Ascorbic acid and N-acetyl cysteine prevent uncoupling of nitric oxide synthase and increase tolerance to ischemia/reperfusion injury in diabetic rat heart

Abstract

Oxidative stress may cause a loss of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), decrease the bioavailability of NO and aggravate ischemia/reperfusion (I/R) injury in diabetic heart. We hypothesized that ascorbic acid (AA) and N-acetyl cysteine (NAC) protect the diabetic heart from I/R injury by increasing BH4/dihydrobiopterin (BH2) ratio and inhibiting uncoupling of NOS. Diabetes mellitus was induced in rats by streptozotocin treatment, and the hearts were isolated and perfused. BH4 and BH4/BH2 ratio decreased in the diabetic heart associated with increased production of superoxide and nitrotyrosine (NT). Treatment with AA or NAC significantly increased BH4/BH2 ratio in the diabetic heart associated with decreased production of superoxide and NT and increased generation of nitrate plus nitrite (NOx). Pre-treatment with AA or NAC before 30 min ischemia followed by 120 min reperfusion improved left ventricular (LV) function and reduced infarct size in the diabetic but not non-diabetic hearts. The NOS inhibitor, L-NAME, inhibited the increase in the generation of superoxide, NT and NOx, but aggravated LV function and increased infarct size in the diabetic heart. L-NAME also abrogated the increase in NOx and improvement of LV function and the infarct size-limiting effect induced by AA or NAC in the diabetic heart. These results suggest that AA and NAC increase BH4/BH2 ratio and prevent NOS uncoupling in the diabetic heart. Resultant increase in the bioavailability of NO renders the diabetic heart toleratant to I/R injury.     

Nitric oxide is involved in establishing the balance between cell cycle progression and cell death in the developing neural tube

Endogenous nitric oxide (NO) has recently been shown to affect cell cycle progression in the neural tube (NT) of the chick embryo. High NO levels trigger entry into S phase basally, while low NO levels facilitate mitosis apically. Here, we further explore the involvement of NO in determining cell numbers in the chick NT. In addition to the effect of short-term (6 h) NOS inhibition, we have observed a concomitant decrease in programmed cell death (PCD). Paradoxically, long-term (12 h) NOS inhibition caused an increase in PCD to compensate for the high proliferation rate under these conditions. Long-term treatment with a NO donor caused a decrease in S phase and increased PCD. The effects produced by the NO donor could be alleviated by folic acid that facilitated entry into S phase and prevented PCD. The effects produced by NOS inhibition (12 h) could be overcome by an embryo extract, used as a source of extracellular survival factors that enhanced proliferation and prevented PCD. Taken together, these data demonstrate that changing endogenous NO levels affect the balance between cell proliferation and PCD in NT of the developing chick embryo.     

NF-kappaB and ERK cooperate to stimulate DNA synthesis by inducing ornithine decarboxylase and nitric oxide synthase in cardiomyocytes treated with TNF and LPS

We previously reported that tumor necrosis factor-alpha (TNF) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CM) via nitric oxide and polyamine biosynthesis. Here we show an involvement of nuclear factor-kappaB (NF-kappaB) in the induction of nitric oxide synthase (NOS) and ornithine decarboxylase (ODC), the key enzyme in polyamine biosynthesis. In addition NF-kappaB activation appears to favor survival of CM by reducing caspase activation. TNF and LPS also stimulate phosphorylation of extracellular signal-regulated kinase (ERK), which is required for the changes in ODC and caspase activity, but not for NOS induction or NF-kappaB activation. In conclusion, these results indicate that NF-kappaB, in cooperation with ERK, plays a pivotal role in the growth stimulating effects of TNF and LPS, leading to the induction of both ODC and NOS and to the reduction of caspase activity.     

Ascorbic acid and N-acetyl cysteine prevent uncoupling of nitric oxide synthase and increase tolerance to ischemia/reperfusion injury in diabetic rat heart

Oxidative stress may cause a loss of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), decrease the bioavailability of NO and aggravate ischemia/reperfusion (I/R) injury in diabetic heart. We hypothesized that ascorbic acid (AA) and N-acetyl cysteine (NAC) protect the diabetic heart from I/R injury by increasing BH4/dihydrobiopterin (BH2) ratio and inhibiting uncoupling of NOS. Diabetes mellitus was induced in rats by streptozotocin treatment, and the hearts were isolated and perfused. BH4 and BH4/BH2 ratio decreased in the diabetic heart associated with increased production of superoxide and nitrotyrosine (NT). Treatment with AA or NAC significantly increased BH4/BH2 ratio in the diabetic heart associated with decreased production of superoxide and NT and increased generation of nitrate plus nitrite (NOx). Pre-treatment with AA or NAC before 30 min ischemia followed by 120 min reperfusion improved left ventricular (LV) function and reduced infarct size in the diabetic but not non-diabetic hearts. The NOS inhibitor, L-NAME, inhibited the increase in the generation of superoxide, NT and NOx, but aggravated LV function and increased infarct size in the diabetic heart. L-NAME also abrogated the increase in NOx and improvement of LV function and the infarct size-limiting effect induced by AA or NAC in the diabetic heart. These results suggest that AA and NAC increase BH4/BH2 ratio and prevent NOS uncoupling in the diabetic heart. Resultant increase in the bioavailability of NO renders the diabetic heart toleratant to I/R injury.     

BMP controls nitric oxide-mediated regulation of cell numbers in the developing neural tube

Balanced cell proliferation and cell death determines neural precursor cell numbers in early stages of neural tube (NT) development. We have previously shown that nitric oxide (NO) regulates cell numbers locally in the NT of eight to 12 somite embryos. Here, we demonstrate that bone morphogenetic protein-4 (BMP-4), which is expressed in the ectoderm and dorsal NT at these developmental stages, induces programmed cell death (PCD) and promotes entry into the S-phase, via nitric oxide synthase (NOS) activity. These effects can be reversed by BMP-4 antagonists, such as follistatin and noggin, or by specific NOS inhibitors, resulting in low NO levels that facilitate mitosis and reduce PCD. Ectopic BMP-4 induction of PCD is restricted to the dorsal NT, whereas promotion of the S-phase is evenly observed across the dorsal-ventral (D-V) axis. Prolonged exposure to either BMP-4 or NOS inhibitors, which results in high or low NO levels, respectively, causes NT defects. The results presented here throw new light on the BMP signaling pathway. The local presence of BMP-4 helps to regulate cell numbers in the developing NT by a NO-mediated pathway, which is essential for normal NT formation.     

Nitric oxide synthase activity and endogenous inhibitors in rats recovered from allergic encephalomyelitis

We have previously reported that in comparison with normal rats, the presence of experimental allergic encephalomyelitis (EAE) leads to decreased endogenous inhibitory activity (EIA) of Ca2+-dependent nitric oxide synthase (NOS) in both brain and serum, and increased expression of protein 3-nitrotyrosine (NT) in brain. In this work we show that animals recovered from the clinical signs of EAE are not different from controls in terms of either brain NOS activity, EIA of NOS, or NT expression. These results suggest that parallel to the reversal of the disease symptoms, a normalization of the production of nitric oxide and related species occurs.     

机械刺激诱导的烟草悬浮细胞一氧化氮产生途径及其与C矿和钙调素的关系

通过提高摇床转速对烟草细胞施加机械刺激（Ms）可诱导其胞内一氧化氮（No）的快速产生和一氧化氮合酶（Nos）活性的提高,这种MS诱导的NO产生可被N0清除剂cPTIO和NOS抑制剂L-NMMA显著抑制。此外,Ca2＋螯合剂EGTA、质膜Ca＋通道阻断剂La3＋、胞内Ca2＋通道拮抗剂钌红,以及钙调素抑制剂CPZ和TFP预处理均不同程度地抑制了机械刺激诱导的烟草细胞NO的产生,而机械刺激过程中钙调素活性显著上升并与NOS活性和NO含量的变化相一致。这些结果暗示着（类）Nos酶催化的精氨酸依赖途径可能是机械刺激诱发烟草细胞NO产生的主要途径,Ca2＋／CAM可能通过调节（类）NOS活性来调控No的产生。     

生物喋呤的生物学效应及其在脓毒症中的意义

研究表明,一氧化氮（ＮＯ）的过度产生可能是诱发脓毒性休克的最后共同通路,而生物喋呤为一氧化氮合酶（ＮＯＳ）重要的辅因子。多种免疫刺激因子均可诱导细胞内ＢＨ４合成显著增加,其可瑟ＮＯＳ紧密结合,调控ＮＯ的合成与释放。本文讨论了ＢＨ４的生物学效应,调控机制及其在脓毒症中的作用,并简要介绍其合成抑制剂在脓毒症防治中的潜在意义。     

The Inhibitory Effects of Nω-Nitro-L-Arginine Methyl Ester on Nitric Oxide Synthase Activity Vary Among Brain Regions in Vivo but Not in Vitro

We studied the effects of intracerebroventricular and intraperitoneal injection and the in vitro effects of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the nitric oxide synthase activities of the cerebellum, brainstem, hypothalamus, hippocampus, and the remainder of the brain after dissections. Male rats were chronically implanted with lateral icv guide cannula. L-NAME was injected in doses of 0.2, 1, and 5 mg intracerebroventricularly, and 50 mg/kg intraperitoneally. L-NAME induced dose-dependent suppression of NOS activities in each brain region. The threshold dose was 0.2 mg; 1 mg L-NAME completely abolished brain nitric oxide synthase activity 90 min after the injection. Brain NOS activities returned to baseline level 48 h after the injection of 5 mg L-NAME. There were significant differences between the sensitivity of various regions to L-NAME after in vivo but not in vitro administration of the enzyme inhibitor. These findings indicate that intracerebroventricular injection of L-NAME is a useful tool for inhibiting brain nitric oxide synthase activities in vivo. The differences between the sensitivity of different brain regions to L-NAME as well as the relative fast recovery of nitric oxide synthase activities must be taken into account when L-NAME is administered intracerebroventricularly to rats.     

尾加压素对新生大鼠心肌细胞一氧化氮合成的影响

应用半定量逆转录－多聚酶链反应法,观察尾加压素（urotensin Ⅱ,UⅡ）对培养的新生SD大鼠心肌细胞内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS)mRNA表达的影响,并测定UⅡ对心肌细胞内一氧化氮合酶（nitric oxide synthase,NOS)活性和一氧化氮（nitric oxide,NO)释放的影响。结果显示：UⅡ抑制培养的新生大鼠心肌细胞eNOS mRNA表达、抑制NOS的活性及NO释放;0.1μmol/L浓度的UⅡ呈时间依赖性抑制心肌细胞NOS的活性及NO生成。上述实验结果提示UⅡ的心血管作用可能与NO合成系统有关。     

Irradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants

Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).     

Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity.

Nitric oxide synthase (NOS) (EC 1.14.23) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. The complex reaction carried out by NOS, which involves NADPH, O2, and enzyme-bound FAD, FMN, and tetrahydrobiopterin (BH4), has only recently begun to be elucidated. Herein we report the characterization of the pterin requirement of murine macrophage NOS. Although purified NOS activity was not dependent on BH4, activity was significantly enhanced by BH4 in a concentration-dependent fashion. NOS purified in the absence of added BH4 was found to contain substoichiometric concentrations of enzyme-bound pterin, where increased concentrations of bound pterin correlated with an increase in activity when assayed in the absence of exogenous BH4. However, NOS purified in the presence of BH4 followed by gel filtration exhibited a 1 mol of pterin:1 mol of NOS 130-kDa subunit stoichiometry and activity that was essentially independent of exogenous BH4. Experiments to probe a redox role for the pterin were carried out using pterin analogues. 6(R,S)-Methyltetrahydropterin was found to increase NOS activity in enzyme purified in the absence of BH4. However, the deaza analogue, 6(R,S)-methyl-5-deazatetrahydropterin, was not only incapable of supporting enzymatic turnover but also inhibited citrulline formation in a concentration-dependent manner. Overall, these results support a role for BH4 in the NOS reaction that involves stabilization of the enzyme and redox chemistry wherein a 1:1 stoichiometry between bound pterin and NOS subunit results in maximum activity.     

The Effect of a Spinal Cord Hemisection on Changes in Nitric Oxide Synthase Pools in the Site of Injury and in Regions Located Far Away from the Injured Site

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury.2. A radioassay detection was used to determine Ca²⁺-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis.3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca²⁺-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca²⁺-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments.4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.     

Inhibition of N-methyl-D-aspartic acid-nitric oxide synthase in rat hippocampal slices by ethanol

The ionotropic glutamatergic receptor system, especially the subtype mediated by N-methyl-D-aspartic acid (NMDA), is known to exhibit special sensitivity to the effect of ethanol. This is due partly to the ability of ethanol to modulate the production of nitric oxide through the NMDA-nitric oxide synthase (NOS) pathway. In this study, we examined the effects of ethanol on basal and NMDA-stimulated NOS activity in rat hippocampal slices by measuring the conversion of [(14)C]-arginine into [(14)C]-citrulline in an incubation system containing the necessary cofactors. Stimulation of hippocampal slices with NMDA (100 microM) enhanced NOS activity by 43% (n = 12). Although ethanol did not alter NOS activity when added to the incubation system during NMDA stimulation, it dose-dependently inhibited NMDA-NOS activity when added to the slices during the 90-min preincubation period. Further assay of NOS activity with brain cytosolic fraction indicated an inhibitory effect of ethanol (200 mM) when the assay was carried out in the absence of exogenous tetrahydrobiopterin (BH4), a redox-active cofactor for NOS. Incubation of brain homogenates resulted in a time-dependent increase in the levels of lipid peroxidation products, but ethanol did not further enhance these products. Taken together, these results provide evidence for the role of BH4 but not oxidative stress in the inhibitory effect of ethanol on NMDA-NOS activity in rat hippocampal slices.     

Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function.

Subsaturating levels of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS), can lead to endothelial dysfunction as a result of decreased production of nitric oxide. Furthermore, insufficient BH(4) can also result in NOS-uncoupled production of reactive oxygen intermediates, such as superoxide anion and hydrogen peroxide. Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH(4) is a primary target for peroxynitrite-catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH(4) are oxidized rapidly by low concentrations of peroxynitrite. Peroxynitrite oxidizes BH(4) to quinonoid 5,6-dihydrobiopterin and a large proportion of the quinonoid isomer readily loses its side chain to form 7,8-dihydropterin which is not a cofactor for nitric oxide synthase. Thus, abnormally low levels of BH(4) can promote a cycle of its own destruction mediated by nitric oxide synthase-dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.     

NADPH-Diaphorase-Stained Neurons after Experimental Epilepsy in Rats

The aim of this study was to determine the neuronal participation of nitric oxide (NO) in experimental epilepsy. To reach this objective, we established the amount of cells presenting nitric oxide synthase (NOS) and the amygdaline concentrations in the -arginine–nitric oxide synthesis pathway. A group of fully epileptic rats, induced by the kindling procedure and that had reached at least 10 generalized seizures, was studied. We evaluated behavioral stages, electroencephalographic activities, and histochemical NOS-positive cells and carried out high-pressure liquid chromatography (HPLC) determinations of arginine, citrulline, and glutamic acid. Our results showed that behavioral and electrographic frequency, and duration of epileptic activities, were increased during the kindling process. Image processing system of NOS cells showed two types of intensities in cell stains in hippocampus, caudate-putamen, and amygdala. When we independently counted the two types of NOS stain cells, a selective increase in the number and density of weak-stained cells was observed, while dark-stained cells did not change in the studied structures. Additionally, arginine, citrulline, and glutamic acid concentrations in amygdala increased in kindled animals. The differential and specific increase in the stained cells expressing the nitric oxide synthase, as well as the increase in concentrations of the -arginine–nitric oxide pathway in amygdala, suggested a relationship with the progressive augmentation in the electrophysiological hyperactivity characteristic of generalized epilepsy.     

Circadian variation of nitric oxide synthase activity in mouse tissue

Endogenous nitric oxide (NO) is an important mediator in the processes that control biological clocks and circadian rhythms. The present study was designed to elucidate if NO synthase (NOS) activity in the brain, kidney, testis, aorta, and lungs and plasma NO_x levels in mice are controlled by an endogenous circadian pacemaker. Male BALB/c mice were exposed to two different lighting regimens of either light-dark 14:10 (LD) or continuous lighting (LL). At nine different equidistant time points (commencing at 09:00h) blood samples and tissues were taken from mice. The plasma and tissue homogenates were used to measure the levels of NO₂+ NO₃^- (NO_x) and total protein. The NO_x concentrations were determined by a commercial nitric oxide synthase assay kit, and protein content was assessed in each homogenate tissue sample by the Lowry method. Nitric oxide synthase activity was calculated as pmol/mg protein/h. The resulting patterns were analyzed by the single cosinor method for pre-adjusted periods and by curve-fitting programs to elucidate compound rhythmicity. The NOS activity in kidneys of mice exposed to LD exhibited a circadian rhythm, but no rhythmicity was detected in mice exposed to LL. Aortic NOS activity displayed 24h rhythmicity only in LL. Brain, testis, and lung NOS activity and plasma NO_x levels displayed 24h rhythms both in LD and LL. Acrophase values of NOS activity in brain, kidney, testis, and lungs were at midnight corresponding to their behavioral activities. Compound rhythms were also detected in many of the examined patterns. The findings suggest that NOS activity in mouse brain, aorta, lung, and testis are regulated by an endogenous clock, while in kidney the rhythm in NOS activity is synchronized by the exogenous signals.     

NADPH-diaphorase and nitric oxide synthase in the canine superior cervical ganglion

By means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we demonstrate that considerable numbers of NADPH-d-positive neurons are distributed throughout the canine superior cervical ganglion (SCG). These neurons also show NOS immunoreactivity. This finding indicates that NADPH-d histochemistry, a simple and reliable technique, can be used as a reliable marker of NOS activity in the sympathetic innervation of canine head and neck. The present findings suggest that the participation of nitric oxide in the SCG differs greatly between species.     

The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1) h(-1)) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.     

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.