Verapamil-stimulated glutathione transport by the multidrug resistance-associated protein (MRP1) in leukaemia cells

Multidrug resistance mediated by the multidrug resistance-associated protei n MRPI is associated with decreased drug accumulation, which is in turn dep endent on cellular glutathione. We have reported that verapamil, an inhibit or of drug transport, caused a decrease in cellular glutathione in CCRF-CEM /E1000 MRP1-overexpressing leukaemia cells (Biochem Pharmacol 55;1283-9, 19 98). We now demonstrate that other inhibitors of MRP1-mediated drug transpo rt (e.g. MK571, indomethacin, genistein, and nifedipine) deplete cellular g lutathione in these leukaemia cells (> 30% decrease; P < 0.01) while having no effect on the parental CCRF-CEM cells. However, treatment with etoposid e or vincristine (at similar molar concentrations) caused a 20% decrease in glutathione. Verapamil-stimulated glutathione transport correlated with MR PI expression in a series of drug-resistant cells, and glutathione was quan titatively recovered in the extracellular media. Further, verapamil-stimula ted glutathione transport was rapid (50% decrease in 10 min), dose-dependen t, and inhibited by vanadate, an inhibitor of ATPase activity, but not by s ulphobromophthalein (BSP) or methionine, inhibitors of hepatic glutathione transporters. Incubation of CCRF-CEM/E 1000 cells in 25 mM glutathione not only showed that verapamil-mediated efflux occurred against the concentrati on gradient, but also demonstrated the MRP1-mediated uptake of glutathione (P < 0.01 compared to the parental CCRF-CEM cells), which was not inhibited by vanadate. These results demonstrate that while MRPI transports glutathi one in the presence of inhibitors of drug transport, there is no convincing evidence for co-transport of glutathione with drug. They further demonstra te that MRPI mediates the facilitated transport of glutathione into the MRP 1-overexpressing CEM/E1000 cells, suggesting that MRP1 may play a major rol e in cellular glutathione homeostasis. (C) 2001 Elsevier Science Inc. All r ights reserved.