Ascorbate transport in cultured cat retinal pigment epithelial cells

Transport of ascorbate by primary cultures of cat retinal pigment epithelial cells (RPE) was studied. Confluent primary cultures were incubated with 10-500 microM L-[carboxyl-14C] ascorbic acid in balanced salt solution (BSS) at 37 degrees C for 1 to 40 min. The uptake of radioactive ascorbate followed saturation kinetics with a Km of 42 microM and Vmax of 117 pmol min-1 microgram-1 DNA. Cells incubated with 10 microM radioactive ascorbate for 40 min showed a ratio of intracellular to extracellular radioactive ascorbate of greater than 40. The transport of ascorbate was sodium- and energy-dependent. Replacement of 150 mM NaCl in BSS with 150 mM LiCl reduced ascorbate uptake significantly. Ouabain, 2,4-dinitrophenol, alpha-D-glucose, 3-O-methyl-D-glucose, and the ascorbate analogues, D-isoascorbate and dehydroascorbate, each inhibited ascorbate uptake into RPE cells. The efflux of radioactivity into the incubation media was slow when cells were preloaded with either 50- or 500 microM radioactive ascorbate, but increased when cells preloaded with 50 microM ascorbate were incubated in the presence of excess non-radioactive ascorbate. These studies demonstrated that a sodium-dependent carrier system is involved in transport of ascorbate in primary cultures of cat RPE.