Interaction of respiratory burst and uptake of dehydroascorbic acid in differentiated HL-60 cells

Abstract

HL-60 cells differentiated with DMSO increased their rates of uptake of ascorbate when they were activated with PMA. The rates observed after this activation were essentially the same as those with dehydroascorbic acid as the original transport substrate. The effect of activation was sensitive to the antioxidant enzymes superoxide dismutase and catalase. When ascorbate was oxidized in situ by chemical or enzymic oxidation, the rates of uptake were similar to those after activation of the cells by phorbol ester; however, in the latter case the extracellular vitamin remained largely in the reduced form and there was very little loss by degradation, whereas after immediate oxidation no more reduced ascorbate could be found outside the cells after a few minutes and a significant part of the total vitamin was lost. The generation of superoxide by xanthine/xanthine oxidase stimulated the uptake of ascorbate much less than the activation by phorbol ester; H(2)O(2) was even less effective. Stimulation of the uptake by phorbol ester was also insensitive to GSH, in contrast with stimulation by the chemical oxidation of ascorbate. Stimulation of ascorbate uptake by phorbol ester was sensitive to the respiratory-burst inhibitor diphenyliodonium as well as the protein kinase C inhibitor staurosporine, indicating the respiratory burst as the cause of stimulation. Activation of the cells by the phorbol ester also stimulated the uptake of dehydroascorbate as the original substrate, in a manner insensitive to antioxidants or inhibitors of the respiratory burst. In all cases the intracellular vitamin was completely in the reduced form. Kinetic characterization by the calculation of maximal velocities and apparent K(m) values and assaying for the dependence of uptake rates on the ionic milieu and for inhibition by glucose analogues and inhibitors of glucose transport revealed that after treatment with phorbol ester the uptake of total vitamin C in differentiated HL-60 cells was largely due to the low-affinity high-capacity glucose transporter. In contrast, in non-stimulated cells reduced ascorbate was taken up by the Na(+)-dependent high-affinity low-capacity ascorbate transporter. This change was probably due to the oxidation of ascorbate and, simultaneously, the recruitment of additional transporter molecules to the cell surface.