application) The long-term objective of this research is to understand the mechanism by which myosin light chain phosphorylation is regulated in smooth muscle. Smooth muscle contraction is primarily regulated by myosin light chain phosphorylation. Since the protein kinase responsible for phosphorylation of myosin is Ca2+/calmodulin dependent myosin light chain kinase, Ca2+ is the primary intracellular messenger and required for initiation of contraction. However, evidence has been accumulated that G-protein mediated pathways play an important role I the regulation of myosin phosphorylation and thus muscle contraction. Hypothesis to be tested in this project is how Rho dependent pathway influences myosin light chain dephosphorylation activity thus regulating smooth muscle contractility. Critical question is how interactions among the key components in the Rho pathway such as Rho G-protein, Rho dependent kinase, myosin phosphatase subunits and myosin, change during stimulation-contraction coupling thus regulating myosin phosphorylation. The project approaches this problem by determining the localization, translocation and interaction of these components in living smooth muscle cells during cell activation by agonists using ultra fast 3D digital fluorescence imaging techniques. The regulatory functions of the key regulatory components on myosin dephosphorylation/cell contraction will be elucidated by transfecting the gene of interest into fully differentiated smooth muscle cultured cells using adenovirus mediated gene transfer technique. The transfected smooth muscle cells will be characterized by determining myosin phosphorylation levels and phosphorylation/dephosphorylation rates in vivo as well as determining mechanical properties of the cells. The itemized specific aims are: 1. Define the translocation of RhoA and Rho kinase during the stimulation of smooth muscle cells. 2. Define the interaction of M110 and M21 phosphatase regulatory subunits with myosin during the stimulation of smooth muscle cells. 3. Define the function of RhoA and Rho-kinase on the phosphorylation of myosin light chain phosphatase subunits. 4. Define the function of RhoA and Rho-kinase on myosin phosphorylation in smooth muscle cells and smooth muscle contractile response.
