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

• Published in: The American journal of physiology Vol. 256; no. 1 Pt 1; p. C44
• Main Authors: Helbig, H, Korbmacher, C, Wohlfarth, J, Berweck, S, Kühner, D, Wiederholt, M
• Format: Journal Article
• Language: English
• Published: United States 01.01.1989
• Subjects: Amphotericin B - pharmacology; Animals; Ascorbic Acid - metabolism; Ascorbic Acid - pharmacology; Biological Transport, Active - drug effects; Cattle; Cells, Cultured; Ciliary Body - drug effects; Ciliary Body - metabolism; Dehydroascorbic Acid - metabolism; Electrochemistry; Epithelium - metabolism; Kinetics; Membrane Potentials; Ouabain - pharmacology; Phloretin - pharmacology; Pigmentation; Sodium - metabolism
• ISSN: 0002-9513
• DOI: 10.1152/ajpcell.1989.256.1.C44

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.