Neuropeptide Y and its receptor analogs differentially modulate the immunoreactivity for neuronal or endothelial nitric oxide synthase in the rat brain following focal ischemia with reperfusion

Summary An intracerebroventricular (icv) injection of neuropeptide Y (NPY) or [Leu³¹, Pro³⁴]-NPY (non-Y2 receptor agonist) given during middle cerebral artery occlusion (MCAO) increases the infarct volume and nitric oxide (NO) overproduction in the rat brain. An icv injection of NPY3-36 (non-Y1 receptor agonist) has no effects, while BIBP3226 (selective Y1 receptor antagonist) reduces the infarct volume and NO overproduction. This study examined the effects of NPY or its receptor analog on the immunoreactivity (ir) for three isoforms of NO synthase (NOS) following 1h of MCAO and 3h of reperfusion. Focal ischemia/reperfusion led to increased ir for neuronal NOS (nNOS) within the ipsilateral caudate putamen and insular cortex. NPY or [Leu³¹, Pro³⁴]-NPY enhanced but BIBP3226 suppressed such increase in the nNOS-ir. Focal ischemia/reperfusion also led to an ipsilateral increase in extent and/or intensity of the ir for endothelial NOS (eNOS) in the caudate putamen and/or parietal cortex. NPY or [Leu³¹, Pro³⁴]-NPY suppressed but BIBP3226 enhanced such change in the eNOS-ir. NPY3-36 did not consistently influence the nNOS-ir or eNOS-ir following MCAO. Specific ir for inducible NOS was undetectable. These opposing effects of NPY-Y1 receptor activation or inhibition on nNOS and eNOS may lead to harmful or beneficial consequences following ischemia/reperfusion.     

大鼠局灶性脑缺血后一氧化氮合酶基因表达的变化

目的:观察大鼠局灶性脑缺血后3种类型一氧化氮合酶(NOS)mRNA表达的变化.方法:大鼠随机分为正常对照组、缺血后2、6、12、24 h组,以逆转录-聚合酶链反应(RT-PCR)法分别检测缺血脑组织NOS基因表达的变化.结果:脑缺血后eNOS、nNOSmRNA表达增强,分别于2、6 h达高峰;iNOS mRNA表达亦增高,但在缺血后12 h达高峰.结论:大鼠脑缺血早期eNOS和nNOS占主要地位,缺血后期iNOS占主要地位.     

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。     

Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient focal cerebral ischemia in mice

The present observations examined the hypothesis that the iNOS expression in the ischemic penumbra after a transient focal ischemic insult is involved in the recruitment of penumbra into infarction. The middle cerebral artery in mice was occluded for 2 h by an intraluminal filament and then recirculated. The measurement of iNOS activity, iNOS protein formation and NO concentration in the ischemic core and penumbra, and the determination of infarct volume were performed at 6, 12, 24 and 48 h after reperfusion. iNOS protein and iNOS enzymatic activity appeared at 6 h, peaked at 24 h, and declined at 48 h in the penumbra after reperfusion. iNOS protein was not detectable in contralateral area and in sham-operated brains. The time course of iNOS protein, enzymatic activity and NO concentration in the penumbra but not in the core matched the process of infarct maturation. Treatment with iNOS inhibitor aminoguanidine (100 mg.kg(-1), i.p.) at 6 and 12 h after reperfusion inhibited iNOS activity by 88.0 +/- 10.4% and reduced NO concentration by 48.5 +/- 8.3% in the penumbra, and lessened infarct size by 48.8 +/- 7.2%. The iNOS activity and NO level in the core were not affected by the administration of aminoguanidine. These results suggest that iNOS expression in the ischemic penumbra is involved in the recruitment of penumbra into infarction and thereby contributing to the enlargement of infarct.     

15-羟二十碳四烯酸下调肺动脉内皮型一氧化氮合酶的活性

15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-HETE）在低氧性肺血管收缩中起着重要作用,低氧肺动脉高压下调内皮型。氧化氮合酶（endothelial nitric oxide synthase,eNOS）,使一氧化氮（nitric oxide,NO）的产量下降,但目前尚无关于15-HETE与eNOS／NO相互作用研究的报道。我们通过Wistar大鼠肺动脉环张力、牛肺动脉内皮细胞NO产量、总eNOS表达及eNOS磷酸化测定等方法对15-HETE与eNOS／NO的相互作用进行研究。首先分离人鼠肺动脉,分为eNOS抑制剂L-NAME组（0．1mmol／L）、去缸管内皮组与内皮完整组,用15-HETE作用夫鼠离体肺动脉环,测定肺动脉张力。结果表明,L-NAME组、去除内皮组与内皮完整组分别比较,15-HETE对血管的收缩作用增强,且都有统计学意义（P〈0．05）。培养牛肺动脉内皮细胞,分别用15-HETE、15-脂氧酶（15-lipoxygenase,15-LO）抑制剂[（cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate,CDC）和（nordihydroguiairetic acid,YDGA）]处理细胞,通过Greiss方法检测亚硝酸盐含量,间接测定NO产量,与对照组比较,1μmol／L 15-HETE明显降低肺动脉内皮细胞NO水平（P〈0．05）,10μmol／L CDC和0．1mmol／L NDGA显著增加NO水平（分别是P〈0．05,P〈0．01）;通过Western blot检测不同时间（5,10,15,20,30,60min）eNOS的表达情况,结果显示,15-HETE的不同作用时间,没有引起eNOS表达的明显不同;用苏氨酸495位点磷酸化eNOS（Thr495）抗体进行免疫沉淀,再用总eNOS抗体和15-LO抗体通过Western blot检测磷酸化型含量,问接测定eNOS活性,结果表明15-HETE增强Thr495磷酸化型eNOS含量。由于Thr495为eNOS抑制性磷酸化位点,因此15-HETE降低eNOS活性。这些数据表明：15-HETE的缩血管作用有eNOS／NO参与,15-HETE可以通过磷酸化Thr495位点降低eNOS活性,并且首次发现磷酸化eNOS（Thr495）和15-LO之间存在蛋白质相互作用。     

Hypothermic storage of coronary endothelial cells reduces nitric oxide synthase activity and expression

Preservation with University of Wisconsin (UW) solution has been implicated in coronary artery endothelial damage and loss of endothelium-dependent vasodilatation. Therefore, the objective of this study was to investigate the effect of this solution on basal nitric oxide (NO) release from porcine coronary endothelial cells (CEC). Cultures were exposed to cold (4 degrees C) storage in UW solution for 6, 8 and 12 h. Parallel cultures were incubated with control medium at 37 degrees C. After treatment, NO release was evaluated by nitrite production, a stable metabolite of NO. Activity of the constitutive endothelial nitric oxide synthase (eNOS) was measured by the conversion [3H]-l-arginine to [3H]-l-citrulline and eNOS protein expression by Western blotting. Nitrite production by control cells was augmented with increasing times of incubation, whereas no change was observed in those cultures preserved with UW solution. Activity of eNOS was significantly decreased compared to the respective control group by cold storage of cells for longer periods than 6 h. Such decrease was correlated with a diminished eNOS protein expression in CEC preserved with UW solution after 8- and 12-h storage. These results suggest that prolonged hypothermic storage of CEC with UW solution does not preserve basal NO release because of a certain loss of eNOS protein, which may contribute to the reported injury of heart transplants after long-term preservation.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.     

Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.     

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.     

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.     

瘦露螽配子发生中一氧化氮合酶的分布

利用还原型烟酰胺腺嘌呤二核苷酸(NADPH)黄递酶组织化学方法,对瘦露螽Phaneroptera gracilis Burmeister配子发生中一氧化氮合酶(nitric oxide synthase, NOS)分布进行了定位研究。结果表明, 一氧化氮合酶阳性反应发生在瘦露螽精子发生中的各级生精细胞的胞质中,成熟精子呈阴性。各级未成熟卵母细胞胞质均呈一氧化氮合酶阳性反应,胞质着色为深蓝黑色,核区不明显。随着卵黄颗粒的逐渐形成,胞质中的一氧化氮合酶阳性产物逐渐减少,直到卵黄颗粒完全形成。卵泡细胞在卵黄颗粒形成之前呈一氧化氮合酶阴性反应,在卵黄颗粒完成后,卵泡细胞的胞质中开始呈一氧化氮合酶阳性反应,直至卵壳的形成。提示一氧化氮参与了瘦露螽配子发生。     

组胺对肺动脉内皮细胞一氧化氮合酶基因表达的影响

本实验研究了组胺对原代培养的肺动脉内皮细胞一氧化氮合酶(nitric oxidCsynthase,NOS)基因表达的影响及分子机制。采用RT-PCR和免疫印迹技术分别检测mRNA和蛋白质的表达水平,用荧光素酶报告基因实验检测eNOS基因转录起始点上游长1.6-kb的启动子活性,用硝酸还原酶法检测NO的产量。结果发现,组胺增强eNOS表达,呈浓度和时间依赖性,10μmol/L组胺处理肺动脉内皮细胞24h可使eNOS mRNA和蛋白质的表达达到高峰,eNOS mRNA水平为正常对照组的160.8±12.2%(P<0.05),蛋白质水平为正常对照组的136.2±11.2%(P<0.05)。特异性CaMK Ⅱ抑制剂KN-93可抑制组胺的这一效应,表明组胺可通过激活CaMK Ⅱ增强肺动脉内皮细胞eNOS基因的表达。报告基因实验表明,10μmol/L组胺处理24h后肺动脉内皮细胞eNOS基因启动子的活性增强,为正常对照组的148.2±33.7%(P<0.05)。组胺可使肺动脉内皮细胞产生NO增加。这些结果表明组胺在转录水平增强肺动脉内皮细胞eNOS基因的表达,并使细胞产生NO增加,这可能是组胺调节肺血管张力的机制之一。CaMK Ⅱ可能是组胺增强肺动脉内皮细胞eNOS基因表达的途径之一。     

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.     

Inhibition of neuronal nitric oxide synthase activity by N1-acetyl-5-methoxykynuramine, a brain metabolite of melatonin

We assessed the effects of melatonin, N(1)-acetyl-N (2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK) on neuronal nitric oxide synthase (nNOS) activity in vitro and in rat striatum in vivo. Melatonin and AMK (10(-11)-10(-3) m), but not AFMK, inhibited nNOS activity in vitro in a dose-response manner. The IC(50) value for AMK (70 microm) was significantly lower than for melatonin (>1 mm). A 20% nNOS inhibition was reached with either 10(-9) m melatonin or 10(-11) m AMK. AMK inhibits nNOS by a non-competitive mechanism through its binding to Ca(2+)-calmodulin (CaCaM). The inhibition of nNOS elicited by melatonin, but not by AMK, was blocked with 0.05 mm norharmane, an indoleamine-2,3-dioxygenase inhibitor. In vivo, the potency of AMK to inhibit nNOS activity was higher than that of melatonin, as a 25% reduction in rat striatal nNOS activity was found after the administration of either 10 mg/kg of AMK or 20 mg/kg of melatonin. Also, in vivo, the administration of norharmane blocked the inhibition of nNOS produced by melatonin administration, but not the inhibition produced by AMK. These data reveal that AMK rather than melatonin is the active metabolite against nNOS, which may be inhibited by physiological levels of AMK in the rat striatum.     

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。     

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.     

Ultrastructural localization of NADPH-diaphorase and colocalization of nitric oxide synthase in endothelial cells of the rabbit aorta

This is the first report on the ultrastructural pattern of distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in endothelial cells, using the rabbit aorta, and its colocalization with the neuronal isoform (type I) of nitric oxide synthase. About 30% of the endothelial cells showed a positive reaction for NADPH-d compared to about 6% for nitric oxide synthase immunoreactivity. Simultaneous double histochemical-immunocytochemical labelling procedures indicate that all of the cells displaying nitric oxide synthase-positive reactivity also contained NADPH-d; the remainder of NADPH-d-positive endothelial cells were negative for this isoform of nitric oxide synthase. Nitric oxide synthase-immunogold labelling was mostly associated with free ribosomes, while NADPH-d activity was distributed largely in patches in the cytoplasm and in association with the cell membrane.     

Retinoic acid decreases nitric oxide production in endothelial cells: a role of phosphorylation of endothelial nitric oxide synthase at Ser(1179)

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser(1179) phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser(116) or eNOS-Thr(497). Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser(1179) phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARalpha antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser(1179) phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.