Influence of bafilomycin A₁ on pH(i) 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 (pH(i)). 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 pH(i) 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. pH(i) was measured using 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein (BCECF). Exposing cultured NPE to K⁺-rich solution caused a pH(i) increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl₂. Bafilomycin A₁ (0.1 μM; an inhibitor of vacuolar H⁺-ATPase) inhibited the pH(i) increase caused by high K⁺. The pH(i) 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 pH(i) increase after an NH4Cl (10 mM) prepulse. However, no detectable pH(i) 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.