Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione

Abstract

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.

A probable relationship between characteristic accumulation of doxorubicin and P-glycoprotein transporter in rat liver

“…Additionally, our suggestion can be supported by the data of the conventional electron microscopy studies demonstrating that no specific organelles such as the lysosomes assemble in the vicinity of the bile capillaries of the hepatocytes. In addition, the similar observations have been made by our recent ICCs for an anti-viral agent oseltamivir (to be published elsewhere) as well as a penicillin analog amoxicillin (Fujiwara et al 2011a, b), on the bile capillaries where both the transporters P-glycoprotein (Morimoto et al 2008;Oo et al 2001;Ose et al 2009) and the multidrug resistance-associated protein Mrp2 (Bodo et al 2003;Ito et al 2004;Paulusma et al 1999;Tsuji et al 1990) for the drugs occur, respectively. Regrettably, double staining for DX and P-glycoprotein was impossible to be performed, since both the fixative GA and protease digestion necessary for DX ICC were not compatible with ICC for P-glycoprotein (Ortiz et al 1999).…”

Human Multidrug Resistance Associated Protein 4 Confers Resistance to Camptothecins

“…Efficient transport of some substrates by several of the MRP protein family members, such as MRP1, MRP2, MRP4, and MRP5 also requires physiological concentrations of the antioxidant GSH, which is cotransported with another substrate (38,50,58). Similarly, in MRP4-overexpressing HepG2 cells used in the present study, GSH depletion by BSO (200 mM) significantly reversed the resistance to bis-POM-PMEA, MTX, and CPTs (Tables I and II).…”

“…However, PgP is excluded as a transporter for CPT resistance in this study by the fact that there was no difference in the IC 50 and accumulation of paclitaxel (a known PgP substrate) in MRP4/HepG2 and V/HepG2 cells (Table III and Fig. 8).…”

“…Paclitaxel seemed to achieve saturable accumulation in both V/HepG2 and MRP4/HepG2 cells within 2 to 5 min and the accumulated drug was 3.2Y4.2 ng/10 6 cells over 120 min. In addition, similar IC 50 …”

“…As background expression of MRP4 is very low in the control cells (V/HepG2) (38), transporters other than MRP4 (e.g., MRP1, MRP2, and MRP5) may play a dominant role in the transport of bis-POM-PMEA, PMEA and MTX. Like MRP4, these transporters can cotransport GSH with their corresponding substrates (49,50). BSO might affect their function by inhibiting GSH synthesis, resulting in different uptake and accumulation and cytotoxicity of PMEA and MTX.…”