Influence of bafilomycin A1 on pHi responses in cultured rabbit nonpigmented ciliary epithelium.
Aqueous humor secretion is in part linked to HCO3- transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pHi). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H(+)-ATPase, the present study was conducted to examine whether vacuolar H(+)-ATPase contributes to pHi regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H(+)-ATPase expression as judged by H(+)-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pHi was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K(+)-rich solution caused a pHi increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl2. Bafilomycin A1 (0.1 microM; an inhibitor of vacuolar H(+)-ATPase) inhibited the pHi increase caused by high K+. The pHi increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl2, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Cl- medium, HCO3(-)-free medium, or Na(+)-free medium. Bafilomycin A1 slowed the pHi increase after an NH4Cl (10 mM) prepulse. However, no detectable pHi change was observed in cells exposed to bafilomycin A1 under control conditions. These studies suggest that vacuolar H(+)-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.
Wu
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Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Kentucky 40292, USA.
Delamere
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002712:Chlorides / Q000494:pharmacology; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000502:physiology; D007700:Kinetics; D018942:Macrolides; D009853:Omeprazole / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D011817:Rabbits; D013552:Swine; D017967:Zinc Compounds / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D002712:Chlorides; D004791:Enzyme Inhibitors; D018942:Macrolides; D017967:Zinc Compounds; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D010042:Ouabain; D000584:Amiloride; C016837:zinc chloride; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D009853:Omeprazole; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium