Regulation of vascular smooth muscle calcium sensitivity

描述(申请人提供)：组织血流量由血管平滑肌(VSM)收缩调节，而VSM收缩又由细胞内游离钙(Ca)水平和收缩蛋白对钙的敏感性的变化来调节。该项目将重点研究调节RhoA激酶(韩国)依赖的钙敏感性的机制。然而，这个项目最新颖的地方是，重点将是了解由钙本身激活的导致韩国诱导的钙敏化的细胞信号系统。钙敏感性的调节是控制血管张力的基本机制，钙敏感性的失调在高血压和血管扩张性休克的血管收缩失败中起着重要作用。我的实验室的长期目标是研究调节VSM钙敏感性和紧张力维持的亚细胞机制，为开发选择性治疗血管收缩障碍的新型治疗药物提供基础知识。我的实验室已经确定，VSM的钙敏感性可以通过一种钙依赖机制来增加。这种钙依赖的钙敏化似乎涉及到ROK和一个非典型的PKC亚型PKcheeta的激活，并且似乎依赖于iPLA2和PI3K的激活。该项目的近期目标是利用生理、生化、药理学、细胞和分子以及形态计量学方法，确定在一个具有良好特征的动脉收缩系统--KCI刺激的兔FA中，调节钙依赖的钙敏感性和紧张力维持的分子机制。这个项目的总体目标是了解维持在尽可能接近生理状态的组织中动脉平滑肌收缩的调节。虽然这种方法有局限性，但通过应用多种方法来评估参与钙依赖的钙敏化的特定信号分子的时空激活，可以得出关于完整、功能组织的VSM中将刺激与收缩联系起来的离散步骤的因果关系的机械性结论。本研究的具体目的是验证一种假设，即韩国和PKcheeta都介导了KCI诱导的FA的钙敏化，并且需要iPLA2和PI3K作为韩国和PKcheeta的上游激动剂。

DESCRIPTION (provided by applicant): Tissue blood flow is regulated by vascular smooth muscle (VSM) contraction, which in turn, is regulated by changes in the levels of cytosolic free calcium (Ca) and the sensitivity of contractile proteins to Ca. This project will focus on mechanisms regulating rhoA kinase (ROK)-dependent Ca sensitivity. However, what is most novel about this project is that an emphasis will be to understand the cell signaling systems activated by Ca itself that cause ROK-induced Ca sensitization. Regulation of Ca sensitivity is a basic mechanism controlling vascular tone, and dysregulation of Ca sensitivity plays a role in hypertension and the "failure" of smooth muscle to contract in vasodilatory shock. The long-term goal of my laboratory is to investigate subcellular mechanisms regulating VSM Ca sensitivity and tonic force maintenance to provide basic knowledge for the development of novel therapeutic agents to treat selectively vascular contractile disorders. My laboratory has determined that Ca sensitivity can be increased in VSM by a Ca-dependent mechanism. This Ca-dependent Ca sensitization appears to involve activation of ROK and an atypical PKC isotype, PKCzeta, and appears to be dependent on iPLA2 and PI3K activation. The immediate goal of this project is to identify, using physiological, biochemical, pharmacological, cell and molecular, and morphometric methodologies, the molecular mechanisms regulating Ca-dependent Ca sensitivity and tonic force maintenance in a well-characterized arterial contractile system, the KCI-stimulated rabbit FA. The overall goal of this project is to understand regulation of arterial smooth muscle contraction in tissues maintained in as near a physiological state as possible. Whereas this approach has limitations, by applying multiple methodologies to assess spatiotemporal activation of specific signaling molecules proposed to participate in Ca-dependent Ca sensitization, mechanistic conclusions can be drawn regarding cause and effect of discrete steps linking stimulus with contraction in the VSM of intact, functional tissues. The Specific Aim of this study will be to test the hypothesis that ROK and PKCzeta both mediate KCI- induced Ca sensitization of FA, and that iPLA2 and PI3K are required as upstream activators of ROK and PKCzeta.