ACTIVE H+ TRANSPORT IN URINARY ACIDIFICATION

H+ and HCO3 are transported across tight urinary epithelia through the intercalated (or mitochondira-rich) cells. In these cells a H+ ATPase in one cell membrane is present in series with a Cl-HCO3 exchanger in the other. We found that there are two forms of these cells, one secretes H+ into the lumen while the other secretes HCO3. These two types of cells have opposite polar distribution of these two transporters. Further, we found that when animals are acid loaded, the HCO3 secreting cell changes its polarity to that of the H+ secreting cell. The aims of this proposal is to purify the H+ ATPase from isolated kidney endocytic vesicles. The catalytic and H+ conducting sectors of this ATPase will be separately purified and reconstituted. Polyclonal and monoclonal antibodies will be raised to these proteins and the biosynthesis and degradation of the ATPase will be analysed. The polar distribution of the H+ ATPase and the Cl:HCO3 exchanger will be studied by immuno-electromicroscopy. Antibodies to the Cl:HCO3 exchanger will be raised using its probably analogues the red cell band 3. The organization of the transport proteins and their relation to the cytoskeleton will be examined before and after acid feeding. Finally, preliminary studies suggest that endocytic removal of the proton ATPase is stimulated by removal of CO2. We will measure the rate of removal of these proton pumps and the role that cell pH and calcium might play in their control. Regulation of acid base balance largely occurs through contol of H+ and HCO3 transport by the renal epithelial cell. The studies outlined in this proposal aim at clarifying the cellular and molecular mechanisms that underlie this process.