Smooth muscles are essential for maintaining homeostasis in all chordate animals. In humans, aberrations in smooth muscle contractile responses have been implicated in hypertension, cardiovascular disease and asthma. Elucidation of the cellular and molecular basis of smooth muscle function is, therefore, crucial for the therapy of a number pathophysiological processes. It is now generally accepted that the phosphorylation of smooth muscle myosin by the Ca2+/calmodulin dependent enzyme myosin light chain kinase is a key regulatory event in the contractile process. However, a number of interesting observations suggest that this may not be the only regulatory mechanism and processes that do not involve myosin light chain kinase activation and/or myosin phosphorylation have been postulated. Therefore, the experiments described in this proposal are designed to obtain a detailed understanding of the role of myosin phosphorylation/dephosphorylation in regulating smooth muscles. Three experimental approaches that are continuations of ongoing research are planned. The thrust of the first series of experiments is to uncover alternative regulatory mechanisms by contracting muscles in the absence of an increase in cell Ca2+ or the activation of myosin light chain kinase. A second approach will be to introduce antibodies into intact living cells to deter- mine the localization of regulatory proteins. In these experiments the direct role of caldesmon, a putative regulatory protein, in regulating smooth muscle contraction will also be determined. The final series of experiments will address the regulation and physiological significance of fetal smooth muscle myosin. This series is based on the observation that protein kinase C, an enzyme implicated in a number of growth-related processes, phosphorylates and thereby activates fetal smooth muscle myosin while having an inhibitory effect on its adult counterpart. Thus, the proposed studies will increase our understanding of the role of myosin phosphorylation and clarify the presence of alternative mechanisms in regulating contractile processes in smooth muscles. This information will not only result in improved therapy for smooth muscle-associated diseases but may also help elucidate the strikingly similar contractile events that occur in non muscle cells.
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