title: 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)
authors.affiliation: Institut für Klinische Physiologie, Klinikum Steglitz der Freien Universität Berlin, Federal Republic of Germany.
authors.forename: H
authors.initials: H
authors.lastname: Helbig
authors.forename: C
authors.initials: C
authors.lastname: Korbmacher
authors.forename: J
authors.initials: J
authors.lastname: Wohlfarth
authors.forename: S
authors.initials: S
authors.lastname: Berweck
authors.forename: D
authors.initials: D
authors.lastname: Kühner
authors.forename: M
authors.initials: M
authors.lastname: Wiederholt
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism* / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002417:Cattle; D002478:Cells, Cultured; D002924:Ciliary Body / Q000187:drug effects / Q000378:metabolism*; D003683:Dehydroascorbic Acid / Q000378:metabolism; D004563:Electrochemistry; D004848:Epithelium / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D010042:Ouabain / Q000494:pharmacology; D010693:Phloretin / Q000494:pharmacology; D010858:Pigmentation; D012964:Sodium / Q000378:metabolism*
chemical_list: C005056:isoascorbic acid; D010042:Ouabain; D000666:Amphotericin B; D012964:Sodium; D001205:Ascorbic Acid; D010693:Phloretin; D003683:Dehydroascorbic Acid