The long term goals of our research are to determine how the synthesis of prostaglandin E2 (PGE2) is regulated in the renal collecting tubule and how PGE2 functions in the collecting tubule to influence solute and water transport. We recently developed a procedure to culture canine cortical collecting tubule (CCCT) cells. CCCT cells exhibit major morphological and biochemical properties expected for the collecting tubule and provide a reasonable in vitro model for examining collecting tubule biochemistry. We have found that CCCT cells form PGE2 in response to arginine vasopressin (AVP), and that PGE2 inhibits AVP-induced cAMP formation. The first specific aim of our proposed research is to determine the mechanism of this inhibition. We will test the hypothesis that PGE2 treatment of CCCT cells causes an intermediate sequence of events which prevents activation of adenylate cyclase by AVP or activates cAMP phosphodiesterase. The second specific aim of our research is to separate and culture principal (light) and intercalated (dark) collecting tubule cells present in CCCT cell populations. This will be accomplished by cell sorting after labeling the dark cells with a monoclonal antibody. Once isolated, dark and light cells will be tested for their capacities to form prostaglandins and cAMP in response to AVP, bradykinin, isoproterenol and PGE2. This work will provide methods for culturing the two types of collecting tubule cells and information on the hormonal responses of these cells. The third specific aim of our proposed research is to determine the immunochemical, physical and kinetic characteristics of PGH:PGE isomerase activities. This enzyme (or group of enzymes) catalyzes the last step in the synthesis of PGE2, the major prostaglandin product of the collecting tubule. We have purified a solubilized PHG:PGE isomerase activity 150 fold by glutathione (GSH)-Sepharose chromatography. Partially purified PGH:PGE isomerase exhibits one major and three minor protein bands on SDS-polyacrylamidegels. Further purification will be performed using a prostaglandin endoperoxide analog as an affinity ligand. Monoclonal antibodies will be generated against the partially purified enzyme and used to determine if all PGH:PGE isomerase activities are catalyzed by proteins which are related immunochemically. Using the purified enzyme, we will define the mechanism by which GSH acts as an activator of the conversion of PGH to PGE.
Showing the most recent 10 out of 11 publications
