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 (Rt), and short circuit current (Isc). In separate experiments the apical and basal membrane voltages (Vap and Vba) and membrane resistance ratio (Rap/Rba) were measured using standard electrophysiological techniques. The average electrical parameters under control conditions were TEP = 3.6 +/- 2.1 mV, Rt = 385 +/- 98 ohm cm2, and Isc = 8.8 +/- 3.6 microA cm-2. Apical bumetanide (10(-5) M) reduced the TEP and Isc an average of 0.6 +/- 0.4 mV and 1.6 +/- 0.8 microA cm-2, respectively. Under open-circuit conditions, we measured a net apical to basal 36Cl flux of 0.30 microEq cm-2 hr-1. Bumetanide reduced this net Cl- flux by 83% by reducing the unidirectional apical-to-basal Cl- flux. Apical bumetanide hyperpolarized Vba from -58 +/- 14 to -66 +/- 17 mV and reduced Rap/Rba 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.