大鼠尾核内一氧化氮对apelin受体mRNA表达的影响

目的研究大鼠尾核内一氧化氮（nitric oxide,NO）的作用机理及其与apelin受体mRNA表达的相关性。方法大鼠尾核内微量注射NO前体L-精氨酸（L-Arg）、N^G-硝基-L-精氨酸甲酯（N^G-nitro-L-arginine methyl ester,L-NAME）和生理盐水（NS）,应用逆转录-聚合酶链反应（RT-PCR）检测大鼠尾核给药4、8、12、24及48h后一氧化氮合酶（neuronal nitric oxide synthase,nNOS）mRNA和apelin受体mRNA表达的变化及二者的相关性。结果注射L-Arg后大鼠尾核nNOS和apelin受体的mRNA表达明显升高,注射L-NAME后大鼠尾核nNOS和apelin受体mRNA表达明显降低,且均有统计学意义。注射L-Arg和L-NAME后,nNOSmRNA和apelin受体mRNA的表达呈正相关。结论在中枢神经系统,尤其是在尾核中,NOS的活性是NO作为神经递质或调质参与包括中枢痛觉调制作用在内的多种生物学作用的关键因素之一,尾核内NO的神经生物学作用可能与apelin受体作用相关。     

中枢神经系统一氧化氮对大鼠的痛觉调制作用

以钾离子透入引起大鼠甩尾的电流强度(mA)作为痛反应指标,采用侧脑室微量注射L-精氨酸(L-Arg)、亚甲基蓝(MB)等,观察大鼠痛阈的变化,分析探讨中枢神经系统中一氧化氮(NO)对大鼠痛觉的调制作用.结果显示:大鼠侧脑室微量注射NO前体及供体物质L-Arg和硝普钠(SNP)均引起明显的痛敏效应.微量注射MB和L-NAME后大鼠痛阈升高非常显著.侧脑室微量注射MB和L-Arg混合液后,大鼠痛阈较单纯注射MB组表现出明显降低的趋势,但与L-Arg组相比大鼠痛阈升高明显.提示:提高中枢内NO水平具有明显的痛敏效应,而降低中枢神经系统NO水平表现显著镇痛作用.中枢神经系统NO对大鼠痛觉的调制作用至少部分是通过NO-cGMP途径实现的.     

肾血管性高血压大鼠头端腹外侧延髓一氧化氮合酶表达的变化

采用免疫组织化学方法观察两肾一夹肾血管性高血压大鼠头端腹外侧延髓(RVLM)神经元型一氧化氮合酶(nNOS)表达的变化.肾血管性高血压大鼠RVLM的nNOS表达增加,说明高血压时该区NO活性加强.NO的前体L-Arg能增强nNOS的表达,nNOS抑制剂L-NAME则抑制nNOS的表达.各组nNOS表达变化的特点在高血压4 w和7 w的动物相同.同一组中高血压晚期(7 w)的nNOS表达和早期(4 w)比较略有增加,但无统计学差异.     

多次鞘内注射不同剂量左旋精氨酸对福尔马林诱导的大鼠行为痛反应的影响(英文)

目的探讨不同剂量的一氧化氮(NO)对炎性痛的作用及其机制。方法大鼠鞘内每天注入一氧化氮的前体左旋精氨酸(L-Arg)10μg(低剂量组),250μg(高剂量组)或生理盐水(对照组),共持续4天。随后在大鼠右后爪掌皮下注射2%甲醛溶液(100μL),并在之后的1h内观察大鼠舔咬后爪的痛行为反应,4h后用免疫细胞化学技术检测大鼠脊髓背角c-Fos蛋白和神经元型一氧化氮合酶(nNOS)的表达。结果福尔马林引起的大鼠舔咬后爪的痛行为反应有两个时相。第一相的急性痛行为反应在各组间无明显差异。对于第二相的慢性痛行为反应累计时间以及脊髓背角c-Fos和nNOS的表达水平,低剂量组较对照组显著降低,而高剂量组较对照组显著升高。结论不同剂量的一氧化氮对脊髓痛觉的调制作用具有双面性,小剂量具有镇痛作用而大剂量具有促痛作用。     

L-NAME加强麻醉大鼠低血压诱发的催产素释放作用

取戊巴比妥麻醉大鼠向侧脑室内分别注射一氧化氮(NO)合酶的底物L-精氨酸和NO合酶抑制剂NG-硝基-L-精氨酸甲酯(L-NAME),用放射免疫法测定血浆中催产素(OT)水平.结果:侧脑室内注射L-精氨酸(100 g/L,10 霯,n=8)和L-NAME(54.0 g/L,5 霯,n=12),对OT的基础分泌无明显影响;侧脑室内注射5 霯 L-NAME(剂量1:27.0 g/L,n=9;剂量2:54.0 g/L,n=8),可进一步增强静脉输注硝普钠引起低血压所诱导的OT分泌升高反应.结果表明L-NAME能加强低血压诱发的OT反射性释放作用,提示NO可能是OT反射性释放的抑制因子.     

一氧化氮合酶在脑缺血再灌注中的双重作用

目的 探讨短暂脑缺血再灌注后大鼠脑内3型一氧化氮合酶(nitric oxide synthase，NOS)的表达及作用，为脑缺血治疗提供理论依据。方法 采用免疫组织化学方法，用3型NOS的多克隆抗体检测大鼠局灶性脑缺血2h再灌注15min及22h NOS在脑内的表达情况。结果 大鼠脑缺血2h再灌注15min，在脑缺血边缘区的血管壁及神经细胞出现内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)上调表达；脑缺血2h再灌注22h，在脑梗死区内表达神经元型一氧化氮合酶(neuronal mitric oxide synthase，nNOS)的神经细胞减少，并出现表达诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)的胶质细胞，同时梗死边缘区血管及神经细胞出现eNOS及iNOS的上调表达。结论 在短暂脑缺血再灌注早期，缺血区周围可能有eNOS相关的保护机制；亚急性期eNOS及iNOS的保护及损伤机制并存；因此，在短暂脑缺血早期恢复灌注后予选择性iNOS抑制剂及促进eNOS活性有可能减少迟发性神经损伤。     

NOS、p38MAPK、Caspase-3介导大鼠脑缺血神经细胞凋亡可能通路的实验研究

目的探讨脑缺血后细胞凋亡发生的可能机制以及神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、p38丝裂原活化蛋白激酶(mitogen activated proteinkinasep38,p38MAPK)和半光氨酸蛋白酶-3(caspase-3)在脑缺血后神经细胞凋亡中的共同作用机制。方法采用线栓法闭塞大鼠大脑中动脉(middle cerebral artery occlusion,MACO)建立脑缺血SD大鼠模型,应用透射电镜观察脑缺血对脑组织超微结构的影响,流式细胞仪方法(FCM)分别定量检测细胞凋亡率,半定量RT-PCR检测nNOS、iNOS,p38MAPK和Caspase-3mRNA表达水平。结果透视电镜下脑缺血6h出现核固缩,缺血12h出现细胞核分裂,缺血24h出现凋亡小体;FCM检测细胞凋亡百分率随着缺血时间延长而增加,缺血72h达到高峰,约70.37%;RT-PCR产物的琼脂糖凝胶电泳显示nNOS、iNOS、p38MAPK和Caspase-3mRNA的特异性片段大小分别为501、342、250和342bp,但mRNA表达量不一致,nNOS mRNA主要在缺血早期表达,iNOS、p38MAPK和Caspase-3mRNA在缺血中晚期表达,并在缺血3~5d,后三种基因的表达量达到高峰。结论脑缺血区域发生典型的神经细胞凋亡现象,nNOS来源的NOS在缺血早期发挥神经毒性作用,iNOS来源的NOS在缺血晚期发挥神经毒性作用;NOS,p38MAPK和Caspase-3三种基因的相互关系可能构成介导缺血神经细胞凋亡的通路之一。     

脑室内注射乙酰胆碱受体抗体IgG对大鼠神经元凋亡及一氧化氮合酶表达的影响

目的研究乙酰胆碱受体抗体(AchRab)对大鼠脑内神经元的损害及一氧化氮合酶(NOS)在损害中所起的作用，探讨重症肌无力(MG)中枢神经系统损害的机制。方法将AchRab IgG或健康人的IgG注入大鼠侧脑室。HE染色、TUNEL法检测细胞凋亡；免疫组化方法观察大鼠皮质、海马及杏仁核神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)表达变化。结果2周后实验组皮质、海马及杏仁核凋亡细胞明显增多，对照组仅见少量凋亡。实验组皮质、海马及杏仁核nNOS神经元数目明显减少。实验组及对照组脑内细胞均来见iNOS表达。结论AchRab脑内注射可诱导神经元凋亡；损伤皮质。海马及杏仁核nNOS神经元；但未能诱导脑内细胞iNOS表达。神经元凋亡损害参与了AchRab对中枢神经损害的机制；nNOS神经元的减少，可能与MG认知功能障碍有密切关系；而神经元的损伤可能与NO的毒性作用无关。     

神经元型一氧化氮合酶的神经生物学研究进展

神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)主要表达于神经元,在星形胶质细胞和神经干细胞中也有一定水平的表达.不同的mRNA拼接形式产生了nNOS蛋白的5种亚型,包括nNOS-α、nNOS-β、nNOS-μ、nNOS-γ和nNOS-2.nNOS单体不具催化活性,二聚体是...     

重症肌无力患者IgG对大鼠脑内NOS不同时间表达的影响

目的观察重症肌无力(myasthenia gravis,MG)患者IgG(AchRab)对大鼠脑内一氧化氮合酶(NOS)表达的影响,探讨NOS在MG中造成中枢神经系统损害的机制.方法将AchRab IgG或健康人的IgG注入大鼠侧脑室,1次/d,连续4次.免疫组化方法观察不同时间点大鼠脑皮质、海马及杏仁核神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)的表达变化.结果侧脑室注射后1周实验组大鼠皮质、海马神经元nNOS表达量明显减少,后2周实验组皮质、海马神经元nNOS表达下降更为明显,同时杏仁核神经元nNOS表达量也减少;实验组及对照组脑内细胞均未见iNOS表达.结论AchRab侧脑室内注射可引起大鼠皮质、海马及杏仁核神经元nNOS表达量减少,且2周内这种减少效应随时间延长而增强,但未能诱导脑内细胞iNOS表达,提示AchRab尚可通过抑制大鼠中枢神经系统nNOS表达,降低脑内正常的一氧化氮浓度,减弱一氧化氮对脑组织的保护作用,增加神经元的易损性.     

Distribution of c-fos mRNA in the brain following intracerebroventricular injection of nitric oxide (NO)-releasing compounds: possible role of NO in central cardiovascular regulation

We injected nitric oxide (NO)-releasing compounds and NO synthase (NOS) inhibitors into the brains of conscious, freely moving rats and measured the effects on mean arterial blood pressure (MAP) and heart rate, as well as on the expression of c-fos mRNA, neuronal NOS (nNOS) mRNA and NADPH-diaphorase, an indicator of NOS activity. When administered i.c.v., the NO donor, NOC-18, caused a significant fall in MAP and heart rate, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), induced a significant rise in MAP. The same dose of NOC-18 or L-NAME when administered i.v. did not affect MAP and heart rate. Centrally administered NOC-18 induced c-fos mRNA expression in several regions of the brain involved in the baroreceptor response, including the nucleus of the solitary tract, the area postrema and the rostral ventrolateral medulla, as well as areas involved in the integration of autonomic, neuroendocrine and behavioural responses, including the medial preoptic area, the organum vasculosum lamina terminalis, the bed nucleus of stria terminalis, the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the central nucleus of amygdala (CeA) and the locus coeruleus. Most of the areas that expressed c-fos also contained nNOS mRNA and/or NADPH-d-positive neurones and fibres. i.c.v. injection of L-NAME induced c-fos mRNA expression in PVN, SON, locus coeruleus and NTS, suggesting a tonic inhibition of neuronal activity by NO or stimulation of neuronal activity by endogenous NO. i.v. injection of NOC-18 or L-NAME did not induce any significant c-fos mRNA expression in rat brain. These results demonstrate that NO acts directly in the brain to reduce the systemic blood pressure, and that the endogenous NO pathway may play a role in cardiovascular and autonomic regulation by modulating neuronal activities in discrete regions of the brain.     

Cardiovascular effects produced by nitric oxide-related drugs in the caudal ventrolateral medulla

The role of nitric oxide in the central control of blood pressure was evaluated by interfering with its local formation in the caudal region of the ventrolateral medulla (CVLM). Urethane anesthetized male Wistar rats were used. Microinjection of L-arginine (L-Arg, 25-100 nmol) produced a hypertensive effect without significant changes in heart rate (HR). Microinjection of N(G)-nitro-L-arginine methyl ester (L-NAME, 7.4 nmol) produced a significant hypotensive effect. Microinjection of L-Arg (50 nmol) combined with L-NAME (7.4 nmol) did not significantly change mean arterial pressure or HR. A similar finding was obtained with microinjection of L-Arg (50 nmol) 5 min after microinjection of methylene blue (5 nmol) into the CVLM. The pressor effect of L-Arg was also abolished by prior i.v. injection of a vasopressin V1 receptor antagonist, but not by prior i.v. injection of prazosin. These results suggest an inhibitory role for local NO in the CVLM and that nitrergic pathways at the CVLM participate in the central regulation of AVP release.     

BMY 14802, a potential antipsychotic drug, increases expression of proneurotensin mRNA in the rat striatum.

Treatment with efficacious antipsychotic drugs, such as haloperidol, increases the concentrations of neurotensin (NT) in the nucleus accumbens and caudate nucleus of the rat. The increases in NT content produced by haloperidol have been shown to be associated with an increase in NT biosynthesis as assessed by the level of expression of proNT mRNA. The potential antipsychotic drug and sigma receptor ligand BMY 14802 has been shown to produce increases in the concentrations of NT in the nucleus accumbens and caudate that are similar to those produced by haloperidol with respect to the magnitude of the increases and the time course over which they occur. This study evaluated the effects of BMY 14802 on the expression of proNT mRNA and NT concentration in the rat striatum. Three hours after a single injection of BMY 14802 (35 mg/kg, i.p.) expression of proNT mRNA was significantly increased in the nucleus accumbens and caudate. Labeling was most intense in the dorsal caudate. NT concentrations were unaltered at this time point. Eighteen hours after injection, significant increases in NT concentration in the nucleus accumbens and caudate were detected. At this time, expression of proNT mRNA was substantially reduced compared to 3 h after treatment although labeling of the dorsal and medial caudate was still greater than that observed in controls.     

Injection of antisense oligos to nNOS into nucleus tractus solitarii increases blood pressure.

We investigated the cardiovascular effects of bilateral microinjection of antisense oligodeoxynucleotides (oligos) into the nucleus tractus solitarii (NTS) to neuronal nitric oxide synthase (nNOS) to suppress the expression of nNOS molecular biologically. In urethane-anesthetized, paralyzed Wistar-Kyoto rats, bilateral microinjection of nNOS antisense oligos (20 pmol in a 50nl volume) into the NTS produced a significant increase in mean arterial blood pressure at 30-60min after injection, compared with rats injected with nNOS sense or scrambled oligos. Immunohistochemical study demonstrated that nNOS immunoreactivity in the rat NTS was suppressed by nNOS antisense oligos. These results indicate that suppression of the nNOS gene using antisense in the NTS increases blood pressure.     

大鼠脑出血早期脑组织神经元型一氧化氮合酶mRNA表达及中药抵当汤干预对其的影响

目的 探讨大鼠脑出血（ICH）早期脑组织神经元型一氧化氮合酶（nNOS）mRNA表达,以及中药抵当汤干预对其的影响。方法 将72只大鼠随机分为ICH组、抵当汤组、NOS抑制剂组和对照组,采用立体定向技术注入自体不凝血建立实验性ICH模型,对照组注入等量的生理盐水;术后6h、24h及72h用Bederson3级标准法对大鼠进行神经功能障碍评定;然后断头取脑,以原位杂交方法检测脑组织中nNOS mRNA阳性细胞的表达。结果 神经功能缺损程度评分术后72h时ICH组与抵当汤组均见明显改善（均P〈0.05）,但抵当汤组的改善明显优于ICH组（P〈0．05）;ICH后6h血肿侧大脑皮质nNOS mRNA阳性细胞的表达较对照组已有明显增高,24h达高峰,72h时下降,但仍明显高于对照组（均P〈0．05）;抵当汤组血肿侧大脑皮质nNOS mRNA阳性细胞表达在各时间点较ICH组明显减少（均P〈0．05）,且与NOS抑制剂组变化规律一致。结论 抵当汤具有与NOS抑制剂相似的作用,通过抑制ICH后脑组织中nNOS mRNA的表达而产生脑保护作用。     

Induction of neuronal nitric oxide synthase by sympathetic denervation is mediated via alpha 2-adrenoceptors in the jejunal myenteric plexus

Nitric oxide (NO) is an important nonadrenergic, non-cholinergic (NANC) inhibitory neurotransmitter in the gastrointestinal tract. In previous studies, neuronal nitric oxide synthase (nNOS) in the jejunal myenteric plexus, a key enzyme responsible for the release of NO, has been demonstrated to increase after splanchinic ganglionectomy (sympathetic nerve transection). The alpha2-adrenoceptor is known to be one of the most important receptors which controls intestinal motility. In the present study, we examined the effect of application of the alpha2-adrenoceptor agonist, clonidine hydrochloride, on nNOS expression in the rat jejunal myenteric plexus after splanchinic ganglionectomy. Clonidine (0.1-1 mg/kg, i.p.) or saline was administered for 5 days after the splanchinic ganglionectomy. The nNOS expression and nNOS mRNA were detected by immunohistochemistry and in situ hybridization for nNOS mRNA, respectively. In the rats treated with vehicle after the splanchinic ganglionectomy, nNOS expression in the myenteric plexus significantly increased compared with sham-operated rats. The increases in nNOS protein and mRNA after splanchinic ganglionectomy were significantly reversed by clonidine treatment. Clonidine-treated naive rats showed no difference in nNOS expression compared with sham-operated rats. These data suggest that nNOS expression in the jejunal myenteric plexus after splanchinic ganglionectomy is regulated by the alpha2-adrenoceptor and that the alpha2-adrenoceptor may play an important role in abnormal intestinal motility following splanchinic ganglionectomy in rat jejunum.     

Nociceptive stimulation increases NO synthase mRNA and vasopressin heteronuclearRNA in the rat paraventricular nucleus

Nociceptive stimulation causes neuroendocrine responses such as arginine vasopressin (AVP) release and activation of the hypothalamo-pituitary-adrenal (HPA) axis. We examined the effects of nociceptive stimulation on the expression levels of neuronal nitric oxide synthase (nNOS) mRNA, heteronuclear (hn)RNA for AVP and AVP mRNA in the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), using in situ hybridization histochemistry. For nociceptive stimulation, formalin (5%) or saline was injected subcutaneously (s.c.) into the bilateral hind paws of rats. The expression of the nNOS gene in the PVN was significantly increased 2 and 6 h after s.c. injection of formalin in comparison with that in untreated and saline injected rats. The expression of the nNOS gene in the SON did not change in the untreated, saline- and formalin-injected rats. The AVP hnRNA in the PVN and SON was also significantly increased 15, 30 min and 2 h after s.c. injection of formalin, though AVP mRNA did not change at any time points that we studied. Plasma concentration of AVP was significantly increased 15 min after s.c. injection of formalin. These results suggest that NO in the PVN may be involved in nociceptive stimulation-induced neuroendocrine responses.     

Bradykinin enhances in vitro procoagulant and antifibrinolytic properties of rat vascular endothelial cells

INTRODUCTION: Bradykinin (BK) is a biologically active peptides that exerts a broad spectrum of pathophysiological effects mainly by producing nitric oxide (NO) and prostacyclin from vascular endothelial cells. A direct effect of BK on vascular endothelial cells regarding the expression of the regulatory proteins of coagulation and fibrinolysis has not been fully elucidated. MATERIALS AND METHODS: The effects of BK on the expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (TPA) in cultured rat aortic endothelial cells (RAECs) were respectively evaluated by Northern blot and chromogenic assay or enzyme-linked immunosorbent assay (ELISA). RESULTS: BK significantly increased the expression of TF and PAI-1 in both mRNA and protein levels, but it did not affect the expression of TFPI. Although BK tended to increase TPA mRNA expression, the observed increase was not statistically significant. Those effects are considered to be mediated by B(2) receptor, because B(2) receptor antagonist (Hoe 140) suppressed those mRNA inductions by BK. Furthermore, since those mRNA inductions by BK were enhanced by nitro-L-arginine-methyl ester (L-NAME) and attenuated by L-arginine (L-Arg), NO was speculated to negatively contribute to the expressions of TF and PAI-1. CONCLUSION: BK was indicated to modify the property of vascular endothelial cells to be procoagulant and antifibrinolytic. Those effects of BK were considered to be the net of its direct effect and the effect negatively mediated by NO.