牛蛙视网膜诱导型一氧化氮合酶免疫组化定位

用免疫组织化学方法研究了诱导型一氧化氮酶（iNOS)在牛蛙视网膜中的表达。结果显示,在正常状态视网膜中,无长突细胞呈弱阳性反应;节细胞层、双极细胞,水平细胞和光感受器内段呈阴性反应,在暗适应状态下,神经节细胞,内核层的无长突细胞呈强阳性反应;一些双极细胞,水平细胞和光感受器内段呈弱阳性反应,提示NO主要在暗适应状态下参与视网膜的信息传递过程。     

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。     

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     

Inducible Nitric Oxide Synthase Up-Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age-matched transgenic mice expressing normal SOD1 and of wild-type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end-stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS-immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region unaffected by mSOD1. Similarly, no up-regulation of iNOS is detected in the spinal cord of age-matched transgenic mice expressing normal SOD1 or of wild-type mice. The time course of the spinal cord gliosis and iNOS up-regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age-matched transgenic mice expressing normal SOD1 and wild-type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up-regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.     

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.     

Cytokine Induction of Inducible Nitric Oxide Synthase in an Oligodendrocyte Cell Line

Abstract : The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen-activated protein kinase (MAPK) activation and cytokine-mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor-α (TNFα), interleukin-1 (IL-1), and interferon-γ (IFNγ), had either minimal (TNFα) or no (IL-1 and IFNγ) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNFα plus IFNγ, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT-PCR analysis, the expression of iNOS. TNFα and IL-1 were able to activate p38 MAPK in a time- and dose-dependent manner and together showed a combinatorial effect. In contrast, IFNγ neither activated on its own nor enhanced the activation of p38 MAPK in response to TNFα and IL-1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination-treated cells in a dose-dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine-treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro-L-arginine methyl ester, had a minimal protective effect against cytokine-mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing.     

糖皮质激素对机械通气大鼠肺组织iNOS、NO表达的影响

目的通过观察糖皮质激素对机械通气大鼠肺组织诱导型一氧化氮合酶（iNOS）及一氧化氮（NO）表达的影响,探讨糖皮质激素对呼吸机所致肺损伤（ventilator induced lung injury,VILI）的干预作用。方法 24只雄性Wistar大鼠随机分为对照组、机械通气组、地塞米松（DXM）干预组。用逆转录-聚合酶链反应（RT-PCR）法检测肺组织iNOS mRNA表达,用免疫组织化学染色法检测肺组织iNOS蛋白表达,用硝酸还原酶法测定肺组织和血浆NO含量。结果机械通气组和DXM干预组大鼠肺组织iNOS mRNA及其蛋白表达水平,以及血浆和肺组织NO含量均明显高于对照组（P〈0.01）;DXM干预组上述指标与机械通气组比较均明显降低（P〈0.01）。结论糖皮质激素可通过抑制肺组织iNOS的表达,减少NO的生成,对机械通气大鼠肺组织具有保护作用。     

软体动物的一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子,参与调节软体动物的嗅觉、运动、取食、机体防御及学习行为。本文从生理、生化、形态定位以及信号转导几方面综述了有关软体动物一氧化氮及其合酶的最新研究进展。     

Inhibition of Nitric Oxide Synthase Blocks N-Methyl-D-Aspartate-, Quisqualate-, Kainate-, Harmaline-, and Pentylenetetrazole-Dependent Increases in Cerebellar Cyclic GMP In Vivo

The synthesis of nitric oxide by brain slices has been demonstrated in several laboratories. In addition, in vitro studies have demonstrated stimulation of nitric oxide synthesis by excitatory amino acid receptor agonists. These data have led to the hypothesis that this readily diffusible "intercellular messenger molecule" acts to generate a cascade effect by activating guanylate cyclase in several cell types and thereby augment levels of the second messenger cyclic GMP (cGMP). Therefore, we evaluated this hypothesis in vivo, by testing the actions of the nitric oxide synthase inhibitor N-mono-methyl-L-arginine (NMMA) on elevations in level of mouse cerebellar cGMP generated by excitatory amino acid receptor agonists. The stimulatory effects of D-serine, quisqualate, and kainate were all found to be antagonized by this enzyme inhibitor. In addition, NMMA antagonized the increases in cerebellar cGMP level elicited by harmaline and pentylenetetrazole, pharmacological agents that augment endogenous excitatory amino acid transmission. Our data are, therefore, the first in vivo demonstration that nitric oxide is an important "messenger molecule" in the cerebellum, mediating the actions of kainate, quisqualate, and N-methyl-D-aspartate receptor agonists on guanylate cyclase. These data are consistent with previous in vitro findings with kainate and N-methyl-D-aspartate.     

Antioxidants Inhibit Angiogenesis In Vivo through Down-regulation of Nitric Oxide Synthase Expression and Activity

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H²O²) scavenger sodium pyruvate decreased, while H²O² increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and l-NAME, but not d-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.     

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.     

Dexamethasone Up-Regulates a Constitutive Nitric Oxide Synthase in Cerebellar Astrocytes but Not in Granule Cells in Culture

Abstract: Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in a time-(>6 h) and concentration-dependent manner (half-maximal effect at 1 n M ). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [³H]arginine to [³H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.     

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.     

Ex Vivo Measurement of Brain Tissue Nitrite and Nitrate Accurately Reflects Nitric Oxide Synthase Activity In Vivo

Abstract: The ex vivo tissue concentration of nitrite and nitrate (NO_x) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor N ^ω-nitro- l -arginine ( l -NA) at doses that completely inhibited both central and peripheral NOS, depleted whole-brain and CSF NO_x by up to 75% but had no effect on plasma NO_x. Selective inhibition of central NOS by intracerebroventricular administration of l -NA methyl ester produced similar decreases in levels of whole-brain NO_x. A residual concentration of NO_x of 10–15 µ M remained in all brain regions even after complete inhibition of brain NOS. Brain NO_x content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NO_x was found to reflect the in vivo efficacy of several different types of NOS inhibitor: l -NA, N ^ω-monomethyl- l -arginine, and 7-nitroindazole. Intraperitoneal administration of the NOS substrate l -arginine increased brain NO_x concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NO_x is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo.     

Protein Kinase C Modulates Calcium Sensitivity of Nitric Oxide Synthase in Cerebellar Slices

Abstract: The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (±)-1-aminocyclopentane- trans -1,3-dicarboxylate ( trans -ACPD) increased cyclic GMP concentration two-fold. The increase was dose-dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans -ACPD were independent of extracellular calcium blocked by N ^ω-nitro- l -arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans -ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA-treated and untreated slices had similar activities at 100 n M free calcium, whereas at 25–70 n M free calcium, NOS in PMA-treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.     

诱导型一氧化氮合酶与疾病

炎症是众多疾病如自体免疫紊乱、神经退行性病变、心血管疾病和癌症发展的病理机制,诱导型一氧化氮合酶在炎症过程中被诱导表达,产生过量的一氧化氮,引发炎症级联反应,进而导致以上多种疾病发生。抑制诱导型一氧化氮合酶表达在体内体外实验及临床使用中均体现抗炎效果和症状改善。本文综述了诱导型一氧化氮合酶在炎症过程中诱导表达及与各类重大疾病联系的最新进展,并展望了诱导型一氧化氮合酶抑制剂作为抗炎治疗策略的前景。     

Involvement of Nitric Oxide in Dopaminergic Transmission in Rat Striatum: An In Vivo Electrochemical Study

Abstract: In vivo electrochemical detection with a Nafion-coated carbon fiber working electrode, which provides information on the spatial and temporal dynamics of dopamine overflow, was used to investigate the involvement of nitric oxide (NO) in the dopaminergic transmission in the striatum of urethane-anesthetized Sprague-Dawley rats. A mixture of N -methyl- d -aspartate (NMDA) and nomifensine, a dopamine uptake blocker, was locally pressure-ejected to elicit a transient dopamine overflow from the dopamine-containing nerve terminals in the striatum. Local application of N ^ω-nitro- l -arginine methyl ester ( l -NAME), which blocks endogenous NO formation, increased the magnitude of dopamine release evoked by a subsequent NMDA and nomifensine application but resulted in no significant alteration in the time course. Furthermore, microejection of l -arginine, an NO precursor, or sodium nitroprusside (SNP), an NO generator, did not cause detectable changes in dopamine level in the striatal extracellular space. However, NMDA-induced dopamine release was profoundly inhibited with l -arginine or SNP pretreatment. In addition, NO affects dopamine uptake in rat striatum. Exogenous dopamine applied through a micropipette, reversibly and reproducibly, elicited an electrochemical signal. The time course of these signals was significantly prolonged by l -NAME treatment. These data suggest that NO is diversely involved in regulating dopaminergic transmission in rat striatum.