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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038976-03
Application #
3355492
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1988-02-01
Project End
1993-01-31
Budget Start
1990-02-01
Budget End
1991-01-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
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del Balzo, U; Rosen, M R; Malfatto, G et al. (1990) Specific alpha 1-adrenergic receptor subtypes modulate catecholamine-induced increases and decreases in ventricular automaticity. Circ Res 67:1535-51
Viamonte, V M; Steinberg, S F; Chow, Y K et al. (1990) Phospholipase C modulates automaticity of canine cardiac Purkinje fibers. J Pharmacol Exp Ther 252:886-93
Han, H M; Robinson, R B; Bilezikian, J P et al. (1989) Developmental changes in guanine nucleotide regulatory proteins in the rat myocardial alpha 1-adrenergic receptor complex. Circ Res 65:1763-73
Steinberg, S F; Kaplan, L M; Inouye, T et al. (1989) Alpha-1 adrenergic stimulation of 1,4,5-inositol trisphosphate formation in ventricular myocytes. J Pharmacol Exp Ther 250:1141-8