Influence of bafilomycin A1 on pHi responses in cultured rabbit nonpigmented ciliary epithelium |
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Authors: | Wu, Qiang Delamere, Nicholas A. |
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Abstract: | Aqueous humorsecretion is in part linked to transport by nonpigmented ciliary epithelium (NPE) cells. During thisprocess, the cells must maintain stable cytoplasmic pH(pHi). Because a recent reportsuggests that NPE cells have a plasma membrane-localized vacuolarH+-ATPase, the present study wasconducted to examine whether vacuolar H+-ATPase contributes topHi regulation in a rabbit NPEcell line. Western blot confirmed vacuolarH+-ATPase expression as judged byH+-ATPase 31-kDa immunoreactivepolypeptide in both cultured NPE and native ciliary epithelium.pHi was measured using2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF).Exposing cultured NPE to K+-richsolution caused a pHi increase weinterpret as depolarization-induced alkalinization. Alkalinization wasalso caused by ouabain or BaCl2. Bafilomycin A1 (0.1 µM; aninhibitor of vacuolar H+-ATPase)inhibited the pHi increase causedby high K+. ThepHi increase was also inhibited byangiotensin II and the metabolic uncoupler carbonyl cyanidem-chlorophenylhydazone but not by ZnCl2,4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid(SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Clmedium, -free medium, orNa+-free medium. BafilomycinA1 slowed thepHi increase after an NH4Cl (10 mM) prepulse. However,no detectable pHi change was observed in cells exposed to bafilomycinA1 under control conditions. Thesestudies suggest that vacuolarH+-ATPase is activated bycytoplasmic acidification and by reduction of the protonelectrochemical gradient across the plasma membrane. We speculate thatthe mechanism might contribute to maintenance of acid-base balance inNPE. |
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