Chloride transport in cultured fetal human retinal pigment epithelium

This study identifies bumetanide-sensitive chloride transport in cultured fetal human retinal pigment epithelium (RPE). Ion flux and electrophysiologic studies were performed on fetal human RPE grown to confluence on microporous culture wells mounted in modified Ussing chambers. Unidirectional transepithelial Cl- fluxes were measured along with the transepithelial potential (TEP), resistance (R(t)), and short circuit current (I-sc). In separate experiments the apical and basal membrane voltages (V-ap and and V-ba) and membrane resistance ratio (R(ap)R(ba)) were measured using standard electrophysiological techniques. The average electrical parameters under control conditions were TEP = 3.6 +/- 2.1 mV, R(t) = 385 +/- 98 ohm cm(2), and I-sc = 8.8 +/- 3.6 mu A cm(-2). Apical bumetanide (10(-5) M) reduced the TEP and I-sc an average of 0.6 +/- 0.4 mV and 1.6 +/- 0.8 mu A cm(-2), respectively. Under open-circuit conditions. we measured a net apical to basal Cl-36 flux of 0.30 mu Eq cm(-2) hr(-1). Bumetanide reduced this net Cl- flux by 83% by reducing the unidirectional apical-to-basal Cl- flux. Apical bumetanide hyperpolarized V-ba from -58 +/- 14 to -66 +/- 17 mV and reduced R(ap)/R(ba) from 1.17 +/- 0.50 to 0.85 +/- 0.38, consistent with blockade of Cl- uptake and reduction of the Cl- equilibrium potential at a basal membrane Cl- channel. Basal bumetanide had no effect on electrical parameters. We identified a bumetanide sensitive Cl- uptake mechanism at the apical membrane which may represent the Na-K-2Cl cotransporter reported in explant and other cultured preparations of mammalian RPE. (C) 1996 Academic Press Limited