This proposal's long term objective is to understand the role(s) of protein kinase C (PKC) isoforms in the regulation of smooth function. Understanding of contractile regulation in smooth muscles is critical to the understanding of smooth muscle contractile pathologies such as asthma, hypertension, coronary vasospasm, and spastic disorders of gastrointestinal and uterine smooth muscles. In addition, this proposal seeks to determine the importance of subcellular compartmentation of signaling enzymes such as PKCs in cell regulation. Stimulation of PKC has been implicated in many important aspects of smooth muscle function, including regulation of calcium ion homeostasis and sensitization of the contractile apparatus to calcium. Since multiple isoforms of PKC exist; identification of the PKCs found in various smooth muscles is an important first step. The gastric myocyte of Bufo Marinus is the most completely studied muscle on the cellular level; therefore identification of PKCs in this cell type with electrophoresis and blotting methods will be performed. Preliminary evidence suggests that the location and translocation of PKC isoforms in response to contractile stimuli is important in smooth muscle contractile regulation. The remainder of the proposed studies will therefore investigate the subcellular locations of PKCs and their relocation in response to contractile stimuli. Immunofluorescence, fluorescent analog cytochemistry, fluorescence resonance energy transfer, and molecular biology techniques, in conjunction with powerful 3D digital imaging methods will be used to track PKCs in resting and stimulated cells. Identification of PKC docking or binding proteins and local substrates will also be performed with biochemical and immunological methods. These studies will provide necessary details to help dissect out the functions of PKCs in smooth muscle regulation.
