During recent years there has been considerable interest in the role that alpha adrenergic receptors play in the regulation of cardiac function and metabolism. The overall objective of this proposal is to define in a comprehensive manner the molecular mechanism(s) of action of the cardiac alpha receptor. Studies will be conducted using the isolated adult ventricular myocyte. The work will focus on two known outcomes of alpha receptor occupation by agonist; i. activation of cyclic AMP degradation and ii. increased hydrolysis of phosphatidylinositol. HPLC- detection will be employed to fully characterize the nature of the sugar phosphates formed by alpha receptor stimulation and the kinetics of their appearance and disappearance in the cell. Stable GTP analogues will be used in broken cell preparations to investigate the role of a GTP-binding protein in the activation of both a cAMP- phosphodiesterase and phospholipase C. Cyclic AMP-phosphodiesterase (PDE) activity will be studied in subcellular fractions prepared from myocytes in order to investigate the nature and cellular location (cytosol, SL, SR etc.) of the alpha-receptor stimulated PDE. SDS polyacrylamide gel electrophoresis and isoelectric focusing will be used to study the nature of the putative G-protein and the possibility that it is a substrate for either cholera or pertussis toxins. Fura 2/AM labeling of myocytes will be employed to study the alpha-receptor regulation of Ca++ release in the myocyte. The work will permit a careful correlation of the time course of inositol phosphate formation, Ca++ release and PDE activation. Regulation of alpha receptor responsiveness will be studied in myocytes treated with phorbol esters to test the hypothesis that desensitization of response occurs due to C-kinase dependent phosphorylation of the G-protein coupling alpha receptors to their effector. These studies will significantly advance our understanding of the regulation of cardiac muscle cell metabolism by alpha receptors. Because much is already known about the regulation of cAMP action in these single muscle cells, it is likely that the proposed work on alpha receptor mechanisms can explain in detail the sequence of events occurring inside cardiac muscle cells when the endogenous agonist norepinephrine stimulates both alpha and beta receptors as surely occurs in vivo. Furthermore, these studies will provide a basic framework in which to assess changes in alpha receptor effects in disease.
