Significant advances have been made in understanding molecular events which transpire in response to binding of catecholamines to Beta-adrenergic receptors, largely because pure populations of cells have become models for studying Beta-adrenergic mechanisms. In contrast, neither a relatively pure cell type nor a cultured cell line possessing large numbers of Alpha-adrenergic receptors has been available as a model to study Alpha-mechanisms. The overall aim of this proposal is to use endocrinological and biochemical techniques to study Alpha-adrenergic mechanisms in a smooth muscle cell line, the DDT1 MF-2 cell. DDT1 Mf-2 cells are grown easily in culture and Alpha 1-adrenergic receptors are present in large numbers (65,000 receptors/cell) which facilitates our aims. Specifically, the objectives of this proposal are: 1) to investigate the physiological response (e.g., Ca++ flux) as a result of catecholamine binding to the Alpha 1-adrenergic receptor, 2) to purify and biochemically characterize the Alpha-adrenergic receptor and 3) produce monoclonal antibodies directed towards the Alpha 1-adrenergic receptor. Our long term objective is to use the DDT1 cell line as a model to study the Alpha-adrenergic receptor and other membrane-associated effector proteins that link receptor to response.