Effects of sulfhydryl inhibitors upon transport of folate compounds into L1210 cells.
Effects of sulfhydryl inhibitors upon transport of folate compounds into L1210 cells.
复制标题
巯基抑制剂对叶酸化合物转运至 L1210 细胞的影响。
DOI：
10.1016/0006-2952(74)90264-0
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发表时间：
1974
影响因子：
5.8
通讯作者：
F. M. Huennekens
中科院分区：
文献类型：
--
作者：
Jeanne I. Rader;Dietrich Niethammer;F. M. Huennekens
图2所示。pcms对甲蝶呤稳态水平的影响。测量蝶呤转运的时间过程(0)，如图1所示。重复实验，16分钟加入50 PM pCMS (0)， pCMS浓度为100 PM。这些结果证明5-甲基四氢叶酸和紫蝶呤通过一个共同的汞敏感系统转运到L1210细胞中，叶酸转运是由一个单独的汞不敏感系统介导的。L1210细胞含有两个转运叶酸化合物的系统，这一结论与Nahas et~ 1的结果一致。~~~从底物竞争实验中得到，也得到了我们的观察结果的支持*，即某些抗紫蝶呤L1210亚系的特征是药物运输系统受损，它们吸收5-甲基四氢叶酸的能力同样存在缺陷；这些突变体以正常速率运输叶酸。然而，两种运输系统的概念是不同的。根据戈德曼的建议，叶酸和紫蝶呤至少在一定程度上具有相同的载体机制。由于5-甲基四氢叶酸是哺乳动物循环中的主要叶酸化合物，它可能是汞敏感运输系统的主要底物。从结构上考虑，预计其他还原性叶酸也会利用该体系。然而，令人惊讶的是，这一系统应该与紫蝶呤共享，因为后者的特征是氧化吡嗪环。5-甲基四氢叶酸甲硫蝶呤体系中pCMS的k值为10 PM。其他巯基抑制剂效果较差：对氯甲苯甲酸酯（35 PM）；p-hydroxymercuribenzoate (45 FM);N-ethylmaleimide(460点);碘乙酸（1950 PM）。pCMS不仅是这种转运系统最有效的抑制剂，而且据报道它也仅限于细胞外空间。”在使用的浓度下，pCMS和巯基乙醇（可以逆转抑制作用）6,7都不会导致细胞破坏或丧失活力。除了区分叶酸化合物转运到L1210细胞的两种系统外，pCMS还可以作为5-甲基四氢叶酸/紫蝶呤系统机制的探针。pCMS对紫蝶呤外排的影响说明了这一点（紫蝶呤是这类实验的首选底物，因为它不被L1210细胞代谢）。如图2所示，紫蝶呤的摄取是双相的。稳态细胞内水平，由图2中的平台表示，是自由的，可交换的紫蝶呤和与二氢叶酸还原酶结合的紫蝶呤的总和。添加pCMS使稳态水平降至1 nmol /lO '细胞，这大约是这些细胞中存在的二氢叶酸还原酶的量。* pCMS似乎使细胞外表面的载体蛋白失活，这允许自由的紫蝶呤流出，而不是流入。与这些结果相反，碘乙酸对稳态水平的紫蝶呤产生相反的作用（图2）。3）. 碘乙酸的加入引起摄取的增加，表明细胞内的一个或两个紫蝶呤池已经扩大。叠氮胺3和vincristiner2在以前的报道中也能对紫蝶呤的稳态水平产生类似的增强作用。这是可能的，这些药物有共同的能力带来增加细胞内NADPH的量。
FIG. 2. Effect ofpCMS upon steady-state level ofamethopterin. The time course of amethopterin transport (0) was measured as described in Fig. 1. In a duplicate experiment, 50 PM pCMS (0) was added at 16 min. pCMS concentration was 100 PM. These results prove that 5-methyl tetrahydrofolate and amethopterin are transported into L1210 cells via a common, mercurial-sensitive system and that folate transport is mediated by a separate, mercurial-insensitive system. The conclusion that L1210 cells contain two systems for transport of folate compounds is in accord with the results of Nahas et~ 1.~~~ obtained from substrate competition experiments, and is also supported by our observation* that certain amethopterin-resistant L1210 sublines characterized by an impaired transport system for the drug are equally defective in their ability to take up 5-methyl tetrahydrofolate; these mutants transport folate at a normal rate. The concept of two transport systems is at variance, however. with the suggestion of Goldman3 that folate and amethopterin share the same carrier mechanism, at least in part. Since 5-methyl tetrahydrofolate is the principal folate compound in the circulation of mammals, 1o It is probably the primary substrate for the mercurial-sensitive transport system. Utilization of this system by other reduced folates would be expected from structural considerations. It is surprising, however, that this system should be shared by amethopterin, since the latter is characterized by an oxidized pyrazine ring. TheK, value of pCMS in the 5-methyl tetrahydrofolateiamethopterin system was 10 PM. Other sulthydryl inhibitors were less efficient:’p-chloromercuribenzoate(35 PM); p-hydroxymercuribenzoate(45 FM); N-ethylmaleimide(460 PM); and iodoacetate (1950 PM). pCMS is not only the most effective inhibitor of this transport system, but it is also reported to be restricted to the extracellular space.” At the concentrations used, neither pCMS nor mercaptoethanol(which can reverse the inhibition) 6, 7 causes cell destruction or loss of viability.In addition to distinguishing between the two systems for transport of folate compounds into L1210 cells, pCMS also serves as a probe for the mechanism of the 5-methyl tetrahydrofolate/amethopterin system. This is illustrated by the effect of pCMS on the efflux of amethopterin(the preferred substrate for experiments of this type because it is not metabolized by L1210 cells). As shown in Fig. 2, the uptake of amethopterin is biphasic. The steady-state intracellular level, represented by the plateau in Fig. 2, is the sum of free, exchangeable amethopterin and that which is bound to dihydrofolate reductase. Addition of pCMS causes the steady-state level to fall to 1 nmole/lO’cells, which is approximately the amount of dihydrofolate reductase present in these cells.* pCMS appears to inactivate the carrier protein at the outer surface of the cell, which allows efflux, but not influx, of the free amethopterin to occur. In contrast to these results, iodoacetate produces the opposite effect on the steady-state level of amethopterin(Fig. 3). Addition of iodoacetate causes an increase in uptake, indicating that one or both of the intracellular amethopterin pools have been enlarged. Azide3 and vincristiner2 have been reported previously to produce a similar enhancement of the steady-state level of amethopterin. It is possible that these agents have in common the ability to bring about an increase in the amount of intracellular NADPH.