一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase

The activation and deactivation of Ca²⁺- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved F?rster resonance energy transfer (FRET), we determined the occurrence of Ca²⁺-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca²⁺ concentrations ([Ca²⁺]_i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca²⁺]_i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

昆虫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 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

Release of nitric oxide and expression of constitutive nitric oxide synthase of human endothelial cells: Enhancement by a 14-membered ring macrolide

A 14-membered ring macrolide, erythromycin, acts not only as an antibacterial but also as an anti-inflammatory agent. We have previously reported that erythromycin modulates neutrophil functions and ameliorates neutrophil-induced endothelial cell damage through the action of cyclic AMP-dependent protein kinase (PKA) and nitric oxide (NO). We investigated the effect of erythromycin on human endothelial cell functions. Erythromycin enhanced intracellular calcium ion concentration ([Ca²⁺]_i) of endothelial cells and NO release from endothelial cells. The enhancement of NO release from endothelial cells by erythromycin was abolished by addition of EGTA in the medium and was partially reduced by addition of H-89, an inhibitor of PKA. These results suggest that erythromycin enhances NO release from endothelial cells through the action of PKA and [Ca²⁺]_i. In addition, constitutive NO synthase (cNOS) protein expression of endothelial cells was dose-dependently enhanced by treatment with erythromycin, which might also contribute to the enhancement of NO release from endothelial cells by erythromycin. The effect of erythromycin as an anti-inflammatory agent might be partially mediated through the enhancement of NO release from endothelial cells and the drug might be a useful tool for the investigation of cNOS of endothelial cells.     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Role of nitric oxide synthase in insect cell radioresistance: an in-silico analysis

Previous studies on various insect cell lines have displayed very high radioresistance in Lepidoptera (butterflies and moths) as compared to mammals as well as other orders of Insecta including Diptera. Since NOS is known to modulate cellular radiation sensitivity, we carried out in silico analysis of Lepidopteran NOS and compared its structural and functional features including the sequence homology, predicted tertiary structure, post-translational phosphorylation and intracellular localization with the other species. Our study demonstrates that Lepidopteran NOS, while carrying significant sequence homology with mammalian nNOS, has structural/ functional features that may enhance resistance to radiation and other stress agents. A higher phosphorylation score of Lepidopteran NOS (0.885±0.02 as against 0.694±0.094 of mammalian NOS; predicted using Net Phos 2.0) was observed at many well-conserved phosphorylation sites, which may reduce NOS activation by stress agents including radiation. Further, the primarily cytoplasmic localization of Lepidopteran NOS (score 23 against 10 of mammalian NOS, derived using WoLFPSORT), aided by higher phosphorylation scores as well as sequence-driven cytoplasmic localizing signals, may significantly reduce amplification of extraneous oxidative damage. Based on these findings, we hypothesize that a primarily cytosolic and less responsive NOS could significantly contribute to radioresistance of Lepidopteran insects as well as their cultured cell lines.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Comparison of effects of nitric oxide synthase (NOS) inhibitors on plasma nitrite/nitrate levels and tissue NOS activity in septic organs

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar-King rats. The rats treated with the three iNOS inhibitors (N-(3-(aminomethyl)benzyl)acetamidine (1400W), (1 S, 5 S, 6 R, 7 R )-2-aza-7-chloro-3-imino-5-methylbicyclo [4.1.0] heptane hydrochloride (ONO-1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose-dependent decreases in plasma NOx levels and NOS activity in the lungs. The non-selective NOS inhibitor (N(G)-methyl-L-arginine (L-NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.     

一氧化氮合酶FMN结合结构域的电子传递及调控机制研究现状

生物体内NO是在一氧化氮合酶（mitric oxide synthase,NOS）催化下生成的,NOS的结构包括C端还原酶域和N端加氧酶域。还原酶域中的FMN结合结构域既可接受来自NADPH-FAD结构域的电子,又可作为提供电子的供体,在调控催化过程中的电子传递方面发挥着重要作用。主要从FMN结合结构域的构象平衡及其对不同亚型NOS的动力学差异的贡献、FMN结合结构域自身的电荷性质以及NOS中其他结构域对FMN结构域的功能调控三个方面进行了论述,以期揭示NOS独特的电子传递催化机制。     

Constitutive expression of mammalian nitric oxide synthase in tobacco plants triggers disease resistance to pathogens

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H₂O₂, with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.     

甲醛炎性痛增强大鼠海马NOS表达和NO生成

目的：观察甲醛炎性痛过程中大鼠痛行为、海马一氧化氮合酶（NOS）活性及一氧化氮（NO）含量的变化以及变化的时程及区域特征。方法：采用辐射热甩尾法测定大鼠痛阈变化;采用NADPH—d组织化学法和硝酸还原酶法分别测定大鼠海马NOS表达和No含量。结果：皮下注射甲醛溶液后,大鼠出现伤害性感受反应及痛阈降低。注射甲醛后6h,海马CA1、CA2～3区及DG区NOS阳性细胞数目、阳性细胞染色深度均显著增加。海马NO含量亦显著增加;注射甲醛后12h时这些改变最为显著,48h时恢复至对照组水平。结论：甲醛炎性痛可诱导海马NOS活性增强及NO生成增多．这种改变可发生在海马各区．并具有一定的时程特征。     

诱导型一氧化氮合酶介导β淀粉样蛋白在体神经毒性

目的：探讨一氧化氮合酶（NOS）及一氧化氮（NO）在β淀粉样蛋白（Aβ）神经毒性和Alzheimer病（AD）发病机制中的介导作用。方法：应用行为学及病理学方法,观察海马注射Aβ1－40对大鼠Y迷宫学习记忆的影响及对局部神经元的损伤作用;观察特异性诱导型一氧化氮合酶（iNOS)抑制剂胍氢酶（AG）及特异性神经元型一氧化氮合酶(nNOS)抑制剂7－硝基吲哚（7－NI）腹腔注射对海马内注射Aβ1－40神经毒性的干预,结果：海马注射Aβ1－40后,大鼠Y迷宫学习记忆能力及海马局部神经元明显受损,特异性iNOS抑制剂AG能够阻止Aβ1－40海马注射对大鼠学习记忆和局部神经元的损伤作用,而特异性nNOS抑制剂7－NI无此干预效应。结论：iNOS/NO参与了在体条件下对Aβ神经毒性的介导,在AD发病机制中具有重要作用。     

诱导型一氧化氮合酶的激活与血压的关系

本实验旨在探讨诱导型一氧化氮合酶（iNOS)的激活与血压之间的关系,三组SD大鼠分别静脉输注不同浓度（0．3％,4％及8％）NaCl溶液以使其处于不同的血压水平,运用同位素标记的L－精氨酸转换成L－Citrulline 的转换率变化及Greiss反应,分别测定不同血压时iNOS的活性及NO的生成量,另四组大鼠包括正常Wistar,正常SD,高盐诱导的高血压（NaHR)及自发性高血压大鼠（SHR）,经测定血压后,取主动脉血管并以Western印迹印交法测定其iNOS蛋白水平,结果表明,血压较低时,SD大鼠iNOS活基本没有改变,而在输入4％和8％NaCl并处于较高血压水平的SD大鼠,其iNOS活性及NO生存均明显升高,。此外Western 印迹表明,两种高血压大鼠主动脉组织iNOS蛋白水平均较正常Wistar及正常SD大鼠高,密度扫描表明,NaHR及SHR主动脉组织iNOS蛋白分别较正常SD大鼠及正常Wistar大鼠升高149％及261％,这一结果提示,诱导型一氧化氮合酶是血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有重要的代偿调节作用,除细胞因子,细菌产物等之外,血压也是调节iNOS表达及活性的重要因素之一。     

The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump

The Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here, we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons.     

Decreased viability of nitric oxide synthase double knockout mice

Nitric oxide acts as an important intracellular messenger in a variety of systems, including reproduction. Previous studies have shown the importance of nitric oxide in embryo development. NO is produced from l-arginine by the enzyme, nitric oxide synthase (NOS), which has three isoforms: endothelial (NOS3), neural (NOS1), and inducible (NOS2). We hypothesize that, because of the importance of NOS in development, at least two NOS isoforms are required in order for normal embryo development to occur. Through the generation of NOS3/NOS2, NOS3/NOS1, and NOS2/NOS1 double knockout mice, we found that while litter size remains unchanged, the expected number of generated double knockout mice varies significantly from what would be predicted by Mendelian genetics. Estrous cycles were similar for both DKO and the wild-type mice, and both groups were deemed fertile by their ability to mate with wild-type (CD-1) mice. Together, these results lead us to conclude that the lack of two NOS isoforms leads to a decreased viability in mice because of a developmental problem in the double knockout embryo.     

An improved histochemical method for measuring nitric oxide synthase activity

The previous quantitative histochemical method for measuring nitric oxide synthase (NOS) activity in tissue sections involved the loss of about 15 per cent of the NOS, presumably from the section into the reaction medium. Two changes are now described. The first is concerned with the preparation in the laboratory of the active reagent, lead ammonium citrate/acetate (LACA). The second change involves an improvement of the procedure for measuring NOS activity. The new method appears to retain all the measurable NOS activity inside the section. Copyright © 1999 John Wiley & Sons, Ltd.     

Production of nitric oxide under Ultraviolet-B irradiation is mediated by hydrogen peroxide through activation of nitric oxide synthase

Excised leaves of kidney bean (Phaseolus vulgaris) were used to investigate the mechanism of NO generation under UV-B stress. We showed that two signaling molecules, NO and H₂O₂, were produced in the irradiated leaves. NO release was blocked by LNNA, an inhibitor of NOS. Application of CAT (EC 1.11.1.6) not only effectively eliminated H₂O₂ in the leaves, but also inhibited the activity of NOS and the emission of NO. In contrast, treatment with exogenous H₂O₂ increased both of those events. Therefore, we suggest that, under UV-B stress, NO production is mediated by H₂O₂ through greater NOS activity.