Studies proposed in this project relate to; 1.) the role of rapidly turning over proteins in the regulation of adenylate cyclase by catecholamines and forskolin, and 2.) fluorescent microscopic localization of beta adrenergic receptors in the myocardium add cultured cells using video intensification microscopy and digital image processing. During the preceding grant period we have identified and characterized phenomena which suggest the presence of adenylate cyclase components or regulatory molecules which are rapidly turning over and involve RNA and protein synthesis. The first putative protein is involved in heterologous hormone refractoriness and is induced by the product of adenylate cyclase, cyclic AMP. The second rapidly turning over protein is necessary for the direct stimulatory actions of forskolin yet is not necessary for catecholamine mediated cyclic AMP accumulation or the potentiative actions of forskolin. In this proposal we propose to isolate, identify and ultimately study the regulation of these proteins using reconstitution assays into digitonin permeabilized cells, a system which maintains whole cell adenylate cyclase activity yet enables the addition or reconstitution of components into native cell membranes. Furthermore we will continue study the expression of mRNA for these cyclase components using Xenopus oocytes for the translation of mRNA isolated from cells undergoing active synthesis of these proteins. Hormone stimulated cyclic AMP accumulation and electrical or ion channel activity in oocytes or oocyte membranes will be used to identify the successful translation of the putative proteins and as an assay for the isolation or enrichment of their respective mRNA species. The second of this project involves the continued development of intensely fluorescent and 125I labeled beta-adrenergic receptor antagonists to accurately and specifically localize beta-adrenergic receptors in living cells. Our first successful derivative, NBD- 125I-iodopindolol maintains high specificity and affinity of binding to the beta receptor and has enabled its localization by high sensitivity video intensification fluorescence microscopy with digital image processing. This approach will be especially useful in studies of hormone refractoriness and receptor internalization in the myocardium and other cells. With this methodology we can evaluate our hypothesis that the dual and distinct actions of isoproterenol in the myocardium to relax and to stimulate the slow inward current is mediated by beta-receptors localized in two distinct regions of the cell.
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