1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

A series of 1,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase. A variety of flexible and restricted basic amine side chain substitutions was explored at the 1-position of the indole ring, while keeping the amidine group fixed at the 5-position. Compounds having N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and 13) and N-(1-(1-methylazepan-4-yl)- side chains (14, 15, (-)-15 and (+)-15) showed increased inhibitory activity for the human nNOS isoform and selectivity over eNOS and iNOS isoforms. The most potent compound of the series for human nNOS (IC(50)=0.02 μM) (S)-12 showed very good selectivity over the eNOS (eNOS/nNOS=96-fold) and iNOS (iNOS/nNOS=850-fold) isoforms.     

1,6-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

A series of 1,6-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). By varying the basic amine side chain at the 1-position of the indole ring, several potent and selective inhibitors of human neuronal NOS were identified. In general compounds with bulkier side chains displayed increased selectivity for nNOS over eNOS and iNOS isoforms. One of the compounds, (R)-8 was shown to reduce tactile hyperesthesia (allodynia) after oral administration (30 mg/kg) in an in vivo rat model of dural inflammation relevant to migraine pain.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

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。     

Chiral linkers to improve selectivity of double-headed neuronal nitric oxide synthase inhibitors

To develop potent and selective nNOS inhibitors, new double-headed molecules with chiral linkers that derive from natural amino acids or their derivatives have been designed. The new structures contain two ether bonds, which greatly simplifies the synthesis and accelerates structure optimization. Inhibitor (R)-6b exhibits a potency of 32 nM against nNOS and is 475 and 244 more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 6b and the two heme propionates in nNOS, but not eNOS, is the structural basis for its high selectivity. This work demonstrates the importance of stereochemistry in this class of molecules, which significantly influences the potency and selectivity of the inhibitors. The structure–activity information gathered here provides a guide for future structure optimization.     

L-精氨酸脂质体对慢性低氧高二氧化碳大鼠一氧化氮及其合酶基因表达的影响

目的:探讨L-精氨酸脂质体(L-Arg)对慢性低氧高二氧化碳大鼠一氧化氮代谢及内皮型一氧化氮合酶基因(ecNOSmRNA)表达的影响.方法:将40只健康雄性SD大鼠随机分为4组,正常对照组(NC组),低氧高二氧化碳4周组(HH组),低氧高二氧化碳加L-Arg4周组(HL组)和低氧高二氧化碳加L-Arg脂质体4周组(HP组).采用硝酸还原酶法测定血浆一氧化氮代谢产物(NOx-)含量,组织原位杂交、光镜和图像分析等方法观察肺细小动脉显微结构以及肺细小动脉ecNOSmRNA的表达变化.结果:①HH组mPAP和RV/(LV S)高于NC组,HP组均明显低于HL组与HH组;②HH组的血浆NO含量显著低于NC组(P＜0.01),HL组与HP组均明显高于HH组(P＜0.01);③HH组的肺细小动脉ecNOSmRNA的平均吸光度值低于NC组(P＜0.05);而HP组明显高于HH组和HL组(P＜0.01);④HP组的肺细小动脉管壁面积/管总面积比值(WA/TA)和中膜厚度(PAMT)均明显低于HH组(P＜0.01),且低于HL组(P＜0.05).结论:L-Arg脂质体较L-Arg有更明显的降低慢性低氧高二氧化碳大鼠肺动脉压和减轻肺血管重建的治疗作用,其机制可能与L-Arg脂质体促进L-Arg的跨膜转运有关.     

Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity

The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K_m for arginine 2.8, 2.0 and 2.3 μM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

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。     

Nω-硝基-L-精氨酸甲酯抑制吗啡镇痛耐受大鼠中脑和脊髓一氧化氮合酶/N-甲基-D-天冬氨酸受体表达上调

采用热甩尾测痛法观察全身应用非特异性一氧化氮合酶（nitric oxide synthase,NOS）抑制剂——N^ω-硝基-L-精氨酸甲酯（L-NAME）对吗啡镇痛耐受形成的影响,并通过观察脊髓和中脑神经元型NOS（nNOS）和N-甲基-D-天冬氨酸（NMDA）受体亚单位表达的变化来阐释NO／NMDA受体在吗啡镇痛耐受形成中的作用。将36只健康成年Sprague-Dawley大鼠平均分为6组（每组6只）：1组为对照组,皮下注射生理盐水1ml;2、3、4、5和6组为处理组,分别皮下注射L-NAME10mg／kg、L-NAME20mg／kg、吗啡10mg／kg、L-NAME10mg／kg＋吗啡10mg／kg、L-NAME20mg／kg＋吗啡10mg／kg,每天2次。在注射前测量大鼠的热甩尾潜伏期（tail-flick latency,TFL）基础值,随后每天第一次给药50min后测量其TFL。第8天最后一次给药80min后（除2组和5组之外）断头取脊髓和中脑,采用RT-PCR技术测量nNOS以及NMDA受体1A（NR1A）和2A（NR2A）亚单位的表达。结果显示,2组大鼠第1天至第7天的TFL与基础值相比无显著差异;3组第7天时的TFL仍显著高于基础值;4组的TFL在第1天时最高,第2至第6天期间逐渐降低,第6天时与基础值相比无显著差异：5组的TFL在实验过程中呈下降趋势,虽然第7天时较第1天有所降低,但是仍然显著高于基础值;6组的TFL变化趋势与5组相同。PT—PCR分析结果显示,与1组相比,3组脊髓和中脑的nNOS mRNA表达显著降低,但NR1A mRNA和NR2A mRNA表达无显著改变;4组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著高于1组。与4组相比,6组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著降低。结果提示,吗啡镇痛耐受大鼠脊髓和中脑的nNOS和NMDA受体表达增加,联合应用L—NAME可抑制长期应用吗啡所致的nNOS表达增加和NMDA受体上调,延缓吗啡镇痛耐受的形成。本研究结果提示,脊髓和中脑的NO／NMDA受体与吗啡镇痛耐受形成密切相关。     

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。     

Activation of pig oocytes using nitric oxide donors

Nitric oxide (NO) plays an important role in intracellular signaling, but its role during the activation of mammalian oocytes is little understood. In our study, in vitro matured pig oocytes were cultured with NO-donors-S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitropruside (SNP). These treatments were able to induce parthenogenetic activation of pig oocytes matured in vitro. The specificity of this effect was confirmed by the activation of oocytes by exogenous endothelial nitric oxide synthase (eNOS) microinjected in the oocyte with its activator calmodulin. Relatively long exposure (10 hr) is needed for activation of pig oocytes with 2.0 mM SNAP. An active NOS is necessary for the NO-dependent activation of pig oocytes because NOS inhibitors L-NMMA or L-NAME are able to inhibit activation of oocytes with NO-donor SNAP. On the basis of our data, we conclude that the NO-dependent activating stimulus seems inadequate because it did not induce the exocytosis of cortical granules. Also, the cleavage of parthenogenetic embryos was very low, and embryos did not develop beyond the stage of eight blastomeres.     

Role of nitric oxide synthase activity in experimental ischemic acute renal failure in rats

To determine the role of nitric oxide (NO) in acute renal failure (ARF), we have studied the time course change activities to activity of nitric oxide synthase (NOS) isoform activities, both calcium dependent and independent NOS, in experimental ischemic ARF. We have also analyzed change activities to activity of the NOS activities in both renal cortex and medulla. Male SD rats (n = 5) were inducted to ARF by ischemia-reperfusion injury and divided into the following groups; Control group (sham operation), Day 0 group, (measurement performed on that day of operation), Day 1 group, (measurement performed one day after induction of ARF), Day 3 group and Day 7 group. Measurement of NOS activity was based on the following principles; NO is synthesized from arginine by nitric oxide synthase (NOS) and NO is converted to NO₂ ^–/NO₃ ^– (NOx) by oxidation. Detection of the final metabolite of NO, NOx was done using flow injection method (Griess reaction). The results were, (1) calcium dependent NOS activity in the cortex and medulla decreased, however it increased in the recovery period in the renal cortex (Cortex; Control, 0.941 ± 0.765, D0, 0.382 ± 0.271, D1, 0.118 ± 0.353, D3, 2.030 ± 0.235, D7, 3.588 ± 2.706, Medulla; Control, 1.469 ± 0.531, D0, 0.766 ± 0.156, D1, 0.828 ± 0.187, D3, 2.078 ± 0.094, D7, 1.289 ± 0.313 mol NOx produced/mg protein/30 min). (2) On the other hand, iNOS activity increased in the early phase of ARF, both in the cortex and medulla, but returned to control values during the recovery phase in cortex and was maintained at higher levels in the medulla (Cortex; Control, 0.333 ± 0.250, D0, 0.583 ± 0.428, D1, 1.167 ± 0.262, D3, 0.250 ± 0.077, D7, 0.452 ± 0.292, Medulla; Control, 0.139 ± 0.169, D0, 0.279 ± 0.070, D1, 1.140 ± 0.226, D3, 0.452 ± 0.048, D7, 0.625 ± 0.048 mol NOx produced/mg protein/30 min). These findings suggest that the role of NOS in ARF are different for the different NOS isoforms and have anatomic heterogeneity.     

Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

Inhibition of nitric oxide synthase (NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos

Aluminum (Al) is toxic to plants when solubilized into Al(3+) in acidic soils, and becomes a major factor limiting plant growth. However, the primary cause for Al toxicity remains unknown. Nitric oxide (NO) is an important signaling molecule modulating numerous physiological processes in plants. Here, we investigated the role of NO in Al toxicity to Hibiscus moscheutos. Exposure of H. moscheutos to Al(3+) led to a rapid inhibition of root elongation, and the inhibitory effect was alleviated by NO donor sodium nitroprusside (SNP). NO scavenger and inhibitors of NO synthase (NOS) and nitrate reductase had a similar inhibitory effect on root elongation. The inhibition of root elongation by these treatments was ameliorated by SNP. Aluminum inhibited activity of NOS and reduced endogenous NO concentrations. The alleviation of inhibition of root elongation induced by Al, NO scavenger and NOS inhibitor was correlated with endogenous NO concentrations in root apical cells, suggesting that reduction of endogenous NO concentrations resulting from inhibition of NOS activity could underpin Al-induced arrest of root elongation in H. moscheutos.     

Enhanced expression of neuronal nitric oxide synthase in islets of exercise-trained rats

It is well known that glucose-stimulated insulin secretion (GSIS) decreases after exercise training. In the present study, we investigated the effects of exercise training (9 weeks of running) on the activity of glucokinase (GK), the production of nitric oxide (NO), and the protein expressions of both glucose transporter-2 (GLUT-2) and NO synthase (NOS) in rat pancreatic islets. Exercise training significantly reduced GSIS, with decreases in GK activity and GLUT-2 protein expression. The NO releases and cGMP contents were higher in the islets of trained rats than in those of control rats. Exercise training enhanced cNOS activity, the protein expression of both neuronal nitric oxide synthase (nNOS) and calmodulin, and NADPH-cytochrome c reductase activity in the homogenates of islets. Thus, exercise training-induced reduction of GSIS would result from, at least in part, decreases in both glucose entry and the first step in glycolytic utilization of glucose. Moreover, exercise training could enhance the protein expression of nNOS, which in turn enhances two catalytic activities of nNOS, an NO production and a cytochrome c reductase activity.     

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。     

不同亚型一氧化氮合酶在脑缺血/再灌注早期的表达变化

目的:观察脑缺血/再灌注(CI/R)早期缺血区脑组织的内皮型一氧化氮合酶(eNOS)与神经型一氧化氮合酶(nNOS)表达的变化。方法:健康wistar大鼠60只,体重200～280g,由中国医科大学动物中心提供,雌雄各半。随机分为6组(n=10):假手术组、缺血1h组、缺血2h组、再灌注0.5h组、再灌注1h组、再灌注2h组。采用线栓法制作大鼠CI/R模型,免疫组化方法检测缺血区脑组织的eNOS与nNOS蛋白表达情况。结果:与假手术组比较,CI/R模型大鼠脑组织血管内皮细胞内eNOS表达在缺血1h内升高,之后到再灌注2h内持续降低。而nNOS的表达在缺血到再灌注2h内持续上升。结论:CI/R模型中缺血区脑组织的eNOS与nNOS的变化趋势不同,表明一氧化氮在缺血性脑损伤病理过程的作用与一氧化氮合酶亚型的变化有关。     

Cellular biosensing system for assessing immunomodulating effects on the inducible nitric oxide synthase (iNOS) cascade

A cellular biosensing system has been constructed to assess the biological safety/toxicity of chemicals. Detection of nitric oxide (NO) by the cellular biosensing system was used as a readout for assessing the immunomodulating effects of various chemicals, because some are known to induce NO synthase (iNOS) activity thereby increasing NO production. The macrophage-like cell line, RAW264.7, was cultured on the electrode coated with a polyion complex layer. The potent immune activating abilities of lipopolysaccharide could be verified by the cellular biosensing system: NO release from cells was detected within 600 ms by double potential step chronoamperometry.     

电针对去卵巢大鼠学习记忆能力及海马神经元型一氧化氮合酶mRNA表达的影响

目的研究电针对去卵巢大鼠学习记忆能力及海马神经元型一氧化氮合酶（nNOS）mRNA表达的影响。方法采用卵巢切除大鼠模型,造成低雌激素记忆障碍,去势2周后进行电针刺激,连续治疗3个月。Morris水迷宫测试空间学习记忆能力,酶联免疫吸附分析（ELISA）检测血清雌二醇（E2）浓度,实时荧光定量PCR检测检测nNOSmRNA的相对表达量。结果与假手术组比较,模型组大鼠逃避潜伏期时间明显延长,跨越平台次数明显减少,血清E2浓度和海马nNOSmR—NA表达显著降低（P〈O．01）;与模型组比较,电针组和假电针组治疗后逃避潜伏期缩短,跨越平台次数增加,血清E2浓度和海马nNOSmRNA表达均显著升高,电针组升高更明显（P〈O．01）。结论电针能够提高去卵巢大鼠学习记忆能力,其机制可能与升高体内雌激素浓度上调海马nNOSmRNA的表达有关。