The canalicular multispecific organic anion transporter (cMOAT), a member of the ATP-binding cassette transporter family, mediates the transport of a broad range of non-bile salt organic anions from liver into bile. cMOAT-deficient Wistar rats (TR-) are mutated in the gene encoding cMOAT, leading to defective hepatobiliary transport of a whole range of substrates, including bilirubin glucuronide. These mutants also have impaired hepatobiliary excretion of GSH and, as a result, the bile flow in these animals is reduced. In the present work we demonstrate a role for cMOAT in the excretion of GSH both in vivo and in vitro. Biliary GSH excretion in rats heterozygous for the cMOAT mutation (TR/tr) was decreased to 63% of controls (TR/TR) (114+/-24 versus 181+/-20 nmol/min per kg body weight). Madin-Darby canine kidney (MDCK) II cells stably expressing the human cMOAT protein displayed >10-fold increase in apical GSH excretion compared with wild-type MDCKII cells (141+/-6.1 pmol/min per mg of protein versus 13.2+/-1.3 pmol/min per mg of protein in wild-type MDCKII cells). Similarly, MDCKII cells expressing the human multidrug resistance protein 1 showed a 4-fold increase in GSH excretion across the basolateral membrane. In several independent cMOAT-transfectants, the level of GSH excretion correlated with the expression level of the protein. Furthermore, we have shown, in cMOAT-transfected cells, that GSH is a low-affinity substrate for the transporter and that its excretion is reduced upon ATP depletion. In membrane vesicles isolated from cMOAT-expressing MDCKII cells, ATP-dependent S-(2,4-dinitrophenyl)glutathione uptake is competitively inhibited by high concentrations of GSH (Ki approximately 20 mM). We concluded that cMOAT mediates low-affinity transport of GSH. However, since hepatocellular GSH concentrations are high (5-10 mM), cMOAT might serve an important physiological function in maintenance of bile flow in addition to hepatic GSH turnover.

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The multidrug resistance protein (MRP) is an ATPdependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we report that dinitrophenyl-S'-glutathione increases ATPase activity in plasma membrane vesicles prepared from the MRP-overexpressing cell line GLC4/ADR. This ATPase stimulation parallels the uptake of DNP-SG in these vesicles. We also show that the (iso)flavonoids genistein, kaempferol and flavopiridol stimulate the ATPase activity of GLC4/ADR membranes, whereas genistin has no effect. The present data are consistent with the hypothesis that certain (iso)flavonoids affect MRPmediated transport of anticancer drugs by a direct interaction with MRP.

The multidrug resistance protein 1 (MRP1) is an ATP-dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we show that flavopiridol, a synthetic flavonoid currently studied in phase 1 trials for its anti-proliferative characteristics, interacts with MRP1 in a potent way. Flavopiridol, as well as other (iso)flavonoids stimulate the ATPase activity of MRP1 in a dose-dependent way at low micromolar concentrations. A new specific monoclonal antibody against MRP1 (MIB6) inhibits the (iso)flavonoid-induced ATPase activity of plasma membrane vesicles prepared from the MRP1 overexpressing cell line GLC 4 /ADR. The accumulation of daunorubicin in GLC 4 /ADR cells is increased by flavopiridol and by other non-glycosylated (iso)flavonoids that interact with MRP1 ATPase activity. However, flavopiridol is the only tested compound that affects the daunorubicin accumulation when present at concentrations below 1 μ M . Glycosylated (iso)flavonoids do not affect MRP1-mediated transport or ATPase activity. Finally, MRP1 overexpressing and transfected cells are resistant to flavopiridol, but not to other (iso)flavonoids tested. These findings may be of relevance for the development of anticancer therapies with flavopiridol. © 1999 Cancer Research Campaign

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 cells. This ATP-dependent uptake into membrane vesicles from GLC4/ADR cells was saturable with a Km of 1.2 +/- 0.2 microM and a Vmax of 560 +/- 80 pmol/mg prot./min. ATP stimulated GS-DNP uptake with a Km of 187 +/- 4 microM. 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 Km of 43 +/- 3 microM and a Vmax 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 on 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.