ADENOSINE RECEPTORS AND ION TRANSPORT IN EPITHELIA

本申请的目的是确定腺苷的作用(S 腺苷受体在调节肾脏离子转运中的作用 和肠上皮细胞。具体地说，我们建议本地化A1 和A2腺苷受体结合到细胞中。 哺乳动物肾单位(肾小球和肾小管段)和回肠 (隐窝和绒毛细胞)使用受体-配体结合技术。 我们将描述腺苷的不同转运途径。 跨越上皮刷状缘和基底外侧膜 并测量间质液体中的腺苷浓度。 我们将确定受体信号转导的机制 在腺苷A1和A2具有功能性的模型上皮中 感受器。方法包括放射配基受体结合研究 使用高活性激动剂和拮抗剂，测量 腺苷环化酶活力、ADP核糖化、聚丙烯酰胺凝胶 GTP结合调节蛋白的电泳法和放射自显影 蛋白质，直肠腺细胞原代培养，~3H-研究 离体膜囊泡中腺苷转运的测量 间质腺苷和细胞内钙浓度， 鲨鱼直肠腺的体外灌流。这项提议将 检验腺苷的外部受体是 存在于肠上皮细胞和肾小管上皮细胞，并且是生理性的 这些上皮细胞中离子转运的调节器。

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.