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. ? ? ?
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