Characterization of the individual transport routes that mediate the influx and efflux of methotrexate in CCRF-CEM human lymphoblastic cells.
Characterization of the individual transport routes that mediate the influx and efflux of methotrexate in CCRF-CEM human lymphoblastic cells.
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发表时间：
1986-04
期刊：
影响因子：
11.2
通讯作者：
G. Henderson;J. M. Tsuji;H. P. Kumar
中科院分区：
文献类型：
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作者：
G. Henderson;J. M. Tsuji;H. P. Kumar
分析了 CCRF-CEM 人淋巴细胞流入和流出甲氨蝶呤的转运途径。获得了甲氨蝶呤单一流入途径的证据：(a) 2 µM [3H]甲氨蝶呤的流入被高浓度的未标记甲氨蝶呤、邻苯二甲酸盐和溴磺酞完全抑制，并且每种阴离子的抑制曲线都是单相的； (b)用甲氨蝶呤的N-羟基琥珀酰亚胺酯预处理细胞也阻止了流入，并且这种抑制在2至50μM的底物浓度范围内是完全的。流入也是饱和的，并以 4.3 pmol/min/mg 蛋白质（37 摄氏度）的最大速率 (Vmax) 进行，在缺乏阴离子的缓冲液中 Kt 为 0.8 µM，在 4-(2-羟乙基)-1-哌嗪乙磺酸盐缓冲盐水中 Kt 为 4.6 µM。 Vmax 与载体蛋白量（0.3 pmol/mg 蛋白）的比率给出了转运系统的周转数为 14.3/min。与流入相反，甲氨蝶呤流出通过三种途径进行，这可以根据它们对特定抑制剂的敏感性来区分。大部分流出是通过甲氨蝶呤流入载体发生的，其特性是根据其对甲氨蝶呤的 N-羟基琥珀酰亚胺酯的敏感性以及其对外部介质中阴离子的需求而确定的。甲氨蝶呤、单磷酸腺苷和磷酸盐均通过该途径刺激流出，并且在接近其抑制甲氨蝶呤流入的 Ki 值的阴离子浓度下，这种刺激是最大的一半。通过对溴磺酞的敏感性确定了第二条流出途径。该途径相对不活跃，并且在添加各种阴离子、葡萄糖或代谢抑制剂后不会显着波动。第三条途径通过其对丙磺舒的敏感性进行定量，其活性在盐水缓冲液中和添加葡萄糖后增加，并被寡霉素抑制。 L1210 小鼠细胞中存在类似的甲氨蝶呤转运途径，尽管这两种细胞系可以通过转运蛋白的量和甲氨蝶呤敏感的溴磺酞外流途径的活性来区分。
The transport routes utilized by CCRF-CEM human lymphoblastic cells for the influx and efflux of methotrexate have been analyzed. Evidence was obtained for a single influx route for methotrexate: (a) Influx at 2 microM [3H]methotrexate was inhibited completely by high concentrations of unlabeled methotrexate, o-phthalate, and bromosulfophthalein, and the inhibition profile with each anion was monophasic; and (b) Pretreatment of the cells with an N-hydroxysuccinimide ester of methotrexate also blocked influx, and this inhibition was complete over a range of substrate concentrations from 2 to 50 microM. Influx was also saturable and proceeded with a maximum rate (Vmax) of 4.3 pmol/min/mg protein (at 37 degrees C) and with a Kt of 0.8 microM in an anion-deficient buffer and 4.6 microM in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonate-buffered saline. The ratio of Vmax to the amount of carrier protein (0.3 pmol/mg protein) gave a turnover number for the transport system of 14.3/min. In contrast to influx, methotrexate efflux proceeded via three routes which could be separated by their sensitivity to specific inhibitors. The major portion of efflux occurred via the methotrexate influx carrier, the identity of which was established from its sensitivity to the N-hydroxysuccinimide ester of methotrexate and by its requirement for anions in the external medium. Methotrexate, adenosine monophosphate, and phosphate each stimulated efflux via this route and this stimulation was half-maximal at anion concentrations that approximated their Ki values for inhibition of methotrexate influx. A second efflux route was identified by its sensitivity to bromosulfophthalein. This route was relatively inactive and did not fluctuate significantly upon addition of various anions, glucose, or metabolic inhibitors. The third route was quantitated by its sensitivity to probenecid and its activity was increased in saline buffers and upon addition of glucose and was inhibited by oligomycin. Similar transport routes for methotrexate are present in L1210 mouse cells, although these two cell lines can be distinguished by the amount of transport protein and by the activity of the bromosulfophthalein-sensitive efflux route for methotrexate.