Regulation of vascular smooth muscle calcium sensitivity

描述（由申请人提供）：组织血流受血管平滑肌（VSM）收缩调节，而血管平滑肌收缩又受胞质游离钙（Ca）水平变化和收缩蛋白对Ca的敏感性调节。本项目将重点研究rhoA激酶（ROK）依赖性Ca敏感性的调节机制。然而，这个项目最新颖的地方在于，重点将放在理解由钙本身激活的细胞信号系统，这些信号系统会导致rok诱导的钙致敏。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时平滑肌收缩“失败”中起作用。我实验室的长期目标是研究调节VSM Ca敏感性和强直性力维持的亚细胞机制，为开发治疗选择性血管收缩性疾病的新型治疗剂提供基础知识。我的实验室已经确定，钙敏感性可以通过钙依赖机制在VSM中增加。这种钙依赖性钙敏化似乎涉及ROK和非典型PKC同型PKCzeta的激活，并且似乎依赖于iPLA2和PI3K的激活。本项目的直接目标是利用生理学、生物化学、药理学、细胞和分子以及形态计量学方法，确定在具有良好特征的动脉收缩系统（kci刺激的兔FA）中调节Ca依赖的Ca敏感性和强直力维持的分子机制。该项目的总体目标是了解在尽可能接近生理状态的组织中动脉平滑肌收缩的调节。尽管该方法存在局限性，但通过应用多种方法来评估参与钙依赖性钙致敏的特定信号分子的时空激活，可以得出关于完整功能组织VSM中连接刺激与收缩的离散步骤的因果机制结论。本研究的具体目的是验证ROK和PKCzeta都介导KCI诱导的FA的Ca敏化，以及iPLA2和PI3K作为ROK和PKCzeta的上游激活剂所必需的假设。

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.