INFLUENCE OF BAFILOMYCIN A(1) ON PH(I) RESPONSES IN CULTURED RABBIT NONPIGMENTED CILIARY EPITHELIUM

Aqueous humor secretion is in part linked to HCO3- transport by nonpig mented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pH(i)). Because a recent report s uggests that NPE cells have a plasma membrane-localized vacuolar H+-AT Pase, the present study was conducted to examine whether vacuolar H+-A TPase contributes to pH(i) regulation in a rabbit NPE cell line. Weste rn blot confirmed vacuolar H+-ATPase expression as judged by H+-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native cil iary 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 alkali nization. Alkalinization was also caused by ouabain or BaCl2. Bafilomy cin A(1) (0.1 mu M; an inhibitor of vacuolar H+-ATPase) inhibited the pH(i) increase caused by high K+. The pH(i) increase was also inhibite d by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chl orophenylhydazone but not by ZnCl2, -acetamido-4'-isothiocyanostilbene -2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfo nic acid (DIDS), omeprazole, low-Cl- medium, HCO3--free medium, or Na-free medium. Bafilomycin A(1) slowed the pH(i) increase after an NH4C l (10 mM) prepulse. However, no detectable pH(i) change was observed i n cells exposed to bafilomycin A(1) under control conditions. These st udies suggest that vacuolar H+-ATPase is activated by cytoplasmic acid ification and by reduction of the proton electrochemical gradient acro ss the plasma membrane. We speculate that the mechanism might contribu te to maintenance of acid-base balance in NPE.