The long range objective is to elucidate the roles of phosphorylation and dephosphorylation of specific vascular proteins in regulating coronary contractility. This study focuses on mechanisms of regulation and function of a vascular phosphatase, PCM-phosphatase (POLYCATION MODULABLE) discovered in this laboratory. PCM-phosphatase probably is the major myosin phosphatase in vascular smooth muscle. The underlying hypothesis is regulation of protein phosphatase activity participates in modulating arterial contractility: the specific hypothesis is PCM-phosphatase consists of a catalytic domain and a modulatory domain containing several regulatory polypeptides (RPP). Selective loss or acquisition of RPP allows for appearance of multiple forms of PCM-phosphatase. Interactions between RPP (recently identified in this laboratory) and an appropriate concentration of cationic effector, such as naturally occurring histone-H1 or synthetic polylysine, alters substrate specificity such that dephosphorylation of myosin is markedly enhanced, whereas dephosphorylation of phosphorylase is suppressed. Bovine aortic and coronary PCM-phosphatase will be characterized with respect to substrate specificity, subunit structure, and susceptibility to modulation of expressed activity by cationic effectors and specific kinases present in vascular smooth muscle. RPP will be characterized and interactions with cationic effectors leading to regulation of phosphatase activity will be elucidated. Effects of RPP and cationic effectors (natural and synthetic) on endogenous PCM-phosphatase actinmyosin interaction are assessed in two contractile model systems at different concentrations of Ca2+: native actomyosin (ATPase) and detergent-skinned coronary artery (isometric force). In both models, extent of actin-myosin interaction will be correlated to extent of myosin light chain phosphorylation. Based on experimental findings obtained, changes in PCM-phosphatase activity, and/or efficacy of cationic effectors in modulating expressed phosphatase activity, will be evaluated during contraction and relaxation of coronary arterial smooth muscle. Experiments employ established procedures which have been used in the laboratory for biochemical studies of protein purification of proteins and enzyme assays, and physiological studies of contractile responses. Since contraction of vascular smooth muscle involves Ca2+ dependent phosphorylation of myosin and other proteins, defects in regulation of protein phosphatase activity may contribute to progressive vascular disease, particularly in hypertension and diabetes.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Experimental Cardiovascular Sciences Study Section (ECS)
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University of Cincinnati
Schools of Medicine
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Steenaart, N A; Ganim, J R; Di Salvo, J et al. (1992) The phospholamban phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme. Arch Biochem Biophys 293:17-24
Rymaszewski, Z; Szymanski, P T; Abplanalp, W A et al. (1992) Human retinal vascular cells differ from umbilical cells in synthetic functions and their response to glucose. Proc Soc Exp Biol Med 199:183-91
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Erdodi, F; Rokolya, A; Di Salvo, J et al. (1988) Effect of okadaic acid on phosphorylation-dephosphorylation of myosin light chain in aortic smooth muscle homogenate. Biochem Biophys Res Commun 153:156-61
Di Salvo, J (1987) Aortic polycation-modulable protein phosphatase(s): structure and function. Prog Clin Biol Res 245:195-206
Gifford, D; Di Salvo, J (1987) Glycosaminoglycans and a newly purified aortic chondroitin proteoglycan block polycationic modulation of protein phosphatase activity. Proc Soc Exp Biol Med 184:64-73
Bialojan, C; Ruegg, J C; DiSalvo, J (1987) A myosin phosphatase modulates contractility in skinned smooth muscle. Pflugers Arch 410:304-12

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