Abstract: | The response ofH+-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pHi) recovery from an acid load. The rate of Na+-independentpHi recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa+-independentH+ extrusion was ATP dependent and K+ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa+-independentH+ extrusion in cultured medullarycells is mediated via H+-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H+-ATPase activity in innermedullary collecting duct cells. Upregulation ofH+-ATPase could play a protectiverole against cell death in severe intracellular acidosis. |