Salerno M, Garnier-Suillerot A
Laboratoire de Physicochimie Biomoléculaire et Cellulaire, ESA CNRS 7033, Université Paris Nord, 74 rue Marcel Cachin, 93017, Bobigny, France.
Eur J Pharmacol. 2001 Jun 1;421(1):1-9. doi: 10.1016/s0014-2999(01)00992-x.
The present study examined how the multidrug resistance protein (MRP1), which is an ATP-dependent anionic conjugate transporter, also mediates the transport of reduced glutathione (GSH) and the co-transport of the cationic drug, daunorubicin, with GSH in living GLC4/Adr cells. To obtain information on the affinity of GSH for the multidrug resistance protein in GLC4/Adr cells, we investigated the GSH concentration dependence of the ATP-dependent GSH efflux. The intracellular GSH concentration was modulated by preincubation of the cells with 25 microM buthionine sulfoximine, an inhibitor of GSH synthetase, for 0-24 h. The transport of GSH was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. Fitting of the obtained data according to the Michaelis-Menten equation revealed a Km of 3.4+/-1.4 mM and a Vmax of 1.5+/-0.2x10(-18) mol/cell/s. The ATP-dependent transport of GSH was inhibited by 3-([[3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl]-[(3-dimethylamino-3-oxopropyl)-thio]-methyl]thio)propanoic acid (MK571), with 50% inhibition being obtained with 1.4 microM MK571. We investigated the GSH concentration dependence of the MRP1-mediated ATP-dependent transport of daunorubicin under conditions where the transport of daunorubicin became saturated. The daunorubicin transport was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued. We were therefore in the situation where GSH acted as an activator: its presence was necessary for the binding and transport of daunorubicin by MRP1. However, GSH was also transported by the multidrug resistance protein. The concentration of GSH that gave half the maximal rate of daunorubicin efflux was 2.1+/-0.8 mM, very similar to the Km value obtained for GSH. In conclusion, the rate of daunorubicin efflux, under conditions where the transport of daunorubicin became saturated, and the rate of GSH efflux determined at any intracellular concentration of GSH were very similar, yielding a 1:1 stoichiometry with respect to GSH and daunorubicin transport. These results support a model in which daunorubicin is co-transported with GSH.
本研究考察了多药耐药蛋白1（MRP1），一种ATP依赖性阴离子共轭转运体，在活的GLC4/Adr细胞中介导还原型谷胱甘肽（GSH）转运以及阳离子药物柔红霉素与GSH共转运的方式。为了获取GLC4/Adr细胞中GSH对多药耐药蛋白的亲和力信息，我们研究了ATP依赖性GSH外排的GSH浓度依赖性。通过用25微摩尔丁硫氨酸亚砜胺（一种GSH合成酶抑制剂）对细胞进行0 - 24小时的预孵育来调节细胞内GSH浓度。GSH的转运与细胞内GSH浓度相关，直至约5毫摩尔，然后趋于平稳。根据米氏方程对所得数据进行拟合，得出米氏常数Km为3.4±1.4毫摩尔，最大反应速度Vmax为1.5±0.2×10⁻¹⁸摩尔/细胞/秒。GSH的ATP依赖性转运受到3 -（[[3 -（2 - [7 - 氯 - 2 - 喹啉基]乙烯基）苯基]-[(3 - 二甲基氨基 - 3 - 氧代丙基）硫代]-甲基]硫代）丙酸（MK571）的抑制，1.4微摩尔MK571可产生50%的抑制率。我们在柔红霉素转运达到饱和的条件下，研究了MRP1介导的柔红霉素ATP依赖性转运的GSH浓度依赖性。柔红霉素的转运与细胞内GSH浓度相关，直至约5毫摩尔，然后趋于平稳。因此，我们得出GSH起到激活剂的作用：其存在对于MRP1结合和转运柔红霉素是必需的。然而，GSH也通过多药耐药蛋白进行转运。使柔红霉素外排达到最大速率一半时的GSH浓度为2.1±0.8毫摩尔，与GSH的Km值非常相似。总之，在柔红霉素转运达到饱和的条件下，柔红霉素外排速率以及在任何细胞内GSH浓度下测定的GSH外排速率非常相似，在GSH和柔红霉素转运方面产生1:1的化学计量关系。这些结果支持了柔红霉素与GSH共转运的模型。
