Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions.
Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions.
复制标题
L1210 细胞中叶酸化合物的载体介导的运输。
DOI：
10.1016/0006-2952(87)90051-7
复制
发表时间：
1987
影响因子：
5.8
通讯作者：
Kisliuk,RL
中科院分区：
文献类型：
--
作者：
Sirotnak,FM;Goutas,LJ;Jacobsen,DM;Mines,LS;Barrueco,JR;Gaumont,Y;Kisliuk,RL
在阴离子缓冲液条件下，L1210 细胞中高纯度[3H]叶酸与[3H]甲氨蝶呤流入的动力学参数的比较显示出明显的不一致。此外，在[3H]甲氨蝶呤转运缺陷的变异L1210细胞中，[3H]叶酸流入的动力学没有变化。在这些变异细胞中，甲氨蝶呤的 Vmax 降低了 17 倍，Km 增加了 3 倍。结果表明，[3H]叶酸流入是由一个系统介导的，该系统对叶酸化合物的亲和力较低，但容量比介导[3H]甲氨蝶呤流入的经典高亲和力系统高20倍。由于后一个系统对[3H]叶酸也表现出非常低的亲和力，因此预计它不会对[3H]叶酸的总流入产生显着贡献。 [3H]叶酸流入的高容量系统与L1210细胞中介导蝶呤流入的系统不同，因为它不受腺嘌呤（一种有效的蝶呤流入抑制剂）的抑制。然而，将细胞暴露于非阴离子缓冲液中的[3H]叶酸会导致初始流入的显着刺激，并且在这些条件下的一部分流入被甲氨蝶呤抑制。这些结果表明阴离子通过其对高亲和力、降低的叶酸/甲氨蝶呤系统的已知影响来调节[3H]叶酸流入的多样性程度。 [14C]5-甲基四氢高叶酸的碳 6 处的非对映异构体共享两个运输系统。对于两个运输系统，天然非对映异构体的流入量是非自然形式的一半。与[3H]叶酸相比，两种非对映体在两种运输系统之间的亲和力差异更大。我们的结果表明，可通过低亲和力/高容量途径有效转运的类似物可用于治疗由于高亲和力/低容量流入系统有缺陷而对甲氨蝶呤耐药的肿瘤。我们还发现，将 L1210 细胞与[3H]叶酸或天然非对映异构体[14C]5-甲基四氢高叶酸一起孵育 10 分钟，会形成不可交换的放射性部分，占总积累的 20-40%。这种不可交换的部分可以通过聚谷氨酸盐以外的代谢物的积累来解释。在与[3H]叶酸一起孵育之前用甲氨蝶呤预加载细胞可防止放射性作为不可交换部分的积累。
Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, theVmax, for methotrexate was reduced 17-fold and theKmwas increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the highaffinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influxKmfor the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20–40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.