Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis

Increasing evidence suggests that depression may be associated with a lack of hippocampal neurogenesis. It is well established that neuronal nitric oxide synthase (nNOS)-derived NO exerts a negative control on the hippocampal neurogenesis. Using genetic and pharmacological methods, we investigated the roles of nNOS in depression induced by chronic mild stress (CMS) in mice. Hippocampal nNOS over-expression was first observed 4 days and remained elevated 21 and 56 days after exposure to CMS. The mice exposed to CMS exhibited behavioral changes typical of depression, and impaired neurogenesis in the hippocampus. The CMS-induced behavioral despair and hippocampal neurogenesis impairment were prevented and reversed in the null mutant mice lacking nNOS gene (nNOS−/−) and in the mice receiving nNOS inhibitor. Disrupting hippocampal neurogenesis blocked the antidepressant effect of nNOS inhibition. Moreover, nNOS−/− mice exhibited antidepressant-like properties. Our findings suggest that nNOS over-expression in the hippocampus is essential for chronic stress-induced depression and inhibiting nNOS signaling in brain may represent a novel approach for the treatment of depressive disorders.     

Blockade of L-type voltage-gated Ca channel inhibits ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.     

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。     

Nitric oxide down-regulates caveolin-1 expression in rat brains during focal cerebral ischemia and reperfusion injury

As a signalling molecule of the integral membrane protein family, caveolin participates in cellular signal transduction via interaction with other signalling molecules. The nature of interaction between nitric oxide (NO) and caveolin in the brain, however, remains largely unknown. In this study we investigated the role(s) of NO in regulating caveolin-1 expression in rat ischemic brains with middle cerebral artery occlusion (MCAO). Exposure to 1 h ischemia induced the increases in neuronal nitric oxide synthase (nNOS) and NO concentration with concurrent down-regulation of caveolin-1 expression in the ischemic core of rat brains. Subsequent 24 h or more reperfusion time led to an increase in inducible NOS (iNOS) expression and NO production, as well as a decline of caveolin-1 protein at the core and penumbra of the ischemic brain. Afterwards, NOS inhibitors and an NO donor were utilized to clarify the link between NO production and caveolin-1 expression in the rats with 1 h ischemia plus 24 h reperfusion. N(G)-nitro-l-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), N(6)-(1-iminoethyl)-lysine (NIL, an iNOS inhibitor), and 7-nitroindazole (7-NI, a nNOS inhibitor) prevented the loss of caveolin-1 in the core and penumbra of the ischemic brain, whereas l-N(5)-(1-iminoethyl)-ornithine (L-NIO, an endothelial NOS inhibitor) showed less effect than the other NOS inhibitors. S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) down-regulated the expression of caveolin-1 protein in normal and ischemic brains. These results, when taken together, suggest that NO modulates the expression of caveolin-1 in the brain and that the loss of caveolin-1 is associated with NO production in the ischemic brain.     

Negative regulation of neurogenesis and spatial memory by NR2B-containing NMDA receptors

Several lines of evidence suggest involvement of NMDA receptors (NMDARs) in the regulation of neurogenesis in adults and the formation of spatial memory. Functional properties of NMDARs are strongly influenced by the type of NR2 subunits incorporated. In adult forebrain regions such as the hippocampus and cortex, only NR2A and NR2B subunits are available to form the receptor complex with NR1 subunit. NR2B is predominant NR2 subunit in any of rat or human neural stem cells (NSCs). Thus, we suppose that NR2B-containing NMDAR should be critical in regulating adult neurogenesis, and thereby playing a role in the formation of spatial memory. In the cultured NSCs derived from the embryonic brain of rats, NR2B subunit-specific NMDAR antagonist Ro25-6981 increased cell proliferation, whereas MK-801, non-selective open-channel blocker of NMDARs, inhibited cell proliferation. Blockade of NR2B-containing NMDAR stimulated neurogenesis in the adult hippocampus and facilitated the formation of spatial memory. The enhanced spatial memory dropped back to base level when the NR2B antagonist-induced neurogenesis was neutralized by 3'-azido-deoxythymidine, a telomerase inhibitor. In addition, blockade of NR2B inhibited neuronal nitric oxide synthase (nNOS) enzymatic activity. In null mutant mice lacking nNOS gene (nNOS−/−), the effects of NR2B antagonist on neurogenesis disappeared. Moreover, nitric oxide donor DETA/NONOate attenuated and nNOS inhibitor 7-nitroindazole enhanced the effect of Ro 25-6981 on NSCs proliferation. Our findings suggest that NR2B-containing NMDAR subtypes negatively regulate neurogenesis in the adult hippocampus by activating nNOS activity and thereby hinder the formation of spatial memory.     

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。     

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。     

Enhancement of nitric oxide production by association of nitric oxide synthase with N-methyl-D-aspartate receptors via postsynaptic density 95 in genetically engineered Chinese hamster ovary cells: real-time fluorescence imaging using nitric oxide sensitive dye

The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N-methyl-D-aspartate (NMDA) receptors, via postsynaptic density 95 (PSD-95) proteins significantly enhances nitric oxide (NO) production. Real-time single-cell fluorescence imaging was applied to measure both NO production and Ca(2+) influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA-R), nNOS, and PSD-95. We examined the relationship between the rate of NO production and Ca(2+) influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca(2+)-sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD-95 (PSD-95-YC3.1). The presence of PSD-95 enhanced the rate of NO production by 2.3-fold upon stimulation with 100 microm NMDA in CHO1(+) cells (expressing NMDA-R, nNOS and PSD-95) when compared with CHO1(-) cells (expressing NMDA-R and nNOS lacking PSD-95). The presence of nNOS inhibitor or NMDA-R blocker almost completely suppressed this NMDA-stimulated NO production. The Ca(2+) concentration beneath the NMDA-R, [Ca(2+)](NR), was determined to be 5.4 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca(2+) concentration around nNOS, [Ca(2+)](NOS), was obtained over the wide range of [Ca(2+)](NOS). This sigmoidal curve had an EC(50) of approximately 1.2 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.     

Loss of hippocampal neuronal nitric oxide synthase contributes to the stress-related deficit in learning and memory

Nitric oxide (NO) has been involved in many pathophysiological brain processes. However, the exact role of NO in the cognitive deficit associated to chronic stress exposure has not been elucidated. In this study, we investigated the participation of hippocampal NO production and their regulation by protein kinase C (PKC) in the memory impairment induced in mice subjected to chronic mild stress model (CMS). CMS mice showed a poor learning performance in both open field and passive avoidance inhibitory task respect to control mice. Histological studies showed a morphological alteration in the hippocampus of CMS mice. On the other hand, chronic stress induced a diminished NO production by neuronal nitric oxide synthase (nNOS) correlated with an increment in gamma and zeta PKC isoenzymes. Partial restoration of nNOS activity was obtained after PKC activity blockade. NO production by inducible nitric oxide synthase isoform was not detected. The magnitude of oxidative stress, evaluated by reactive oxygen species production, after excitotoxic levels of NMDA was increased in hippocampus of CMS mice. Moreover, ROS formation was higher in the presence of nNOS inhibitor in both control and CMS mice. Finally, treatment of mice with nNOS inhibitors results in behavioural alterations similar to those observed in CMS animals. These findings suggest a novel role for nNOS showing protective activity against insults that trigger tissue toxicity leading to memory impairments.     

一氧化氮对心肌的抑制性保护作用

内皮型与神经型一氧化氮合酶(eNOS,nNOS)在心肌细胞内持续表达,而细胞应激可引起诱导型NOS(iNOS)表达.心肌细胞结构型eNOS与nNOS源性NO,在生理条件下对心肌主要发挥4方面的抑制作用:减缓心肌细胞搏动频率,轻度抑制心肌细胞收缩功能,加速心肌细胞舒张并增加顺应性,以及轻度抑制线粒体电子传递而增强氧利用效...     

Poster Sessions CP13: Hypoxia,Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

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.     

The localization of neuronal nitric oxide synthase may influence its role in neuronal precursor proliferation and synaptic maintenance

Neuronal nitric oxide synthase (nNOS) is implicated in some developmental processes, including neuronal survival, differentiation, and precursor proliferation. To define the roles of nNOS in neuronal development, we utilized the olfactory system as a model. We hypothesized that the role of nNOS may be influenced by its localization. nNOS expression was developmentally regulated in the olfactory system. During early postnatal development, nNOS was expressed in developing neurons in the olfactory epithelium (OE), while in the adult its expression was restricted to periglomerular (PG) cells in the olfactory bulb (OB). At postnatal week 1 (P1W), loss of nNOS due to targeted gene deletion resulted in a decrease in immature neurons in the OE due to decreased proliferation of neuronal precursors. While the pool of neuronal precursors and neurogenesis normalized in the nNOS null mouse by P6W, there was an overgrowth of mitral or tufted cells dendrites and a decreased number of active synapses in the OB. Cyclic GMP (cGMP) immunostaining was reduced in the OE and in the glomeruli of the OB at early postnatal and adult ages, respectively. Our results suggest that nNOS appears necessary for neurogenesis in the OE during early postnatal development and for glomerular organization in the OB in the adult. Thus, the location of nNOS, either within cell bodies or perisynaptically, may influence its developmental role.     

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.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Phosphorylation of neuronal nitric oxide synthase at Ser in the dentate gyrus of rat brain after transient forebrain ischemia

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser⁸⁴⁷ in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser¹⁴¹²in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser¹⁴¹² after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24 h, after 15 min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser¹⁴¹², PKA, and p-PKA at Thr¹⁹⁷ were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser¹⁴¹² significantly increased between 1 and 6 h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser¹⁴¹² and PKA at Thr¹⁹⁷ occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr¹⁹⁷, which then phosphorylates nNOS at Ser¹⁴¹² in the subgranular layer of the dentate gyrus.     

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。     

缺血再灌注对小鼠肠神经丛nNOS 和iNOS表达的影响

目的观察缺血再灌注后小鼠回肠神经型一氧化氮合酶(neuron alnitric oxide synthase,nNOS)和诱导型一氧化氮合酶(induciblenitric oxide synthase,iNOS)的表达,探讨肠缺血再灌注损伤(ischemia-reperfusion injury,IRI)的发生机制。方法采用小鼠肠系膜上动脉缺血再灌注模型,根据不同再灌注时间对小鼠随机分1d组、3d组、5d组、7d组、对照组和假手术组,用SP法检测小鼠回肠nNOS和iNOS的表达情况。结果与对照组和假手术组相比较,nNOS在再灌注1d后开始在肌间神经丛持续高表达(P<0.01);而iNOS在再灌注3d后开始在肌间神经丛持续高表达(P<0.05)。结论nNOS和iNOS在肠缺血再灌注后的表达增强,提示一氧化氮及一氧化氮合酶与肠神经节细胞在缺血再灌注中的损伤有着密切关系。