Prediction of hepatobiliary transport of drugs : Contribution of carrier-mediated transport in the detoxication of xenobiotics

胆道排泄在外源性药物的解毒中起着重要作用。在本研究中，我们阐明了正常SD大鼠和胆管膜上多特异性有机阴离子转运体（cMOAT）遗传缺陷的eisai高胆红素血症大鼠（EHBR）胆汁排泄有机阴离子（包括偶联代谢产物）的机制。二硝基苯酚和E3040葡萄糖醛酸盐的谷胱甘肽偶联物在离体胆管膜囊（CMV）中的atp依赖性摄取仅在SD大鼠中观察到，在EHBR中没有观察到。相比之下，从SD和EHBR分离的CMV中，在ATP存在的情况下，没有刺激E3040硫酸盐进入CMV。这些结果表明葡萄糖醛酸是cMOAT的底物，而不是硫酸盐。此外，我们还研究了cMOAT的分子特征。我们着眼于(1)cMOAT的底物特异性与多药耐药相关蛋白（MRP）相似，多药耐药相关蛋白是位于多药耐药肿瘤细胞上的一种初级活性转运蛋白（MRP）；(2)一系列初级活性转运蛋白具有保守的atp结合盒（ABC）区域，并制备了人类MRP羧基末端ABC的退化PCR引物。从SD大鼠肝脏cDNA中扩增出421 bp的片段。对SD大鼠肝脏的poly (A)+RNA进行Northern blot分析，发现与该片段杂交的mRNA有5kb和8.5kb两种。相比之下，EHBR的poly (A)+ RNA没有与该片段杂交。这些结果表明：(1)该特定区域的表达受损可能与EHBR中高胆红素血症的发病机制有关，该区域可能编码部分cMOAT。

The biliary excretion plays an important role in the detoxication of xenobiotics. In the present study, we clarified the mechanism for the biliary excretion of organic anions including conjugative metabolites in normal SD rats and in Eisaihyper-bilirubinemic rats (EHBR) whose multispecific organic anion transporter on the bile canalicular membrane (cMOAT) is hereditarily defective. ATP-dependent uptake of glutathione conjugate of dinitrophenol and E3040 glucuronide into the isolated bile canalicular membrane vesicles (CMV) was observed only in SD rats, but not in EHBR.In contrast, E3040 sulfate uptake into CMV was not stimulated in the presence of ATP in CMV isolated from both SD and EHBR.These results suggest that glucuronide, but not sulfate, can be the substrate for cMOAT.Furthermore, we examined the molecular feature of cMOAT.We fixed our eyes upon the fact (1) that the substrate specificity of cMOAT resembles that of multidrug resistance associated protein (MRP), a primary active transporter located on the multidrug resistance tumor cells and (2) that a series of the primary active transporters possess conserved ATP-binding cassette (ABC) region, and prepared the degenerated PCR primer for the carboxy-terminal ABC of human MRP.A 421 bp fragment was amplified from the SD rat liver cDNA.Northern blot analysis of poly (A)+RNA from SD rat liver revealed the presence of 5 kb and 8.5kb mRNA species which hybridized to this fragment. In contrast, poly (A)+ RNA from EHBR did not hybridize to this fragment. These results suggest (1) that the impaired expression of this particular region might be related to the pathogenesis of hyperbilirubinemia in EHBR and that this region might encode part of cMOAT.