Anthracyclines modulate multidrug resistance protein (MRP) mediated organic anion transport

We studied the ATP-dependent uptake of dinitrophenyl-glutathione (GS-DNP) into plasma membrane vesicles derived from parental GLC4 cells and from multidrug resistant GLC4/ADR cells. The: latter have a high expression of the multidrug resistance protein (MRP). Uptake of GS-DNP into membrane vesicles from GLC4/ADR cells was highly stimulated by the addition of ATP, compared to the uptake into membrane vesicles from GLC4 soils, This ATP-dependent uptake into membrane vesicles from GLC4/ADR cells was saturable with a K-m of 1.2 +/- 0.2 mu M and a V-max of 560 +/- 80 pmol/mg prot./min. ATP stimulated GS-DNP uptake with a K-m of 187 +/- 4 mu M. This uptake was specifically inhibited by a polyclonal serum raised against a fusion protein containing a segment of MRP, The ATP-dependent uptake of GS-DNP was not only inhibited by organic anions, such as oxidized glutathione (GSSG), methotrexate (MTX) and some bile acids, but also by non-anionic natural product drugs, such as anthracyclines, vinca alkaloids and etoposide (VP-16), Uptake of GSSG and MTX into membrane vesicles from GLC4/ADR cells could be stimulated by ATP. The ATP-dependent uptake of GSSG had a K-m of 43 +/- 3 mu M and a V-max of 900 +/- 200 nmol/mg protein/min, The ATP-dependent uptake of GS-DNP seemed to be non-competitively inhibited by the anthracycline daunorubicin (DNR), whereas the ATP-dependent GSSG uptake seemed to be competitively inhibited by DNR. A substrate binding site an MRP is proposed that comprises a pocket in which both DNR and GS-DNP or GSSG bind in random order to different, only partly overlapping sites. In this pocket binding of a second compound is influenced by the compound which was bound first.