Na⁺-Linked Active Transport of Ascorbate into Cultured Bovine Retinal Pigment Epithelial Cells: Heterologous Inhibition by Glucose

Abstract

The transport of ascorbate into cultured bovine retinal pigment epithelial (RPE) cells is reported. Primary or subcultured RPE cells were incubated in the presence of 10–500 μM L-[carboxyl-¹⁴C]-ascorbate for various periods of time. Accumulation of ascorbate into RPE cells followed a saturable active transport with a K_m of 125 μM and a V_max of 28 pmole/μg DNA/min. RPE intracellular water was calculated to be 0.8 pL/cell, and the transported cellular ascorbate concentration was 7.5±0.8 mM. Replacement of 150 mM NaCl in the incubation media with choline-Cl strongly inhibited (80±8%) ascorbate uptake into cultured RPE cells. Although the depletion of cellular ATP by 2, 4-dinitrophenol and the inhibition of Na⁺-K⁺-ATPase by ouabain reduced ascorbate transport into RPE significantly, active transport of ascorbate was not entirely inhibited by these metabolic inhibitors. The ascorbate analogue, D-isoascorbate, competitively inhibited ascorbate transport into cultured RPE with a K_i of 12.5 mM. Cells grown in the presence of 5 to 50 mM α-D-glucose in the growth media did not differ in their ability to transport ascorbate. In contrast, the presence of α-D-glucose or its nonmetaboliiable analogues, 3–0-methyl-glucose, α-methyl-glucose, and 2-deoxy-glucose, but not L-glucose or β-D-fructose, in the incubation media inhibited ascorbate transport. myo-Inositol (10 or 20 mM) also inhibited ascorbate transport into RPE cells.

The active uptake of ascorbate into cultured RPE cells was primarily coupled to the movement of sodium ion down its electrochemical gradient. A bifunctional, cotransport carrier possessing an ascorbate-binding site and a sodium-binding site may be involved in the ascorbate uptake system. The inhibition of ascorbate uptake by sugars appeared to be heterologous in nature, occurring between two distinct carrier systems, both of which were dependent on the sodium ions.

Key Words:

• Large-scale RPE culture
• Na⁺-dependent
• ascorbate
• active transport
• glucose heterologous inhibition
• myo-inositol