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.
- Cytoplasmic pH
- Hydrogen adenosinetriphosphatase
- Sodium/hydrogen exchanger
ASJC Scopus subject areas
- Cell Biology