The principal goal of this project is to examine how catecholamine-responsive cells regulate their number of alpha- and beta-adrenergic receptors. Recent work in my laboratory using radioligand binding techniques and functional assays has shown that two cell types, BC3H-1 muscle cells and MDCK renal tubular epithelial cells, co-express alphal-and beta2-adrenergic receptors. These two cell types thus offer a unique opportunity to test directly whether alpha- and beta-adrenergic receptors present in the same cell are separate molecular species; to resolve this issue we propose experiments of solubilized receptors analyzed by density gradient centrifugation, gel filtration chromatography, and SDS polyacrylamide gel electrophoresis. Moreover, we will compare the """"""""life cycle"""""""" of these two types of receptors in the same cell by using BC3H-1 and MDCK cells to assess biosynthesis and degradation of alphal- and beta2-adrenergic receptors. We will establish two separate methods--one involving """"""""heavy amino acids"""""""" and the other receptor-specific, irreversible antagonists--in order to define the kinetics and mechanisms of receptor formation and turnover. In additional studies of mechanisms regulating receptor metabolism, we will use inhibitors of processes implicated in such regulation and will monitor the fate of receptors labeled with irreversible antagonists. In further experiments we will relate the appearance of surface receptors to the acquisition of functional activity and will define the contribution of changes in receptor synthesis and degradation to agonist-mediated """"""""down regulation."""""""" The experiments in this proposal have been designed to assess alpha- and beta-adrenergic receptor metabolism in target cells using methods that only minimally perturb cellular function. Accordingly, the results are likely to yield new and potentially important information regarding cellular events that receptors on catecholamine-responsive cells. Because of the wide range of tissues capable of responding to catecholamines and the importance of adrenergic drugs in therapy of many clinical disorders, the results are likely to prove applicable not only to the two principal cell types that we will study--muscle and kidney epitheliuim--but also to a large number of other cells that possess functional adrenergic receptors. Information gained in these studies should have particular relevance to cardiovascular and pulmonary diseases.
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