Burke T, Lee S, Ferguson P J, Hammond J R
Department of Pharmacology and Toxicology, University of Western Ontario, Canada.
J Pharmacol Exp Ther. 1998 Sep;286(3):1333-40.
The uptake of [3H]formycin B by Ehrlich ascites tumor cells was examined in both normal Na+ buffer (physiological) and nominally Na+-free buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subtypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues by these cells. Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure transport kinetics in energetically competent cells. Initial studies established that formycin B inhibited [3H]uridine uptake by the ei (equilibrative inhibitor-insensitive) and es (equilibrative inhibitor-sensitive) transporters of Ehrlich cells with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]formycin B had Km values of 111 +/- 52 and 635 +/- 147 microM for uptake by the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]formycin B was significantly higher in the Li+ buffer (es-mediated Vmax = 65 +/- 10 pmol/microliter . sec) than in the Na+ buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . sec); this may reflect trans-acceleration of [3H]formycin B uptake by elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitabine (2',2'-difluorodeoxycytidine) with the equilibrative and concentrative nucleoside transporters. Gemcitabine, which has shown considerable potential for the treatment of solid tumors, was a relatively poor inhibitor of [3H]formycin B uptake via the equilibrative transporters (IC50 approximately 400 microM). In contrast, gemcitabine was a potent inhibitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.
在正常钠离子缓冲液（生理缓冲液）和名义上无钠离子的缓冲液（用锂离子进行等渗替代）中，研究了艾氏腹水瘤细胞对[3H]福米司汀B的摄取情况。进行这些研究是为了进一步表征艾氏细胞的平衡核苷转运体亚型，并评估钠离子依赖性浓缩转运机制对这些细胞中核苷类似物细胞内积累的贡献。福米司汀B在哺乳动物细胞中代谢缓慢，因此可作为底物用于测量有能量活性细胞中的转运动力学。初步研究表明，福米司汀B抑制艾氏细胞的ei（平衡抑制剂不敏感）和es（平衡抑制剂敏感）转运体对[3H]尿苷的摄取，其Ki值分别为48±28和277±25微摩尔/升。同样，[3H]福米司汀B通过ei和es转运体摄取的Km值分别为111±52和635±147微摩尔/升。当在存在钠离子的情况下进行测定，并加入100纳摩尔的硝基苄硫基肌苷以防止通过es转运体的外排时，[3H]福米司汀B的细胞内浓度超过初始培养基浓度3倍以上，表明存在钠离子依赖性转运体的活性。有趣的是，[3H]福米司汀B在锂离子缓冲液中的初始摄取速率（es介导的Vmax = 65±10皮摩尔/微升·秒）显著高于在钠离子缓冲液中的摄取速率（Vmax = 8.4±0.9皮摩尔/微升·秒）；这可能反映了低钠离子环境导致细胞内腺苷水平升高，从而使[3H]福米司汀B的摄取发生反式加速。然后使用该模型评估吉西他滨（2',2'-二氟脱氧胞苷）与平衡和浓缩核苷转运体的相互作用。吉西他滨在实体瘤治疗中显示出相当大的潜力，它对通过平衡转运体摄取[3H]福米司汀B的抑制作用相对较弱（IC50约为400微摩尔/升）。相比之下，吉西他滨是艾氏细胞钠离子依赖性核苷转运体的强效抑制剂（IC50 = 17±5纳摩尔/升）。这些结果表明，钠离子依赖性核苷转运体的细胞表达/活性可能是吉西他滨细胞毒性和临床疗效的重要决定因素。
