the long term objectives of this research program are to determine the form(s) of protein kinase C (PKC) which exists in bovine carotid artery and tracheal smooth muscle, and to define the functions of the enzyme(s) in the regulation of smooth muscle contraction. The first major aim is to isolate and characterize the different isoforms of PKC from these two types smooth muscle. First, the specific isoforms will be isolated by standard chromatographic methods. these enzymes will then be characterized by use of Northern and Western blot analysis. functional characterization will involve an analysis of the kinetic properties of the isoforms using a natural membrane (inside-out vesicles of human erythrocyte membranes) to define the effects of Ca2+, diacylglycerol and phorbol esters and other lipids on their ability to phosphorylate an intrinsic membrane protein (the Ca2+ pump) and soluble substrates. The effect of agonists such as histamine and norepinephrine on the cellular distribution of PKC using both cell fractionation and immunocytochemistry will also be carried out. Additionally, the relationship of PKC activity to the phosphorylation of four critical proteins: myosin light chain, desmin, caldesmo and protein #4, a low molecular weight (20 kDa) cytosolic protein, will be examined. The second major aim is to define the role of PKC in smooth muscle cell function. This effort will involve three different approaches. First, studies in intact tissue, alpha-toxin skinned muscle, and isolated muscle cells will be performed to further define the relationship between PKC activity, Ca2+ metabolism, protein phosphorylation and contraction. Second, studies of pharmacologic as well as peptide and protein inhibitors of PKC will be conducted. The technique of reversible permeabilization will be employed to introduce these protein and peptide inhibitors into the cells. Third, studies of the influence of phosphoprotein phosphatase inhibitor will be carried out under a number of circumstances. by these combined approaches, it is hoped to define more clearly the role PKC in smooth muscle contraction. The results of this work should provide new insights into the cellular and molecular basis of smooth muscle contraction which could point the way to a better understanding of the altered states of smooth muscle contraction seen in asthma, hypertension and cerebral vascular spasm.
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