Influences of glutathione on anionic substrate efflux in tumour cells expressing the multidrug resistance-associated protein, MRP1.

The ATP-dependent transport of natural product drugs, e.g. vincristine, by multidrug resistance-associated protein (MRP1) requires reduced glutathione (GSH), whilst that of anionic substrates does not. The present results suggest, however, that GSH can modulate transport of anionic species. Efflux of fluorescent anionic substrates was measured from adherent MRP1-expressing human multidrug-resistant lung tumour cells, COR-L23/R, and drug-sensitive parental cells. As expected, much greater efflux of calcein, methylfluorescein-glutathione (GS-MF), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was observed from the resistant cells. Unexpectedly, lowering GSH levels in COR-L23/R cells by inhibiting GSH synthesis with buthionine sulfoximine decreased efflux of calcein and of GS-MF (3-fold and 1.6-fold) but not efflux of BCECF. Transport of the anionic conjugate dinitrophenyl-glutathione ([(3)H]DNP-SG) was investigated by following its uptake into inside-out plasma membrane vesicles prepared from the MRP1-expressing cells. At least 90% of the ATP-dependent uptake was blockable by the anti-MRP1 antibody QCRL-3 and 100 microM vincristine inhibited uptake but only in the presence of 1--3 mM GSH, suggesting MRP1 to be the protein primarily responsible for this transport. Agents shown to reduce efflux of calcein from resistant cells, i.e. indomethacin, MK-571, and probenecid, also inhibited [(3)H]DNP-SG uptakes, consistent with MRP1 being responsible for export of calcein. At concentrations achievable within cells, GSSG (70 microM) inhibited uptake whereas GSH (1 and 3 mM) enhanced uptake. We suggest that variations in both GSH and GSSG levels within cells may affect MRP1-mediated anion transport.