Arachidonic acid is one of the most versatile molecules in Biology. It serves as the initial substrate for three major metabolic pathways (cyclooxygenase, lipoxygenase, cytochrome P450) resulting in its conversion to over l00 biologically active lipids. While metabolites from all of these pathways have diverse and potent biologic effects, predominantly as autocoids, a central role for metabolites from the cyclooxygenase pathway in regulating renal function has been most firmly established. A particularly striking feature of the actions of PGE2, the major renal cyclooxygenase product, is its multiple regulatory influences on both the renal circulation, and on certain hormonally regulated transport processes, i.e. sodium absorption in the medullary thick limb of Henle, sodium absorption In the collecting duct, and osmotic water flow resulting from the action of arginine vasopressin in the collecting duct. In an effort to explain the molecular basis for these multiple actions, we have completed studies focused on the collecting duct. These studies have led us to postulate that three separate PGE2 receptors are involved in the regulation of Na+ and water transport in this segment of the nephron. The individual components of this three receptor model conform to the work of others who, using bioassays of smooth muscle contraction or relaxation in certain tissues, have identified three prostaglandin receptors called EP1, EP2, and EP3. Over the past year, critical new information has emerged identifying prostaglandin receptors as seven membrane-spanning G protein- coupled receptors. The genes for EP1, EP2, and EP3 have now been cloned (the human EP1 by our collaborator, Dr. Colin Funk, at Vanderbilt and the rabbit renal EP3 receptor by another collaborator, Dr. Richard Breyer, at Vanderbilt), thus enabling us to molecularly characterize these receptors in the kidney. We propose a set of complementary techniques which will characterize the specific receptor-coupled transport events regulated by agonists for these three receptors. Our studies will focus mostly on the medullary thick ascending limb of Henle and the cortical collecting duct of the rabbit. These functional studies will be complemented by several molecular approaches (in situ hybridization with receptor specific riboprobes, Northern hybridization and nuclease protection assays for receptor mRNA, and immunolocalization using anti-receptor specific antibodies) designed to localize specific receptor types to either apical or basolateral cell membranes of transporting epithelia. Our overall strategy also includes identification of receptor-specific agonists and antagonists that might become useful as models for the development of new therapeutic agents as well as tools for understanding specific receptor structure/function relationships (i.e. binding pocket characteristics which define ligand specificity). We also hope to extend these studies to mapping the EP receptors to specific human chromosomes. Finally, the results of these studies should allow us to pose and solve potentially important pathophysiologic questions about altered regulation or structure of the EP receptors.
Showing the most recent 10 out of 90 publications
