ucn-1028-c and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

The effect of hypotonicity on H+-ATPase activity was examined in cultured inner medullary collecting duct (mIMCD-3) cells. mIMCD-3 cells were grown to confluence, loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and assayed for H+-ATPase activity measured as the Na+- and K+-independent intracellular pH (pHi) recovery following an acid load. Exposure of mIMCD-3 cells to a hypotonic solution (150 mosmol/kgH2O) increased pHi recovery by approximately 350% (P < 0.0001). This effect was inhibited by diethylstilbestrol (an inhibitor of H+-ATPase) and was not dependent on external K+, indicating lack of involvement of H+-K+-ATPase. H+-ATPase activation was acute, independent of cell calcium, and was not secondary to Cl- channel activation. The magnitude of H+-ATPase upregulation was dependent on the osmolarity of the media, with maximum stimulation at 150 mosmol/kgH2O. H+-ATPase upregulation in hypotonicity was significantly blocked in the presence of staurosporine or calphostin C or in cells pretreated with phorbol 12-myristate 13-acetate (PMA), indicating involvement of protein kinase C. Hypotonicity inhibited the Na+/H+ exchanger activity in mIMCD-3 cells, indicating that its stimulatory effect is specific to H+-ATPase. In conclusion, a novel regulatory mechanism of H+-ATPase by hypotonicity is described. The increased H+-ATPase activity in hypotonicity may be responsible for increased HCO-3 reabsorption and maintained acid-base homeostasis in hyposmolar states.

Topics: Ammonium Chloride; Animals; Cell Line; Cells, Cultured; Enzyme Activation; Fluoresceins; Hydrogen-Ion Concentration; Kidney Medulla; Kidney Tubules, Collecting; Kinetics; Naphthalenes; Osmolar Concentration; Protein Kinase C; Proton-Translocating ATPases; Staurosporine; Tetradecanoylphorbol Acetate; Tetraethylammonium