Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells.
The response of H+-ATPase to lethal acid stress is unknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cycles of lethal acid stress. Cells were grown to confluence on coverslips, loaded with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, and monitored for intracellular pH (pHi) recovery from an acid load. The rate of Na+-independent pHi recovery from an acid load in mutant cells was approximately fourfold higher than in parent cells (P < 0.001). The Na+-independent H+ extrusion was ATP dependent and K+ independent and was completely inhibited in the presence of diethylstilbestrol, N,N'-dicyclohexylcarbodiimide, or N-ethylmaleimide. These results indicate that the Na+-independent H+ extrusion in cultured medullary cells is mediated via H+-ATPase and is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remained unchanged in mutant cells compared with parent cells. We propose that lethal acid stress results in increased H+-ATPase activity in inner medullary collecting duct cells. Upregulation of H+-ATPase could play a protective role against cell death in severe intracellular acidosis.
Amlal
H
H
Department of Medicine, University of Cincinnati School of Medicine, and Veterans Affairs Medical Center, Ohio 45267-0585, USA.
Wang
Z
Z
Soleimani
M
M
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004054:Diethylstilbestrol / Q000494:pharmacology; D004791:Enzyme Inhibitors; D005033:Ethylmaleimide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000201:enzymology*; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D011190:Potassium Cyanide / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000096:biosynthesis*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects
D004791:Enzyme Inhibitors; D007093:Imidazoles; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; C035235:Sch 28080; D004024:Dicyclohexylcarbodiimide; D004054:Diethylstilbestrol; D006180:Proton-Translocating ATPases; C027917:tetramethylammonium; D011190:Potassium Cyanide; D005033:Ethylmaleimide