Lee C W
Department of Physiology, National University of Singapore.
Biochem J. 1994 Jun 1;300 ( Pt 2)(Pt 2):407-12. doi: 10.1042/bj3000407.
The dose-response curves for the inhibition of equilibrative uridine transport by dilazep, dipyridamole and nitrobenzylthioinosine (NBMPR) in undifferentiated HL-60 cells were biphasic. Some 70% of the transport activity was inhibited with IC50 values of 0.7, 1 and 7 nM respectively. No inhibition of the remaining 30% of transport activity was observed until the dilazep, dipyridamole and NBMPR concentrations exceeded 1, 0.1 and 3 microM respectively. Exposure to phorbol 12-myristate 13-acetate (PMA) for 48 h, to induce monocytic differentiation, caused a 20-fold decrease in Vmax. of both NBMPR-sensitive and NBMPR-insensitive equilibrative uridine transport. The decrease in NBMPR-sensitive uridine transport induced by PMA corresponded to a decrease in NBMPR binding sites. A 30% decrease in specific NBMPR binding sites occurred within 6 h of PMA exposure, and could be prevented by uridine and thymidine at concentrations as low as 100 microM, and by staurosporine at 40 nM. However, the protective effects of these compounds diminished with prolonged PMA exposure. No protection was observed with uracil. Exogenous protein kinase C (PKC) in the presence of ATP and PMA decreased the number of specific NBMPR-binding sites in purified HL-60 cell plasma membranes. These results suggest that a PKC-induced conformational change in substrate-binding/transporting site may be responsible for the decrease in NBMPR-sensitive nucleoside transport during PMA-induced monocytic differentiation of HL-60 cells.
在未分化的HL-60细胞中，双嘧达莫、潘生丁和硝基苄硫肌苷（NBMPR）对平衡型尿苷转运的抑制作用的剂量反应曲线呈双相。约70%的转运活性被抑制，IC50值分别为0.7、1和7 nM。直到双嘧达莫、潘生丁和NBMPR的浓度分别超过1、0.1和3 microM时，才观察到对其余30%转运活性的抑制。用佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）处理48小时以诱导单核细胞分化，导致NBMPR敏感和NBMPR不敏感的平衡型尿苷转运的Vmax均下降20倍。PMA诱导的NBMPR敏感的尿苷转运减少与NBMPR结合位点的减少相对应。在PMA暴露6小时内，特异性NBMPR结合位点减少30%，低至100 microM的尿苷和胸苷以及40 nM的星形孢菌素可阻止这种减少。然而，随着PMA暴露时间延长，这些化合物的保护作用减弱。尿嘧啶未观察到保护作用。在ATP和PMA存在下，外源性蛋白激酶C（PKC）减少了纯化的HL-60细胞质膜中特异性NBMPR结合位点的数量。这些结果表明，PKC诱导的底物结合/转运位点的构象变化可能是PMA诱导HL-60细胞单核细胞分化过程中NBMPR敏感的核苷转运减少的原因。
