The aims of this proposal are: a) to understand the functional relationship between Beta-adrenergic receptor and adenylate cyclase in intact cells and b) to determine the effects that agonist-induced changes in the Beta-adrenergic receptor have on intact cell adenylate cyclase activity. It is planned to use primarily S49 cultured lymphoma cells for these studies, but other cell types (WI-38 and VA13 fibroblasts and turkey erythrocytes) are available in this laboratory for purposes of comparison. The hypothesis that catecholamine-specific desensitization in S49 cells is explicable entirely in terms of changes in receptor state and number will be examined in detail. This will be achieved by: a) quantitating changes in the rate of intracellular cAMP synthesis that occur with time of agonist treatment and relating those changes with the effects of the agonist on the receptor and b) comparing the effects of reduced receptor availability and of desensitization on intracellular adenylate cyclase activity. Methodology includes: a) inhibition by catecholamines of 125I-iodopindolol binding to Beta-adrenergic receptors as an assay for catecholamine affinity to the receptor, b) a newly developed technique in which 125I-iodopindolol binding data is used to determine the rate constants of association and dissociation of unlabeled antagonists and c) the use of intracellular cAMP turnover data to calculate the time courses of adenylate cyclase desensitization from time courses of cAMP accumulation. Knowledge of the mechanism of Beta-adrenergic receptor desensitization and of its consequences on adenylate cyclase activity should lead to a more rational approach to the design and utilization of Beta-adrenergic agonists and antagonists for such conditions as heart disease and asthma.
