Na(+)-K(4)-Cl- cotransport in cultured cells derived from human retinal pigment epithelium

Unidirectional fluxes of Rb+ and Cl- were measured in cultured human retinal pigment epithelium (HRPE) to characterize the Na(+)-K(+)-Cl-cotransport system in this epithelial tissue. In HRPE, the ouabain-sensitive fraction of Rb+ influx comprises 65% of the total Rb+ influx. Bumetanide [inhibition constant (KI) = 160 nM] inhibited the residual ouabain-insensitive fraction of Rb+ influx by 70%. The bumetanide-sensitive fraction of Rb+ influx was dependent on the presence of both extracellular Na+ and Cl-. Cl- influx was similarly inhibited by bumetanide and also dependent on extracellular Na+ and Rb+. The stoichiometry of bumetanide-sensitive Cl- influx to Rb+ influx was 2:1. Elevation of extracellular osmolarity (by 30%) caused a 2.5-fold increase in Na(+)-K(+)-Cl- cotransport activity in cultured HRPE. The present study provides direct evidence for the occurrence of Na(+)-K(+)-Cl- cotransport-mediated cation and anion transport in HRPE.