The issues of transport multiplicity and energetics pertaining to methotrexate efflux in L1210 cells addressed by an analysis of cis and trans effects of inhibitors.
The issues of transport multiplicity and energetics pertaining to methotrexate efflux in L1210 cells addressed by an analysis of cis and trans effects of inhibitors.
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DOI：
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发表时间：
1991-03
期刊：
影响因子：
11.2
通讯作者：
F. Sirotnak;D. F. O'Leary
中科院分区：
文献类型：
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作者：
F. Sirotnak;D. F. O'Leary
研究描述解决有争议的问题的[3 H]MTX在L1210细胞的外排途径的多样性。我们研究了外排的多样性的条件下，做和不保持细胞ATP在生理水平。在ATP-充满细胞，结果描绘丙磺舒，溴磺酞，维拉帕米，可替代的路线，占近90%的[3 H]MTX流出。[3 H]MTX通过该途径的外排仅在反式方向被溴磺酞抑制，仅在顺式和反式方向同时存在时被丙磺舒抑制，并且仅在顺式方向被维拉帕米抑制。这种叶酸类似物在ATP充满细胞中的剩余流出似乎是由一碳还原叶酸系统（MTX流入途径）介导的，因为它不受溴磺酞或维拉帕米的抑制，但受MTX的N-羟基琥珀酰亚胺酯（MTX流入的特异性抑制剂）和比抑制ATP依赖性流出所需浓度高10倍的丙磺舒的抑制。在这些条件下，MTX不反式刺激[3 H]MTX流出。此外，没有证据表明[3 H]MTX外排存在假定的溴磺酞不敏感、丙磺舒可降解途径。在不含D-葡萄糖和含10 mM叠氮化钠的培养基中孵育60分钟，细胞ATP耗竭至90-95%的程度，[3 H]MTX的总体外排显著减少，似乎仅由MTX内流途径介导。内流的[3 H]MTX的顺式和反式抑制丙磺舒，并在这些条件下的外排显着抑制的N-羟基琥珀酰亚胺酯的MTX和反式刺激的MTX。总之，这些研究的结果与L1210细胞中甲氨蝶呤转运的模型一致[Dembo et al.，J. Membrane Biol.，78：9-17，1984]在作者的实验室中仅基于ATP-充满和耗尽的L1210细胞中[3 H]MTX流入和流出的动力学分析。因此，这些新的结果确定了一个单一的ATP依赖性外排途径的溴磺酞，丙磺舒，维拉帕米在L1210细胞中的条件下，保持ATP水平最大的可降解的路线。
Studies are described addressing the controversial issue of the multiplicity of efflux routes for [3H]MTX in L1210 cells. We examined efflux multiplicity under conditions that do and do not maintain cellular ATP at the physiological level. In ATP-replete cells, the results delineate a probenecid-, bromosulfophthalein-, and verapamil-inhibitable route that accounts for nearly 90% of [3H]MTX efflux. Efflux of [3H]MTX by this route is inhibited by bromosulfophthalein in the trans orientation only, inhibited by probenecid only when present simultaneously in the cis and trans orientation and inhibited by verapamil only in the cis orientation. The remaining efflux of this folate analogue in ATP-replete cells appears to be mediated by the one-carbon, reduced folate system (MTX influx route), in that it is not inhibited by bromosulfophthalein or verapamil but is inhibited by the N-hydroxysuccinimide ester of MTX, a specific inhibitor of MTX influx, and a 10-fold higher concentration of probenecid than that required to inhibit ATP-dependent efflux. Under these conditions, MTX did not trans-stimulate [3H]MTX efflux. Also, no evidence was obtained for a putative bromosulfophthalein-insensitive, probenecid-inhibitable route for [3H]MTX efflux. In cells depleted to the extent of 90-95% of their ATP by 60-min incubation in medium in the absence of D-glucose and with 10 mM sodium azide, overall efflux of [3H]MTX was markedly reduced and appears to be mediated solely by the MTX influx route. Influx of [3H]MTX was both cis and trans inhibited by probenecid, and efflux under these conditions was markedly inhibited by the N-hydroxysuccinimide ester of MTX and trans-stimulated by MTX. Overall, the results of these studies are consistent with a model for methotrexate transport in L1210 cells derived [Dembo et al., J. Membrane Biol., 78: 9-17, 1984] in the authors' laboratory based solely upon a kinetic analysis of [3H]MTX influx and efflux in ATP-replete and depleted L1210 cells. As such, these new results identify a single ATP-dependent efflux route as the bromosulfophthalein-, probenecid-, and verapamil-inhibitable route in L1210 cells under conditions that maintain ATP levels at maximum.