Amlal, Hassane, Akhil Goel, and Manoocher Soleimani. Activation of H ϩ -ATPase by hypotonicity: a novel regulatory mechanism for H ϩ secretion in IMCD cells. Am. J. Physiol. 275 (Renal Physiol. 44): F487-F501, 1998.-The effect of hypotonicity on H ϩ -ATPase activity was examined in cultured inner medullary collecting duct (mIMCD-3) cells. mIMCD-3 cells were grown to confluence, loaded with 2Ј,7Јbis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and assayed for H ϩ -ATPase activity measured as

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... the Na ϩ -and K ϩ -independent intracellular pH (pH i ) recovery following an acid load. Exposure of mIMCD-3 cells to a hypotonic solution (150 mosmol/kgH 2 O) increased pH i recovery by ϳ350% (P Ͻ 0.0001). This effect was inhibited by diethylstilbestrol (an inhibitor of H ϩ -ATPase) and was not dependent on external K ϩ , indicating lack of involvement of H ϩ -K ϩ -ATPase. H ϩ -ATPase activation was acute, independent of cell calcium, and was not secondary to Cl Ϫ channel activation. The magnitude of H ϩ -ATPase upregulation was dependent on the osmolarity of the media, with maximum stimulation at 150 mosmol/kgH 2 O. H ϩ -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 ϩ /H ϩ exchanger activity in mIMCD-3 cells, indicating that its stimulatory effect is specific to H ϩ -ATPase. In conclusion, a novel regulatory mechanism of H ϩ -ATPase by hypotonicity is described. The increased H ϩ -ATPase activity in hypotonicity may be responsible for increased HCO 3 Ϫ reabsorption and maintained acidbase homeostasis in hyposmolar states. proton-adenosinetriphosphatase; inner medulla; hypotonicity; sodium/proton exchange; acid-base balance The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.