Ascorbate stimulates ferricyanide reduction in HL-60 cells through a mechanism distinct from the NADH-dependent plasma membrane reductase

Abstract

The impermeable oxidant ferricyanide is reduced by the plasma membrane redox system of HL-60 cells. The rate of reduction is strongly enhanced by ascorbate or dehydroascorbate. The aim of this study was to determine the mechanism by which ascorbate and dehydroascorbate accelerate ferricyanide reduction in HL-60 cells. Addition of ascorbate or dehydroascorbate to cells in the presence of ferricyanide led to the intracellular accumulation of ascorbate. Control experiments showed that extracellular ascorbate was rapidly converted to dehydroascorbate in the presence of ferricyanide. These data suggest that intracellular ascorbate originates from extracellular dehydroascorbate. Accumulation of ascorbate was prevented by inhibitors of dehydroascorbate transport into the cell. These compounds also strongly inhibited ascorbate-stimulated ferricyanide reduction in HL-60 cells. Thus, it is concluded that the stimulation of ferricyanide reduction is dependent on intracellular accumulation of ascorbate. Changing the α-tocopherol content of the cells had no effect on the ascorbate-stimulated ferricyanide reduction, showing that a nonenzymatic redox system utilizing α-tocopherol was not involved. p-Chloromercuribenzenesulfonic acid strongly affected ferricyanide reduction in the absence of ascorbate, whereas the stimulated reaction was much less responsive to this compound. Thus, it appears that at least two different membrane redox systems are operative in HL-60 cells, both capable of reducing ferricyanide, but through different mechanisms. The first system is the ferricyanide reductase, which uses NADH as its source for electrons, whereas the novel system proposed in this paper relies on ascorbate.