Nitric oxide and in vivo brain arteriolar relaxation

描述(申请人提供)：该研究项目是一个扩展项目 一项关于一氧化氮在控制脑血管扩张中的作用的项目 在小动脉水平。在这项竞争性续签申请中的研究将 主要发生在脑血管平滑肌的地址调控机制 (VSM)在环状GMP和环状AMP水平。该项目的一个基本前提 环核苷酸通路不是作为单独的实体发挥作用， 而是通过一种通常被称为“串音”的过程进行互动。这个 这一提议的中心假设是，通过对串扰的调节， VSM中，每个环核苷酸都能够调节血管扩张作用 通过与磷酸二酯酶(PDE)的相互作用。该项目 将解决两个基本假设。首先，通过一个周期控制串扰 核苷酸在PDE水平上是通过减少其水解物的水解度而发生的 对应者。其次，水解性功能的降低可能通过直接的 环核苷酸或其激酶对PDE本身或途径的作用 促进PDE的蛋白激酶相关的亚细胞隔离远离 存在于细胞质中的大量环核苷酸。三个具体目标 将根据在大脑VSM中发现的3个主要PDE- CAMP-“偏好”PDE3、cAMP特异性PDE4和cGMP特异性PDE5。 内源性cAMP和cGMP产量的原发增加将由 腺苷和鸟苷酸环化酶激活剂，分别为Forsklin和YC-1。 在这些研究中将使用两个实验模型：已建立的封闭模型 颅窗，活体显微镜系统，用于监测脑膜 麻醉大鼠和原代培养大鼠小动脉内径的变化 软脑膜血管平滑肌。这两个型号都将用于测试第一个 假设。这些研究将涉及到使用精心挑选的选择性 酶抑制剂和激活剂。在大多数情况下，这些选择性一直是 得到了我们的验证。此外，确认一次循环的能力 在这些存在的情况下，影响另一种的核苷酸含量 药理操作，将使用培养系统获得。 将使用pial VSM电池和高分辨率解决隔离问题 免疫荧光检测技术。这些研究通常会涉及 3核内与胞浆内相对表达的评估 PDE，在没有和存在增加的情况下 对应的环核苷酸。环核苷酸激酶的作用将是 在这方面也应加以解决。这些研究将提供重要的新的 关于控制生理过程的机制的信息 脑功能正常与存活--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.