BIOCHEMICAL MECHANISMS OF SMOOTH MUSCLE CONTRACTION

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In smooth muscle, phosphorylation of myosin light chain (P-light chain) by Ca+-and calmodulin-dependent myosin light chain kinase is obligatory for actin activation of myosin Mg2+-ATPase activity. Thus, myosin phosphorylation may be essential for smooth muscle contraction. The long-range goals of the research described in this application are to determine the regulatory properties of the myosin phosphorylation system and to evaluate the relationships between P-light chain phosphorylation and mechanical performance (force, maximum shortening velocity) in bovine trachealis and coronary arteries. Reversible hyperpermeabilization will be used to introduce specific inhibitors of myosin light chain kinase or calmodulin-independent myosin light chain kinase into smooth muscle cells to affect P-light chain phosphorylation. Involvement of protein kinase C in myosin P-light chain phosphorylation and regulation of mechanical properties will also be assessed. Biochemical studies will be extended to cultured smooth muscle cells. Inositol 1,4,5-triphosphate may be a second messenger for pharmacological agonists that mobilize internal stores of ca2+. Therefore, the kinetic properties of P-light chain phosphorylation will be measured in relation to formation of inositol 1,4,5-triphosphate and to sarcoplasmic Ca2+ concentrations. The relative roles of decreased sarcoplasmic Ca2+ concentrations vs. myosin light chain kinase phosphorylation as biochemical mechanisms for cyclic nucleotide inhibiton of P-light chain phosphorylation will be analyzed. The biochemical properties of myosin light chain kinase isozymes from gizzard and bovine tracheal smooth muscles will be compared to the properties of the skeletal muscle isozymes. Functional domains within a kinase will be probed by limited proteolysis and identification of specific peptides that bind calmodulin, ATP analogs, and monoclonal antibodies. The catalytic properties of purified mammalian smooth muscle myosin light chain kinase will be analyzed with synthetic peptide substrates to define amino acid determinants in the primary sequence that may account for marked substrate specificity. These investigations will provide information on the biochemical properties of smooth muscle myosin ligh chain kinases which will be related to other calmodulin-dependent enzymes and other protein kinases.