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.     

Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity

The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.     

Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase

The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.     

The untranslated region of exon 2 of the human neuronal nitric oxide synthase (NOS1) gene exerts regulatory activity

Expressional dysregulation of the human neuronal nitric oxide synthase (NOS1) gene represents an important mechanism in the pathogenesis of certain neuronal disease states. The structure and regulation of the human NOS1 gene is highly complex based on cell type- and stimulus-dependent usage of multiple exon 1 variants. Here we demonstrate that the untranslated region of exon 2 exerts promoter and enhancer functions as well, facilitated in large part by cooperative interaction of two conserved adjacent CREB/AP-1 binding sites. In human neuronal A673 cells, NOS1 expression is stimulated by several compounds which act through these sites, but also stimulate the combined promoter region of exons 1f and 1g. While stimulation of NOS1 expression by dibutyryl-cAMP is mediated by protein kinase A (blocked by H-89), the antiepileptic drug valproic acid is likely to activate phosphatidylinositol-3 kinase (inhibited by LY 294002).     

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.     

Inhibition of neuronal nitric oxide synthase by N-phenacyl imidazoles.

Nitric oxide (NO) mediates a series of physiological processes, including regulation of vascular tone, macrofage-mediated neurotoxicity, platelet aggregation, learning and long-term potentiation, and neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in several pathological diseases. Accordingly, more potent inhibitors, more selective for neuronal nitric oxide synthase (nNOS) than endothelial NOS (eNOS) or inducible NOS (iNOS), could be useful in the treatment of cerebral ischemia and other neurodegenerative diseases. We recently described the synthesis of a series of imidazole derivatives. Among them N-(4-nitrophenacyl) imidazole (A) and N-(4-nitrophenacyl)-2-methyl-imidazole (B) were considered selective nNOS inhibitors. In the present study the action mechanism of compounds A and B was analyzed. Spectral changes observed in the presence of compound A indicate that this inhibitor exerts its effect without interaction with heme iron. Moreover compounds A and B, inhibit nNOS "noncompetitively" versus arginine, but "competitively" versus BH(4).     

Down-regulation of adrenal neuronal nitric oxide synthase mRNAs and proteins after deoxycorticosterone acetate-salt treatment in rats

The aim of this study was to evaluate the possible changes of adrenal neuronal nitrite oxide synthase (nNOS) messenger RNA (mRNA) and protein of rats after deoxycorticosterone acetate (DOCA)-salt treatment. We determined adrenal nNOS expression in 12 vehicle-treated and 13 DOCA-salt-treated rats by in situ hybridization, immunohistochemistry, and multiplex RT-PCR methods. Adrenal nNOS was also detected by Western blot in five vehicle-treated and five DOCA-salt-treated rats. The results showed that adrenal nNOS mRNA and nNOS immunoreactivities were mainly localized in the medulla and some in the regions of zona glomerulosa. DOCA-salt treatment inactivated nNOS mRNA and peptide expression prominent in the adrenal medulla and slight in the zona glomerulosa. The relative quantities of nNOS mRNA in the adrenals of the DOCA-salt-treated group was 8.8-fold decreased. At the same time, the relative quantities of steroid acute regulatory protein mRNA and phenylethanolamine N-methyltransferase mRNA in the adrenals of the DOCA-salt-treated group were significantly decreased. Western blots showed that total adrenal nNOS were 3.7-fold down-regulated after DOCA-salt treatment. Our results indicated that the down-regulation of adrenal nNOS synthesis might be associated with the inactivation of adrenal function in face of volume expansion.     

Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase.

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.     

Kinetic studies on the successive reaction of neuronal nitric oxide synthase from L-arginine to nitric oxide and L-citrulline

Rat neuronal nitric oxide synthase (nNOS) was heterologously expressed in Escherichia coliand purified. The conversion of L-arginine to N(omega)-hydroxy-L-arginine and further to L-citrulline in one cycle of the reaction of the purified nNOS was measured with the reaction rapid quenching method using (3)H-L-arginine as the substrate. It was found that most of the produced (3)H-N(omega)-hydroxy-L-arginine was successively hydroxylated to (3)H-L-citrulline without leaving the enzyme. From the analysis of time courses, the rate constants for each reaction step, and also for the dissociation of the intermediate, were estimated at various temperature in which the rates for the first and the second reactions were not much different each other but the rate for the dissociation of (3)H-N(omega)-hydroxy-L-arginine from the enzyme was significantly slow. Under the steady-state reaction condition, almost all of the nNOS was estimated to be active from the amount of burst formation of L-citrulline in the pre-steady state. The rate constant for the dissociation of the product L-citrulline from nNOS was calculated from the combination of results of the rapid quenching experiments and the metabolism of L-arginine in the presence of an excess amount of substrate, which was the smallest among all the rate constants in one cycle of the nNOS reaction. The activation energies for all the reaction steps were determined from the temperature dependence of the rate constants, which revealed that the rate-determining step of the nNOS reaction in the steady state was the dissociation of the product L-citrulline from the enzyme.     

Upregulation of neuronal nitric oxide synthase in skeletal muscle by swim training

Exercise enhances cardiac output and blood flow to working skeletal muscles but decreases visceral perfusion. The alterations in nitric oxide synthase (NOS) activity and/or expression of the cardiopulmonary, skeletal muscle, and visceral organs induced by swim training are unknown. In sedentary and swim-trained rats (60 min twice/day for 3-4 wk), we studied the alterations in NOS in different tissues along with hindquarter vasoreactivity in vivo during rest and mesenteric vascular bed reactivity in vitro. Hindquarter blood flow and conductance were reduced by norepinephrine in both groups to a similar degree, whereas N(G)-nitro-L-arginine methyl ester reduced both indexes to a greater extent in swim-trained rats. Vasodilator responses to ACh, but not bradykinin or S-nitroso-N-acetyl-penicillamine, were increased in swim-trained rats. Ca(2+)-dependent NOS activity was enhanced in the hindquarter skeletal muscle, lung, aorta, and atria of swim-trained rats together with increased expression of neuronal NOS in the hindquarter skeletal muscle and endothelial NOS in the cardiopulmonary organs. Mesenteric arterial bed vasoreactivity was unaltered by swim training. Physiological adaptations to swim training are characterized by enhanced hindquarter ACh-induced vasodilation with upregulation of neuronal NOS in skeletal muscle and endothelial NOS in the lung, atria, and aorta.     

Regulation of the successive reaction catalyzed by rat neuronal nitric oxide synthase

The rat neuronal nitric oxide synthase (nNOS) catalyzes two monooxygenase reactions successively from L-arginine (L-Arg) to L-citrulline (L-Cit) via N(omega)-hydroxy-L-arginine (OH-Arg) without most of OH-Arg leaving the substrate-binding site. In the steady-state reaction conditions, the amount of OH-Arg produced is about 1/30-1/50 that of L-Cit. We found in this study using nNOS purified from an Escherichia coli expression system that the ratio of the amount of OH-Arg to L-Cit (OH-Arg/L-Cit) increased to about 1 at low concentration of NADPH. In one cycle of the nNOS reaction, the decrease in NADPH concentration was found to reduce the rates of two monooxygenase reactions but had little effect on the rate constant of OH-Arg dissociation from the enzyme. The addition of NADP(+), the competitive inhibitor for NADPH, caused the decrease in the rates of monooxygenase reactions in a single cycle of the reaction and the increase in the ratio of OH-Arg/L-Cit in the steady state. At low CaM concentrations, the ratio of OH-Arg/L-Cit was about the same as that at high CaM. In a single cycle of the nNOS reaction, the rate of monooxygenation was not altered by the CaM concentration but the amount of metabolized L-Arg decreased with the decrease in CaM concentration, showing that the amount of active nNOS was regulated by complex formation between nNOS and CaM. It becomes clear that there are two regulatory mechanisms for the successive reaction of nNOS. One controls the rates of monooxygenations and the other controls the amount of active species of nNOS.     

Studies of neuronal nitric oxide synthase inactivation by diverse suicide inhibitors.

N(G)-Amino-l-arginine, N(5)-(1-iminoethyl)-l-ornithine, N(6)-(1-iminoethyl)-l-lysine, and aminoguanidine were studied for the mechanisms by which they produce suicidal inactivation of the neuronal nitric oxide synthase isoform (nNOS). All of the inactivators that were amino acid structural analogs targeted the heme residue at the nNOS active site and led to its destruction as evidenced by the time- and concentration-dependent loss of the nNOS heme fluorescence, which reflects the disruption of the protoporphyrin-conjugated structure. The loss of heme was exclusively associated with the dimeric population of the nNOS. This inactivator-mediated loss of the nNOS heme never reached more than 60%, suggesting that only half of the dimeric heme is involved in catalytic activation of mechanism-based inactivators studied. Aminoguanidine-induced nNOS inactivation produced covalent modification of the nNOS protein chain with a stoichiometry of 0.8 mol of aminoguanidine per mole of the nNOS monomer. Specific covalent modification by aminoguanidine was exclusively associated with the oxygenase domain of the nNOS. The mechanisms by which N(6)-(1-iminoethyl)-l-lysine and aminoguanidine inactivate the nNOS and iNOS do not differ between the isoforms. The selectivity of these inactivators toward the iNOS isoform is a reflection of their much lower partition ratios, which were determined to be 0.16 +/- 0. 1 for N(6)-(1-iminoethyl)-l-lysine and 12 +/- 1.5 for aminoguanidine in case of the iNOS isoform while the same inactivators produced the partition ratios of 17 +/- 2 and 206 +/- 4, respectively, for the nNOS isoform.     

Specific transcriptional enhancement of inducible nitric oxide synthase by targeted promoter demethylation

The ability to specifically reactivate epigenetically silenced genes would have great utility in experimental studies and potential therapeutic value. Here, we describe the specific targeting of thymidine DNA glycosylase (TDG), an enzyme involved in the mechanism of methylcytosine demethylation, to the promoter of Nos2, a gene silenced by methylation in fibroblasts, using artificial zinc finger DNA binding domains. Individual targeted TDG constructs had a small effect on Nos2 expression and methylation, but simultaneous targeting of a quartet of TDG constructs significantly restored responsiveness to LPS and IFN stimuli in association with marked cytosine demethylation at the promoter and CpG island; catalytically inactive TDG complexes had no effect. Whole-genome expression microarray and pathway analysis found only 42 genes that were affected by targeted TDG constructs; the majority are likely downstream of the effect on Nos2. This study therefore shows highly specific, directed reactivation of a single, silenced gene by targeting of a demethylase to the promoter.