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)

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

“…Although AH efflux from HepG2 cells seemed to be facilitated, the transport mechanism involved was clearly distinguished from mammalian glucose\DHA transport (Table 3). The inhibition of AH efflux from HepG2 cells by phloretin and DIDS agrees with previous studies on other cells [33][34][35][36]. The putative AH transport mechanism might have characteristics in common with DIDS-sensitive anion channels but further work is required to support this hypothesis.…”

A model to measure lymphatic drainage from the eye

“…Most aqueous humor proteins are intrinsic glycoproteins of the vitreous, which are secretory products of the inner epithelial layer of the ciliary body (Haddad et al, 1991). Anti-oxidant substances found in the aqueous humor such as glutathione and ascorbate, are also important as they protect against light-induced oxidative damage (Krupin and Civan, 1996;Helbig et al, 1989). Also, molecules that are important in extracellular matrix maintenance, such as collagenase, have been found in human aqueous humor.…”

Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters.

“…As sucrose has no transport mechanism, its clearance from the serum was slow. Cellular transport is a clearance mechanism for ascorbic acid and dehydroascorbic acid, with the participation of glucose transporters in the case of dehydroascorbic acid and possibly a Na ϩ -ascorbate cotransporter in the case of ascorbic acid (19)(20)(21)(22)(23)(24)(25). The rapid clearance of dehydroascorbic acid from the serum likely reflected the large number of glucose transporters available.…”

“…In these cells, dehydroascorbic acid is transported across the cell membrane and accumulated in the reduced form, ascorbic acid, which is not transportable through the bidirectional glucose transporters (16)(17)(18). Ascorbic acid may also be transported through a Na ϩ -ascorbate cotrans-porter that is reported to be present in the small intestine, renal epithelial cells, osteoblasts, adrenomedullary chromaffin cells, and fibroblasts (19)(20)(21)(22)(23)(24)(25). Sodium-sensitive uptake of ascorbic acid was also demonstrated by expression studies in Xenopus oocytes injected with poly(A) ϩ RNA extracted from rabbit kidney cortex (26).…”