Abstract.We have previously shown that the multidrug resistance protein (MRP) mediates the ATPdependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes.The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATPdependent conjugate transport in canalicular membranes from TR-mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transportdeficient TR-mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.XCRETION into bile is a major pathway for the elimination of endogenous and xenobiotic lipophilic compounds from the mammalian organism. Drugmetabolizing transferases in the hepatocyte convert many of these compounds into amphiphilic anionic conjugates with glutathione, glucuronate, or sulfate. Excretion of these conjugates across the hepatocyte canalicular membrane into bile is mediated by a primary-active ATP-dependent export pump which has been characterized functionally and termed multispecific organic anion transporter (MOAT; 1 Oude Elferink and Jansen, 1994), non-bile acid organic anion transporter (Arias et al., 1993), glutathione S-conju-

The ATP-dependent transport of the three 3H-labeled, amphiphilic cations quinidine, N-(n-pentyl)-quinidinium, and N-(4',4'-azo-n-pentyl)-21-deoxyajmalinium was studied in rat canalicular plasma membrane vesicles. N-Alkylation of quinidine with an n-pentyl residue resulted in a permanently charged cationic substrate for ATP-dependent transport which exhibited a IO-fold higher transport rate relative to quinidine. The K, value was 0.4 PM for N-(n-pentyl)-quinidinium and 5 PM for quinidine. The permanently cationic and photolabile derivative of ajmaline, N-(4',4'-azo-n-pentyl)-21-deoxyajmalinium, was also an efficient substrate and served to label canalicular membrane proteins with molecular masses of 143 kDa and 108 kDa. ATP-dependent transport of the permanently charged amphiphilic cations was inhibited by the P-glycoprotein inhibitors and substrates quinidine, verapamil, and daunorubicin. The data demonstrate that N-alkylation of quinidine and ajmaline results in most efficient substrates for mdrl P-glycoprotein-mediated ATP-dependent transport.

The Dubin-Johnson syndrome is characterized by an patocytes into the bile. 3,4 This defect results in predomiinherited defect in the secretion of amphiphilic anionic nantly conjugated hyperbilirubinemia and a characterconjugates from hepatocytes into the bile. We have re-istic secondary rise of intravenously administered cently identified the membrane protein mediating the sulfobromophthalein in blood plasma following its conadenosine triphosphate (ATP)-dependent transport of jugation with glutathione in hepatocytes and transport glutathione and glucuronate conjugates as a multidrug-of the conjugate from hepatocytes to the blood. 3-9 Macresistance protein (MRP) and localized it to the canalicu-roscopic examination of the liver from patients with lar as well as to the lateral hepatocyte plasma memDubin-Johnson syndrome shows it to be intensely pigbrane. In the present study we show the selective mented with an appearance described as grossly black 2 absence of the canalicular isoform of MRP (cMRP) from the hepatocytes in a patient with Dubin-Johnson syn-or dark blue. 10 Several most useful animal models of drome by double-label immunofluorescence and confo-this defect in human hepatobiliary transport have been cal laser scanning microscopy using antibodies di-described and extensively studied. 3,[11][12][13][14][15][16] In the transrected against MRP and dipeptidyl-peptidase IV port-deficient GY/TR 0 mutant Wistar rat 11 a selective (DPPIV). Another isoform of MRP was detected, how-canalicular defect in the ATP-dependent transport of ever, in the lateral hepatocyte membrane of the pa-glutathione S-conjugates and other nonbile salt organic