Estrogen signaling in microvascular arteries: parturition reduces vasodilation by reducing 17β-estradiol and nNOS

Few studies have examined the potential effects of childbirth on the responses of the female vasculature--especially the resistance microvasculature of non-reproductive tissues. In the present study we have investigated the response of mesenteric microvascular resistance vessels to estrogen (E2), an important vasoactive hormone. Vessels were obtained from either nulliparous or postpartum female Sprague-Dawley rats, and isometric tension studies were performed. We found that E2 induced a concentration-dependent, endothelium-independent relaxation of microvessels precontracted with 10(-5) M phenylephrine; however, E2-induced relaxation was reduced by nearly half in vessels from postpartum animals compared to nulliparous controls. Inhibiting nitric oxide synthase activity with 10(-4) M L-NMMA or L-NPA (which exhibits selectivity for type 1 or nNOS) attenuated the relaxation effect of E2 on arteries from nulliparous animals. In contrast, L-NPA had little effect on arteries from postpartum animals, suggesting a reduced influence of nNOS after parturition. Moreover, expression of nNOS protein in microvessels was decreased 39% in the postpartum state compared to arteries from nulliparous animals. We propose that the impaired E2-induced relaxation response of microvessels from postpartum animals reflects a downregulation of NO production due to lower nNOS expressed in vascular smooth muscle cells. We measured a 73% decrease in serum E2 levels in the postpartum state compared to nulliparous animals. Because E2 has been shown to increase nNOS protein expression, we propose that lower E2 levels after parturition decrease expression of nNOS, leading to a reduced vasodilatory capacity of resistance microvessels.     

Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOSα to enteric varicosity membrane

Nitrergic neurotransmission at the smooth muscle neuromuscular junctions requires nitric oxide (NO) release that is dependent on the transport and docking of neuronal NO synthase (nNOS) α to the membrane of nerve terminals. However, the mechanism of translocation of nNOSα in actin-rich varicosities is unknown. We report here that the processive motor protein myosin Va is necessary for nitrergic neurotransmission. In wild-type mice, nNOSα-stained enteric varicosities colocalized with myosin Va and its tail constituent light chain 8 (LC8). In situ proximity ligation assay showed close association among nNOSα, myosin Va, and LC8. nNOSα was associated with varicosity membrane. Varicosities showed nitric oxide production upon stimulation with KCl. Intracellular microelectrode studies showed nitrergic IJP and smooth muscle hyperpolarizing responses to NO donor diethylenetriamine-NO (DNO). In contrast, enteric varicosities from myosin Va-deficient DBA (for dilute, brown, non-agouti) mice showed near absence of myosin Va but normal nNOSα and LC8. Membrane-bound nNOSα was not detectable, and the varicosities showed reduced NO production. Intracellular recordings in DBA mice showed reduced nitrergic IJPs but normal hyperpolarizing response to DNO. The nitrergic slow IJP was 9.1 ± 0.7 mV in the wild-type controls and 3.4 ± 0.3 mV in the DBA mice (P < 0.0001). Deficiency of myosin Va resulted in loss of nitrergic neuromuscular neurotransmission despite normal presence of nNOSα in the varicosities. These studies reveal the critical importance of myosin Va in nitrergic neurotransmission by facilitating transport of nNOSα to the varicosity membrane.     

nNOS in canine lower esophageal sphincter: colocalized with Cav-1 and Ca2+-handling proteins?

Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cav) in canine lower esophageal sphincter. The main Cav was Cav-1. It appeared to be colocalized at the cell periphery, in punctate sites, with immunoreactivity to antibodies against different COOH- and NH2-terminal epitopes of neuronal nitric oxide (NO) synthase (nNOS). One COOH-terminal-directed antibody, made in guinea pig, was used to colocalize other immunoreactivities. Those that apparently colocalized with nNOS were L-Ca2+ channels, the PM Ca2+ pump, and, in part, calreticulin and calsequestrin. The large-conductance Ca2+-activated K+ (BK(Ca)) channels were located in discrete peripheral sites, some with Cav. Immunoreactivities not fully colocalized with nNOS were to the sarcoplasmic reticulum Ca2+ pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through BK(Ca) channels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several Ca2+-handling molecules are localized at the PM with and/or near Cav. This may allow intracellular calcium concentration levels to be controlled differently than those in the cytosol near caveolae.     

人胎海马内nNOS神经元的形态学观察

目的研究胎儿海马内nNOS神经元的分布、形态的变化以及意义.方法采用ABC免疫细胞化学法结合DAB显色技术研究人胎儿海马内nNOS神经元的定位和分布.结果人胎脑13周开始有少量的nNOS的表达,20周后随着胎龄的增加含nNOS阳性神经元数量逐渐增加,于28周海马内nNOS神经元数量最多,随后数量逐渐减少;阳性神经元胞体体积随着胎龄的增加逐渐增大;根据细胞形态nNOS阳性神经元可分两种类型,其比例随着胎龄的变化而变化.结论人胎海马存在有nNOS阳性神经元的表达,且呈两种不同的形态.     

眼镜蛇毒对大鼠延髓nNOS表达的影响

目的 探讨眼镜蛇毒对大鼠延髓nNOS表达的影响.方法 采用免疫组织化学方法,观察并比较nNOS阳性神经元在眼镜蛇毒组、生理盐水组、正常对照组大鼠延髓的表达.结果 眼镜蛇毒组大鼠延髓外侧网状核nNOS阳性神经元明显多于生理盐水组和正常对照组(P＜0.01),细胞平均灰度值明显降低(P＜0.01).结论 眼镜蛇毒对大鼠延髓nNOS表达有上调作用.     

神经元型一氧化氮合酶(nNOS)与疼痛

一氧化氮(NO)是神经元细胞内一种新型的神经递质,它由一氧化氮合酶(NOS)催化而成。在神经系统中神经元型一氧化氮合酶(nNOS)是NO合成的关键酶。大量研究表明,nNOS可调节多种生理和病理过程诸如炎性痛和神经病理性疼痛。该文通过介绍nNOS的结构、分布和影响nNOS活性的因素,阐述了nNOS在病理性疼痛中的重要作用,为此可通过调节nNOS表达来达到调节生理和病理过程。     

眼镜蛇毒对大鼠弓状核nNOS表达的影响

目的探讨眼镜蛇毒对大鼠弓状核神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)表达的影响。方法采用免疫组织化学方法分别显示眼镜蛇毒组、生理盐水组和正常对照组弓状核的nNOS表达。结果与生理盐水组和正常对照组相比,眼镜蛇毒组弓状核nNOS阳性神经元数量明显减少,细胞平均灰度值明显升高(P0.05)。结论眼镜蛇毒能够使大鼠弓状核nNOS表达降低。     

运动性疲劳对大鼠乳头体nNOS表达的影响

目的：探讨乳头体中神经元型一氧化氮合酶（nNOS）与运动性疲劳的关系。方法：建立运动性疲劳动物模型,用免疫组织化学方法检测乳头体内侧核内侧部（mmm）、乳头体内侧核外侧部（mml）、乳头体内侧核后部（mmp）和乳头体外侧核（1m）等部位nNOS的表达。结果：疲劳组大鼠mm／n处nNOS阳性神经元数量为（4．504±2．84）cells／U,面积为（179．81±130．15）μm^2,均大于对照组（P〈0．05,P（0．01）;疲劳组大鼠mml处nNOS阳性神经元数量为（43．7±6．93）cells／U,面积为（5208．63±1253．20）μm^2,均大于对照组（P〈0．01）;疲劳组大鼠mmp处nNOS阳性神经元数量为（8．88±4．26）cells／U,面积为（202．75±109．67）μm^2,均大于对照组（P〈0．05）;疲劳组大鼠lm处nNOS阳性神经元数量为（27．2±6．94）cells／U,面积为（2763．23±1107．35）μm^2,均大于对照组（P〈0．01）;疲劳组大鼠mmp处和lm处nNOS阳性神经元灰度值分别为54．274±14．86和72．454±28．07,均大于对照组（P〈0．01,P〈0．05）。结论：乳头体nNOS神经元与运动性疲劳密切相关,NO可能在乳头体对疲劳应激反应的调节中发挥重要作用。     

Is there a role for neuronal nitric oxide synthase (nNOS) in cytokine toxicity to pancreatic beta cells?

Nitric oxide (NO), produced by the action of the inducible NO synthase, plays a crucial role in cytokine toxicity to pancreatic beta cells during type 1 diabetes development. It was the aim of this study to analyze the role of the neuronal NOS (nNOS) in proinflammatory cytokine-mediated beta cell toxicity. Expression of different isoforms of nitric oxide synthase in insulin-secreting INS1E cells and rat islets was analyzed by quantitative real-time PCR and Western blotting. The expression of nNOS in insulin-secreting INS1E cells was similar to that found in rat brain, while two other isoforms, namely the endothelial eNOS and inducible iNOS were not expressed in untreated cells. IL-1β alone or in combination with TNF-α and/or IFNγ induced iNOS but not eNOS expression. In contrast, nNOS expression was strongly decreased by the mixture of the three proinflammatory cytokines (IL-1β, TNF-α and IFNγ) both on the gene and protein level in INS1E cells and rat islet cells. The effects of cytokines on glucose-induced insulin-secretion followed the pattern of nNOS expression reduction and, on the other hand, of the iNOS induction. The data indicate that a low level of nitric oxide originating from the constitutive expression of nNOS in pancreatic beta cells is not deleterious. In particular since proinflammatory cytokines reduce this expression. This nNOS suppression can compensate for NO generation by low concentrations of IL-1β through iNOS induction. Thus, this basal nNOS expression level in pancreatic beta cells represents a protective element against cytokine toxicity.     

脑缺血再灌注大鼠模型eNOS和nNOS的变化

目的通过对缺血再灌注早期eNOS与nNOS表达情况的观察,探讨NO在脑缺血再灌注损伤中发挥神经毒性作用时是否出现一氧化氮合酶(NOS)不同亚型的变化。方法采用线栓法制作大鼠脑缺血再灌注模型,激光多普勒灌流监测仪测血流来判断模型是否成功,Western blot方法检测eNOS与nNOS变化。结果血管内皮细胞内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。结论大鼠脑缺血再灌注模型中eNOS与nNOS的变化趋势不同。表明NO在缺血性脑损伤的病理过程的发挥作用与NOS亚型的变化有关。     

慢性激活MrgC受体上调CGRP表达的nNOS机制

目的：检查持续应用BAMS-22对体外组织培养感觉神经节合成钙调素基因相关肽（cGRP）的影响。方法：将体外培养的大鼠三叉神经节和背根神经节经BAM8—22和L-NAME处理后,用酶联免疫法测定CGRP的表达含量变化。结果：与对照组相比,连续4天给予SNSR的选择性激动剂BAM8-22,CGRP的合成会增加。联合给予BAM8—22和NOS的非选择性抑制剂L-NAME,CGRP的表达随不同剂量的L-NAME引起不同程度的上调。结论：持续激活SNSR能使感觉神经节合成CGRP增多,是在体动物慢性激活SNSR后吗啡镇痛作用降低的细胞学机制。     

慢性激活MrgC 受体上调CGRP表达的nNOS机制*

摘要目的：检查持续应用BAM8-22 对体外组织培养感觉神经节合成钙调素基因相关肽(CGRP)的影响。方法：将体外培养的大鼠 三叉神经节和背根神经节经BAM8-22 和L-NAME 处理后,用酶联免疫法测定CGRP 的表达含量变化。结果：与对照组相比,连 续4 天给予SNSR 的选择性激动剂BAM8-22,CGRP 的合成会增加。联合给予BAM8-22 和NOS 的非选择性抑制剂L-NAME, CGRP的表达随不同剂量的L-NAME 引起不同程度的上调。结论：持续激活SNSR 能使感觉神经节合成CGRP增多,是在体动物 慢性激活SNSR 后吗啡镇痛作用降低的细胞学机制。     

Hypertension:健康人的血压是由酶nNOS调节的

正在一项新的研究中,来自英国伦敦国王学院的研究人员发现调节血压的一氧化氮(NO)是在神经组织中而不是在血管壁中形成的。这一发现可能导致人们开发出更加有效地治疗高血压的方法。相关研究结果在线发表在Hypertension期刊上,论文标题为"Blood Pressure in Healthy Humans Is Regulated by Neuronal NO Synthase"。之前的研究已证实NO在调节血压中发挥着重     

眼镜蛇毒对大鼠脑桥前庭神经核nNOS表达的影响

目的 观察眼镜蛇毒对大鼠脑干神经元型一氧化氮合酶(nNOS)表达的影响,探讨其对前庭神经核的作用.方法 采用免疫组织化学方法,观察nNOS阳性细胞在眼镜蛇毒组、生理盐水组、正常对照组大鼠前庭神经核中的表达,并对其作比较.结果 蛇毒组前庭神经核nNOS表达与正常对照组、生理盐水组比较有明显下调(P＜0.01);蛇毒组前庭神经核nNOS阳性细胞灰度值与正常对照组、生理盐水组比较明显增高(P＜0.01),蛇毒对前庭神经核nNOS阳性细胞形态影响不明显.结论 眼镜蛇毒对前庭神经核nNOS表达呈下调作用.     

锌对急性缺氧小鼠海马NOS和nNOS水平的影响

目的：观察锌对急性缺氧小鼠海马一氧化氮合酶（nitric oxide synthase,NOS）和神经元型一氧化氮合酶（neuronal NOS,nNOS）阳性神经元的影响,以探讨锌抗脑缺氧的作用机制。方法：复制小鼠急性缺氧模型,采用NADPH-d组织化学和nNOS免疫组织化学方法,研究给锌组和不给锌组急性缺氧小鼠海马各分区NOS和nNOS阳性神经元数量的变化。结果：给锌组比不给锌组小鼠缺氧耐受时间显著延长,差异有显著性（P〈0．05）;海马及其CA1区NOS和nNOS阳性神经元的数量明显减少,差异有显著性（P〈0．05）。结论：急性缺氧时锌通过减少海马NoS和nNOS水平而发挥其抗脑缺氧作用。     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

腹腔注射LPS 对大鼠海马nNOS 和iNOS 表达的影响

目的：探讨外周免疫刺激对大鼠海马nNOS和iNOS表达的影响。方法：腹腔注射LPS600μg／kg建立免疫激发大鼠模型,对照组注射等量的生理盐水,2．5h后用RT—PCR技术,观察两组大鼠海马nNOS mRNA和iNOS mRNA的表达变化,检测OD值并进行统计学分析。结果：腹腔注射LPS组大鼠海马的nNOS和iNOS的mRNA水平较对照组高。结论：海马在大鼠机体的免疫应激反应中起重要调节作用,iNOS和nNOS来源的N0是该调节过程中的重要信使分子。     

噬菌体展示筛选得到一种能修饰nNOS活性的多肽

多种疾病的病理过程涉及脑型一氧化氮合酶（nNOS）的活性改变。为了寻找特异的nNOS调控分子,将nNOS的钙调素结合区在大肠杆菌（E. coli）中表达,表达蛋白经纯化后作为靶分子用于12肽噬菌体展示库筛选,得到10个对nNOS具有特异性抑制作用的克隆。DNA测序显示一个氨基酸共同序列：KPHHHPMPPQVS,将其命名为NPI。合成的NPI多肽对从小鼠脑新鲜制备的nNOS具有抑制作用（抑制率为25%-35%）,而对失去部分活性的nNOS却表现为剂量依赖的恢复活性效应。本文结果提出一种研发nNOS调节药物的新方法-噬菌体展示技术,同时NPI对nNOS活性的双向调节功能也为认识机体自身nNOS活性的调控过程提供新的启示。