河北环毛蚓神经系统 一氧化氮合酶的组织化学定位

用依赖还原型辅酶Ⅱ的黄酶组织化学方法,研究了环节动物门寡毛纲种类河北环毛蚓（Pheretima tschiliensis)神经系统k 一氧化氮合酶(NOS)阳性细胞及阳性纤维的分布,结果表明,河北环毛蚓神经系统中脑神经节背侧有大量细胞呈现NO强阳性反应,胞体和突起染色明显。咽下神经中偶尔能见少数染色较浅的神经元。在脑神经节腹内侧、围咽神经、 咽下神经节外侧部及腹神经链中都有一氧化氮合酶阳性纤维存在脸染色很深,实验结果表明,在环节动物中作为信息分子的一氧化氮已广泛存在于神经系统中。     

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。     

东亚三角头涡虫一氧化氮合酶的组织化学定位初步研究

采用一氧化氮合酶（ＮＯＳ）组织化学定位方法研究了三角涡虫的ＮＯＳ的分布。结果表明,三角涡虫的咽部具有ＮＯＳ分布,在神经系统中未发现ＮＯＳ分布。推测咽部ＮＯＳ可能与其摄食有关。     

脑血管的一氧化氮合酶分布的组织化学研究

本研究用ＮＡＤＰＨ－ｄ组织化学方法观察了脑表面及脑实质（皮质、纹状体）血管壁的ＮＯＳ阳性细胞。结果发现,在正常脑大于５０μｍ的血管壁上可见有内皮型一氧化氮合酶（ｅＮＯＳ）阳性内皮细胞,并有神经元型一氧化氮合酶（ｎＮＯＳ）阳性细胞紧贴于管壁或发出突起布于管壁。在神经毒损伤的纹状体中,可见有诱导型一氧化氮阳性胶质细胞,终足附于脑血管壁上,而周围未受损伤的脑实质中未见有诱导型一氧化氮合酶阳性的胶质细胞     

大白鼠中缝核一氧化氮合酶阳性神经元的组织化学观察

中脑和脑桥部中缝核被认为与睡眠有直接和间接关系的重要脑结构。本文用一氧化氮合酶（ＮＯＳ）组织化学结合荧光组织化学方法证实在中缝核群中,ＮＯＳ阳性神经元主要定位于这两个脑部的中缝核内,ＮＯＳ产生的ＮＯ能使脑血管扩张,参与脑血流的调节。提示这二个脑部中缝核内的ＮＯＳ阳性神经元可能作为多种因素之一,参于睡眠状态下基本脑血流的维持     

虎斑颈槽蛇脑一氧化氮合酶NADPH-d组织化学定位研究

运用NADPH—d组织化学方法,对虎斑颈槽蛇脑的一氧化氮合酶进行了定位研究。结果表明：阳性反应主要位于脑的大脑半球、视上核、室旁核、联合下器官、内侧纵束、室旁灰质、蓝斑以及网状结构等区域,比较讨论了与其他脊椎动物的异同。分析一氧化氮作为神经信号分子除了参与各类动物共有的一些机能的调节以外,不同类群或种类中还参与某些特定生理功能的调节。     

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。     

蓖麻毒素诱导肝癌细胞生成一氧化氮合酶——免疫组织化学研究

应用免疫组织化学方法观察不同浓度蓖麻毒素作用于肝癌细胞不同时间后,对iNOS的诱导作用。结果显示,未受蓖麻毒素作用的肝癌细胞胞浆iNOS呈阴性反应;当受到蓖麻毒素诱导后,才能在胞浆内合成,不同浓度的蓖麻毒素对肝癌细胞诱导iNOS的产生没有明显差别,而蓖麻毒素诱导iNOS的表达有明显的时间依赖性,2h没有表达,4h才出现,随着时间的延长,到8h时仍然有明显的表达,由于iNOS的合成涉及基因转录,蛋白质合成等过程,故需诱导数小时后显示酶活性,但一经诱导生成,酶活性持续时间长,蓖麻毒素诱导iNOS的产生在抗癌应用中具有重要的价值。     

大鼠烧伤复合内毒素脏器损伤中诱导型一氧化氮合酶的免疫组织化学定位及意义初探

选用烧伤复合内毒素血症大鼠模型,应用免疫组化ＬＳＡＢ法观察肝、肾组织中诱导型一氧化氮合酶（ｉＮＯＳ）的定位分布与含量变化。结果表明：在肝损伤中,ｉＮＯＳ主要定位于肝窦内皮细胞、枯否细胞和肝细胞胞浆,在损伤严重时,定位于多发性、溶解性坏死灶内残留的窦内皮细胞、巨噬细胞、中性粒细胞以及坏死灶基质内。在肾损伤中,ｉＮＯＳ主要定位于肾小管上皮细胞胞浆。并且ｉＮＯＳ与ＴＮＦ－α、ＩＬ－６的蛋白和基因表达密切相关。提示：ｉＮＯＳ及合成产物ＮＯ在烧伤复合内毒素性脏器损伤中起着重要作用,并推测ＮＯ可能是单核／巨噬细胞激活反应的信号传导分子之一     

柞蚕幼虫期神经系统一氧化氮合酶的NADPH-d组织化学

运用NADPH-d组织化学整体染色方法研究柞蚕Antheraea pernyi Gu rin-Meneville幼虫神经系统中一氧化氮合酶阳性细胞的分布、数量及形态特征。结果表明,柞蚕各龄期幼虫中枢神经的脑及各神经节中都有一氧化氮合酶阳性反应,阳性神经元根据其形态大小和染色特性可分为A,B,C3种类型:A型细胞沿神经节中线分布,阳性反应较强,胞体长径约30~70μm,各神经节中的数量恒定。B型细胞多分布于神经节周边部分,阳性反应较弱,胞体长径约8~20μm,各神经节中的数量变化较大,随着龄期增加有减少的趋势。C型细胞分布于咽下神经节和第8腹神经节,在3种细胞中阳性反应最强,胞体长径约16~50μm。     

棕色田鼠小肠肌间神经丛NOS的组织化学观察

用NDP-黄递酶组织化学法研究了NOS在棕色田鼠（Microtus mandarinus)小肠肌间神经丛的分布,同样的方法对大白鼠进行了实验比较。结果发现,棕色田鼠小肠肌间祖辈 经丛NOS阳性神经元形态各异,大小悬殊数倍;在神经节和阳性神经纤维中可见阳性神经元呈“串珠”状和“U”型排列。10倍镜下记数100个视野,观察到阳性神经元平均密度为42.8个/mm^2,每个视野内平均有神经节12.4个,每个神经节内平均有NOS阳性神经元8.5个,占其神经节内神经元总数的27.2%,棕色田鼠为野生草食性动物,其NOS在小肠肌间神经丛的分布与其他杂食性啮齿类有相同之处,但也存在着种属差异。     

Biochemical Characterization and Histochemical Localization of Nitric Oxide Synthase in the Nervous System of the Snail, Helix pomatia

Abstract: Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca²⁺ and NADPH and was inhibited by N ^G-nitro- l -arginine ( l -NNA). Proteins purified by 2',5'-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca²⁺- and NADPH-dependent NOS activity, which was partially inhibited by EGTA and l -NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue.     

一氧化氮合酶的若干研究进展

一氧化氮合酶(NOS)是一氧化氮(NO)生物学与医学研究的重要内容.近年来,对NOS酶本质及其生化与分子生物学特性甚至某些分子遗传学方面的认识都在迅速发展和深化.研究表明,干预NOS-NO途径的某些环节,如酶激活、NO合成、释放与转运甚至有关酶的编码基因及其表达,将为某些临床问题的解决提供新的思路和手段．     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

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.     

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。     

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.