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.     

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.     

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     

Modulation of the Nitric Oxide/BH4 Pathway Protects Against Irradiation-Induced Neuronal Damage

Neurochemical Research - The kynurenine pathway (KP, IDO/Kyn pathway) is an important metabolic pathway related to many diseases. Although cranial radiotherapy is the mainstay in metastatic tumors...     

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.     

神经型一氧化氮合酶的活性调控

一氧化氮是重要的信使分子,在生物体内参与众多生理及病理过程。生物体内存在着复杂的一氧化氮合酶活性调控机制以精确调控一氧化氮的生成。在神经系统中,一氧化氮主要由神经型一氧化氮合酶催化生成。神经型一氧化氮合酶的活性主要受到翻译后水平上钙离子和钙调蛋白的调控,其调控方式包括二聚化、多位点的磷酸化和去磷酸化,以及主要由PDZ结构域介导的蛋白质-蛋白质相互作用。一氧化氮本身对其合酶的活性具有负反馈调控作用。近年来的研究提示,细胞质膜上的脂筏微区在神经性一氧化氮合酶的活性调控中也起到重要的调节作用。     

Substrate Inhibition of Nitric Oxide Synthase in Pulmonary Artery Endothelial Cells in Culture

The effects of arginine on nitric oxide synthase (NOS) activity and NO production were studied in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 0–100 μM arginine increased NO production, detected as nitrite in the culture medium, in a dose-dependent manner. In contrast, incubation with concentrations of arginine in excess of 100 μM resulted in a reversible dose-dependent inhibition of NO production, even though intracellular arginine content increased in these cells. The NOS enzyme kinetics were studied in a total membrane preparation and in purified NOS protein and revealed that theK_mof arginine as a substrate for NOS is 3–5 μM, theV_maxoccurred at 100 μM arginine, and substrate inhibition occurred at >100 μM arginine. Oxyhemoglobin, carboxy-PTIO, catalase, SOD, citrulline, hydroxyarginine, and -arginine did not change NOS kinetics. These results indicate that substrate inhibition of eNOS exists in porcine PAEC in vitro.     

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.     

Induction of Nitric Oxide Synthase in Glial Cells

Primary astrocyte cultures, C6 glioma cells, and N18 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL-6 fibroblasts. Treatment of astrocyte cultures for 16-18 h with lipopolysaccharide (LPS) induced NOS-like activity that was L-arginine and NADPH dependent, Ca2+ independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%, with a loss of 44% of NOS-like activity. C6 cells treated with LPS also showed Ca(2+)-independent and L-arginine-dependent NOS-like activity. N18 cells demonstrated constitutive Ca(2+)-dependent NOS-like activity that was not enhanced by LPS induction. These data indicate that NOS-like activity can be induced in microglia, astrocytes, and a related glioma cell line as it can in numerous other cell types, but not in neuron-like N18 cells.     

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.     

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.     

Inhibition of Depolarization-Induced Nitric Oxide Synthase Activation by NS-7, a Phenylpyrimidine Derivative, in Primary Neuronal Culture

Abstract: Neuronal nitric oxide synthase (NOS) is considered to be involved in the pathogenesis of ischemic brain damage. In the present study, the effect of a novel neuroprotective phenylpyrimidine derivative, 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), on depolarization-stimulated NOS activity was examined in cultured neurons of mouse cerebral cortex. Various depolarizing stimuli such as veratridine, KCl, and N -methyl- d -aspartate increased the NOS activity determined by cyclic GMP formation. NS-7 concentration-dependently inhibited both the veratridine- and KCl-induced NOS activation with IC₅₀ values of 9.3 and 9.6 µ M , respectively. The reversal of KCl-evoked NOS activity by NS-7 was also observed under blockade of both ionotropic glutamate receptors and the Na⁺ channel with MK-801, 6-cyano-7-nitroquinoxaline-2,3-dione, and tetrodotoxin. In contrast, NS-7, even at 100 µ M , did not affect N -methyl- d -aspartate-stimulated NOS activity, nor did it have any influence on NOS activity determined in the soluble fraction of rat hippocampus. Because NS-7 has already been shown to block both Na⁺ and Ca²⁺ channels, the present findings suggest that this compound inhibits depolarization-induced NOS activation by reducing Ca²⁺ influx through blockade of Na⁺ and Ca²⁺ channels in primary neuronal culture.     

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.     

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.     

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.     

Regulation of the Manganese Superoxide Dismutase and Inducible Nitric Oxide Synthase Gene in Rat Neuronal and Glial Cells

Abstract: Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O₂⁻) and nitric oxide (NO^•). Neurotoxicity mediated by NO^• may result from the reaction of NO^• with O₂, leading to formation of peroxynitrite (ONOO⁻). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age-dependent variation of mRNA expression, we used glial cells derived from newborn, 3-, 21-, and 95-day-old rat brains. Interleukin-1β, interferon-γ (IFN-γ), bacterial lipopolysaccharide (LPS), and tumor necrosis factor-α showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN-γ increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.