Endothelial nitric oxide synthase is myristylated.

The enzyme responsible for the synthesis of endothelium-derived relaxing factor and/or nitric oxide in the endothelium has been described as a particulate enzyme, whereas other isoforms of nitric oxide synthase are soluble enzymes. Here we are reporting that endothelial cells metabolically incorporate myristate (C14), but not palmitate (C16), into nitric oxide synthase. We are postulating that the endothelial-derived nitric oxide synthase is a particulate enzyme because of the fatty acid acylation of the protein which 'anchors' the enzyme into the membrane either directly or via another membrane-bound protein.     

Neuronal nitric oxide synthase is resistant to ethanol.

To test for a possible role of nitric oxide (NO) in the neurotoxicity of ethanol, we studied the effects of ethanol on the neuronal NO synthase (nNOS) both in vitro and in vivo. Ethanol, up to 200 mM, did not change the NOS activity in the cerebellar homogenate or the production of NO by the cultured cerebellar granule cells. The number of NADPH diaphorase-positive cells in the culture did not change after the exposure to 200 mM ethanol in vitro. The NOS activity in the various brain regions of mice remained similar to the controls after the acute (3 g/kg) and the chronic (33 g/kg/day, 3.5 days) administration of ethanol. N(omega)-nitro-L-arginine, a NOS inhibitor, did not affect the ethanol-withdrawal behavior. These results indicate that nNOS is resistant to ethanol at clinically relevant concentrations and that ethanol affects the NO-operated system in the brain through a pathway other than that of nNOS.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Inducible nitric oxide synthase expression is inhibited by myeloperoxidase.

Nitric oxide (NO) plays key roles in vasodilation and host defense, yet the overproduction of NO by inducible nitric oxide synthase (iNOS) at inflammatory sites can also be pathogenic. Here, we investigate the role of MPO in modulating the induction of iNOS by IFNgamma/LPS (IL). In monocyte-macrophages (Mvarphi) treated with IL, MPO gene expression was found to be downregulated as iNOS was upregulated. In Mvarphi from MPO-knockout (KO) mice, the induction of iNOS by IL was earlier and higher than in MPO-positive cells, suggesting MPO is inhibitory. Consistent with that interpretation, the addition of purified MPO enzyme to cultured macrophages inhibited iNOS induction by IL. In addition, an inhibitor of MPO enzyme, 4-aminobenzohydrazide, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. Similarly, taurine, a scavenger of MPO-generated HOCl, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. MPO affects an early event, suppressing iNOS induction when added within 2h of IL, but not when added several hours after IL. The suppression by MPO was alleviated by NO donor, sodium nitroprusside, suggesting the suppression results from scavenging of NO by MPO. This interpretation is consistent with earlier reports that MPO consumes NO, and that low levels of NO donor augment induction of iNOS by IFNgamma/LPS. The implication of these findings is that MPO acts as gatekeeper, suppressing the deleterious induction of iNOS at inflammatory sites by illegitimate signals. The combined signaling of IFNgamma/LPS overrides the gatekeeper function by suppressing MPO gene expression.     

Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine.

The objective of this study was to determine whether constitutive nitric oxide (NO) synthase from rat cerebellum could be regulated by the two products of the reaction, NO and L-citrulline, utilizing L-arginine as substrate. NO synthase activity was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine in the presence of added cofactors. The rate of citrulline formation in enzyme reaction mixtures was non-linear. Addition of superoxide dismutase (SOD; 100 units) inhibited NO synthase activity and made the rate of product formation more non-linear, whereas addition of oxyhemoglobin (HbO2; 30 microM) increased NO synthase activity, made the rate of product formation linear and also abolished the effect of SOD. Added NO (10 microM) inhibited NO synthase activity and this inhibition was potentiated by SOD and abolished by HbO2. Added L-citrulline (1 mM) did not alter NO synthase activity. The two NO donors, S-nitroso-N-acetylpenicillamine (200 microM) and N-methyl-N'-nitro-N-nitrosoguanidine (200 microM) mimicked the inhibitory effect of NO and inhibition of NO synthase activity by NO was reversible. These observations indicate clearly that NO formed during the NO synthase reaction or added to the enzyme reaction mixture causes a reversible inhibition of NO synthase activity. Thus, NO may function as a negative feedback modulator of its own synthesis.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Hyperactivity caused by a nitric oxide synthase inhibitor is countered by ultra-wideband pulses.

Potential action of ultra-wideband (UWB) electromagnetic field pulses on effects of N(G)-nitro- L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on nociception and locomotor activity was investigated in CF-1 mice. Animals were injected IP with saline or 50 mg/kg L-NAME and exposed for 30 min to no pulses (sham exposure) or UWB pulses with electric field parameters of 102+/-1 kV/m peak amplitude, 0.90+/-0.05 ns duration, and 160+/-5 ps rise time (mean+/-S.D.) at 600/s. Animals were tested for thermal nociceptive responses on a 50 degrees C surface and for spontaneous locomotor activity for 5 min. L-NAME by itself increased mean first-response (paw lift, shake, or lick; jump) and back-paw-lick response latencies and mean locomotor activity. Exposure to UWB pulses reduced the L-NAME-induced increase in back-paw-lick latency by 22%, but this change was not statistically significant. The L-NAME-induced hyperactivity was not present after UWB exposure. Reduction and cancellation of effects of L-NAME suggest activation of opposing mechanism(s) by the UWB pulses, possibly including increase of nitric oxide production by NOS. The action, or actions, of UWB pulses appears to be more effective on locomotor activity than on thermal nociception in CF-1 mice.     

Mitochondrial nitric oxide synthase

Nitric oxide (NO*) can bind to and inhibit the terminal enzyme of the mitochondrial respiratory chain, cytochrome c oxidase (complex IV). In vivo, NO* is made by the NO* synthase (NOS) family of enzymes, and considerable debate has recently arisen regarding a NOS inside mitochondria (termed 'mtNOS'). Such an enzyme is an intriguing proposition, since it affords unique organelle-based regulatory mechanisms for NO* synthesis, and has considerable implications for mitochondrial function. This review serves to discuss some of the current issues regarding mtNOS, such as its isoform identity, the availability of co-factors and substrates within the organelle, and potential physiological vs. pathological roles for the enzyme, all within the broader context of mitochondrial regulation by NO*.     

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.     

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.     

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K_ATP channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.     

Non-quantal acetylcholine release is increased after nitric oxide synthase inhibition

After anticholinesterase treatment, depolarization of the postsynaptic muscle membrane by about 5 mV develops due to non-quantally released acetylcholine from the motor nerve terminal and can be revealed as hyperpolarization by the addition of curare (H-effect). The H-effect increases significantly to 8.7 mV after inhibition of NO-synthase by L-nitroarginine methylester (L-NAME) whilst no changes in the amplitude and frequency of quantal miniature endplate potentials are observed.     

Antifibrotic role of inducible nitric oxide synthase.

Long-term treatment in rats with l-NAME, an isoform-non-specific inhibitor of nitric oxide synthase (NOS), leads to fibrosis of the heart and kidney, suggesting that nitric oxide (NO) may play a role in preventing tissue fibrosis. In this process, a likely target of NO is the quenching of reactive oxygen species (ROS) through peroxynitrite formation, and one possible source for this NO is inducible NOS (iNOS). Using Peyronie's disease (PD) tissue from both human specimens and from a rat model of PD as the source of fibrotic tissue, we investigated if NO derived from iNOS could act as such an antifibrogenic defense mechanism by determining whether: (a) tunical ROS and iNOS are increased in PD; and (b) the long-term inhibition of iNOS activity decreases the NO/ROS balance in the tunica albuginea thereby promoting collagen deposition. It was determined that in the human PD plaque, iNOS mRNA and protein, ROS, collagen, and the peroxynitrite marker, nitrotyrosine, were all increased in comparison to the normal tunica. In the rat model of PD, the fibrotic plaque also showed significant increases in iNOS mRNA and protein, nitrotyrosine, ROS as measured by heme oxygenase-1, and collagen when compared with the normal control tunica. When a selective inhibitor of iNOS, L-NIL, was given to rats with the PD-like plaque, this resulted in a decrease in nitrotyrosine levels but intensified ROS levels and collagen deposition. These data demonstrate that: (a) iNOS induction occurs in both the human and rat PD fibrotic plaque; and (b) that the NO derived from iNOS appears to counteract ROS formation and collagen deposition. Because the inhibition of iNOS activity leads to a decrease in the NO/ROS ratio, thereby favoring the development of fibrosis, it is proposed that iNOS induction in this tissue may be a protective mechanism against fibrosis and abnormal wound healing.     

Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase.

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.     

Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine

Mouse brain mitochondria have a nitric oxide synthase (mtNOS) of 147 kDa that reacts with anti-nNOS antibodies and that shows an enzymatic activity of 0.31-0.48 nmol NO/min mg protein. Addition of chlorpromazine to brain submitochondrial membranes inhibited mtNOS activity (IC50 = 2.0 +/- 0.1 microM). Brain mitochondria isolated from chlorpromazine-treated mice (10 mg/kg, i.p.) show a marked (48%) inhibition of mtNOS activity and a markedly increased state 3 respiration (40 and 29% with malate-glutamate and succinate as substrates, respectively). Respiration of mitochondria isolated from control mice was 16% decreased by arginine and 56% increased by NNA (Nomega-nitro-L-arginine) indicating a regulatory activity of mtNOS and NO on mitochondrial respiration. Similarly, mitochondrial H2O2 production was 55% decreased by NNA. The effect of NNA on mitochondrial respiration and H2O2 production was significantly lower in chlorpromazine-added mitochondria and absent in mitochondria isolated from chlorpromazine-treated mice. Results indicate that chlorpromazine inhibits brain mtNOS activity in vitro and can exert the same action in vivo.     

Brain catecholaminergic and tryptophan responses to restraint are attenuated by nitric oxide synthase inhibition

Increases in the brain concentrations of tryptophan and in serotonin (5-HT) metabolism are commonly observed in animals under stress. Previous experiments indicated that the increase in brain tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) observed in response to administration of endotoxin (lipopolysaccharide, LPS) and interleukin-1 (IL-1) were largely prevented by pretreatment with N-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase (NOS). Therefore we tested whether the increases in tryptophan and 5-HT metabolism observed following restraint and footsthock were similarly affected. Mice were injected with L-NAME (30 mg/kg) or saline and restrained for 40 min. Restraint caused increases in concentrations of tryptophan and the catabolites of dopamine (DA), norepinephrine (NE) and 5-HT in the medial prefrontal cortex, hypothalamus, and brain stem. The L-NAME pretreatment significantly attenuated, but did not prevent, the changes in tryptophan and catecholamine metabolism, with a very small effect on the increase in plasma corticosterone. When mice pretreated with L-NAME were subjected to 30 min footshock, the NOS inhibitor had no statistically significant effects on the increases in DA, NE and 5-HT metabolism, but tended to attenuate the increases in tryptophan. We interpret these results to indicate that NOS plays a relatively small role in the cerebral neurochemical responses to restraint and footshock, but the role in the restraint-induced changes was greater than that in the footshock-induced ones. The attenuation of the restraint-related effects on the catecholamines most probably reflects a contribution to the CNS responses from peripheral vascular changes which are likely to be limited by the inhibition of NOS.     

Hemodilutional anemia is associated with increased cerebral neuronal nitric oxide synthase gene expression.

Severe hemodilutional anemia may reduce cerebral oxygen delivery, resulting in cerebral tissue hypoxia. Increased nitric oxide synthase (NOS) expression has been identified following cerebral hypoxia and may contribute to the compensatory increase in cerebral blood flow (CBF) observed after hypoxia and anemia. However, changes in cerebral NOS gene expression have not been reported after acute anemia. This study tests the hypothesis that acute hemodilutional anemia causes cerebral tissue hypoxia, triggering changes in cerebral NOS gene expression. Anesthetized rats underwent hemodilution when 30 ml/kg of blood were exchanged with pentastarch, resulting in a final hemoglobin concentration of 51.0 +/- 1.2 g/l (n = 7 rats). Caudate tissue oxygen tension (Pbr(O(2))) decreased transiently from 17.3 +/- 4.1 to 14.4 +/- 4.1 Torr (P < 0.05), before returning to baseline after approximately 20 min. An increase in CBF may have contributed to restoring Pbr(O(2)) by improving cerebral tissue oxygen delivery. An increase in neuronal NOS (nNOS) mRNA was detected by RT-PCR in the cerebral cortex of anemic rats after 3 h (P < 0.05, n = 5). A similar response was observed after exposure to hypoxia. By contrast, no increases in mRNA for endothelial NOS or interleukin-1beta were observed after anemia or hypoxia. Hemodilutional anemia caused an acute reduction in Pbr(O(2)) and an increase in cerebral cortical nNOS mRNA, supporting a role for nNOS in the physiological response to acute anemia.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。