarginine (ADMA) on the forearm arteriolar bed of healthy volunteers.
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Adult↗
Involvement of endogenous nitric oxide in the regulation of rat intestinal motility in vivo.
The effect of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on the motility of the small intestine in an acute model in the anaesthetised rat was determined by changes in jejunal intraluminal pressure. L-NAME (0.5-10 mg kg-1 i.v.) caused a dose-dependent increase in intraluminal pressure and initiated phasic intestinal contractions. These responses were inhibited by concurrent administration of L-arginine (200 mg kg-1 i.v.) but not by D-arginine (200 mg kg-1). The increase in jejunal motility induced by L-NAME was attenuated by atropine (4 mg kg-1), although even high doses of atropine (16 mg kg-1) did not abolish these responses. This indicates that although there are interactions between NO and muscarinic cholinergic mechanisms, other processes are also involved in these contractile events following administration of L-NAME. These observations in the rat suggest that endogenous NO plays a role in the modulation of intestinal motility in vivo.
Animals↗
Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes.
Incubation of rabbit articular chondrocytes with interleukin-1 beta caused time-dependent expression of NO synthase, determined as nitrite, after a lag period of 6h. The synthesis of nitrite was concentration-dependent and was inhibited by cycloheximide and NG-monomethyl-L-arginine, but not by dexamethasone or hydrocortisone. The synthesis of NO in the 100,000g supernatant of activated chondrocytes was inhibited by EGTA, but not by the calmodulin inhibitors W-13 or trifluoperazine. The synthesis of NO was half-maximal at approximately 20nM free Ca2+. Endotoxin also induced the expression of this NO synthase. Thus, rabbit articular chondrocytes express a novel inducible NO synthase which is Ca(2+)-dependent, and whose induction is not prevented by glucocorticoids.
Amino Acid Oxidoreductases↗
Nitric oxide mediates rat mucosal vasodilatation induced by intragastric capsaicin.
The role of endogenous nitric oxide (NO) in the gastric mucosal vasodilatation induced by acute intragastric perfusion with capsaicin or close-arterial infusion of rat alpha-calcitonin gene-related peptide (CGRP) was evaluated in the pentobarbitone-anaesthetised rat using laser Doppler flowmetry (LDF). The mucosal vasodilatation induced by intraluminal capsaicin (160 microM) was dose dependently reduced by the inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME; 1-5 mg kg-1 i.v.), effects reversed by concurrent administration of L-arginine (100 mg kg-1 i.v.). L-NAME (2 mg kg-1) induced a small reduction in the mucosal vasodilatation induced by close-arterial infusion of rat alpha-CGRP (50 pmol kg-1 min-1). These findings indicate a role of NO in the gastric vasodilatation induced by stimulation of sensory neurones with intragastric capsaicin.
Amino Acid Oxidoreductases↗
Interleukin-8 inhibits the induction of nitric oxide synthase in rat peritoneal neutrophils.
The effect of interleukin(IL)-8 and leukotriene B4 (LTB4) on the induction of nitric oxide (NO) synthase in rat peritoneal neutrophils (PMN) ex vivo was studied. IL-8, but not LTB4, caused concentration-dependent inhibition of the induction of a Ca(2+)-independent NO synthase ex vivo. The effect of IL-8 was not attributable to the synthesis of an inhibitor of this enzyme. These findings suggest complex regulatory control of the induction of NO synthase by cytokines.
Amino Acid Oxidoreductases↗
Nitric oxide from endothelium and smooth muscle modulates responses to sympathetic nerve stimulation: implications for endotoxin shock.
The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of noradrenergic nerves was studied in isolated rings of rat iliac arteries. TNS produced frequency-dependent contractions in all vessels. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) significantly enhanced TNS responses in intact vessels, but not in those in which the endothelium had been removed. However, in endothelium-denuded rings incubated for 8 hours, L-NMMA increased the contractions induced by nerve stimulation, an effect which was prevented by treatment with dexamethasone or cycloheximide, and enhanced by incubation with lipopolysaccharide and gamma-interferon. Addition of L-arginine reversed the effect of L-NMMA in intact rings; however, it significantly decreased below control values TNS-induced contractions in vessels without endothelium. The results indicate that a) the arterial response to noradrenergic nerve stimulation is modulated by NO originating either in endothelial cells or in smooth muscle cells after induction of NO synthase activity, and b) once NO synthase is induced, the limiting step in NO production is the availability of the substrate L-arginine. An overproduction of vascular NO in the presence of endotoxin or other inflammatory stimuli may prevent the vascular response to sympathetic stimuli and contribute to the vasodilation observed in inflammation or endotoxic shock.
Acetylcholine↗
Nitric oxide mediates tumor necrosis factor-alpha cytotoxicity in endothelial cells.
Tumor necrosis factor alpha (TNF-alpha) exerts multiple actions on endothelial cells including among others the expression of pro-coagulant activity and adhesion molecules, and secretion of cytokines. We now show that TNF-alpha induces a time- and dose-dependent cytotoxic effect on cultured bovine aortic endothelial cells. This TNF-induced cytotoxicity, which is preceded by increased production of nitric oxide (NO), is significantly decreased by the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO). Dexamethasone, which prevents the expression of cytokine-induced NO synthase in endothelial cells, also inhibits TNF-alpha-dependent cytotoxicity. The results indicate that NO is involved in the cytotoxic effect of TNF-alpha on endothelial cells.
Amino Acid Oxidoreductases↗
L-arginine transport is increased in macrophages generating nitric oxide.
Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-gamma. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0.14 +/- 0.04 mM and Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO.
Animals↗
Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa.
Cells isolated from the rat gastric mucosa were resolved into two fractions on a percoll density gradient, and into five fractions using counterflow centrifugation (elutriation). Ca(2+)-dependent nitric oxide synthase (NOS) activity was found in the high density percoll fraction but not in the parietal cell enriched low density fraction. This activity was inhibited by NG-monomethyl-L-arginine with an IC50 of 3.7 microM. Cells in the elutriator fraction rich in mucous-epithelial cells exhibited the highest NOS activity, while the smaller cell fractions had no detectable NOS yet had the highest basal release of prostaglandin E2. The parietal cell enriched elutriator fraction again had low NOS activity. The activity of a constitutive NOS in the mucous-cell fraction may indicate a role of NO in the regulation of epithelial cell integrity or secretion.
Amino Acid Oxidoreductases↗
Nitric oxide synthase inhibitors cause sustained, but reversible, hypertension and hindquarters vasoconstriction in Brattleboro rats.
Homozygous male Brattleboro rats were given a solution of NG-monomethyl-L-arginine (L-NMMA; 1 mg ml-1) to drink for a period of 7 days. There was a persistent elevation of mean arterial blood pressure, accompanied by a significant hindquarters vasoconstriction. Within 9 h of withdrawal of L-NMMA all variables were not different from pre-L-NMMA values. Brattleboro rats (n = 3) which had been drinking NG-nitro-L-arginine methyl ester (L-NAME) solution (0.05 mg ml-1) for 5-6 months showed an increased blood pressure which reversed to normal within 48 h after withdrawing the L-NAME. Thus, inhibition of nitric oxide synthesis leads to long-lasting, but reversible, hypertension.
Amino Acid Oxidoreductases↗
Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure.
Nitric oxide (NO), synthesised from L-arginine, contributes to the regulation of blood pressure and to host defence. We describe in-vitro and in-vivo evidence that NO synthesis can be inhibited by an endogenous compound, NG,NG-dimethylarginine (asymmetrical dimethylarginine, ADMA). In man, this inhibitor is found in plasma and more than 10 mg is excreted in urine over 24 h. However, in patients with end-stage chronic renal failure, who have little or no urine output, elimination is blocked and circulating concentrations of the inhibitor rise sufficiently to inhibit NO synthesis. Accumulation of endogenous ADMA, leading to impaired NO synthesis, might contribute to the hypertension and immune dysfunction associated with chronic renal failure.
Adult↗
Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.
A murine macrophage cell line, J774, expresses high levels of the enzyme nitric oxide synthase (NOS) and produces large amounts of nitric oxide (NO) when activated with recombinant interferon (IFN)-gamma and a low concentration of LPS (10 ng/ml). Both the expression of NOS and the production of NO were inhibited by recombinant IL-10 in a dose-dependent manner. The inhibition was effective only when the cells were pretreated with IL-10; addition of IL-10 at the same time or after IFN-gamma activation was without effect. These results demonstrate that IL-10, a product of Th2 (helper T lymphocyte 2) cells, can antagonise the function of IFN-gamma, a product of Th1 cells, by modulating the mechanism of synthesis of nitric oxide in the macrophages.
Amino Acid Oxidoreductases↗
Modulation of acute inflammation by endogenous nitric oxide.
The role of endogenous nitric oxide (NO) in acute inflammation was investigated using two inhibitors of NO synthase (NG-nitro-L-arginine methyl ester(L-NAME) and NG-monomethyl-L-arginine (L-NMMA)) as well as L- or D-arginine. The effect of test compounds was studied on the carrageenin-induced increase in vascular permeability in rat skin and in dextran- and carrageenin-induced paw oedema. Both L-NAME and L-NMMA dose dependently inhibited the increase in vascular permeability and oedema formation. L- but not D-arginine increased these inflammatory responses and reversed the inhibitory effects of L-NAME and L-NMMA. In dexamethasone-treated rats L-arginine enhanced the dextran-induced oedema and the early phase of carrageenin-induced oedema but did not modify the inhibition by dexamethasone of the late phase of carrageenin-induced oedema. These results suggest that endogenous NO is released at the site of acute inflammation and modulates oedema formation. Depending on the time course or on the type of inflammation, NO may be predominantly generated by the constitutive or by the inducible NO synthase.
Acute Disease↗
Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide.
Both nitric oxide (NO) and superoxide are generated by macrophages, neutrophils and endothelial cells. It has been postulated that the generation of these two radicals under physiological conditions can lead to the formation of peroxynitrite and (as a result of the homolytic lysis of this molecule) the production of hydroxyl radicals. We have used 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), a sydnonimine capable of generating both NO and superoxide simultaneously, to test this hypothesis. SIN-1 (1 mM) generated superoxide and NO at rates of 7.02 microM/min and 3.68 microM/min respectively in phosphate-buffered saline, pH 7.2, at 37 degrees C. Incubation of SIN-1 with both deoxyribose and sodium benzoate resulted in the formation of malondialdehyde (MDA). In addition, the incubation of SIN-1 with sodium benzoate resulted in the production of compounds with fluorescence emission spectra characteristic of hydroxylated products. Both the production of MDA and the generation of fluorescent compounds were inhibited by the hydroxyl radical scavenger mannitol. In all the above respects, SIN-1 mimicked the production of hydroxyl radicals from the ascorbate-driven Fenton reaction. Catalase had no effect on the SIN-1-dependent generation of MDA, and superoxide dismutase was partially inhibitory. SIN-1 produces an oxidant with the properties of the hydroxyl radical by a mechanism clearly different to that of the Fenton reaction. We conclude that the simultaneous production of NO and superoxide from SIN-1 results in the formation of hydroxyl radicals.
Benzoates↗
Catalase inhibits nitric oxide synthesis and the killing of intracellular Leishmania major in murine macrophages.
Mouse peritoneal macrophages activated with interferon-gamma (IFN-gamma) and lipopolysaccharide produce substantial amounts of nitric oxide (NO), which correlates with the elimination of the intracellular protozoan parasite Leishmania major. Both the production of NO and the leishmanicidal function of the activated macrophages can be significantly inhibited by catalase in a dose- and time-dependent manner. These results could not be interpreted by the reduction of H2O2 by catalase since the removal of H2O2 by the addition of glutathione peroxidase had no effect on the NO synthesis or the leishmanicidal function of activated macrophages. Furthermore, catalase did not affect the induction of NO synthase in IFN-gamma-activated macrophages. In contrast, the inhibition of NO synthesis and leishmanicidal activity by catalase was reversed in a dose-dependent manner by the addition of tetrahydrobiopterin, a cofactor of NO synthase. Taken together, these results not only further support the central role of NO as the cytotoxic moiety, but also suggest that hydrogen peroxide may interfere with NO production by affecting the levels of cofactor needed for its synthesis.
Animals↗
Enhancers of nonspecific immunity induce nitric oxide synthase: induction does not correlate with toxicity or adjuvancy.
A range of bacterial products and related synthetic compounds, either alone or in combination, enhance nonspecific resistance to infection and tumors. These compounds, which vary in their other properties such as pyrogenicity, toxicity and adjuvancy, were used to assess the hypothesis that nonspecific resistance is mediated by induction of the L-arginine: nitric oxide (NO) pathway. The results obtained show that agents which stimulate nonspecific immunity, such as endotoxin, muramyl dipeptide (MDP) and combinations of monophosphoryl lipid A (MPL) with either trehalose dimycolate (TDM) or MDP cause a substantial induction of Ca(2+)-independent NO synthase in murine lung. In contrast, agents which do not stimulate nonspecific resistance, such as either MPL or TDM alone or threonyl MDP (ThrMDP), do not induce NO synthase. This difference in the ability of MDP and ThrMDP to induce NO synthase in the lung in vivo was also manifest in peritoneal macrophages in vivo as well as being evident in the greater than 100-fold greater potency of MDP in inducing the enzyme in vitro in lung slices. In contrast to the good correlation between induction of NO synthase and induction of nonspecific resistance, no correlation was observed with either the toxic effects of these agents or their adjuvancy.
Acetylmuramyl-Alanyl-Isoglutamine↗