The long-term goal of this proposal is to investigate the molecular regulation of smooth muscle contractile proteins. Two general hypothesis will be tested. Hypothesis 1: Myosin phosphorylation regulates the number of active crossbridges in vascular and nonvascular smooth muscle. Hypothesis 2: the actin-binding proteins caldesmon and filamin contribute to an actin-linked regulatory system operating in concert with the myosin-linked phosphorylation system.
The specific aims which will be addressed by these studies are: (1) to determine whether myosin phosphorylation functions as a simple switch to activate force-generating crossbridges, (2) to determine whether noncycling, attached """"""""latch-bridges"""""""" exist in vascular and/or nonvascular smooth muscle, (3) to determine if the regulation by myosin phosphorylation is mechanistically similar for vascular (both tonic and phasic) and nonvascular smooth muscle, (4) to determine if the presence of a """"""""latch-state"""""""" contributes to the mechanical differences (e.g., different rates of isotonic shortening velocity) observed for different smooth muscle types, (5) to determine if the actin-binding proteins caldesmon and filamin contribute to an actin-linked regulatory system in smooth muscle, and (6) to investigate the possible involvement of caldesmon and filamin in the regulation of """"""""latch-bridge"""""""" formation in vascular and nonvascular smooth muscle.
These specific aims will be approached experimentally by utilizing both intact and skinned preparations from porcine carotid, rat portal vein, and rat uterine smooth muscle. The relationship betwen myosin phosphorylation and isometric force, stiffness, and shortening velocity will be determined for both intact and skinned muscles. Experiments with the skinned muscle preparation involving the addition of purified myosin light chain phosphatase, myosin light chain kinase, thiophosphorylated myosin light chains, caldesmon, filamin, and inactive proteolytic fragments of caldesmon and filamin will be performed to examine both myosin-linked and actin-linked regulatory mechansims. In general, these studies should provide data essential to the understanding of the molecular regulation of vascular and nonvascular smooth muscle contraction. This in turn is important because of the important role blood vessel disease plays in human morbidity and mortality.
