The goal of the work outlined in this application is to further understanding of the signalling systems in vascular smooth muscle that result in an alteration of contractility. In particular, studies are proposed to investigate the roles of mitogen-activated protein kinase (MAPK) and caldesmon in vascular smooth muscle contraction. The high molecular weight form of caldesmon (h-caldesmon) is a potent, smooth muscle specific, inhibitor of actomyosin ATPase activity, in vitro, that binds both actin and myosin. h-Caldesmon is phosphorylated in resting porcine carotid arteries and the stoichiometry of phosphorylation increases in response to pharmacological stimulation suggesting that this process may be regulatory for contraction. h- Caldesmon is phosphorylated on two sites in the carboxyl-terminus of the protein; these sites are modified by mitogen-activated protein kinase (MAPK), in vitro. The hypothesis to be tested is that the activation of MAPK is the contractile phenotype of smooth muscle results in the phosphorylation of h-caldesmon and an alteration of contractile function. Two sub-hypotheses to this are that the phosphorylation of h- caldesmon by MAPK 1:alters actomyosin kinetics, either directly or via an interaction with another protein, and 2) allows for structural changes within the cell that are required for prolonged alterations of stress. In order to investigate this hypothesis, the following specific aims will be pursued: 1. To measure MAPK activity, h-caldesmon phosphorylation and force upon pharmacological stimulation of porcine carotid arteries. Agonists will include neurohumoral agents and growth factors. 2. To measure MAPK activity and h-caldesmon phosphorylation in porcine carotid arteries subjected to stretch. MAPK activity and h-caldesmon phosphorylation levels will be measured as a function of mechanical load applied to the muscle. 3. To determine the biochemical effects of h-caldesmon phosphorylation by MAPK on a)actin and myosin binding to h-caldesmon, b) actin polymerization, and c) actomyosin ATPase activity. Studies in this aim will investigate the direct biochemical effects of h-caldesmon phosphorylation on the contractile apparatus. MAPK plays a role in cultured cell growth and proliferation; however, cells in the contractile phenotype of vascular smooth muscle do not normally undergo cell division. These studies are unique in investigating involvement of MAPK in smooth muscle contractility, the primary function of this tissue.
