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.     

Regulation of multimeric structure of NO-synthase as a new factor of organogenesis

The effect of NO on organogenesis in Drosophila is discussed. A new model of regulation of the activity of NO-producing enzyme, NO synthase is described, which takes into account endogenous synthesis of its reduced isoform. The reduced isoform of NO synthase is capable of suppressing the enzymatic activity of full-sized NO synthase during formation of a heterodimer in vivo and in vitro. The reduced form of this enzyme inhibits the antiproliferative effect of the full-sized NO synthase isoform during formation of eye structure in Drosophila by affecting the pathways of cell cycle regulation. The reduced form of NO synthase is an endogenous dominant-negative factor of regulation of the NO synthase activity in development of Drosophila.     

Effect of bilirubin on the activity and thermokinetic characteristics of brain (synaptosomal) membrane NO synthase

NO synthase activity was found in the plasma (synaptosomal) membrane particles isolated from the homogenate of adult rat brain (without cerebellum) under conditions preventing the protease attack and formation of reactive oxygen species. The NO synthase discovered exhibited some properties of a neuronal constitutive integral membrane enzyme and was inhibited by N-nitro-L-arginine. NO synthase activity decreased when bilirubin entered the synaptosomal membrane in vitro. Bilirubin caused the shift of the transition temperature in the temperature dependence of NO synthase activity in Arrhenius plots. The incorporation of bilirubin into synaptosomal membranes resulted in an increase in the apparent activation energy for NO synthase within a temperature range of 10-30 degrees C. The membrane NO synthase was susceptible to the photodynamic effect of membrane-bound bilirubin molecules. Monomeric human serum albumin without organophilic ligands exerted a protective effect on NO synthase in bilirubin-containing membrane particles.     

Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo.

Inhibition of nitric oxide (NO) synthase activity by L-NG-Nitroarginine (NO2Arg) in brain preparations is not reversed by dialysis and is enhanced by prolonged preincubation of NO2Arg with the enzyme. By contrast, the weaker inhibition by NO2Arg of macrophage NO synthase is fully reversible. NO2Arg inhibits NO synthase activity in the brain after i.p. administration of 5 or 50 mg/kg. This in vivo inhibition also appears to be irreversible. The potent in vivo inhibition of central NO synthase by NO2Arg may facilitate studies of the physiologic function of NO as a neuronal messenger.     

Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock.

The relationship between vascular tone and the induction by endotoxin of a nitric oxide (NO) synthase was studied in vitro in rings of rat thoracic aorta. In rings with and without endothelium there was a time-dependent induction of NO synthase accompanied by both spontaneous and L-arginine-induced relaxation and by reduced contractility to phenylephrine. These effects, which were attributable to the presence of endotoxin in the Krebs' buffer, were attenuated by cycloheximide, polymyxin B and inhibitors of NO synthase. Furthermore, dexamethasone inhibited the induction of NO synthase and the consequent effects on vascular tone. These findings indicate that prevention of the induction of NO synthase by glucocorticoids may be an important component of their therapeutic action.     

Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.     

Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4-II-E cells overexpressing regucalcin

The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4-II-E cells was investigated. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 microM) and calmodulin (2.5 microg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, inhibited the effect of calcium (10 microM) addition in increasing NO synthase activity, indicating the existence of Ca(2+)/calmodulin-dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10(-8) or 10(-7) M) in the reaction mixture without or with Ca(2+)/calmodulin addition. The effect of regucalcin (10(-7) M) in decreasing NO synthase activity was also seen in the presence of TFP (50 microM) or EGTA (1 mM). The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca(2+)/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild-type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4-II-E cells.     

Regulation of Multimeric Structure of NO-synthase as a New Factor of Organogenesis

The effect of NO on organogenesis in Drosophila is discussed. A new model of regulation of the activity of NO-producing enzyme, NO synthase is described, which takes into account endogenous synthesis of its reduced isoform. The reduced isoform of NO synthase is capable of suppressing the enzymatic activity of full-sized NO synthase during formation of a heterodimer in vivo and in vitro. The reduced form of this enzyme inhibits the antiproliferative effect of the full-sized NO synthase isoform during formation of eye structure in Drosophila by affecting the pathways of cell cycle regulation. The reduced form of NO synthase is an endogenous dominant-negative factor of regulation of the NO synthase activity in development of Drosophila.     

The degree of cerebal oxidative stress in rats during ischemia-reperfusion combined with a modulaion of L-arginine-NO system

We estimated the cerebral oxidative stress during ischemia-reperfusion of the brain in conditions of modulated L-arginine-NO system in rats. In 128 rats, the changes in lipid peroxidation products (conjugated dienes, malondialdehyde, Schiff bases) and antioxidant defense factors (retinal, a-tocopherol, SH-groups) were studied. Analysis of the inhibitor (administration of NO synthase substrate L-arginine, non-selective inhibitor Nw-nitro-L-arginine methyl ester, selective inhibitor of neuronal NO synthase 7-nitroindasole, or selective inhibitor of inducible NO synthase S-methyl-isothiourea) showed the NO-dependent nature of oxidative stress in rats with brain ischemia-reperfusion. NO generated by different NO synthase isoenzymes played different roles: hyperactivation of the neuronal NO synthase was associated with oxidative stress during both brain ischemia-reperfusion periods, and the higher inducible NO synthase activity had such association only during the later period.     

Nitric oxide inhibits methionine synthase activity in vivo and disrupts carbon flow through the folate pathway

Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B(12)) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of [(14)C]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [(14)C]formate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway.     

Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum.

The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum.     

Early loss of the tyrosyl radical in ribonucleotide reductase of adenocarcinoma cells producing nitric oxide.

Nitric oxide (NO) has been previously shown to inhibit crude preparations of ribonucleotide reductase, a key enzyme in DNA synthesis, and to destroy the essential tyrosyl free radical in pure recombinant R2 subunit of the enzyme. In R2-overexpressing TA3 cells, a decrease in the tyrosyl radical was observed by whole-cell EPR spectroscopy, as soon as 4 h after NO synthase induction by immunological stimuli. Complete loss of the tyrosyl EPR signal occurred after 7 h in cells cultured at a high density. Disappearance of the tyrosyl radical was prevented by N omega-nitro-L-arginine, a specific inhibitor of NO synthesis, and by oxyhemoglobin, which reacts rapidly with NO. It was reproduced by S-nitrosoglutathione, a NO-releasing molecule. Stable end products of NO synthase metabolism did not affect the radical. Immunoblot analysis of the R2 subunit indicated that expression of the protein was not influenced by NO synthase activity. These results establish that NO, or a labile product of NO synthase, induces the disappearance of the R2-centered tyrosyl radical. Since the radical is necessary for ribonucleotide reductase activity, its destruction by NO would contribute markedly to the antiproliferative action exerted by macrophage-type NO synthase.     

Nitric oxide in physiology and pathology

Summary Nitric oxide (NO) can exert a multitude of biological actions. NO, formed froml-arginine by a calcium-dependent enzyme (NO synthase) plays a key physiological role in regulating vascular tone and integrity. NO, formed by a constitutive neuronal isoform of NO synthase, likewise plays an important neuromodulator role. By contrast, high levels of NO can be generated following induction of a calcium-independent isoform of NO synthase. This excessive production of NO can provoke hypotension such as that observed in septic shock, and can exert cytotoxic actions leading to tissue injury and inflammation. Selective inhibitors of this inducible isoform thus have therapeutic potential in a number of disease states.     

Nitric oxide voltammetric measurements in the rat brain after gamma irradiation

The effects of a lethal gamma irradiation were investigated on cerebral NO-ergic system by using a voltammetric method in freely moving rats. It is reported that the cortical NO concentration increases right from the end of the radiation exposure (15 Gy) and reaches a maximal magnitude (+120%) 24 h later. A dose-effect relationship from 2 to 15 Gy for gamma-ray exposure has also been observed. The effects, obtained with either an NO synthase inhibitor nonselective for the different NO synthase isoforms or an NO synthase inhibitor selective for the constitutive isoform, suggest that the radiation-induced increase in NO is likely to be dependent on the inducible NO synthase isoform. Moreover, experiments performed under ex vivo conditions showed that the cortical mRNA level for Ca(++)-independent NO synthase, the brain NOS activity, and urinary nitrites/nitrates increased significantly 24 h after gamma-ray exposure. These results demonstrate that a supralethal whole-body irradiation alters the NO-ergic pathways. The increase in NO obtained under such conditions might constitute a good index of central nervous system radiosensitivity during the acute phase of the radiation syndrome.     

Up-regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

We have examined the effects of sex hormones on calcium-dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17β-estradiol and testosterone for 8–48 h. Treatment with 17β-estradiol enhanced calcium-dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti-human endothelial NO synthase antibody clarified that increased NO production by 17β-estradiol treatment was accompanied by increased NO synthase protein. Our results provide evidence that human endothelial NO synthase can be regulated by estrogens.     

Role of Nitric Oxide Synthase in the Light-Induced Development of Sporangiophores in Phycomyces blakesleeanus

Blue light controls the development of sporangiophores in the zygomycete Phycomyces blakesleeanus Burgeff. Light represses the production of microsporangiophores and enhances the development of macrosporangiophores. Inhibition of the biosynthesis of tetrahydrobiopterin, a cofactor of NO synthase, inhibits this photomorphogenesis. Light induces production of citrulline from arginine in the mycelium and in sporangiophores. The citrulline-forming activity is dependent on NADPH, independent of calcium, and inhibited by NO synthase inhibitors. It is reduced in tetrahydrobiopterin-depleted mycelium. Light induces emission of NO from the developing fungus in the same order of magnitude as citrulline formation from arginine. The NO donor sodium nitroprusside can replace the light effect on sporangiophore development, and inhibitors of NO synthase repress it. We suggest that a fungal NO synthase is involved in sporangiophore development and propose its participation in light signaling.     

Nitric Oxide Synthase in Bovine Superior Cervical Ganglion

Abstract: We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by N ^G-methyl-L-arginine (NMA), and the inhibition was reversed by L-arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2',5'-ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca²⁺/calmodulin dependent and required NADPH and tetrahydrobiopterin as co-factors. Superoxide dismutase protected and oxyhemo-globin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155-kDa protein band. Immunohisto chemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine-β-hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine-induced stimulation of soluble guanylyl cyclase.     

Inhibition of NO synthase activity in nervous tissue leads to decreased motor activity in the rat

The nitric oxide/cGMP system has been shown to play a crucial role in the mechanism of learning and memory. The aim of the present study was to investigate whether the inhibition of NO synthase in brain regions leads to alterations of spontaneous behavior in rats. Male Wistar rats were treated with NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) at the dose of 40 mg/kg/day. After 4 weeks of L-NAME treatment, NO synthase activity was significantly decreased by 75% in the cerebellum, by 71% in the cerebral cortex and by 72% in the thoracic spinal cord. Decreased NO synthase activity in the nervous tissue was associated with decreased motor horizontal and vertical activities as well as by lowered frequency of sniffing, cleaning and defecation. It is concluded that the inhibition of NO synthase activity has a suppressive effect on spontaneous behavior of rats.     

Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin.

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca2(+)-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.