The long-term objective of this proposal is to understand the role of protein kinases in regulating smooth muscle function. Our recent studies reveal that Ca2+ signalling in smooth muscle is subject to powerful feedback regulation, mediated at least in part by protein kinases. The focus of the studies proposed in this application is thus to investigate the role of protein kinases, specifically CaM dependent protein kinase II and protein kinase C, in regulating Ca2+ entry and Ca2+ removal from the smooth muscle cell. To this end studies are proposed in intact enzymatically disaggregated smooth muscle cells from the stomach of Bufo marinus using peptides to increase or decrease the activity of a specific protein kinase and investigate the effect on: 1) inward Ca2+ currents; 2) ryanodine sensitive Ca2+ release from internal stores; and, 3) Ca2+ removal from the cytoplasm by Ca2+ pumps and the Na+/Ca2+ exchanger. We will begin with physiological studies at the single cell level measuring transmembrane currents using patch clamp methods and [Ca2+] using fluorescent indicators. The second phase of this study will utilize biochemical and molecular biological approaches to determine the protein targets that are responsible for changes in Ca2+ handling induced by these protein kinases. The final stage of this investigation will focus on an assessment of the intracellular localization of both the proteins that control Ca2+ influx and efflux as well as the protein kinases that regulate them with the aim of determining if the specificity of the action of the kinase results from translocation of the kinases to their targets upon activation or their docking at those sites both at rest and upon activation. Fluorescence immunocytochemistry in fixed cells and fluorescent protein analogs microinjected into live cells will be used in conjunction with novel 3D digital imaging methods to address these questions. The studies promise to increase our understanding of Ca2+ signalling in smooth muscle, and thus how contraction is regulated in both normal smooth muscle as well as in derangements of smooth muscle such as hypertension, asthma, and disorders of gastrointestinal and uterine motility.
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