Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells

Abstract

The high level of ascorbic acid (AA) in the aqueous humor of many mammals suggests an active transport of AA across the double-layered ciliary epithelium from blood to aqueous humor. We used [14C]AA to study AA uptake in bovine pigmented ciliary epithelial cells in tissue culture. We observed a 40-fold intracellular accumulation of AA, which was dependent on extracellular Na+. With labeled dehydroascorbate (DHA, the oxidized form of the vitamin) in the medium, there was a 20-fold intracellular accumulation of the label. However, the time course of DHA uptake was different compared with AA uptake and was not Na+ dependent, suggesting different transport systems for AA and DHA. AA uptake was inhibited by 1 mM phloretin and in the presence of isoascorbate. Furthermore, AA uptake was markedly reduced when intracellular Na+ was elevated by preincubation with ouabain or amphotericin B. With increasing AA concentration, Na+-dependent AA uptake exhibited first-order saturation kinetics with half-maximal uptake at 76 microM AA. Na+ dependence of AA uptake revealed a sigmoidal curve of Na+-dependent AA uptake vs. Na+ concentration with a half-maximal AA uptake at 45.4 mM Na+. The slope of the Hill plot from these data was 1.94, suggesting a transport system translocating two or more Na+ for one AA. This stoichiometry implies electrogenicity of the transporter. We, therefore, measured membrane potentials using conventional microelectrodes. Addition of 200 microM AA resulted in a depolarization of the membrane voltage by 4.9 +/- 0.5 mV (n = 22), which was absent in Na+ free medium and was markedly reduced by phloretin.(ABSTRACT TRUNCATED AT 250 WORDS)