MODULATION OF VASCULAR CONTRACTION BY PHOSPHATASES

这项建议的长期目标是确定肌凝蛋白的作用

The long-term goals of this proposal are to determine the role of myosin light chain phosphorylation in the regulation of vascular smooth muscle mechanical properties and to investigate the specific enzyme or enzymes which dephosphorylate myosin. The specific aims that will be undertaken to investigate myosin dephosphorylation in vascular smooth muscle are: 1) to purify and characterize the phosphatase activities in vascular smooth muscle which demonstrate activity toward isolated myosin light chains or intact myosin; 2) to classify myosin phosphatase activities with respect to other known substrates and to examine important relationships among phosphatases using biochemical and immunochemical techniques; 3) to determine the relationship between the stoichiometry of myosin light chain phosphorylation and the mechanical parameters, isometric force and lightly-loaded shortening velocity, in chemically-skinned vascular smooth muscle; 4) to examine the effects of specific phosphatase inhibitors on myosin phosphorylation and mechanical properties (ie. isometric force and lightly-loaded shortening velocity) in chemically-skinned vascular smooth muscle; and 5) to examine the effects of purified myosin phosphatases on mechanical properties of chemically-skinned vascular smooth muscle. Myosin or myosin light chain phosphatases will be purified by standard protein isolation methods and extensively characterized. A major attempt will be made to determine whether multisubunit phosphatases share common subunits with other enzymes displaying different substrate specificities. In addition, the actin binding properties of purified phosphatases will be investigated. Ultimately, purified phosphatases will be added directly to small muscle baths containing glycerinated (ie. chemically-skinned) vascular smooth muscle. The effects on myosin light chain phosphorylation levels will be determined by glycerol-urea gel radioimmunoblot. In addition, force and lightly-loaded shortening velocity will be monitored using and electromagnetic ergometer. In this manner, we will determine which mechanical parameter is sensitive to changes in myosin light chain phosphorylation and the quantitative relationship of this sensitivity. It is anticipated that studies will be conducted over five years by a Physician-Scientist. In general, these studies are deemed to be important because of the important role blood vessel disease plays in human morbidity and mortality. Our proposal will investigate the basic mechanism of contraction in vascular smooth muscle.