: The overall aim of this research is to elucidate the cellular mechanisms that mediate agonist-stimulated vascular smooth muscle contraction and relaxation. To achieve these goals, we must identify the specific signaling molecules and decipher their role in signal transduction pathways that lead to smooth muscle contraction and relaxation. Major objectives of the current proposal are to elucidate the molecular mechanisms and physiological significance of arachidonic acid (AA)-induced Ca2+-sensitization in regulation of vascular smooth muscle contraction. Ca2+-sensitization describes a mechanism through which an excitatory agonist induces significant smooth muscle contraction under constant free Ca2+. Much evidence indicates that AA, released by various Ca2+-sensitizing agonists, may contribute to this G-protein mediated Ca2+-sensitization. However, the phospholipase A2 (PLA2) responsible for AA release, the G protein that regulates AA release, and the molecular mechanism through which AA induces Ca2+-sensitization remain unclear. We propose to test the hypothesis that AA, released by ras-mediated activation of Ca2+-independent iPLA2, contributes to the agonist-induced Ca2+-sensitization by acting at multiple sites: rho-kinase (ROK). protein kinase C (PKC) and myosin phosphatase (MLCP).
Specific aims are (1) To test the hypothesis that iPLA2 mediates the GTPgammaS-induced, Ca2+-independent AA release and Ca2+-sensitization in vascular smooth muscle. (2) To test the hypothesis that ras mediates agonist-induced activation of iPLA2 and Ca2+-independent AA release in regulation of Ca2+-sensitization of smooth muscle contraction. (3) To test the hypothesis that in vascular smooth muscle tissue, AA inhibits myosin phosphatase by acting at multiple sites: ROK, PKC and MLCP. a-toxin or beta-escin-permeabilized rabbit femoral arteries or portal veins will be used to determine the effects of selective activation and inhibition of AA pathway on agonist- and GTPgammaS-induced activation of ras, ROK, PKC, inhibition of myosin phosphatase and Ca2+-sensitization of force and MLC20 phosphorylation. AA pathway will be selectively activated by adding free AA or overexpressing iPLA2, and selectively inhibited by using a selective iPLA2 inhibitor (BEL) or antisense oligonucleotide. Understanding the cellular signal transduction pathways in vascular smooth muscle will help to identify the cause(s) of cardiovascular diseases. This information may lead to strategic drug design and/or molecular biological approaches targeted at messenger molecules that provide new treatments for cardiovascular diseases.
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