The long-range goal is to determine how differences in actin-myosin interaction in smooth muscle from the esophagus may account for the mechanical properties of this muscle that lead to such specialized functions as active conduct versus sphincter. A major hypothesis to be tested is that isoenzymes of actin, interacting with key actin-binding proteins (i.e. caldesmon), play a major role in determining the kinetics of actin-myosin interaction. In order to achieve the long-range goal and test the specific hypothesis, six specific aims are proposed: 1) To prepare the predominant actin isoforms from the body (Gamma-actin) and sphincter (Alpha-actin) of the cat esophagus and compare the two with respect to activation of myosin ATPase and binding of heavy meromyosin (HMM); 2) To purify the actin-binding protein, caldesmon, from the body and sphincter of the esophagus and conduct comparative biochemical studies; 3) To examine the effects of caldesmon on acto-HMM ATPase, binding of HMM to actin and upon selected steps in the acto-HMM ATPase cycle; 4) To investigate several factors that may reverse the effects of caldesmon on acto-HMM ATPase and upon binding of HMM to actin such as calmodulin, phosphorylation and ATP-regenerating systems; 5) To examine the relationship between isometric force, shortening velocity and myosin light chain phosphorylation in intact and chemically-skinned esophageal smooth muscle; and 6) To study the effects of agents that may reverse the effects of caldesmon on acto-HMM ATPase in both intact and chemically-skinned esophageal smooth muscle. A variety of biochemical techniques, including purification of actin, myosin and caldesmon, measurement of actomyosin ATPase activity and analysis of binding of caldesmon and heavy meromyosin to actin will be used to address specific aims 1-44. Intact and chemically-skinned esophageal smooth muscle preparations will be used for specific aims 5-6. At a basic scientific level, these studies are likely to provide important new information about the contractile mechanism in smooth muscle. Moreover, a more fundamental insight into clinical disorders of esophageal motility (i.e. reflux) may be a relevant biomedical consequence of these studies.
