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ABCC2/Abcc2 is a member of the ABC transporter family expressed mainly in the liver bile canalicular membrane and involved in the excretion of various kinds of organic anions from hepatocytes into bile. During the drug development process, species differences in the pharmaco- and toxicokinetics of candidate drugs are a major problem. It is possible that ABCC2/Abcc2 transport activity as well as inhibitor sensitivity could lead to a number of phenomena (e.g. a difference in the biliary excretion clearance, a delay in the elimination half-life from the circulating blood and toxic side effects on ABCC2 -mediated drug-drug interactions, such as drug-induced hyperbilirubinemia). From this point of view, it is useful to be able to predict during preclinical development if certain compounds of interest are substrates and/or modulators of ABCC2. Although an in vivo animal model or an in vitro model expressing ABCC2 are useful assay systems, these have some limitations as far as predicting the transport profile of compounds in vivo is concerned. I will present an overview of the species differences in the tissue distribution, function, and also characteristic transport properties of ABCC2/Abcc2 mainly in an in vitro experimental model.

Retinol-binding protein (RBP) is a specific transport protein which carries retinol in the circulation. RBP concentration in plasma and liver of rats following a large dose of acetaminophen (APAP) intraperitoneally was examined. The RBP concentration in plasma decreased significantly at 12 hr after the APAP administration, while the plasma albumin concentration was affected a little. Western blot and northern blot analyses showed marked changes in RBP but not in albumin. Thus, RBP was suggested to be more sensitive for the acute drug-induced hepatotoxicity than albumin. The decrease of RBP concentration in plasma was suggested to be caused by the dysfunction of RBP synthesis in the liver.

Multidrug resistance-associated protein 2 (MRP2), along with MRPI, transports glutathione and glucuronide conjugates as well as non-conjugated organic anions. In the present study, we examined the role of charged amino acids in the transmembrane domains of rat Mrp2, conserved among MRP families, by means of sitedirected mutagenesis. A single amino acid mutation from Lys to Met at 325 (K325M), from Arg to Leu at 586 (R586L) and from Asp to Asn at 329 (D329N) resulted in a marked reduction in the transport activity of glutathione-S conjugates (2, 4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C4 (LTC4)) without affecting that of the subsequent metabolites of LTC4 (LTD4, LTE4 and LTF4) or glucuronide conjugates (17β-estradiol 17-β-D-glucuronide (E217βG) and E3040-glucuronide). In contrast to the reduced affinity for DNP-SG and LTC4, the affinity for E217βG was increased several-fold in these mutants, suggesting that amino acids at 325 and 586 may play an important role in distinguishing between glutathione and glucuronide conjugates. Furthermore, R1096L acquired the ability to transport TC, which is not transported by Mrp2. Since Mrp3, which transports TC, has a neutral amino acid at 1084 corresponding to this position, the charge-dependence on the transport ability of TC was further investigated in both Mrp2 and Mrp3. In the case of Mrp2, substitution of Arg at 1096 by Lys (R1096K) and Val (RIO96V) also resulted in the acquisition of the ability to transport TC. In contrast, substitution of Leu at 1084 of rat Mrp3 by Lys (L1084KMrp3) resulted in the loss of transport activity for both E217βG and TC, although transport activity for these compounds was unaffected by substitution by Val (L1084VMrp3) or Met (L1084MMrp3). This suggests that the charged moieties at 1096 of Mrp2 and 1084 of Mrp3 do not simply govern the transport ability for TC; Arg at 1096 acts as an obstacle to the transport of TC in rat Mrp2, whereas a neutral amino acid at the corresponding position in Mrp3 (Leu at 1084) is required for transport of both TC and E217βG.

MRP and cMOAT are the organic anion transporters involved in the efflux of organic anions from cells. We have already found two novel cDNA sequences (MLP-1 and -2: MRP like protein) which show high homology with MRP and cMOAT. Liver expression of MLP-2 was observed only in mutant rats (EHBR) whose cMOAT is hereditarily defective, suggesting that MLP-2 is an inducible transporter. MLP-2 is induced in normal rat liver by injection of phenobarbital, ANIT and bile duct ligation. In normal rats, MLP-2 is expressed mainly in the duodenum and jejunum. To clarify the nature of the organic anion transport mechanism in the intestine, we used Caco-2 cells as an intestinal model. DNP-SG and estradiol 17 β-D-glucuronide were taken up by BBMV prepared from Caco-2 in an ATP-dependent manner. Northern blot analysis suggested high expression of human cMOAT and MLP-2 and low expression of MRP in cultured Caco-2 cells.

Several organic anions, including the glutathione- and glucuronide-conjugates, are excreted into the bile via a canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats such as Eisai hyperbilirubinemic rat (EHBR). In the present study, we cloned cMOAT from the SD rat liver cDNA library using the ATP-binding cassette region as a probe which was amplified by PCR with the degenerated primers for human multidrug resistance associated protein (hMRP). cMOAT was encoded by 4623 by cDNA with a homology of 53.0 % and 46.3 % with hMRP at the cDNA and deduced amino acid level, respectively. By screening the library, three kinds of cDNA species for cMOAT with the same open reading frame and different 3′-untranslated region lengths were isolated. The Northern blot analysis of poly A+ RNA from the liver with the entire open reading frame of cMOAT as a probe revealed that the expression of plural bands (approximately 5, 6 and 8 kb) was defective in EHBR and this may be due to the presence of these cDNA species. RT-PCR and subsequent sequence analysis were performed using RNA specimens from EHBR liver. The analysis revealed that one base pair replacement within the open reading frame resulted in the introduction of the premature stop codon. The impaired expression of this particular protein is related to the pathogenesis of hyperbilirubinemia in the mutant rats. Although the expression of cMOAT is defective in EHBR, our recent kinetic studies suggested the presence of ATP-dependent organic anion transporters other than cMOAT in EHBR. Using the degenerated PCR primers described previously, we could amplify two kinds of PCR product (MRP like protein (MLP-1 and MLP-2)) from EHBR liver. The nucleotide homology of ABC region between MLP-1, 2, MRP and cMOAT was approximately 60-70%. Northern blot analysis indicated that MLP-1 hybridized with a single 6 kb transcript in the liver of SD rats and EHBR. In contrast, MLP-2 hybridized to a single 5 kb transcript in the liver and the expression was rather induced in EHBR. These results may suggest the possibility that MLP-1 and MLP-2 are novel transporters for organic anions.