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)

The objective of this study was to elucidate the mechanism by which nitric oxide (NO) inhibits NO synthase. Previous studies revealed that NO inhibits unpurified preparations of NO synthase. In the present study, the mechanism by which NO inhibits purified neuronal NO synthase from rat cerebellum was examined. The rate of L-citrulline formation from L-arginine was non-linear despite the presence of excess substrate and cofactors and was further inhibited by 30% by 200 units/ml superoxide dismutase. In contrast, 30 microM oxyhemoglobin increased NO synthase activity by 2-fold and made the reaction rate linear. These observations were consistent with the hypothesis that enzymatically generated NO inhibits NO synthase activity. Exogenous NO (0.1-10 microM) (but not NO2, nitrite, or nitrate) also inhibited NO synthase, and enzyme inhibition was not competitive with L-arginine. NO synthase inhibition by NO and other heme ligands supports the view that heme is involved in the catalytic activity of NO synthase. Oxyhemoglobin prevented but could not reverse enzyme inhibition by NO. NO synthase inhibition by NO was markedly diminished and reversed, however, by tetrahydrobiopterin (50 microM) or a tetrahydrobiopterin-regenerating system, and the latter made the reaction rate linear. In contrast, NO synthase inhibition by NO was markedly enhanced by heme oxidants (10 microM methylene blue; 3 microM ferricyanide), and these oxidants directly inhibited NO synthase activity. These observations suggest that NO interacts with enzyme-bound ferric heme to inhibit NO synthase activity. In support of this view, NO inhibited enzyme activity in the absence of turnover, when the heme iron is in the ferric state, and this inhibition was reversed by tetrahydrobiopterin. Therefore, the oxidation state of heme iron appears to be one important determinant for the inhibitory action of NO, and tetrahydrobiopterin may increase NO synthase activity by diminishing the inhibitory action of NO.
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PMID:Nitric oxide inhibits neuronal nitric oxide synthase by interacting with the heme prosthetic group. Role of tetrahydrobiopterin in modulating the inhibitory action of nitric oxide. 752 Apr 40

Nitric oxide (NO) synthesis during experimental endotoxemia has been shown to have both deleterious and beneficial effects. In the present study, we analyzed the in vivo production and the regulatory role of NO in the shock syndrome induced by staphylococcal enterotoxin B (SEB) in mice. First, we found that intraperitoneal administration of 100 micrograms SEB in BALB/c mice induced a massive synthesis of NO as indicated by high serum levels of nitrite (NO2-) and nitrate (NO3-) peaking 16 h after SEB injection. The inhibition of NO2- and NO3- release in mice injected with anti-tumor necrosis factor (TNF) and/or anti-interferon gamma (IFN-gamma) monoclonal antibody (mAb) before SEB challenge revealed that both cytokines were involved in SEB-induced NO overproduction. In vitro experiments indicated that NO synthase (NOS) inhibition by N-nitro-L-arginine methyl ester (L-NAME) enhanced IFN-gamma and TNF production by splenocytes in response to SEB. A similar effect was observed in vivo as treatment of mice with L-NAME resulted in increased IFN-gamma and TNF serum levels 24 h after SEB challenge, together with persistent expression of corresponding cytokine mRNA in spleen. The prolonged production of inflammatory cytokines in mice receiving L-NAME and SEB was associated with a 95% mortality rate within 96 h, whereas all mice survived injections of SEB or L-NAME alone. Both TNF and INF-gamma were responsible for the lethality induced by SEB in L-NAME-treated mice as shown by the protection provided by simultaneous administration of anti-IFN-gamma and anti-TNF mAbs. We conclude the SEB induces NO synthesis in vivo and that endogenous NO has protective effects in this model of T cell-dependent shock by downregulating IFN-gamma and TNF production.
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PMID:The protective role of endogenously synthesized nitric oxide in staphylococcal enterotoxin B-induced shock in mice. 752 Apr 69

This study was carried out to determine the role of reactive nitrogen intermediates in Trypanosoma cruzi infection. In vitro, splenocytes obtained during the acute phase of infection produced elevated amounts of nitric oxide (NO) that were correlated with the resistance or susceptibility of the animals. In vivo, the levels of NO2- plus NO3- in plasma during the later phase of infection were higher in C57BL/6 mice than in BALBL/c mice. The treatment of infected C57BL/6 mice with inhibitors of NO synthase increased parasitemia and mortality. Finally, we found that the NO donor drug S-nitroso-acetyl-penicillamine is able to kill trypomastigotes in vitro in the absence of any other cells, suggesting a direct NO-mediated killing of T. cruzi.
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PMID:Nitric oxide is involved in control of Trypanosoma cruzi-induced parasitemia and directly kills the parasite in vitro. 752 7

Since early growth response-1 (Egr-1) is required for macrophage differentiation and nitric oxide (NO) is immunosuppressive, we hypothesized that NO would reduce Egr-1 expression in rat lung macrophages. The inflammatory stimuli interferon-gamma and lipopolysaccharide induced an early, transient increase in Egr-1 mRNA (> 5-fold at 2 h) and a sustained, high level of inducible NO synthase mRNA (> 100-fold from 4 to 24 h). The NO metabolites nitrite and nitrate rose > 10-fold in medium from stimulated versus unstimulated cells over 24 h. Concomitant with elevated nitrogen oxides, Egr-1 mRNA levels declined to 80% below unstimulated cells at 24 h. This decline was blocked by an inhibitor of NO production, NG-monomethyl-L-arginine. Further, the NO donor S-nitroso-N-acetylpenicillamine inhibited Egr-1 expression in a dose-dependent manner, producing complete inhibition at 0.5 mM. The effect of S-nitroso-N-acetylpenicillamine was not due to reduced macrophage viability. We conclude that Egr-1 induction precedes inducible NO synthase induction in stimulated rat macrophages and that subsequent NO production reduces macrophage expression of Egr-1. We propose that this mechanism is used to regulate macrophage differentiation in human immunodeficiency virus infection and other inflammatory states.
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PMID:Nitric oxide reduces early growth response-1 gene expression in rat lung macrophages treated with interferon-gamma and lipopolysaccharide. 752 82

Although nitric oxide (NO) appears to be one of the oxidation products of L-arginine catalyzed by NO synthase (NOS; EC 1.14.13.39), past studies on the measurement of NO in cell-free enzymatic assays have not been based on the direct detection of the free NO molecule. Instead, assays have relied on indirect measurements of the stable NO oxidation products nitrite and nitrate and on indirect actions of NO such as guanylate cyclase activation and oxyhemoglobin oxidation. Utilizing a specific chemiluminescence assay, we report here that the gaseous product of L-arginine oxidation, catalyzed by both inducible macrophage and constitutive neuronal NOS, is indistinguishable from authentic NO on the basis of their physicochemical properties. NO gas formation by NOS was dependent on L-arginine, NADPH, and oxygen and inhibited by NG-methyl-L-arginine and cyanide anion. Superoxide dismutase (SOD) caused a marked, concentration-dependent increase in the production of free NO by mechanisms that were unrelated to the dismutation of superoxide anion or activation of NOS. These observations indicate that free NO is formed as a result of NOS-catalyzed L-arginine oxidation and that SOD enhances the generation of NO without directly affecting NO itself. SOD appears to elicit a novel biological action, perhaps accelerating the conversion of an intermediate in the L-arginine-NO pathway such as nitroxyl (HNO) to NO.
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PMID:Formation of free nitric oxide from l-arginine by nitric oxide synthase: direct enhancement of generation by superoxide dismutase. 752 87

Previous studies have suggested that glomerular mesangial cells produce nitric oxide (NO), using measurements of the NO decomposition products, NO2- and NO3-. We have now directly measured NO in the headspace above rat mesangial cell cultures, using a chemiluminescence analyzer. In addition, we examined mesangial cell RNA for inducible NO synthase (iNOS). We found no detectable NO in the headspace or iNOS mRNA in unstimulated mesangial cells. However, after four hours of incubation with LPS (10 micrograms/ml), iNOS mRNA was apparent and after six hours, significant increases in NO were detected. Both of these parameters continued to increase for at least 24 hours. Significant increases in NO2-/NO3- in the media and cGMP in the mesangial cells were also detected after 24 hours of incubation with LPS. The induction of iNOS mRNA by LPS was markedly inhibited by actinomycin D and dexamethasone, as was the accumulation of NO2-/NO3- in the media. Cycloheximide significantly inhibited NO2-/NO3- in the media of LPS-treated cells, but had little effect on induction of iNOS mRNA by LPS. We conclude that rat mesangial cells possess an iNOS, with activity and regulation similar to that described in macrophages. Furthermore, we demonstrate the activity of this enzyme by direct measurement of NO and its decomposition products, NO2- and NO3-. We suggest that production of NO by glomerular mesangial cells could occur, even when macrophage infiltration is not present, and could, thereby, modulate glomerular and tubular functions within the kidney.
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PMID:Inducible nitric oxide synthase mRNA and activity in glomerular mesangial cells. 752 75

NO is a major messenger molecule which regulates immunity and vascular tone, serves as a neurotransmitter and participates in wound healing. It has also been known to be increased in vivo by thermal injury. Urinary nitrate excretion and tissue levels of NO synthase activity were measured after a non-lethal thermal injury. Urinary nitrate excretion decreased by 90% 24-48 h after injury, but dramatically increased by 10-fold thereafter for 30-40 days after injury and remained elevated until the wound healed. This response was inhibited by a competitive inhibitor of NO synthase. These rates of urinary nitrate excretion suggest that NO production is dramatically affected by injury in a pattern that is distinct from that observed after lipopolysaccharide administration. On the basis of these findings, we suggest that the hyperdynamic cardiovascular and hypermetabolic responses seen to continue weeks after thermal injury could be a result of the autocrine and paracrine effects of NO generated locally within the tissues in addition to that generated by inflammatory cells.
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PMID:Nitric oxide production is intensely and persistently increased in tissue by thermal injury. 752 6

A novel spectrophotometric nitrite (NO2-)/nitrate (NO3-) assay system for a small quantity (5 microliter) of dialysate sample obtained by in vivo brain microdialysis was developed based on the diazotization reaction. The system has the advantage of in vivo consecutive measurement, high precision, good reproducibility, technical simplicity, relatively short resolution time (2.5-20 min), and wide availability. The NO3- level in the rat striatum was found to be 3 times higher than the NO2- level. A nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester, reduced striatal NO2-/citrulline formation in a dose-related manner and increased arginine, indicating that the tissue NO2- level detected by this assay system adequately reflects the striatal NO synthase activity.
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PMID:A novel in vivo assay system for consecutive measurement of brain nitric oxide production combined with the microdialysis technique. 753 Mar 52

Tumor necrosis factor-alpha (TNF-alpha) inhibits release of nitric oxide (NO) in vitro by stimulating the degradation of constitutive NO synthase (cNOS III) mRNA. However, TNF-alpha is believed to be the cytokine mediator of the hypotension and upregulation of inducible NO synthase (iNOS II) produced by gram-negative bacterial endotoxin (LPS). Some in vivo effects of TNF-alpha are opposite to those which occur in vitro. This study tested the hypothesis that in vivo administration of exogenous TNF-alpha and endogenously released TNF-alpha induce iNOS II activity and inhibit cNOS III activity, and thereby mediate the acute phase effects of LPS on blood pressure and the NO system in the rat. We show that LPS produces acute phase hypotension in ketamine anesthetized rats. The hypotension was associated with elevation of biologically active TNF-alpha in plasma, increased production of RNI (NO2- and NO3- anion) in rat neutrophils (PMN) and suppression of RNI production by A23187 (1 microM) stimulated thoracic aorta (RTA) ex vivo. TNA-alpha (10(6) U/ml, iv) did not produce acute phase hypotension but initially raised arterial blood pressure and heart rate (HR), did not increase RNI production by PMN, and inhibited RNI production by A23187 stimulated RTA ex vivo. Pretreatment of rats with the immunex monomeric soluble P75 receptor binding protein for TNF-alpha (TNFsr, 0.5 mg/kg, iv) 15 min prior to LPS administration decreased circulating TNF-alpha from 92,137 +/- 12,456 U/ml to undetectable levels as determined by the L929 bioassay. However, LPS-induced increases in RNI in PMN was enhanced and LPS-induced decreases in RNI production by RTA was inhibited by TNFsr. Thus, in vivo administration of TNF-alpha does not mimic the hemodynamic and NO-inducing effects of LPS. However, TNF-alpha mediates in part LPS-induced inhibition of RNI production by RTA. Thus, endogenous TNF-alpha is not required for LPS-induced acute phase hypotension or iNOS II activity. The importance of TNF-alpha in sepsis resides in systems other than iNOSII and blood pressure.
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PMID:In vivo administration of endotoxin and tumor necrosis factor-alpha produce different effects on constitutive and inducible nitric oxide synthase activity in rat neutrophils and aorta ex vivo. 753 Mar 65

Nitric oxide appears to act as a novel intercellular messenger activating soluble guanylyl cyclase to cause an increase in cGMP in target cells. In the nervous system, NMDA receptor activation has often been shown to result in activation of NO synthase, and many of the actions of NMDA are thought to be mediated by NO. We have recently combined intracerebral microdialysis with a sensitive assay for the NO oxidation products nitrite and nitrate, to assess NO release directly in awake, freely-moving animals. In the present study, we have applied this method to the hippocampus, where we have found that local NMDA application increases NO release. This was prevented by prior administration of an NMDA receptor antagonist or a nitric oxide synthase inhibitor. These pretreatments also reduced the basal extracellular nitrite and nitrate levels, suggesting that there may be a tonic glutamate-induced NO production in the hippocampus in awake, freely-moving animals. Previous work on NMDA-induced increases in cGMP suggested a possible role for noradrenaline. We found that pretreatment with the alpha 1A antagonists WB4101 or 5-methyl-urapidil, reduced the basal levels of NO oxidation products, however, NMDA still induced an increase in extracellular NO in the presence of these alpha 1A antagonists. In contrast, prior lesion of the central noradrenergic system with DSP4 prevented the increase in hippocampal NO release seen following local NMDA application. These results indicate that in vivo microdialysis can be used to monitor NO release in the hippocampus in response to NMDA receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:NMDA-dependent nitric oxide release in the hippocampus in vivo: interactions with noradrenaline. 753 17


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