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

Bagrij, T Klokouzas, A Hladky, SB Barrand, MA

T. Bagrij et al., Influences of glutathione on anionic substrate efflux in tumour cells expressing the multidrug resistance-associated protein, MRP1, BIOCH PHARM, 62(2), 2001, pp. 199-206

39

INGLESE

Article

Pharmacology & Toxicology

BIOCHEMICAL PHARMACOLOGY

0006-2952 → ACNP

62

2

2001

199 - 206

ISI

0006-2952(20010715)62:2<199:IOGOAS>2.0.ZU;2-C

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 sug gest, however, that GSH can modulate transport of anionic species. Efflux o f 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, methylfluores cein-glutathione (GS-MF), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyf luorescein (BCECF) was observed from the resistant cells. Unexpectedly: low ering GSH levels in COR-L23/R cells by inhibiting GSH synthesis with buthio nine sulfoximine decreased efflux of calcein and of GS-MF (3-fold and 1.6-f old) but not efflux of BCECF. Transport of the anionic conjugate dinitrophe nyl-glutathione ([H-3]DNP-SG) was investigated by following its uptake into inside-out plasma membrane vesicles prepared from the MRP1-expressing cell s. At least 90% of the ATP-dependent uptake was blockable by the anti-MRP1 antibody QCRL-3 and 100 muM vincristine inhibited uptake but only in the pr esence of 1-3 mM GSH, suggesting MRP1 to be the protein primarily responsib le for this transport. Agents shown to reduce efflux of calcein from resist ant cells, i.e. indomethacin, MK-571, and probenecid, also inhibited [H-3]D NP-SG uptakes, consistent with MRP1 being responsible for export of calcein . At concentrations achievable within cells, GSSG (70 muM) inhibited uptake whereas GSH (1 and 3 mM) enhanced uptake. We suggest that variations in bo th GSH and GSSG levels within cells may affect MRP1-mediated anion transpor t. (C) 2001 Elsevier Science Inc. All rights reserved.