Activation of H+-ATPase by hypotonicity: a novel regulatory mechanism for H+ secretion in IMCD cells.
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.
Department of Medicine, University of Cincinnati School of Medicine, and Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0585, USA.
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D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004789:Enzyme Activation; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007679:Kidney Medulla / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D009281:Naphthalenes / Q000494:pharmacology; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019789:Tetraethylammonium / Q000494:pharmacology
D005452:Fluoresceins; D009281:Naphthalenes; D000643:Ammonium Chloride; D019789:Tetraethylammonium; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D011493:Protein Kinase C; D006180:Proton-Translocating ATPases; D019311:Staurosporine; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate