Mechanisms and interaction of vinblastine and reduced glutathione transport in membrane vesicles by the rabbit multidrug resistance protein Mrp2 expressed in insect cells

The present study examined how the multidrug resistance protein (MRP) 2, which is an ATP-dependent anionic conjugate transporter, also mediates transport of the chemotherapeutic cationic drug vinblastine (VBL). We show that ATP-dependent [H-3] VBL (0.2 mu M) uptake into membrane vesicles from Sf9 cells infected with a baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) was similar to vesicles from mock-infected Sf9 cells (Sf9-mock) but could be stimulated by reduced glutathione (GSH) with a half-maximum stimulation of 1.9 +/- 0.1 mM. At 5 mM GSH, initial ATP-dependent [H-3] VBL uptake rates were saturable with an apparent K-m of 1.5 +/- 0.3 mu M. The inhibitory effect of VBL on Mrp2-mediated ATP-dependent transport of the anionic conjugate [H-3] leukotriene C-4 was potentiated by increasing GSH concentrations. Membrane vesicles from Sf9-Mrp2 cells exhibited a similar to 7-fold increase in initial GSH uptake rates compared with membrane vesicles from Sf9-mock cells. Uptake of [H-3] GSH was osmotically sensitive, independent of ATP, and was trans-inhibited by GSH. The anionic conjugates estradiol-17 beta-D-glucuronide and leukotriene C-4 cis-inhibited [H-3] GSH uptake but only in the presence of ATP. Whereas ATP-dependent [H-3] VBL uptake was stimulated by GSH, VBL did not affect [H-3] GSH uptake. Our results show that GSH is required for Mrp2-mediated ATP-dependent VBL transport and that Mrp2 transports GSH independent of VBL.