The overall organization of urinary acidification has been defined by a variety of studies of the intact kidney and individual nephrons in vivo, yet relatively little is known about the transport processes across the individual cell membranes--their biochemical nature, their rate-limiting factors, and their organization within the epithelial cell layer. The proposed research extends our work on the control of the rate of urinary acidification in the turtle urinary bladder and focuses on the nature of the transport processes across the cell membranes, i.e. the proton pump at the luminal membrane and the efflux of bicarbonate across the serosal membrane. The proton pump characteristics will be analyzed according to a kinetic model consisting of two components: a catalytic unit responsible for active H+ translocation and a membrane channel with a finite resistance to H+ flow. This model simulates the observed relationship between transport rate (JH) and Delta MuH in the linear region and will be tested in the nonlinear regions at large and small Delta MuH. In addition to kinetic factors, JH is regulated by the number of H+ pumps present in the luminal membrane of the carbonic anhydrase(CA) containing cell population. The role of endocytosis and exocytosis will be examined by morphometric techniques during CO2 stimulation of JH with and without agents inhibiting cytoskeletal function. In a combined biochemical and morphologic approach to the isolation of the H+ pumps, we will test the hypothesis that a distinctive rod-shaped intramembrane particle that occurs in abundance in the luminal and vesicular membranes of the CA cells contains components of the H+-translocating ATPase. Freeze fracture studies of membrane fractions will be correlated with the activity of oligomycin- and ouabain-resistant ATPase. In related efforts, we will examine two HC03- transport systems which depend on Cl- and occur in the same epithelium, one in series with the H+ pump and one parallel to the H+ pump. The possibility will be explored that the parallel HCO3- secretory system occurs in a subpopulation of CA cells and is composed of the same transport elements in a different arrangement across the two cell membranes. These studies should advance the understanding of the cellular mechanisms of urinary acidification and provide new insights into epithelial acid-base transport in general.
