Characterization of ATP-dependent monoglucuronosyl bilirubin transport across rat canalicular membrane vesicles.

Bilirubin conjugates are secreted from hepatocytes into bile. Monoglucuronosyl bilirubin is an endogenous substrate for the multidrug resistance protein 2, which is located in rat hepatocyte canalicular membrane. We have characterized this ATP-dependent transport using rat canalicular membrane vesicles. Monoglucuronosyl bilirubin, 3H-labeled in the glucuronosyl moiety, was synthesized enzymatically using recombinant UDP-glycosyltransferase 1A1, and was stabilized with ascorbate. The rate for ATP-dependent transport of monoglucuronosyl bilirubin (at 0.5 µM) was 7.3+/-1.1 pmol mg protein(-1) min(-1) and the K(m) value was 1.3+/-0.4 µM. This is the first time to demonstrate this kinetic constant of monoglucuronosyl bilirubin for the rat hepatocyte canalicular membrane. The K(m) value is similar to one for recombinant rat multidrug resistance protein 2.

Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6.

Bilirubin, the end product of heme catabolism, is taken up from the blood circulation into the liver. This work identifies a high-affinity transport protein mediating the uptake of bilirubin and its conjugates into human hepatocytes. Human embryonic kidney cells (HEK293) permanently expressing the recombinant organic anion-transporting polypeptide 2 (human OATP2, also known as LST-1 or OATP-C; symbol SLC21A6) showed uptake of [(3)H]monoglucuronosyl bilirubin, [(3)H]bisglucuronosyl bilirubin, and [(3)H]sulfobromophthalein with K(m) values of 0.10, 0.28, and 0.14 microm, respectively. High-affinity uptake of unconjugated [(3)H]bilirubin by OATP2 occurred in the presence of albumin and was not mediated by another basolateral hepatic uptake transporter, human OATP8 (symbol SLC21A8). OATP2 and OATP8 differed by their capacity to extract substrates from albumin before transport. In comparison to the high-affinity transport by OATP2, OATP8 transported [(3)H]sulfobromophthalein and [(3)H]monoglucuronosyl bilirubin with lower affinity, with K(m) values of 3.3 and 0.5 microm, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a K(i) value of 112 nm, but did not inhibit transport mediated by OATP8. Human OATP2 may play a key role in the prevention of hyperbilirubinemia by facilitating the selective entry of unconjugated bilirubin and its glucuronate conjugates into human hepatocytes.

ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2.

BACKGROUND: Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2). This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney. METHODS: Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of [3H]PAH, and the net ATP-dependent transport into inside-out vesicles was determined. Comparative studies were performed with membrane vesicles containing recombinant human MRP1. RESULTS: Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively. The Km value for PAH was 880 micromol/L. The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively. The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L. Ochratoxin A was itself a substrate for MRP2. CONCLUSIONS: PAH is a good substrate for the ATP-dependent export pump MRP2. The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.

Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance.

The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family. Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized. Orthologs of the human MRP isoforms have been detected in many different organisms. Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane. Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome. Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents. Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention.

Export pumps for anionic conjugates encoded by MRP genes.

Several members of the multidrug resistance protein (MRP) family mediate the ATP-dependent transport of amphiphilic anions across membranes. The substrate specificity of recombinant human MRP1 has been most extensively defined by use of inside-out membrane vesicles. Substrates include the glutathione S-conjugate leukotriene C4, 17 beta-glucuronosyl estradiol, glucuronosyl bilirubin, glutathione disulfide, in addition to the fluorescent lipophilic anion Fluo-3. These substances are also substrates for the apical isoform MRP2, also termed canalicular multispecific organic anion transporter, cMOAT, which shares only 49% amino acid sequence identity with MRP1. The K(m) of leukotriene C4 for MRP2 is 10-fold higher than for MRP1, and the K(m) of 17 beta-glucuronosyl estradiol is 4.8-fold higher for MRP2 than for recombinant human MRP1. Human as well as rat MRP2 confer multidrug resistance to polarized MDCKII cells permanently expressing the recombinant glycoprotein in their apical plasma membrane. Resistance of cells transfected with human and rat MRP2 to etoposide was enhanced 5-fold and 3.8-fold, and resistance to vincristine was enhanced 2.3-fold and 6.0-fold, respectively. Conjugate-transporting members of the MRP family with a related sequence and a similar function have been detected recently. In addition to several MRP isoforms (MRP1-6) and orthologs in mammals (human, rat, rabbit, mouse), MRP family members have been identified in the nematode Caenorhabditis elegans, in the yeast Saccharomyces cerevisiae, and in the plant Arabidopsis thaliana. These conjugate export pumps of the MRP family play a widespread role in detoxification, drug resistance, and, because of the role in the export of glutathione disulfide by MRP1 and MRP2, in the defense against oxidative stress.

Transport of monoglucuronosyl and bisglucuronosyl bilirubin by recombinant human and rat multidrug resistance protein 2.

The secretion of bilirubin conjugates from hepatocytes into bile represents a decisive step in the prevention of hyperbilirubinemia. The bilirubin conjugates, monoglucuronosyl bilirubin (MGB) and bisglucuronosyl bilirubin (BGB), were previously suggested to be endogenous substrates for the apical multidrug resistance protein (MRP2), a member of the adenosine triphosphate (ATP)-binding cassette family of transporters (symbol ABCC2), also termed canalicular multispecific organic anion transporter. We have characterized this ATP-dependent transport using membrane vesicles from human embryonic kidney (HEK) cells expressing recombinant rat as well as human MRP2. MGB and BGB, (3)H-labeled in the glucuronosyl moiety, were synthesized enzymatically with recombinant UDP-glucuronosyltransferase 1A1, and stabilized with ascorbate. Rates for ATP-dependent transport of MGB and BGB (0.5 micromol/L each) by human MRP2 were 183 and 104 pmol x mg protein(-1) x min(-1), respectively. K(m) values were 0.7 and 0.9 micromol/L for human MRP2, and 0.8 and 0.5 micromol/L for rat MRP2, with MGB and BGB as substrates, respectively. Leukotriene C(4) and 17beta-glucuronosyl estradiol, which are both known high-affinity substrates for human MRP2, inhibited [(3)H]MGB transport with IC(50) values of 2.3 and 30 micromol/L, respectively. Cyclosporin A competitively inhibited human and rat MRP2-mediated transport of [(3)H]MGB, with K(i) values of 21 and 10 micromol/L, respectively. Our results provide direct evidence that recombinant MRP2, cloned from rat as well as human liver, mediates the primary-active ATP-dependent transport of the bilirubin conjugates MGB and BGB.

Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells.

The multidrug resistance protein MRP1 functions as an ATP-dependent conjugate export pump and confers multidrug resistance. We cloned MRP2 (symbol ABCC2), a MRP family member localized to the apical membrane of polarized cells. Stable expression of MRP2 in transfected human embryonic kidney (HEK-293) and Madin-Darby canine kidney (MDCK) cells was enhanced by inhibitors of histone deacetylase. In polarized MDCK cells, both rat and human MRP2 were sorted to the apical plasma membrane. An antibody raised against the amino terminus of rat MRP2 recognized the recombinant protein on the apical surface of nonpermeabilized cells, providing direct evidence for the extracellular localization of the amino terminus of MRP2. ATP-dependent transport by recombinant human and rat MRP2 was measured with membrane vesicles from stably transfected cells. The Km value of human MRP2 was 1.0 +/- 0.1 microM for leukotriene C4 and 7.2 +/- 0.7 microM for 17beta-glucuronosyl estradiol; the Km values of human MRP1 were 0.1 +/- 0.02 microM for leukotriene C4 and 1.5 +/- 0.3 microM for 17beta-glucoronosyl estradiol. Thus, the conjugate-transporting ATPases MRP2 and MRP1 differ not only by their domain-specific localization but also by their kinetic properties. Drug resistance conferred by recombinant MRP2 was studied in MDCK and HEK-293 cells using cell viability assays. Expression of human and rat MRP2 enhanced the resistance of MDCK cells to etoposide 5.0-fold and 3.8-fold and to vincristine 2.3- and 6.0-fold, respectively. Buthionine sulfoximine reduced resistance to these drugs. Human MRP2 overexpressed in HEK-293 cells enhanced the resistance to etoposide (4-fold), cisplatin (10-fold), doxorubicin (7.8-fold), and epirubicin (5-fold). These results demonstrate that MRP2 confers resistance to cytotoxic drugs.

Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF-B.

The polarized rat hepatoma/human fibroblast hybrid cell line, WIF-B, forms apical vacuoles into which cholephilic substances are secreted. We studied expression, localization, and function of the apical conjugate export pump, Mrp2, in WIF-B cells. Mrp2, the apical isoform of the multidrug resistance protein, alternatively termed canalicular Mrp (cMrp) or canalicular multispecific organic anion transporter (cMoat), is a 190-kd membrane glycoprotein mediating adenosine triphosphate (ATP)-dependent transport of glucuronides, glutathione S-conjugates, and other amphiphilic anions across the hepatocyte canalicular membrane into bile. Expression of the rat mrp2 gene in WIF-B cells was shown by reverse-transcription polymerase chain reaction (PCR), followed by sequencing of the amplified 789-bp fragment. Immunoblotting, using antibodies reacting with the amino-terminal or with the carboxyl-terminal sequence of rat Mrp2, detected the 190-kd glycoprotein in WIF-B cell homogenates. Immunofluorescence microscopy localized Mrp2 to the apical membrane domain. Preloading of WIF-B cells with a membrane-permeable ester of the calcium-dependent fluorescent indicator, Fluo-3, was followed by Mrp2-mediated secretion of the amphiphilic anion, Fluo-3, into the apical vacuoles. This transport was potently inhibited by cyclosporin A added to the culture medium. Direct measurements of ATP-dependent transport into Mrp2-containing plasma membrane vesicles in comparison with Mrp2-deficient vesicles established that Fluo-3 is transported by Mrp2 with a Km value of 3.7 micromol/L. Our results indicate that the polarized WIF-B cells express the rat ortholog of the apical conjugate-transporting ATPase, Mrp2. The function of Mrp2 as well as the action of inhibitors can thus be analyzed by use of the fluorescent amphiphilic anion, Fluo-3.

ATP-dependent transport of glutathione S-conjugates by the multidrug resistance protein MRP1 and its apical isoform MRP2.

The membrane proteins mediating the ATP-dependent transport of glutathione S-conjugates and related amphiphilic anions have been identified as the multidrug resistance proteins MRP1 and MRP2. These 190-kDa membrane glycoproteins were cloned in recent years and shown to be unidirectional, ATP-driven, export pumps with an amino acid identity of 49% in humans. MRP1 is detected in the plasma membrane of many cell types, including erythrocytes; whereas MRP2, also termed canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, such as the hepatocyte canalicular membrane and kidney proximal tubule luminal membrane. Physiologically important substrates of both transporters include glutathione S-conjugates, such as leukotriene C4, as well as bilirubin glucuronides. 17 beta-glucuronosyl estradiol and glutathione disulfide. Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space. The substrate specificity of MRP1 and MRP2 studied in inside-out oriented membrane vesicles is very different from MDR1 P-glycoprotein. MRP1 and MRP2 may be termed conjugate transporting ATPases, functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress.

Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione.

The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions. The present study details for the first time MRP1-mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan. In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan. In addition, we show that membranes from chlorambucil-resistant GST-alpha-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1. With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1. The transport rates measured in the resistant cells were only two- to three-fold higher than those measured in the control cells. These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines.

ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2.

Bilirubin is secreted from the liver into bile mainly as monoglucuronosyl and bisglucuronosyl conjugates. We demonstrate for the first time that ATP-dependent transport of both bilirubin glucuronides is mediated by the multidrug resistance protein (MRP1) as well as by the distinct canalicular (apical) isoform MRP2, also termed cMRP or cMOAT (canalicular multispecific organic anion transporter). In membrane vesicles from MRP1-transfected HeLa cells mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin (each at 0.5 microM) were transported with rates of 5.3 and 3.1 pmol/min per mg of protein respectively. Rat hepatocyte canalicular membrane vesicles, which contain Mrp2 (the rat equivalent of MRP2), transported mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin at rates of 8.9 and 8.5 pmol/min per mg of protein, whereas membrane vesicles from mutant liver lacking Mrp2 showed no transport of the conjugates. In membrane vesicles from human hepatoma Hep G2 cells, which predominantly expressed MRP2, transport rates were 8.3 and 4.4 pmol/min per mg of protein for monoglucuronosylbilirubin and bisglucuronosylbilirubin respectively. ATP-dependent transport of the glutathione S-conjugate -3H-leukotriene C4, an established high-affinity substrate for MRP1 and MRP2, was inhibited by both bilirubin glucuronides with IC50 values between 0.10 and 0.75 microM. The ratios of leukotriene C4 transport and bilirubin glucuronide transport, determined in the same membrane vesicle preparation, indicated substrate specificity differences between MRP1 and MRP2 with a preference of MRP2 for the glucuronides.

Transport of glutathione conjugates and glucuronides by the multidrug resistance proteins MRP1 and MRP2.

The search for the membrane proteins mediating the ATP-dependent transport of conjugates with glutathione, glucuronate, or sulfate has led to the identification of the multidrug resistance proteins MRP1 and MRP2. Both 190-kDa membrane glycoproteins were cloned in the recent years and shown to be unidirectional ATP-driven export pumps with an amino acid identity of 49% in human. MRP1 is detected in the plasma membrane of many cell types, including erythrocytes, whereas MRP2, also termed canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, particularly to the hepatocyte canalicular membrane. Physiologically important substrates of both transporters include glutathione S-conjugates such as leukotriene C4, bilirubin glucuronides, 17 beta-glucuronosyl estradiol, dianionic bile salts such as 6 alpha-glucuronosyl hyodeoxycholate, and glutathione disulfide. Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space. The substrate specificity of MRP1 and MRP2 is very different from MDR1 P-glycoprotein. MRP1 and MRP2 may be termed conjugate transporting ATPases functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress.

Identification of the biosynthetic leukotriene C4 export pump in murine mastocytoma cells as a homolog of the multidrug-resistance protein.

A membrane glycoprotein of 190 kDa has been identified previously by photoaffinity labeling as a candidate for the ATP-dependent export pump for leukotriene C4 in mastocytoma cells [Leier, I., Jedlitschky, G., Buchholz, U. & Keppler, D. (1994) Eur. J. Biochem. 220, 599-606]. The present study indicates that this protein represents the murine homolog of the human multidrug resistance protein (MRP). In immunoblot analyses several polyclonal anti-MRP antibodies and one monoclonal antibody recognized the protein of 190-kDa in plasma membranes of mastocytoma cells. Immunoprecipitation after photoaffinity labeling with [3H]leukotriene C4 precipitated the labeled 190-kDa glycoprotein. Deglycosylation by glycopeptide N-glycosidase F of mastocytoma membrane proteins was performed in comparison with membranes from MRP-overexpressing cells and resulted in a reduction of the molecular mass of 190 kDa by about 20 kDa in all membrane preparations. The expression of the murine mrp gene in the mastocytoma cells was analyzed by amplification and sequencing of two mrp cDNA fragments in the first nucleotide binding domain (182 bp) and in a domain proximal to the 3'-end (291 bp). The deduced amino acid sequences of these fragments were identical with murine Mrp and 86.7% and 89.7% identical with the corresponding sequences of human MRP. These results indicate that the ATP-dependent release of leukotriene C4 by murine mastocytoma cells is mediated by murine Mrp.

Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes.

The identification of the multidrug resistance protein (MRP) as a conjugate export pump in several cell types suggested its involvement in the long-known glutathione-S-conjugate transport across erythrocyte membranes. We investigated the ATP-dependent transport of glutathione S-conjugates in human erythrocyte and erythroleukemia cell membrane vesicles using the endogenous conjugate leukotriene C4 (LTC4), known to be a high-affinity substrate for MRP, in addition to S-(2,4-dinitrophenyl)glutathione. The kinetic parameters, including the Km value for LTC4 of 118 +/- 5 nM and the inhibition constants for transport of both substrates for the quinoline-based inhibitor MK 571, were similar to those obtained for transport mediated by recombinant MRP. Direct photoaffinity labeling of human erythrocyte membranes with [3H]LTC4 revealed a major binding protein of about 190 kDa which was immunoprecipitated by an anti-MRP serum. The radiolabeling of this protein was specifically suppressed by the transport inhibitor MK 571. Several additional anti-MRP sera detected the protein of about 190 kDa in human erythrocyte and erythroleukemia cell membranes. These data identify for the first time the glutathione-S-conjugate transporting protein in erythrocyte membranes.

ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump.

We have previously shown that the multidrug resistance protein (MRP) mediates the ATP-dependent membrane transport of the endogenous glutathione conjugate leukotriene C4 (LTC4) and of structurally related anionic conjugates of lipophilic compounds [Jedlitschky, Leier, Buchholz, Center and Keppler (1994) Cancer Res. 54, 4833-4836; Leier, Jedlitschky, Buchholz, Cole, Deeley and Keppler (1994) J. Biol. Chem. 269, 27807-27810]. We demonstrate in the present study that MRP also mediates the ATP-dependent transport of GSSG, as shown in membrane vesicles from human leukaemia cells overexpressing MRP (HL60/ADR cells) or HeLa cells transfected with an MRP expression vector (HeLa T5 cells) in comparison with the respective parental or control cells. The Km value for ATP-dependent transport of GSSG was 93 +/- 26 microM (mean value +/- S.D., n=5) in membrane vesicles from HeLa T5 cells. GSH, at a concentration of 100 microM, was not a substrate for any significant ATP-dependent MRP-mediated transport. The transport of GSSG was competitively inhibited by LTC4, by the leukotriene D4 receptor antagonist 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3- oxopropyl)-thio}-methyl]thio)propanoic acid (MK 571) and by S-decylglutathione, with K1 values of 0.3, 0.6 and 0.7 microM respectively. These studies identify MRP as the membrane glycoprotein which mediates the ATP-dependent export of GSSG from these cells.

Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump.

Previous studies have identified the ATP-dependent export of glutathione conjugates as a physiological function of the multidrug resistance protein (MRP). The involvement of MRP in the transport of endogenous and xenobiotic conjugates was investigated further using membrane vesicles from MRP-transfected HeLa cells. The ATP-dependent transport of the glutathione conjugates [(3)H]leukotriene C(4), S-(2,4-dinitrophenyl)-[(3)H]glutathione, and (3)H- labeled oxidized glutathione was characterized by determination of the transport efficiency V(max):K(m) amounting to 1031, 114, and 7.1 ml multiplied by min(-1), respectively. Additional endogenous substrates for MRP-mediated transport included the steroid conjugate 17 beta- glucuronosyl [(3)H]estradiol and the bile salt conjugates [6 alpha-(14)C]glucuronosylhyodeoxycholate and 3 alpha-sulfatolithocholyl [(3)H]taurine. The K(m) value of MRP for 17-beta-glucuronosyl [(3)H]estradiol was 1.5 +/- 0.3 microM, with a V(max):K(m) ratio of 42 ml multiplied by mg protein(-1) multiplied by min(-1), and a K(i) value of 0.7 microM for the leukotriene receptor antagonist MK 571. MRP-mediated ATP-dependent transport was observed for the anticancer drug conjugates glucuronosyl [(3)H]etoposide and monocholoro-mono[(3)H]glutathionyl melphalan, but not for unmodified [(14)C]doxorubicin, [(3)H]daunorubicin, or [(3)H]vinblastine. Our results establish that MRP functions as an ATP-dependent export pump not only for glutathione conjugates but also for glucuronidated and sulfated endogenous as well as exogenous compounds.

The function of the multidrug resistance proteins (MRP and cMRP) in drug conjugate transport and hepatobiliary excretion.

The MRP gene encodes a 190-kDa integral membrane glycoprotein which functions as a primary-active ATP-dependent export pump for amphiphilic anions. The MRP gene-encoded conjugate export pump and its canalicular isoform represent the transport activity which has been described earlier as multispecific organic anion transporter, non-bile acid organic anion transporter, glutathione S-conjugate export pump, or leukotriene export pump. Analyses of the substrate specificity of the human MRP pump were performed in plasma membrane vesicles from MRP-overexpressing drug-selected cells (7) and cells transfected with an MRP expression vector (8). Substrates for MRP include thioether-linked conjugates of lipophilic compounds with glutathione, cysteinyl glycine, cysteine, and N-acetyl cysteine, but also glutathione disulfide, and glucuronate conjugates such as etoposide glucuronide. This broad-specificity ATP-dependent export pump is not only overexpressed in several multidrug resistant tumor cells and tissues, but is also present in most normal cells and tissues. The expression of cMRP and MRP in human liver and of cMrp and its homolog Mrp in rat liver was demonstrated by reverse transcription PCR, cDNA sequencing, and immunoblotting (13). The important function of the cMRP gene-encoded broad-specificity conjugate export pump in hepatobiliary excretion is illustrated by the selective absence of this canalicular isoform from the hepatocyte canalicular membrane in transport-deficient mutant rats. This altered lack of cMrp is the basis for the hereditary detect of the hepatobiliary excretion of anionic conjugates in the mutant animals (13). The absence of this canalicular Mrp in the mutants is analogous to the defect in the human Dubin-Johnson syndrome which is characterized by an impaired excretion of conjugated anions across the canalicular membrane.

Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.

We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent 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 ATP-dependent 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 transport-deficient 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.