It is widely accepted that an increase in [Ca2+] initiates vascular smooth muscle contraction. In addition, it is now known that the sensitivity of the contractile proteins to Ca2+ can be modulated. Agonist stimulation of vascular smooth muscle increases myofilament Ca2+ sensitivity by a G- protein dependent pathway; the steps in this pathway are however unknown. This proposal is designed to determine the pathway by which G-protein activation leads to an alteration in Ca2+ sensitivity. Specifically, this proposal will test the hypothesis that agonist activation of the smooth muscle cell initiates the following cascade of events; G-protein dependent activation of protein kinase C (PKC); PKC dependent activation of mitogen activated protein kinase kinase (MEK); MEK dependent activation of mitogen activated protein (MAP) kinase; down regulation of MLC phosphatase activity by MAP kinase catalyzed phosphorylation; and elevation of MLC phosphorylation levels at any given [Ca2+]. The following specific aims will be used to test this hypothesis; 91) To determine if activation of PKC increases while its inhibition decreases myofilament Ca2+ sensitivity; (2) To determine if MEK and MAP kinase activities are increased during agonist activation and if inhibition of PKC abolishes the increase; and (3) To determine if MLC phosphatase is phosphorylated during agonist activation and whether or not the phosphorylation site is a proline-directed serine. These studies will provide the necessary training with which to pursue my long term goal of becoming an independent academic scientist investigating vascular physiology.
