Qian Y M, Song W C, Cui H, Cole S P, Deeley R G
Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada K7L 3N6.
J Biol Chem. 2001 Mar 2;276(9):6404-11. doi: 10.1074/jbc.M008251200. Epub 2000 Dec 1.
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) transporter that transports a range of hydrophobic xenobiotics, as well as relatively hydrophilic organic anion conjugates. The protein is present at high levels in testicular Leydig and Sertoli cells. Studies with knockout mice suggest that MRP1 may protect germ cells from exposure to some cytotoxic xenobiotics, but potential endobiotic substrates in this organ have not been identified. Previously, we have shown certain D-ring, but not A-ring, estrogen glucuronides can act as competitive inhibitors of MRP1 mediated transport, suggesting that they are potential substrates for the protein. In the case of 17 beta-estradiol-17 beta-d-glucuronide, this has been confirmed by direct transport studies. The Leydig cell is the major site of estrogen conjugation in the testis. However, the principal products of conjugation are A-ring estrogen sulfates, which are then effluxed from the cell by an unknown transporter. To determine whether MRP1/mrp1 could fulfill this function, we used membrane vesicles from MRP1-transfected HeLa cells to assess this possibility. We found that estradiol and estrone 3-sulfate alone were poor competitors of MRP1-mediated transport of the cysteinyl leukotriene, leukotriene C(4). However, in the presence of reduced glutathione (GSH), their inhibitory potency was markedly increased. Direct transport studies using [(3)H]estrone 3-sulfate confirmed that the conjugated estrogen could be efficiently transported (K(m) = 0.73 microm, V(max) = 440 pmol mg(-)1 protein min(-)1), but only in the presence of either GSH or the nonreducing alkyl derivative, S-methyl GSH. In contrast to previous studies using vincristine as a substrate, we detected no reciprocal increase in MRP1-mediated GSH transport. These results provide the first example of GSH-stimulated, MRP1-mediated transport of a potential endogenous substrate and expand the range of MRP1 substrates whose transport is stimulated by GSH to include certain hydrophilic conjugated endobiotics, in addition to previously identified hydrophobic xenobiotics.
多药耐药蛋白1（MRP1）是一种ATP结合盒（ABC）转运蛋白，可转运一系列疏水性外源性物质以及相对亲水性的有机阴离子共轭物。该蛋白在睾丸间质细胞和支持细胞中高水平表达。对基因敲除小鼠的研究表明，MRP1可能保护生殖细胞免受某些细胞毒性外源性物质的影响，但尚未确定该器官中的潜在内源性底物。此前，我们已表明某些D环而非A环雌激素葡糖醛酸苷可作为MRP1介导转运的竞争性抑制剂，这表明它们是该蛋白的潜在底物。对于17β-雌二醇-17β-D-葡糖醛酸苷，这已通过直接转运研究得到证实。间质细胞是睾丸中雌激素共轭的主要部位。然而，共轭的主要产物是A环雌激素硫酸盐，然后通过未知转运蛋白从细胞中流出。为了确定MRP1/mrp1是否能履行这一功能，我们使用来自转染了MRP1的HeLa细胞的膜囊泡来评估这种可能性。我们发现，单独的雌二醇和雌酮3-硫酸盐是MRP1介导的半胱氨酰白三烯、白三烯C4转运的较弱竞争者。然而，在存在还原型谷胱甘肽（GSH）的情况下，它们的抑制效力显著增加。使用[³H]雌酮3-硫酸盐的直接转运研究证实，共轭雌激素可以被有效转运（米氏常数K(m)=0.73微摩尔，最大转运速度V(max)=440皮摩尔毫克⁻¹蛋白分钟⁻¹），但仅在存在GSH或非还原性烷基衍生物S-甲基GSH的情况下。与先前使用长春新碱作为底物的研究不同，我们未检测到MRP1介导的GSH转运有相应增加。这些结果提供了GSH刺激的、MRP1介导的潜在内源性底物转运的首个实例，并将GSH刺激转运的MRP1底物范围扩大到包括某些亲水性共轭内源性物质，以及先前确定的疏水性外源性物质。
