scholarly journals Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase

2000 ◽  
Vol 348 (3) ◽  
pp. 579-583 ◽  
Author(s):  
Ernst R. WERNER ◽  
Hans-Jörg HABISCH ◽  
Antonius C. F. GORREN ◽  
Kurt SCHMIDT ◽  
Laura CANEVARI ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Phenylalanine Hydroxylase ◽  
Neuronal Nitric Oxide Synthase ◽  
Dihydropteridine Reductase ◽  
Redox Active ◽  
Competitive Inhibitors ◽  
Oxide Synthase ◽  
Active Contribution ◽  
Stimulation Of

Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H4biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N5-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1), the well-studied classical H4biopterin-dependent reactions. H4biopterin substituted at N5 with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N5 occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N2ʹ-methyl H4biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N5-methyl H4biopterin, N5-formyl H4biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N5-substituted derivatives were redox-active: N5-methyl- and N5-hydroxylmethyl H4biopterin, but not N5-formyl- and N5-acetyl H4biopterin, reduced 2,6-dichlorophenol indophenol. N5-Substituted H4biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and, depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N5-methyl- and N5-hydroxymethyl H4biopterin inhibited phenylalanine hydroxylase, whereas N5-formyl- and N5-acetyl H4biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H4biopterin. They are compatible with a novel, non-classical, redox-active contribution of H4biopterin to the catalysis of the nitric oxide synthase reaction.

scholarly journals Optogenetic Stimulation Reduces Neuronal Nitric Oxide Synthase Expression After Stroke

Neurosurgery ◽  
2019 ◽  
Vol 66 (Supplement_1) ◽  
Author(s):  
Arjun Vivek Pendharkar ◽  
Daniel L Smerin ◽  
Lorenzo Gonzales ◽  
Eric Wang ◽  
Sabrina L Levy ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Motor Cortex ◽  
Functional Recovery ◽  
Neuronal Nitric Oxide Synthase ◽  
Optogenetic Stimulation ◽  
Oxide Synthase ◽  
Nnos Expression ◽  
Stimulation Of ◽  
Nnos Inhibition

Abstract INTRODUCTION Poststroke optogenetic stimulation has been shown to enhance neurovascular coupling and functional recovery. Neuronal nitric oxide synthase (nNOS) has been implicated as a key regulator of neurovascular response in acute stroke but its role in subacute recovery remains unclear. Here we investigate nNOS expression in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examine the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. METHODS Transgenic Thy1-ChR2-YFP male mice (10-12 wk) were used. Stereotaxic surgery was performed to implant a fiber cannula in the cLCN and animals underwent intraluminal middle cerebral artery suture occlusion (30 min). Optogenetic stimulation began at poststroke (PD) day 5 and continued until PD14. Sensorimotor tests were used to assess behavioral recovery at PD4, 7, 10, and 14. At PD15, primary motor cortex from both ipsi- and contralesional motor cortex (iM1, cM1) were dissected. nNOS mRNA and protein levels were examined using quantitative polymerase chain reaction and western blot. In another set of studies, nNOS inhibitor ARL 17477 dihydrochloride (10 mg/kg, intraperitoneally) was administered daily between PD5-14 and functional recovery was evaluated using sensorimotor tests. RESULTS cLCN stimulated stroke mice demonstrated significant improvement in speed (cm/s) on the rotating beam task at PD10 and 14 day (P < .05, P < .001 respectively). nNOS mRNA and protein expression was significantly and selectively decreased in cM1 of cLCN stimulated mice (P < .05). The reduced nNOS expression in cM1 was negatively correlated with improved recovery (R2 = −0.839, Pearson P = .009). nNOS inhibitor-treated stroke mice exhibited a significant functional improvement in speed at PD10, when compared to stroke mice receiving vehicle (saline) (P < .05). CONCLUSION Our results suggest that nNOS may play a maladaptive role in poststroke recovery. Optogenetic stimulation of cLCN and systemic nNOS inhibition produce functional benefits after stroke.

scholarly journals Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase

1996 ◽  
Vol 320 (1) ◽  
pp. 193-196 ◽  
Author(s):  
Ernst R. WERNER ◽  
Eva PITTERS ◽  
Kurt SCHMIDT ◽  
Helmut WACHTER ◽  
Gabriele WERNER-FELMAYER ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Reductase Inhibitor ◽  
Neuronal Nitric Oxide Synthase ◽  
Redox Cycling ◽  
No Synthase ◽  
Dihydropteridine Reductase ◽  
Basal Activity ◽  
Potent Antagonist ◽  
Oxide Synthase

The binding of tetrahydropteridines with 6-di- and trihydroxypropyl side chains to recombinant rat neuronal nitric oxide (NO) synthase (EC 1.14.13.39) was determined by competition with 6R-[3´-3H]-5,6,7,8-tetrahydro-L-erythro-biopterin (6R-[3´-3H]H4biopterin). Although all but one of the derivatives exhibited only poor affinities (Ki 50 µM), the 4-amino analogue of 6R-H4biopterin was a potent antagonist of 6R-H4biopterin binding (Ki 13.2 nM). The 4-amino analogue of 6R-H4 biopterin inhibited NO synthase stimulation by the natural cofactor 6R-H4biopterin with an IC50 of 1 µM without affecting the basal activity observed in the absence of added 6R-H4biopterin. Because the 4-amino analogue of 6R-H4biopterin also inhibited dihydropteridine reductase (EC 1.6.99.7; IC50 20 µM), our results support the hypothesis that redox cycling of H4biopterin might be required for the NO synthase reaction.

Inhibition of neuronal nitric oxide synthase by 6-nitrocatecholamines, putative reaction products of nitric oxide with catecholamines under oxidative stress conditions

2001 ◽  
Vol 356 (1) ◽  
pp. 105-110 ◽  
Author(s):  
Anna PALUMBO ◽  
Giuseppe ASTARITA ◽  
Marco d'ISCHIA
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Enzyme Activation ◽  
H2o2 Production ◽  
Reaction Products ◽  
Competitive Inhibitors ◽  
Cytochrome C Reduction ◽  
Ic50 Values ◽  
Oxide Synthase

6-Nitrodopamine and 6-nitronoradrenaline (6-nitronorepinephrine), putative products of the nitric oxide (NO)-dependent nitration of dopamine and noradrenaline, are reported to be reversible, competitive inhibitors of neuronal nitric oxide synthase (nNOS) with Ki values of 45 and 52μM respectively. The nitrocatecholamines inhibited H2O2 production in the absence of l-arginine and tetrahydrobiopterin (BH4) (the IC50 values for 6-nitrodopamine and 6-nitronoradrenaline were 85 and 55μM respectively) but without affecting cytochrome c reduction. The apparent Ki values for nitrocatecholamine inhibition of enzyme activation by BH4 were 18μM for 6-nitrodopamine and 40μM for 6-nitronoradrenaline. Both nitrocatecholamines antagonized the dimerization of nNOS induced by BH4 and by l-arginine, the effect being reversed by BH4 (more than 10μM) and l-arginine (e.g. 100μM). Overall, these results suggest that nitrocatecholamines interfere with nNOS activity by binding to the enzyme in the proximity of the substrate and BH4-binding sites near the haem group.

scholarly journals Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase

2000 ◽  
Vol 348 (3) ◽  
pp. 579 ◽  
Author(s):  
Ernst R. WERNER ◽  
Hans-Jörg HABISCH ◽  
Antonius C.F. GORREN ◽  
Kurt SCHMIDT ◽  
Laura CANEVARI ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Phenylalanine Hydroxylase ◽  
Dihydropteridine Reductase ◽  
Oxide Synthase

Peroxynitrite Reduction of Calmodulin Stimulation of Neuronal Nitric Oxide Synthase

1996 ◽  
Vol 9 (2) ◽  
pp. 484-491 ◽  
Author(s):  
Andreas F. R. Hühmer ◽  
Nancy Counts Gerber ◽  
Paul R. Ortiz de Montellano ◽  
Christian Schöneich
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Oxide Synthase ◽  
Stimulation Of

Neuronal nitric oxide synthase generates superoxide from the oxygenase domain

2001 ◽  
Vol 360 (1) ◽  
pp. 247-253 ◽  
Author(s):  
Hirohito YONEYAMA ◽  
Akira YAMAMOTO ◽  
Hiroaki KOSAKA
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Wild Type ◽  
Superoxide Generation ◽  
Superoxide Formation ◽  
Redox Active ◽  
Oxygenase Domain ◽  
Oxide Synthase ◽  
Reductase Domain

When l-arginine is depleted, neuronal nitric oxide synthase (nNOS) has been reported to generate superoxide. A flavoprotein module construct of nNOS has been demonstrated to be sufficient for superoxide production. In contrast, nNOS was reported not to be involved in superoxide formation, because such formation occurred with a mixture of the boiled enzyme and redox-active cofactors. We aimed to resolve these controversial issues by examining superoxide generation, without the addition of redox-active cofactors, by recombinant wild-type nNOS and by C415A-nNOS, which has a mutation in the haem proximal site. In a superoxide-sensitive adrenochrome assay, the initial lag period of C415A-nNOS was increased 2-fold compared with that of native nNOS. With ESR using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, prominent signals of the superoxide adduct were obtained with wild-type nNOS, whereas an enzyme preparation boiled for 5min did not produce superoxide. Higher concentrations of NaCN (10mM) decreased superoxide formation by 63%. Although the activity of the reductase domain was intact, superoxide generation from C415A-nNOS was decreased markedly, to only 10% of that of the wild-type enzyme. These results demonstrate that nNOS truly catalyses superoxide formation, that this involves the oxygenase domain, and that full-length nNOS hinders the reductase domain from producing superoxide.

scholarly journals Cyclooxygenase 2 Promotes Cell Survival by Stimulation of Dynein Light Chain Expression and Inhibition of Neuronal Nitric Oxide Synthase Activity

2000 ◽  
Vol 20 (22) ◽  
pp. 8571-8579 ◽  
Author(s):  
Yu-Wen E. Chang ◽  
Rolf Jakobi ◽  
Ann McGinty ◽  
Marco Foschi ◽  
Michael J. Dunn ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Pc12 Cells ◽  
Neuronal Nitric Oxide Synthase ◽  
Dynein Light Chain ◽  
No Donors ◽  
Differentiated Pc12 Cells ◽  
Cox 2 ◽  
Oxide Synthase ◽  
Stimulation Of

ABSTRACT Cyclooxygenase 2 (COX-2) inhibits nerve growth factor (NGF) withdrawal apoptosis in differentiated PC12 cells. The inhibition of apoptosis by COX-2 was concomitant with prevention of caspase 3 activation. To understand how COX-2 prevents apoptosis, we used cDNA expression arrays to determine whether COX-2 regulates differential expression of apoptosis-related genes. The expression of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase [PIN]) was significantly stimulated in PC12 cells overexpressing COX-2. The COX-2-dependent stimulation of DLC expression was, at least in part, mediated by prostaglandin E2. Overexpression of DLC also inhibited NGF withdrawal apoptosis in differentiated PC12 cells. Stimulation of DLC expression resulted in an increased association of DLC/PIN with neuronal nitric oxide synthase (nNOS), thereby reducing nNOS activity. Furthermore, nNOS expression and activity were significantly increased in differentiated PC12 cells after NGF withdrawal. This increased nNOS activity as well as increased nNOS dimer after NGF withdrawal were inhibited by COX-2 or DLC/PIN overexpression. An nNOS inhibitor or a membrane-permeable superoxide dismutase (SOD) mimetic protected differentiated PC12 cells from NGF withdrawal apoptosis. In contrast, NO donors induced apoptosis in differentiated PC12 cells and potentiated apoptosis induced by NGF withdrawal. The protective effects of COX-2 on apoptosis induced by NGF withdrawal were also overcome by NO donors. These findings suggest that COX-2 promotes cell survival by a mechanism linking increased expression of prosurvival genes coupled to inhibition of NO- and superoxide-mediated apoptosis.

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