The purpose of this investigation is to evaluate the role of a guanine nucleotide regulatory protein (N protein) in alpha 1- adrenergic biochemical and pharmacological responses. The experimental models to be employed include rat cardiac myocytes in primary culture, neonatal and adult ventricular myocardium, and canine Purkinje fibers. In these systems, the nature of the pharmacologic response to alpha-agonists appears to be related to a 41K ADP-ribosylatable substrate for pertussis toxin. Acquisition of this substrate, as a function of myocardial innervation, is associated with the conversion of the chronotropic response to alpha 1-adrenergic catecholamines from plus to minus. Functional linkage of this protein to the alpha 1-adrenergic response is indicated by experiments demonstrating that exposure of innervated myocytes to pertussis toxin to ADP-ribosylate and inactivate the 41K substrate completely reverses the alpha 1-adrenergic agonists do not alter cAMP levels in primary cultures of cardiac cells by they do stimulate inositol phospholipid breakdown and inositol- phosphate formation. A substantial component of the inositol lipid response is also coupled by a 41K ADP-ribosylatable pertussis toxin substrate.
The specific aims of this research proposal are to identify, using two-dimensional protein separation and immunochemical techniques, the pertussis toxin substrate which couples alpha 1-adrenergic receptor activation to negative chronotropy in innervated myocardial tissue; to perform a detailed investigation using HPLC techniques of the effect of alpha 1- adrenergic receptor activation on inositol phospholipid metabolism and to characterize the control of this pathway by N proteins; and to elucidate the actions of alpha 1-adrenergic catecholamines upon cellular calcium homeostasis using fluorescence microscopy (with the intracellular fluorescent calcium indicator, fura-2) and 45Ca flux studies and to assess the dependence of the calcium response on inositol phosphate metabolites generated via alpha 1-adrenergic receptor activation and N proteins. The studies are designed to enhance our understanding of the cellular actions of the alpha 1- adrenergic catecholamines and to provide a framework with which normal adrenergic responsiveness in health and altered adrenergic function in disease can be appreciated more completely.
