CHLORIDE TRANSPORT IN CULTURED FETAL HUMAN RETINAL-PIGMENT EPITHELIUM

This study identifies bumetanide-sensitive chloride transport in cultu red fetal human retinal pigment epithelium (RPE). Ion flux and electro physiologic studies were performed on fetal human RPE grown to conflue nce 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 a verage 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 op en-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 bumet anide 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 bloc kade of Cl- uptake and reduction of the Cl- equilibrium potential at a basal membrane Cl- channel. Basal bumetanide had no effect on electri cal parameters. We identified a bumetanide sensitive Cl- uptake mechan ism at the apical membrane which may represent the Na-K-2Cl cotranspor ter reported in explant and other cultured preparations of mammalian R PE. (C) 1996 Academic Press Limited