Nitric oxide and in vivo brain arteriolar relaxation

描述（由申请人提供）：本研究计划是一个扩展 一氧化氮在脑血管舒张控制中的作用 在小动脉水平。本次竞争性更新申请中的研究将 地址控制机制主要发生在脑血管平滑肌 (VSM)在环磷酸鸟苷和环磷酸腺苷水平。项目的一个基本前提是 环核苷酸途径不是作为独立的实体发挥作用， 而是通过一般标记为“串扰”的过程相互作用。“那 该建议的中心假设是，通过串扰调节 VSM，每个环核苷酸能够调节血管舒张作用 通过与磷酸二酯酶（PDE）的相互作用来合成其对应物。项目 我将提出两个基本假设。首先，通过一个周期的串扰控制， 核苷酸，在PDE水平上，通过减少其水解而发生， 对应物。第二，水解功能的降低可通过直接水解而发生。 环核苷酸或其激酶对PDE本身的作用或通过 促进PDE的激酶相关的亚细胞隔离，使其远离 胞质溶胶中环状核苷酸的大量聚集。三个具体目标 将根据在脑VSM中发现的3种主要PDE-- cAMP-“偏好”PDE 3、cAMP特异性PDE 4和cGMP特异性PDE 5。 内源性cAMP和cGMP产生的主要增加将由以下引起： 腺苷酸和鸟苷酸环化酶激活剂，分别为毛喉素和YC-1。 在这些研究中将使用两个实验模型：已建立的封闭 颅窗，设计用于监测软脑膜的活体显微镜系统 麻醉大鼠和原代培养大鼠的小动脉直径变化 软脑膜血管平滑肌这两个模型将用于测试第一个 假说.这些研究将涉及使用精心挑选的选择性 酶抑制剂和活化剂。在大多数情况下，这些选择性一直是 由我们验证。此外，确认一个循环的能力， 核苷酸影响其他的内容，在这些存在下， 药理学操作，将使用培养系统获得。 隔离问题将使用软脑膜VSM细胞和高分辨率 免疫荧光检测技术。这些研究通常涉及 评估3种蛋白的相对核内与胞浆表达 在不存在和存在的情况下， 对应的环核苷酸。环核苷酸激酶的作用将 也要在这方面加以解决。这些研究将提供重要的新 关于控制生理过程的机制的信息， 正常的脑功能和存活-脑小动脉扩张。

DESCRIPTION (provided by the applicant): This research program is an extension of a project regarding the role of NO in the control of cerebral vasodilation at the arteriolar level. The studies in this competing renewal application will address control mechanisms occurring chiefly in cerebral vascular smooth muscle (VSM) at the level of cyclic GMP and cyclic AMP. A basic premise of the project is that the cyclic nucleotide pathways do not function as separate entities, but rather interact through a process generically labeled as "crosstalk." The central hypothesis of this proposal is that, through crosstalk regulation in VSM, each cyclic nucleotide is capable of regulating the vasodilating actions of its counterpart via interactions with phosphodiesterases (PDEs). The project will address two basic hypotheses. First, crosstalk control by one cyclic nucleotide, at the PDE level, occurs by reducing the hydrolysis of its counterpart. Second, the reduction in hydrolytic function may occur by a direct action of the cyclic nucleotide or its kinase on the PDE itself or via promoting a kinase-related subcellular sequestration of the PDE away from the bulk pool of cyclic nucleotides residing in the cytosol. Three specific aims will be addressed, based upon the 3 principal PDEs found in cerebral VSM-the cAMP-"preferring" PDE3, the cAMP-specific PDE4, and the cGMP-specific PDE5. Primary increases in endogenous cAMP and cGMP production will be elicited by the adenylyl and guanylyl cyclase activators, forskolin and YC-1, respectively. Two experimental models will used in these studies: the established closed cranial window, intravital microscopy system designed for monitoring pial arteriolar diameter changes in anesthetized rats and primary cultures of rat pial vascular smooth muscle. Both models will be used to test the first hypothesis. Those studies will involve the use of carefully chosen selective enzyme inhibitors and activators. In most cases, those selectivities have been validated by us. In addition, confirmation of the ability of one cyclic nucleotide to influence the content of the other, in the presence of these pharmacologic manipulations, will be obtained using the culture system. Sequestration issues will be addressed using pial VSM cells and high-resolution immunofluoresence detection techniques. Those studies will generally involve assessments of the relative intra-nuclear vs cytosolic expression of the 3 PDEs, in the absence and the presence of increases in the levels of the counterpart cyclic nucleotide. The roles of the cyclic nucleotide kinases will also be addressed in this regard. These studies will provide important new information regarding the mechanisms controlling a physiologic process vital to normal brain function and survival-cerebral arteriolar dilation.