Laggner H, Goldenberg H
Institute of Medical Chemistry, University of Vienna, Waehringerstrasse 10, A-1090 Wien, Austria.
Biochem J. 2000 Jan 15;345 Pt 2(Pt 2):195-200.
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.
用二甲基亚砜（DMSO）诱导分化的HL-60细胞在用佛波酯（PMA）激活后，其抗坏血酸摄取速率增加。这种激活后观察到的摄取速率与以脱氢抗坏血酸作为原始转运底物时的速率基本相同。激活作用对抗氧化酶超氧化物歧化酶和过氧化氢酶敏感。当抗坏血酸通过化学或酶促氧化在原位被氧化时，摄取速率与用佛波酯激活细胞后的摄取速率相似；然而，在后一种情况下，细胞外维生素大部分仍处于还原形式，降解损失很少，而在立即氧化后，几分钟后细胞外就找不到更多还原型抗坏血酸了，并且总维生素的很大一部分损失了。黄嘌呤/黄嘌呤氧化酶产生的超氧化物对抗坏血酸摄取的刺激作用远小于佛波酯的激活作用；过氧化氢（H₂O₂）的作用甚至更小。与抗坏血酸化学氧化的刺激作用相反，佛波酯对摄取的刺激作用对谷胱甘肽（GSH）也不敏感。佛波酯对抗坏血酸摄取的刺激作用对呼吸爆发抑制剂二苯基碘鎓以及蛋白激酶C抑制剂星形孢菌素敏感，表明呼吸爆发是刺激的原因。佛波酯对细胞的激活也刺激了以脱氢抗坏血酸作为原始底物的摄取，其方式对抗氧化剂或呼吸爆发抑制剂不敏感。在所有情况下，细胞内维生素完全处于还原形式。通过计算最大速度和表观米氏常数（Kₘ）值进行动力学表征，并测定摄取速率对离子环境的依赖性以及葡萄糖类似物和葡萄糖转运抑制剂的抑制作用，结果表明，用佛波酯处理后，分化的HL-60细胞中总维生素C的摄取主要归因于低亲和力高容量的葡萄糖转运蛋白。相比之下，在未刺激的细胞中，还原型抗坏血酸是通过钠依赖性高亲和力低容量的抗坏血酸转运蛋白摄取的。这种变化可能是由于抗坏血酸的氧化，同时细胞表面募集了额外的转运蛋白分子。
