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细胞溶质游离钙（Ca）水平变化和收缩蛋白对Ca的敏感性调节。该项目将重点关注调节rhoA激酶（ROK）依赖性Ca敏感性的机制。然而，这个项目最新颖的是，重点将是了解由钙本身激活的细胞信号系统，导致ROK诱导的钙致敏。Ca敏感性的调节是控制血管张力的基本机制，Ca敏感性的失调在高血压和血管舒张性休克中平滑肌收缩的“失败”中起作用。本实验室的长期目标是研究调节VSM Ca敏感性和张力维持的亚细胞机制，为开发选择性治疗血管收缩性疾病的新型治疗药物提供基础知识。我的实验室已经确定，钙敏感性可以增加VSM钙依赖性机制。这种Ca依赖性Ca敏化似乎涉及ROK和非典型PKC同种型PKCzeta的活化，并且似乎依赖于iPLA 2和PI3K活化。本项目的直接目标是确定，使用生理学，生物化学，药理学，细胞和分子，和形态计量学方法，调节钙依赖性钙敏感性和紧张力维持在一个良好的特征动脉收缩系统，氯化钾刺激的兔FA的分子机制。本项目的总体目标是了解尽可能接近生理状态的组织中动脉平滑肌收缩的调节。然而，这种方法有局限性，通过应用多种方法来评估时空激活的特定信号分子提出参与钙依赖性钙敏化，机械结论可以得出有关的原因和影响的离散步骤连接刺激与收缩的VSM的完整的，功能性组织。本研究的具体目的是检验以下假设：ROK和PKCzeta均介导KCl诱导的FA钙致敏作用，并且需要iPLA 2和PI3K作为ROK和PKCzeta的上游激活剂。

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.