Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase

Abstract: In contrast to the predominantly participate, Ca²⁺/calmodulin-dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca²⁺ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca²⁺/calmodulin-dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF-525A of the NO synthase activity in these subcellular fractions (IC₅₀= 90 μ M ) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum-bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450-type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.     

Role of Nitric Oxide in Methamphetamine Neurotoxicity: Protection by 7-Nitroindazole, an Inhibitor of Neuronal Nitric Oxide Synthase

Abstract: The role of nitric oxide (NO^•) in the neurotoxic effects of methamphetamine (METH) was evaluated using 7-nitroindazole (7-NI), a potent inhibitor of neuronal nitric oxide synthase. Treatment of mice with 7-NI (50 mg/kg) almost completely counteracted the loss of dopamine, 3,4-dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity observed 5 days after four injections of 10 or 7.5 mg/kg METH. With the higher dose of METH, this protection at 5 days occurred despite the fact that combined administration of METH and 7-NI significantly increased lethality and exacerbated METH-induced dopamine release (as indicated by a greater dopamine depletion at 90 min and 1 day). Combined treatment with 4 × 10 mg/kg METH and 7-NI also slightly increased the body temperature of mice as compared with METH alone. Thus, the neuroprotective effects of 7-NI are independent from lethality, are not likely to be related to a reduction of METH-induced dopamine release, and are not due to a decrease in body temperature. These results indicate that NO^• formation is an important step leading to METH neurotoxicity, and suggest that the cytotoxic properties of NO^• may be directly involved in dopaminergic terminal damage.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Calmodulin-induced Conformational Control and Allostery Underlying Neuronal Nitric Oxide Synthase Activation

Nitric oxide synthase (NOS) is the primary generator of nitric oxide signals controlling diverse physiological processes such as neurotransmission and vasodilation. NOS activation is contingent on Ca^2 +/calmodulin binding at a linker between its oxygenase and reductase domains to induce large conformational changes that orchestrate inter-domain electron transfer. However, the structural dynamics underlying activation of full-length NOS remain ambiguous. Employing hydrogen–deuterium exchange mass spectrometry, we reveal mechanisms underlying neuronal NOS activation by calmodulin and regulation by phosphorylation. We demonstrate that calmodulin binding orders the junction between reductase and oxygenase domains, exposes the FMN subdomain, and elicits a more dynamic oxygenase active site. Furthermore, we demonstrate that phosphorylation partially mimics calmodulin activation to modulate neuronal NOS activity via long-range allostery. Calmodulin binding and phosphorylation ultimately promote a more dynamic holoenzyme while coordinating inter-domain communication and electron transfer.     

The Heart Is an Early Target of Anthrax Lethal Toxin in Mice: A Protective Role for Neuronal Nitric Oxide Synthase (nNOS)

Anthrax lethal toxin (LT) induces vascular insufficiency in experimental animals through unknown mechanisms. In this study, we show that neuronal nitric oxide synthase (nNOS) deficiency in mice causes strikingly increased sensitivity to LT, while deficiencies in the two other NOS enzymes (iNOS and eNOS) have no effect on LT-mediated mortality. The increased sensitivity of nNOS−/− mice was independent of macrophage sensitivity to toxin, or cytokine responses, and could be replicated in nNOS-sufficient wild-type (WT) mice through pharmacological inhibition of the enzyme with 7-nitroindazole. Histopathological analyses showed that LT induced architectural changes in heart morphology of nNOS−/− mice, with rapid appearance of novel inter-fiber spaces but no associated apoptosis of cardiomyocytes. LT-treated WT mice had no histopathology observed at the light microscopy level. Electron microscopic analyses of LT-treated mice, however, revealed striking pathological changes in the hearts of both nNOS−/− and WT mice, varying only in severity and timing. Endothelial/capillary necrosis and degeneration, inter-myocyte edema, myofilament and mitochondrial degeneration, and altered sarcoplasmic reticulum cisternae were observed in both LT-treated WT and nNOS−/− mice. Furthermore, multiple biomarkers of cardiac injury (myoglobin, cardiac troponin-I, and heart fatty acid binding protein) were elevated in LT-treated mice very rapidly (by 6 h after LT injection) and reached concentrations rarely reported in mice. Cardiac protective nitrite therapy and allopurinol therapy did not have beneficial effects in LT-treated mice. Surprisingly, the potent nitric oxide scavenger, carboxy-PTIO, showed some protective effect against LT. Echocardiography on LT-treated mice indicated an average reduction in ejection fraction following LT treatment in both nNOS−/− and WT mice, indicative of decreased contractile function in the heart. We report the heart as an early target of LT in mice and discuss a protective role for nNOS against LT-mediated cardiac damage.     

Striatal Malonate Lesions Are Attenuated in Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Intrastriatal administration of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions by a secondary excitotoxic mechanism. To investigate the role of nitric oxide (NO^•) in the pathogenesis of the lesions we examined malonate toxicity in mice in which the genes for neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) were disrupted. Malonate striatal lesions were significantly attenuated in the nNOS mutant mice, and they were significantly increased in the eNOS mutant mice. Malonate-induced increases in levels of 2,3- and 2,5-dihydroxybenzoic acid/salicylate, markers of hydroxyl radical generation, were significantly attenuated in the nNOS knockout mice. Malonate-induced increases in 3-nitrotyrosine, a marker for peroxynitrite-mediated damage, were blocked in the nNOS mice, whereas a significant increase occurred in the eNOS mice. These findings show that NO^• produced by nNOS results in generation of peroxynitrite, which plays a role in malonate neurotoxicity.     

Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.     

The Potential Role of Nitric Oxide Synthase in Survival and Regeneration of Magnocellular Neurons of Hypothalamo-Neurohypophyseal System

Previous investigations from this laboratory have demonstrated that hypophysectomy induces up-regulation of neuronal nitric oxide synthase (nNOS) in magnocellular neurons of the mammalian hypothalamo-neurohypophyseal system (HNS). Accompanied by this upregulation of nNOS, both neuronal regeneration and degeneration are also observed in this system following hypophysectomy. The specific aim of this study was to determine the potential role of nNOS upregulation in neuronal survival and regeneration after hypophysectomy in the adult Sprague–Dawley (SD) rat by using a competitive nitric oxide synthase blocker, N(G)-nitrol-l-arginine methyl ester (l-NAME). We found that l-NAME treatment effectively blocked the regeneration of magnocellular neurons of the rodent hypothalamus as observed in the lumen of the third cerebral ventricle following hypophysectomy. However, l-NAME had no effect on the survival of magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei after hypophysectomy. These results suggest that the induced increase of nNOS expression enhance the regenerative ability of magnocellular neurons of the HNS following hypophysectomy.     

Anomalous Increase in Nitric Oxide Synthase Activity by Certain Nitric Oxide-Generating Compounds in Intact Neuronal Cells

Abstract: It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E-115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of l -[³H]arginine into l -[³H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN-1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN-1 and SNP markedly stimulated l -[³H]citrulline formation in a time- and concentration-dependent manner when cells were incubated with the compounds for >1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E-115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN-1 or SNP in intact cells was dependent on the presence of extracellular Ca²⁺, suggesting that it might be due to increased Ca²⁺ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN-1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken-cell preparations and in intact cells. Thus, modulation of NOS activity by NO-releasing compounds is not dependent on cyclic GMP formation and might not be related in a simple fashion to NO generation. Alternatively, activation of guanylate cyclase and stimulation of NOS activity might require different redox species of NO. Our present findings might be of clinical relevance in relation to long-term use of NO-generating compounds as therapeutic agents.     

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

Nitric oxide (NO) has emerged as a central signaling molecule in plants and animals. However, the long search for a plant NO synthase (NOS) enzyme has only encountered false leads. The first works describing a pathogen-induced NOS-like plant protein were soon retracted. New hope came from the identification of NOS1, an Arabidopsis thaliana protein with an atypical NOS activity that was found to be targeted to mitochondria in roots. Although concerns about the NO-producing activity of this protein were raised (causing the renaming of the protein to NO-associated 1), compelling data on its biological role were missing until recently. Strong evidence is now available that this protein functions as a GTPase that is actually targeted to plastids, where it might be required for ribosome function. These and other results support the argument that the defective NO production in loss-of-function mutants is an indirect effect of interfering with normal plastid functions and that plastids play an important role in regulating NO levels in plant cells.A major revolution in biology took place by the early 1990s after the discovery that nitric oxide (NO), a free radical, was not a toxic by-product of oxidative metabolism but had a fundamental role as a signaling molecule regulating normal physiological processes in animal cells (Culotta and Koshland, 1992). A role of this volatile molecule in plant defense responses was subsequently reported, and it is now well established that NO is also a key player in the regulation of different plant developmental processes, including germination, root growth, vascular differentiation, stomatal closure, and flowering (Lamattina et al., 2003; Wendehenne et al., 2004; Crawford and Guo, 2005). Animal cells synthesize NO primarily by the activity of NO synthase (NOS) enzymes. There are several NOS isoforms, but all of them catalyze the same basic reaction: a NADPH-dependent oxidation of l-Arg to NO and l-citrulline. By contrast, the synthesis of NO in plant cells remains a matter of debate. The first reported mechanism to make NO in plants was the reduction of nitrite to NO catalyzed (with low efficiency) by nitrate reductase (NR), a cytosolic enzyme that normally reduces nitrate to nitrite (Yamasaki et al., 1999). But the contribution of NR to NO synthesis is still controversial.The analysis of the Arabidopsis thaliana nia1 nia2 double mutant, which shows substantially reduced NR activity levels, has shown that such activity is required for NO synthesis during flowering (Seligman et al., 2008), auxin-induced lateral root development (Kolbert et al., 2008), and abscisic acid (ABA)-induced stomatal closure (Desikan et al., 2002; Bright et al., 2006) but not during infection (Zhang et al., 2003), salicylic acid treatment (Zottini et al., 2007), or mechanical stress (Garces et al., 2001). Furthermore, foliar extracts of the mutant show the same capacity to produce NO as wild-type plants when nitrite is exogenously supplied (Modolo et al., 2005). These results indicate that additional mechanisms to reduce nitrite into NO exist in plant cells and that the decreased capability for NO synthesis of mutant plants with defective NR activity might result from their reduced nitrite levels (Modolo et al., 2005). Other enzymatic sources for nitrite-dependent NO synthesis exist in the plasma membrane (Stohr et al., 2001) and mitochondria (Planchet et al., 2005), whereas nonenzymatic production of NO from nitrite has been shown to occur in acidic and reducing environments, such as the apoplasm (Bethke et al., 2004) and plastids (Cooney et al., 1994). The highly reduced levels of l-Arg in the nia1 nia2 mutant (Modolo et al., 2006) might also compromise its ability to produce NO. This amino acid is a substrate for the production of polyamines, compounds that have been proposed to participate in NO synthesis (Tun et al., 2006). Additionally, plants have been found to synthesize NO by an Arg-dependent NOS activity similar to that present in animal cells, as detailed in the next section.     

Neuronal Nitric Oxide Synthase and Calmodulin-Dependent Protein Kinase IIα Undergo Neurotoxin-Induced Proteolysis

Abstract: Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [ N -methyl- d -aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid α-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-α (CaMPK-IIα) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified μ- and m-calpain produced fragmentation patterns for CaMPK-IIα and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-α are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIα and nNOS. The degradation of CaMPK-IIα, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.     

逆转录病毒载体介导诱导型NO合酶在神经细胞中表达

为了深入研究诱导型一氧化氮合酶基因表达产物在阿片耐受和依赖中作用,采用脂质体介导基因转染技术,将ｉＮＯＳ ｃＤＮＡ重组逆转录病毒载体导入ＮＧ１０８－１５神经细胞,获得Ｇ４１８抗性克隆,命名为ＮＧ－ＬＮＣＸｉＮＯＳ细胞。ＤＮＡ印迹杂交,ＰＣＲ扩增及ＲＴ－ＰＣＲ和蛋白质免疫印迹杂交分析,证实ＮＧ－ＬＮＣＸｉＮＯＳ细胞有外源ｉＮＯＳ基因整合,转录和表达;ＮＡＤＰＨ黄递酶（ＮＡＤＰＨ ｄｉａｐｈｏｒａｓｅ     

胰岛素对PC12细胞神经型一氧化氮合酶表达及活性的影响

为探讨胰岛素对神经细胞中神经型一氧化氮合酶（nNOS）的表达及活性的影响,应用流式细胞术、原位杂交、电子自旋共振等技术方法研究胰岛素对PC12细胞中神经型一氧化氮合酶的影响.胰岛素作用PC12细胞9 h 后,神经型一氧化氮合酶的免疫荧光强度显著升高,且呈浓度依赖关系,其最大效应为对照的(155±13)%(P＜0.01, n=3, t-test).加入胰岛素(16 mU/L, 6 h)也能够显著上调nNOS mRNA的表达,为对照的(182±13)%(P＜0.01, n=3, t-test).另外加入胰岛素(16 mU/L)作用9 h后,神经型一氧化氮合酶的活性也显著升高,为对照的(167±15)%(P＜0.01, n=4, t-test).由上述结果可知,胰岛素对PC12细胞的神经型一氧化氮合酶的表达及活性有上调作用.     

The Role of 5-HT1A Receptors and Neuronal Nitric Oxide Synthase in a Seizur Induced Kindling Model in Rats

Neurochemical Research - Dentate gyrus (DG) has a high density of 5-HT1A receptors. It has neural nitric oxide synthase (nNOS), which is involved in neural excitability. The purpose of this study...     

Role of Nitric Oxide Synthase in the Infarct-Limiting Effect of Normobaric Hypoxia

Journal of Evolutionary Biochemistry and Physiology - The study was carried out in male Wistar rats. Animals were randomly divided into normoxic control groups and groups exposed to normobaric...     

Inhibition of Neuronal Nitric Oxide Synthase by 7-Nitroindazole Protects Against MPTP-Induced Neurotoxicity in Mice

Abstract: Several studies suggest that nitric oxide (NO^•) contributes to cell death following activation of NMDA receptors in cultured cortical, hippocampal, and striatal neurons. In the present study we investigated whether 7-nitroindazole (7-NI), a specific neuronal nitric oxide synthase inhibitor, can block dopaminergic neurotoxicity seen in mice after systemic administration of MPTP. 7-NI dose-dependently protected against MPTP-induced dopamine depletions using two different dosing regimens of MPTP that produced varying degrees of dopamine depletion. At 50 mg/kg of 7-NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP-induced depletions of both homovanillic acid and 3,4-dihydroxyphenylacetic acid. 7-NI had no significant effect on dopamine transport in vitro and on monoamine oxidase B activity both in vitro and in vivo. One mechanism by which NO^• is thought to mediate its toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO⁻), which then may nitrate tyrosine residues. Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3-nitrotyrosine, which was attenuated by treatment with 7-NI. Our results suggest that NO^• plays a role in MPTP neurotoxicity, as well as novel therapeutic strategies for Parkinson's disease.     

Norepinephrine Increases Cyclic GMP Levels in Cerebellar Cells from Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Cyclic GMP is an important intracellular messenger in the nervous system that may mediate cellular forms of neuronal plasticity. Previous studies show that most neurotransmitters stimulate cyclic GMP levels by the activation of nitric oxide synthase (NOS). In this study, we report that in primary cell cultures from the cerebellum of neuronal NOS knockout mice, norepinephrine stimulates an increase in cyclic GMP content. This increase is seen in both granule cell and astrocyte cultures and is not blocked by inhibitors of NOS or by inhibition of soluble guanylyl cyclase. These results suggest a novel pathway by which norepinephrine enhances cyclic GMP levels in the nervous system.     

西伯利亚鲟和鲫鱼脑型一氧化氮合酶cDNA片段克隆及序列分析

实验根据脑型一氧化氮合酶氨基酸高度保守序列设计一对简并引物,采用RT-PCR方法扩增并克隆了西伯利亚鲟(Acipenser baeri)和鲫鱼(Carassius auratus)的脑型一氧化氮合酶cDNA片段。西伯利亚鲟cD-NA片段为356 bp,鲫鱼cDNA片段为377 bp。氨基酸序列同源分析发现,西伯利亚鲟与斑马鱼(Brachydaniorerio)的脑型一氧化氮合酶同源性为70%,鲫鱼与斑马鱼的同源性为97.6%。