MECHANISM AND ROLE OF CYCLIC GMP AND CALCIUM IN HEART

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The objectives of the research are to study 1) the mechanism of action of Ca 2 ion pertaining to protein phosphorylation, and its regulation by phospholipid and calmodulin, 2) phosphorylations of cardiac proteins stimulated by Ca 2 ion, cGMP and/or cAMP and their interrelations, 3) possible modifications in properties of sarcolemma and sarcoplasmic reticulum to bind, transport and/or uptake Ca 2 ion as results of Ca 2 ion-, cGMP-and/or cAMP-dependent phosphorylations of their proteins, and 4) possible effects of cardioactive agents and physio-pathologic states of the heart on the parameters mentioned in 1), 2) and 3). Phospholipid-sensitive and calmodulin-sensitive Ca 2 ion-dependent protein kinases will be purified to homogeneity from the bovine heart (and/or brain) by procedures including controlled-pore glass, phosphatidylserine-Affigel 102 and calmodulin-Sepharose 4B chromatographies. Phosphorylations of endogenous proteins in the rat and/or guinea pig heart, stimulated by Ca 2 ion, cGMP and/or cAMP and radiolabeled by exogenous or endogenous (gamma-32P)ATP, will be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. Isolation and purification of endogenous substrate proteins will be done by absorbing the (35S)thiophosphorylated proteins on organomercury-affinity gel (Affigel 501) and specifically eluting them with dithiothreitol. Distribution of protein kinases and their substrates in the myocardium, and effects of physiopathologic states on these parameters, will be studied by radioimmunolabeling these proteins separated on polyacrylamide gels with the antibody/(125I)protein A technique followed by autoradiography.