Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.13.39 (NO synthase)
 15,778 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide (NO) is an important signal substance in cell-cell communication and can induce relaxation of blood vessels by activating guanylate cyclase in smooth muscle cells (SMCs). NO is synthesized from L-arginine by the enzyme NO synthase, which is present in endothelial cells. It was recently shown that SMCs may themselves produce NO or an NO-related compound. We have studied NO production and its effects on energy metabolism in cultured rat aortic smooth muscle cells. It was observed that the cytokines, interferon-gamma and tumor necrosis factor-alpha, synergistically induced an arginine-dependent production of NO in these cells. This was associated with an inhibition of complex I (NADH: ubiquinone oxidoreductase) and complex II (succinate: ubiquinone oxidoreductase) activities of the mitochondrial respiratory chain, suggesting that NO blocks mitochondrial respiration in these cells. Lactate accumulated in the media of the cells, implying an increased anaerobic glycolysis, but there was no reduction of viability. An NO-dependent inhibition of mitochondrial respiration and a switch to anaerobic glycolysis would reduce energy production of the SMCs. This would in turn reduce the contractile capacity of the cell and might represent another NO-dependent vasodilatory mechanism. It could be of particular importance in inflammation, since cytokines released by inflammatory cells may induce autocrine NO production in SMCs.
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PMID:Interferon-gamma and tumor necrosis factor synergize to induce nitric oxide production and inhibit mitochondrial respiration in vascular smooth muscle cells. 139 84

1. A closed system was developed for perfusing J774 macrophages in columns. The cells were perfused for up to 100 h, at which time they were still viable. 2. Stimulation with increasing concentrations (0.01-10 micrograms ml-1) of bacterial lipopolysaccharide (LPS) caused the cells to produce increasing amounts of nitrite in the perfusion medium. This production was time-dependent, reaching a plateau by 48-50 h. 3. The nitrite accumulation caused by 0.1 microgram ml-1 of LPS was augmented by priming the cells for 2 h with increasing amounts of interferon-gamma. The nitrite accumulation also reached a plateau under these conditions. 4. N-iminoethyl-L-ornithine (L-NIO, 30 microM) completely inhibited the accumulation of nitrite whereas dexamethasone (0.3 microM) caused 60-70% inhibition. 5. Perfusion of the cells without L-arginine prevented the nitrite accumulation. Replacement of this amino acid after 20 or 50 h of perfusion led to a rapid generation of nitrite, the levels of which continued to increase for the duration of the experiment. 6. Thus, the perfusion system can be used to study the kinetics of the activation of the NO synthase and most likely other parameters in J774 cells and probably other cells in culture. An observation already of interest is that the 'disappearance' of the NO synthase after its activation can be prevented or reduced by removal of L-arginine from the medium.
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PMID:A perfusion system for the long term study of macrophage activation. 142 83

Low density lipoproteins (LDL) oxidatively modified by macrophages have been shown to be atherogenic in ex vivo studies. We studied the potential role of nitric oxide (NO), a free radical produced by macrophages, in LDL modification. Human LDL (1 mg/ml) were incubated with mouse peritoneal macrophages in Ham's F-10 medium. The cells were then stimulated by interferon-gamma and tumor necrosis factor-alpha to increase their production of NO from 1.3 to 12.2 microM in 24 h, as measured by nitrite. Lipid peroxidation of LDL, as measured by thiobarbituric acid-reactive materials (TBARS), was reduced in stimulated cells in a time-dependent manner. At 24 h, the decrease was about 27%. In the presence of an NO synthase inhibitor (NG-aminophomoarginine), the generation of NO was diminished and the protection against LDL lipid peroxidation was reversed. The extent of LDL protein modification was also assessed by examining its electrophoretic mobility. It was found that macrophage NO reduced the change in LDL electromobility. These data indicate that the production of NO may inhibit the oxidative modification of LDL with cytokine-stimulated macrophages. We suggest that NO plays a protective role in limiting macrophage-induced LDL modification.
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PMID:A protective role for nitric oxide in the oxidative modification of low density lipoproteins by mouse macrophages. 150 77

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.
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PMID:Catalase inhibits nitric oxide synthesis and the killing of intracellular Leishmania major in murine macrophages. 153 80

Incubation of vascular endothelial cells with S.typhosa endotoxin and interferon-gamma caused a time- and concentration-dependent reduction in the viability of the cells. The cytotoxic effect was inhibited in a concentration-dependent manner by NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthesis, and by the glucocorticoids dexamethasone and hydrocortisone, two inhibitors of the induction of NO synthase. These findings indicate that in these cells the cytotoxic effect of endotoxin is mediated by the NO synthesized by an inducible NO synthase. This induction of NO synthase in vascular endothelial cells may represent a mechanism of local endothelial damage during endotoxin shock and other immunologically based conditions.
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PMID:The role of nitric oxide in endothelial cell damage and its inhibition by glucocorticoids. 159 73

Vascular endothelial cells contain a constitutive nitric oxide (NO) synthase that is Ca2(+)-dependent. In addition, we have found that these cells express, after activation with interferon-gamma and lipopolysaccharide, an inducible Ca2(+)-independent NO synthase that is distinct from the constitutive enzyme. The generation of NO by this enzyme was detectable after a lag period of 2 hr, reached a maximum between 6 and 12 hr, and was maintained for the duration of the experiment (48 hr). The expression of the inducible NO synthase was inhibited by the protein synthesis inhibitor cycloheximide, a compound that had no direct effect on the activity of either of the two enzymes. Furthermore, hydrocortisone and dexamethasone, but not progesterone, inhibited the expression of the inducible enzyme, without directly affecting the activity of either enzyme, without directly affecting the activity of either enzyme. The effect of these steroids was inhibited in a concentration-dependent manner by cortexolone, a partial agonist of glucocorticoid receptors. Thus, the inhibition of the induction of an NO synthase by glucocorticoids is a receptor-mediated event involving the inhibition of the synthesis of mRNA for de novo synthesis of this enzyme. The induction of this NO synthase may contribute to the pathophysiology of immunologically based conditions. Furthermore, the inhibition of this induction by anti-inflammatory steroids may explain some of the therapeutic and adverse effects of these compounds.
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PMID:Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. 170 14

The cytosol fraction of J774-1 murine macrophages activated with lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) was found to nitrosate a wide range of secondary and tertiary amines. The reaction was dependent on L-arginine and NADPH. The optimal pH for nitrosation was 7.2-7.3. Nitrosation was inhibited by arginine derivatives such as NG-monomethyl-L-arginine and NG-nitro-L-arginine, well-known inhibitors of nitric oxide (NO) synthase. These results indicate that nitrosation is mediated by NO synthase, which catalyzes formation of NO and L-citrulline from L-arginine. Nitrosamine formation also required oxygen and was inversely correlated with the basicity of nitrosatable amines. The nitrosation was inhibited by oxyhemoglobin, an NO trapping agent, and enhanced by superoxide dismutase, which stabilizes NO. LPS + IFN-gamma induced approximately 500-600 times greater nitrosation activity than that of non-activated macrophages. Macrophages treated with LPS alone exhibited 3-4 times greater nitrosation activity than untreated macrophages, whereas macrophages treated with IFN-gamma alone did not show enhanced nitrosation activity. A combination of the cytosols from macrophages treated with LPS alone and IFN-gamma alone did not nitrosate morpholine as rapidly as the cytosol of macrophages treated with both compounds together. The activity for forming L-citrulline and nitrite/nitrate from L-arginine was markedly induced by treatment with either LPS alone or LPS + IFN-gamma but not with IFN-gamma. Those results suggest that some other factor(s) in addition to NO synthase is involved for efficient nitrosation by the macrophage cytosol. This factor(s) was not induced in macrophages by either LPS- or IFN-gamma alone, but was induced only in the presence of the two compounds.
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PMID:L-arginine-dependent formation of N-nitrosamines by the cytosol of macrophages activated with lipopolysaccharide and interferon-gamma. 171 76

We have previously reported that cultured murine brain endothelial cells (MBE) produce large quantities of nitric oxide (NO) after activation with interferon-gamma in combination with any of several immunoactivators including: bacterial endotoxin, tumor necrosis factor and interleukin-1. Since endothelial cells are the first example of a cell-type which may possess both a constitutive and an inducible type of NO synthase, it was of interest to compare the requirements of these two enzyme activities. Induction of NO synthesis in MBE by cytokines was abolished by the protein synthesis inhibitor, cycloheximide, and by 2,4-diamino-6-hydroxypyridine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo synthesis of tetrahydrobiopterin (THB). In the presence of DAHP, NO synthesis was restored by sepiapterin (SEP), a substrate for the alternative pathway of THB synthesis occurring via pterin salvage. Moreover, SEP increased NO synthesis to greater than 150% of control values, suggesting that THB availability is rate-limiting for NO synthesis by cytokine-induced MBE. Methotrexate, an inhibitor of the pterin salvage pathway of THB synthesis, completely reversed the stimulation of NO synthesis by sepiapterin. Thus, cytokine-induced MBE NO synthase appears to have an absolute requirement for THB as cofactor. In additional studies we found that NO synthesis by cytokine-activated MBE was inhibited by NG-monosubstituted arginine analogs with a rank-order of potency NH2 greater than CH3 greater than NO2, in contrast with the rank-order of NO2 greater than NH2 greater than CH3 previously described for inhibition of the constitutive endothelial cell enzyme. Using a kinetic assay for NO synthase activity, based on oxidation of myoglobin heme-iron, we have found that these rank orders of potency also apply to cytosol preparations of cytokine-induced and untreated endothelial cells, respectively. Further differences between constitutive and cytokine-induced NO synthase were observed with regard to calmodulin requirements. Whereas constitutive NO synthase was potently inhibited by the calmodulin antagonists mellitin and trifluoperazine, cytokine-induced NO synthase was unaffected. In summary, NO synthesis by cytokine-activated MBE is THB-dependent, calmodulin-independent and inhibited by NG-substituted arginine analogs with a rank-order profile distinct from that for untreated endothelial cells but identical to that for cytokine-activated macrophages.
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PMID:Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. 171 27

Inbred strains of mice differ considerably in their innate resistance to leishmanial infection. BALB/c mice are highly susceptible to cutaneous leishmaniasis caused by Leishmania major, whereas CBA mice are resistant. We now show that this resistance correlates with the ability of macrophages to synthesize nitric oxide (NO) following activation with interferon-gamma or tumor necrosis factor alpha. Furthermore, the larger amounts of NO generated by resistant macrophages are related to higher levels of NO synthase activity, a difference which is not attributable to the number or the affinity of the receptors for interferon-gamma on these cells. The level of NO synthesis by activated macrophages was also correlated to the resistance in a number of other inbred mouse strains tested; macrophages from the resistant B10.S, C57BL and C3H mice produced significantly higher levels of NO than the macrophages from the susceptible BALB.b and DBA/2 mice.
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PMID:Resistance to Leishmania major infection correlates with the induction of nitric oxide synthase in murine macrophages. 172 Oct 24

We investigated whether calcium channel antagonists would alter the induction of nitric oxide (NO) synthesis by bacterial lipopolysaccharide (LPS) alone or in combination with interferon-gamma (IFN gamma) in cultured J774 macrophages, rat vascular smooth muscle cells, rat renal mesangial cells, and rat cardiac myocytes. The induction of NO synthesis was determined by measuring nitrite, the stable end-product. The dihydropyridine calcium channel antagonists, nifedipine, manidipine, nitrendipine, benidipine, barnidipine, perdipine, and nilvadipine all reduced the LPS-induced nitrite production in a dose-dependent manner, each with a differing half-maximal inhibitory concentration, in cultured J774 macrophages. Nifedipine also inhibited nitrite production in vascular smooth muscle cells, mesangial cells, and cardiac myocytes. The half-maximal inhibitory concentrations of nifedipine were ranked as follows: smooth muscle cells < mesangial cells < cardiac myocytes. Diltiazem, at nontoxic concentrations, had no effect on the nitrite formation in the three cell types. Verapamil markedly increased the formation of nitrite in cardiac myocytes in response to LPS and IFN gamma, but not in vascular smooth muscle or mesangial cells. Exposure of cardiac myocytes to LPS and IFN gamma caused the expression of NO synthase mRNA that was significantly increased by verapamil. Thus, certain calcium channel antagonists modulate NO synthesis by altering the induction of NO synthase.
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PMID:Effects of calcium channel antagonists on the induction of nitric oxide synthase in cultured cells by immunostimulants. 747 30


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