The aim of this application is to identify the role(s) of adenosine and adenosine receptors in the regulation of ion transport in renal and intestinal epithelia. Specifically, we propose to localize A1 and A2 adenosine receptors to cells from isolated segments of the mammalian nephron (glomeruli and tubular segments) and ileum (crypt and villus cells) using receptor-ligand binding techniques. We will characterize separate transport pathways for adenosine across brush-border and basdolateral membranes of epithelial cells and measure adenosine concentrations in interstitial fluid. We will determine the mechanisms of receptor signal transduction in a model epithelium with functional A1 and A2 adenosine receptors. Methods include radioligandreceptor binding studies with high activity agonists and antagonists, measurements of adenylate cyclase activity, ADP ribosylation, polyacrylamide gel electrophoresis and autoradiography of GTP binding regulatory proteins, primary culture of rectal gland cells, studies of 3H- adenosine transport in isolated membrane vesicles, measurements of interstitial adenosine and intracellular calcium concentrations, and in vitro perfusion of shark rectal glands. The proposal will examine the hypothesis that external receptors for adenosine are present on enterocytes and renal tubular cells and are physiologic regulators of ion transport in these epithelia.
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