Vitamin C recycling and function in human monocytic U-937 cells

The uptake, recycling, and function of ascorbic acid was evaluated in cultu red U-937 monocytic cells. Dehydroascorbic acid, the two-electron oxidized form of the vitamin, was taken up on the glucose transporter and reduced to ascorbate to a much greater extent than ascorbate itself was accumulated b y the cells. In contrast to dehydroascorbic acid, ascorbate entered the cel ls on a sodium- and energy-dependent transporter. Intracellular ascorbate e nhanced the transfer of electrons across the cell membrane to extracellular fenicyanide. Rates of ascorbate-dependent ferricyanide reduction were satu rable, fivefold greater than basal rates, and facilitated by intracellular recycling of ascorbate. Whereas reduction of dehydroascorbic acid concentra tions above 400 mu M consumed reduced glutathione (GSH), even severe GSH de pletion by 1-chloro-2,4-dinitrobenzene was without effect on the ability of the cells to reduce concentrations of dehydroascorbic acid likely to be in the physiologic range (< 200 mu M). Dialyzed cytosolic fractions from U-93 7 cells reduced dehydroascorbic acid to ascorbate in an NADPH-dependent man ner that appeared due to thioredoxin reductase. However, thioredoxin reduct ase did not account for the bulk of dehydroascorbic acid reduction, since i ts activity was also decreased by treatment of intact cells with 1-chloro-2 ,4-dinitrobenzene. Thus, U-937 cells loaded with dehydroascorbic acid accum ulate ascorbate against a concentration gradient via a mechanism that is no t dependent on GSH or NADPH, and this ascorbate can serve as the major sour ce of electrons for transfer across the plasma membrane to extracellular fe rricyanide, (C) 1999 Elsevier Science Inc.