Download Hepatic Secretion of Conjugated Drugs and Endogenous Substances

SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000
Hepatic Secretion of Conjugated Drugs and Endogenous
Substances
DIETRICH KEPPLER, M.D. and JÖRG KÖNIG, Ph.D.
ABSTRACT Conjugate export pumps of the multidrug resistance protein (MRP) family mediate the ATPdependent secretion of anionic conjugates across the canalicular and the basolateral hepatocyte membrane into bile
and sinusoidal blood, respectively. Xenobiotic and endogenous lipophilic substances may be conjugated with glutathione, glucuronate, sulfate, or other negatively charged groups and thus become substrates for export pumps of
the MRP family. The apical isoform, MRP2 (gene symbol ABCC2), has been localized to the apical membrane of several polarized epithelia and particularly to the canalicular membrane of hepatocytes. Absence of functionally active
MRP2 glycoprotein from this membrane domain prevents the secretion of many anionic conjugates into bile.
Prototypic endogenous substrates of high affinity for recombinant human MRP2 include bisglucuronosyl bilirubin,
monoglucuronosyl bilirubin, and the glutathione S-conjugate leukotriene C4. Several mutations in the human MRP2
gene have been identified that lead to the absence of MRP2 from the canalicular membrane and to the conjugated
hyperbilirubinemia of Dubin-Johnson syndrome. MRP2-mediated conjugate export represents a decisive final step
in the detoxification of drugs, toxins, and endogenous substances. The basolateral isoform, MRP3 (gene symbol
ABCC3), is upregulated in MRP2 deficiency and in extrahepatic cholestasis. MRP3 mediates the ATP-dependent
transport of anionic conjugates, particularly of glucuronides and sulfoconjugates, across the basolateral hepatocyte
membrane into sinusoidal blood. The inverse regulation of MRP3 and MRP2 expression under many conditions is
consistent with their distinct localization and with a compensatory role of MRP3 in the hepatic secretion of anionic
conjugates during impaired transport into bile.
KEY WORDS: conjugate export pumps, Dubin-Johnson syndrome, MRP transporters MRP2, MRP3
Hepatocytes actively convert exogenous and endogenous substances into anionic conjugates with glutathione, glucuronate, sulfate, or other negatively
charged moieties. This conjugation of lipophilic sub-
stances precedes their transport into the extracellular
space. Under physiologic conditions, these conjugates
are secreted across the canalicular membrane into bile
by an ATP-dependent conjugate export pump.1–6 The
Objectives
Upon completion of this article, the reader should be able to 1) summarize the role of the conjugate export pumps of the multidrug resistance
protein (MRP) family in the hepatic handling of endogenous and xenobiotic substances, 2) summarize the prototypic substrates of the apical
conjugate export pump MRP2, 3) summarize the molecular basis of Dubin-Johnson syndrome, and 4) discuss the compensatory role of the
basolateral conjugate export pump MRP3 in cholestasis and Dubin-Johnson syndrome.
Accreditation
The Indiana University School of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide
continuing medical education for physicians.
Credit
The Indiana University School of Medicine designates this educational activity for a maximum of 1.0 hours credit toward the AMA Physicians
Recognition Award in category one. Each physician should claim only those hours of credit that he/she actually spent in the educational activity.
Disclosure
Statements have been obtained regarding the authors’ relationships with financial supporters of this activity. There is no apparent conflict of
interest related to the context of participation of the authors of this article.
From the Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Reprint requests: Dr. D. Keppler, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany. E-mail:
[email protected]
Copyright © 2000 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.: +1(212) 584-4662.
0272-8087,p;2000,20,03,265,272,ftx,en;sld00073x
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SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000
molecular identification and cloning of this canalicular
conjugate export pump (MRP2)7–9 was a consequence
of the discovery that the multidrug resistance protein 1
(MRP1) transports similar substrates, including glutathione S-conjugates, glucuronides, and sulfoconjugates.10–13 Mutant rat strains deficient in the hepatobiliary secretion of conjugates and additional organic
anions14,15 were shown to lack the multidrug resistance
protein 2 (MRP2) in the canalicular membrane8,9 due to
mutations in the rat MRP2 gene, leading to premature
termination codons.8,16 Observations in these mutant
rats prove the concept that anionic conjugates of many
lipophilic substances cannot exit across the plasma
membrane into bile in the absence of the ATPdependent export pump MRP2. The lack of this protein
prevents the secretion into bile of substances such as the
glutathione S-conjugate leukotriene C415 and N-acetyl
leukotriene E4,17 both high-affinity substrates for
MRP2.3,18 Evidently, there is no alternative transport
protein in the canalicular membrane for the secretion of
these conjugates. It is important to note, however, that
many conjugates formed in the hepatocytes can be
transported into the sinusoidal space, particularly under
pathophysiologic conditions associated with an impaired function of MRP2.17,19 The recent localization of
another member of the MRP family, MRP3, to the basolateral membrane of hepatocytes20,21 suggests that basolateral isoforms of the MRP family can support the secretion of conjugates from hepatocytes into blood.
In this article we consider the role of MRP family
members in the hepatocellular secretion of anionic substances formed in the conjugation phase (phase II) of
detoxification of drugs and endogenous substances.
Moreover, we review consequences of mutations in the
MRP2 gene leading to the Dubin-Johnson syndrome in
humans.
CONJUGATE EXPORT PUMPS
OF THE MRP FAMILY
Members of the MRP family have been identified
in a variety of different organisms, including yeast,
nematodes, plants, and mammals. The major function of
this ATP-binding cassette (ABC) transporters is the export of anionic conjugates from cells. In humans, the
MRP family currently comprises six characterized
members, known as MRP1 (symbol ABCC1); MRP2
(ABCC2), also known as canalicular MRP or canalicular multispecific organic anion transporter; MRP3
(ABCC3); MRP4 (ABCC4); MRP5 (ABCC5); and
MRP6 (ABCC6). The deduced amino acid numbers
range from 1,325 amino acids for MRP4 to 1,545 amino
acids for MRP2 (Table 1). The length of the proteins
and the membrane topology discriminates the MRPs
from the MDR Pglycoproteins. In contrast to the typical
six plus six transmembrane segment topology described
for members of the MDR family,22,23 four of the six current members of the MRP family exhibit an additional
aminoterminal membrane-spanning domain represented
by an extension of approximately 200 amino acids.24 In
addition to computational methods, the topology of
MRP1 and MRP2 has been studied by mutational analyses, limited proteolysis experiments,25 and epitope insertion studies.26–28 Both transporters are predicted to
consist of a P-glycoprotein-like core structure with two
ATP-binding domains and two transmembrane regions,
in addition to a third transmembrane region located
TABLE 1. Members of the MRP Family with Amino Acid Identities
Relative to MRP2
Identity
(%)
Accession
Number
1531
1545
1527
1325
1437
1503
48
100
46
36
36
38
NM_004996
X96395
Y17151
AF071202
AF104942
AF076622
MRP2
1564
82
Z49144
Rat
MRP2
MRP3
MRP6
1541
1523
1502
78
46
36
X96393
U73038
AB010466
Mouse
MRP1
MRP2
MRP5
1528
1543
1436
48
77
36
AF022908
AF227274
AB019003
Species
Protein
Symbol
Chromosome
Human
MRP1
MRP2
MRP3
MRP4
MRP5
MRP6
ABCC1
ABCC2
ABCC3
ABCC4
ABCC5
ABCC6
16p13.1
10q24
17q21.3
13q32
3q27
16p13.1
Rabbit
ABCC, ABC subfamily C.
Amino Acids
267
HEPATIC SECRETION—KEPPLER, KÖNIG
amino-proximal in front of the core sequence. A remarkable topologic feature of MRP1 and MRP2 represents their amino-terminus, which was predicted to be
extracellular. This was first described for MRP2 on the
basis of topology prediction programs9 and subsequently experimentally established both for MRP1 by
epitope insertion studies29 and for MRP2 by direct immunofluorescence microscopy.18
MRP1 and MRP2 are the best characterized members of the MRP family, and both share a similar substrate specificity.30 MRP1, the founding member of the
family, was cloned from a drug-selected human lung
cancer cell line.31 The MRP1 gene spans approximately
200 kilo-base pairs (kbp) and is located on chromosome
16p13.12-13; the gene contains 31 exons.32 The human
MRP2 protein consists of 1,545 amino acids and is encoded by a gene located on chromosome 10q23q24.9,18,19,33–36 The MRP2 gene spans approximately 45
kbp and contains 32 exons ranging in size from 56 to
255 bp.37 Each nucleotide binding domain of the MRP2
gene is encoded by three exons. The comparison of the
genomic organization of MRP237,38 with the genomic
organization of the MRP1 gene32 displays remarkable
similarities indicated by the size and number of exons
and by 21 identical splice sites when viewed on the
amino acid level.37 Recently, the genomic organization
of the MRP3 gene became accessible as a genomic cosmid clone (GenBank accession AC004590). The MRP3
gene contains 31 exons. Interestingly, MRP1, MRP2,
and MRP3 have 21 identical splice sites when viewed
on an amino acid alignment. Despite the fact that these
three MRP isoforms share only a relatively low degree
of amino acid identity, a close evolutionary relationship
of these transporters is indicated by their similar genomic organization.
The identification of MRP3, MRP4, MRP5, and
MRP6 was mainly based on the analysis of the expressed sequence tag database39 followed by the cloning
of partial cDNA sequences40 and subsequently of the
complete cDNA of the respective transporter. MRP1–5
are encoded by genes located on different chromosomes
(Table 1). Among the more recently identified MRP isoforms (MRP3–5), MRP3 is best characterized with respect to its tissue-specific expression, its transport function, and its localization.20,21,41–43 Unlike the substrates
for MRP1 and MRP2, glutathione S-conjugates are poor
substrates for MRP3.42 On the other hand, other
glucuronosyl conjugates, including 17-glucuronosyl
estradiol, are substrates for MRP1, MRP2, and MRP3.
In contrast to MRP1 and MRP2 from rat and human, rat
MRP3 is able to transport sulfated and nonsulfated bile
salts.42 Moreover, MRP3 is also able to confer drug resistance against several epipodophyllotoxins and methotrexate.21 The tissue distribution of MRP3 exhibits
similarities with the tissue distribution of MRP2, and
both proteins are expressed in liver and colon.20,21,30
APICAL CONJUGATE EXPORT
PUMP MRP2
MRP2 has been the second member of the MRP
family to be cloned, localized, and functionally characterized.7–9,16,18,19,30,33–36,44–49 Up to now, it is the only
conjugate export pump detected in the apical (canalicular) membrane domain of hepatocytes. MRP2 has also
been localized to the apical membrane of kidney proximal tubules,47,48 of intestinal epithelia, and various polarized cells in culture.49–52 It should be noted that other
known members of the human MRP family have been
localized to the basolateral domain of polarized cells.30
The localization of MRP2 is dynamic as shown by the
endocytic retrieval and exocytic insertion of the protein
in rat hepatocytes.53–57 Endocytic retrieval followed by
downregulation of MRP2 gene expression is also observed in rat hepatocytes after endotoxin-induced
cholestasis56,57 and after duct ligation.55 The endotoxininduced decrease of MRP2 in the canalicular membrane
explains the well-known impairment of the excretion of
MRP2 substrates into bile in endotoxemia. This is exemplified by the early and potent inhibition of cysteinyl
leukotriene secretion across the canalicular membrane
into rat bile after endotoxin administration.58,59
The substrate specificity of MRP2 has been elucidated in a stepwise fashion, starting from hepatobiliary elimination studies in MRP2-deficient mutant
rats,6,14,15,60 leading to measurements of ATP-dependent
transport using inside-out-oriented canalicular membrane vesicles derived from normal and MRP2-deficient
mutant rat liver,2–7,9 and finally by transport measurements using recombinant MRP2 from human and other
species.18,30,36,46 Based on the work with recombinant
human MRP2 studied in membrane vesicles, the prototypic high-affinity substrates include monoglucuronosyl
bilirubin, bisglucuronosyl bilirubin, and the endogenous
glutathione S-conjugate leukotriene C4 (Table 2). A
large number of glutathione S-conjugates, glucuronides,
and sulfoconjugates of drugs and other xenobiotics are
also substrates for MRP2, although the affinity for most
drug conjugates is much lower than for the prototypic
TABLE 2. Selected Substrates for the Apical
Conjugate Export Pump MRP2 (Human
Recombinant)
Substrate
Monoglucuronosyl bilirubin
Bisglucuronosyl bilirubin
Leukotriene C4
S-glutathionyl
2,4-dinitrobenzene
17-Glucuronosyl estradiol
para-Aminohippurate
Km Value
(µM)
0.7
0.9
1.0
6.5
7.2
880
Reference
Kamisako et al., 199944
Kamisako et al., 199944
Cui et al., 199918
Evers et al., 199836
Cui et al., 199918
Leier et al., 200062
268
SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000
endogenous substrates.61 In addition, a number of nonconjugate amphiphilic anions, such as the lipophilic
pentaanion Fluo349,52 and the monoanionic paraaminohippurate,62 are MRP2 substrates.
Because MRP2 shares only 48% identical amino
acids with MRP1 (Table 1), it cannot be anticipated that
known inhibitors for MRP163 will also interfere with
MRP2-mediated transport. Inhibitors acting on MRP2,
although less potently than on MRP1,11 include the
quinoline derivative MK5719 and cyclosporin A.52
Selective inhibitors for MRP2 may be of interest to
overcome MRP2-mediated drug resistance18,36 and to
enhance the intestinal absorption of drugs that are
MRP2 substrates and are otherwise transported back
into the intestinal lumen.
MEMBERS OF THE MRP FAMILY
LOCALIZED TO THE BASOLATERAL
MEMBRANE
The founding member of the MRP family, MRP1,31
has been localized to the basolateral membrane of polarized pig kidney cells after transfection with human
MRP1 cDNA.64 In hepatocytes, however, the expression
of MRP1 mRNA is extremely low31 and the immunoreactive proteins detected at the basolateral hepatocyte
membrane by use of antibodies, raised against human
MRP1,7,19 could only be fully identified after the more
recent cloning of the isoforms MRP320,21 and
MRP6.30,65–67 MRP3 and MRP6 have been localized to
the basolateral membrane of human hepatocytes and
other polarized epithelial cells.20,21,30,65 MRP6 is constitutively and highly expressed in hepatocytes and kidney,66 but its substrate specificity has not yet been elucidated. MRP3 is low under normal conditions in
hepatocytes and upregulated in cholestatic liver disease.20,21,41–43 Under many conditions, MRP3 is regulated inversely when compared with MRP2.68 It is of in-
terest that MRP3 has also been localized to the basolateral membrane of cholangiocytes21 and intestinal epithelia where it may contribute to the enterohepatic circulation of bile salts.42,43,69
MRP2 DEFICIENCY IN
DUBIN-JOHNSON SYNDROME
Some mutations in the MRP2 gene are associated
with the absence of the MRP2 protein from the hepatocyte canalicular membrane.34,37 Several different mutations in the MRP2 gene have been discovered in humans34,37,38,70 and rats.8,16 The Dubin-Johnson syndrome
in humans is an autosomal recessively inherited disorder characterized by conjugated hyperbilirubinemia and
pigment deposition in the liver.71–73 The deficient transport of monoglucuronosyl bilirubin and bisglucuronosyl
bilirubin and other anionic conjugates from hepatocytes
into bile is caused by the absence or the functional impairment of the MRP2 protein in the canalicular membrane.19,34,37,74 So far, however, mutations in the MRP2
gene leading to a functionally deficient protein inserted
into the hepatocyte canalicular membrane have not been
identified. Furthermore, we have neither detected truncated MRP2 protein in the hepatocytes from a DubinJohnson syndrome patient with a stop codon in exon 23
nor in a patient with a 6-nucleotide deletion in exon
30.37 Current knowledge on the sites of mutations in the
coding sequence and in splice sites of the MRP2 gene in
Dubin-Johnson syndrome together with the exon–intron
organization of the human MRP2 gene are depicted in
Figure 1. Determination of the exon–intron organization
of the gene has been a prerequisite for the elucidation of
mutations underlying Dubin-Johnson syndrome.37,38
The currently known mutations in the MRP2 gene are
scattered preferentially over the 3-proximal half of the
mRNA including the exons encoding both nucleotidebinding domains (Fig. 1).
FIG. 1. Mutations in the MRP2 gene leading to Dubin-Johnson syndrome. The genomic organization of the human MRP2 gene
is characterized by 32 exons and a size of the total gene of about 45 kbp.37 The exon–intron boundaries (GenBank accession
AJ132244), the number of exons, and both ATP-binding domains are indicated. Arrows indicate the sites of mutations currently identified. F1–F4 correspond to mutations identified in Fukuoka,38,70 S1 indicates a mutation identified in Saga,75 H1 and H2 correspond
to mutations studied in Heidelberg,37 A1 is an identical mutation as H1 and was first described in Amsterdam,34 and T1 designates the
mutation present in a large group of Iranian Jews analyzed in Tel Aviv.76
HEPATIC SECRETION—KEPPLER, KÖNIG
Established mutations in patients with DubinJohnson syndrome include a nonsense mutation leading
to a premature termination codon,34,37 a missense mutation affecting the first nucleotide-binding domain,38,70 a
deletion mutation leading to the loss of two amino acids
in the second nucleotide-binding domain,37 splice junction mutations leading to exon deletions and premature
termination codons,38,70,75 and a mutation causing an
isoleucin to phenylalanine exchange in position 1173.76
Furthermore, mutations were identified in two wellcharacterized hyperbilirubinemic rat strains, which
have been considered as animal models of the human
Dubin-Johnson syndrome, the GY/TR mutant rat8 and
the Eisai hyperbilirubinemic rat.16 These mutations introduce premature termination codons at codon 401 and
855 in GY/TR and Eisai hyperbilirubinemic rat mutant
rats, respectively. In both mutant livers, however, no
truncated proteins were detected,9 and the MRP2
269
mRNA was below detectability as analyzed by Northern
blotting.8,9,16 The introduction of premature termination
codons may lead to a decrease in the level of mRNA by
a mechanism termed “nonsense mediated decay.”77 In
the case of a stop codon 5 of the last splice site, this is
recognized during translation, and the mRNA is subjected to decay.77 It is likely that the absence of the
MRP2 protein from the hepatocyte canalicular membrane in certain cases of Dubin-Johnson syndrome19,37,74
is also a consequence of the rapid degradation of the
mutated mRNA. Other mutations in the MRP2 gene
may lead to a reduced stability of the protein, may affect
the interaction of the MRP2 protein with chaperone proteins, may influence trafficking and apical localization
of the protein, or may lead to an apically localized but
functionally deficient MRP2 protein. These alternatives
should be considered and possibly investigated for each
of the mutations in the MRP2 gene, unless mutations
FIG. 2. Localization and function of the conjugate export pumps MRP2 and MRP3 in normal and MRP2-deficient hepatocytes. (Top) The uptake of various substances across the basolateral membrane, followed by conjugation, and MRP2-mediated export across the apical (canalicular) membrane is shown. (Bottom) Situation in extrahepatic cholestasis or MRP2 deficiency with the
compensatory function of MRP3 mediating export of conjugates across the basolateral membrane into sinusoidal blood.
270
leading to a premature termination codon allow for a
simpler interpretation of the consequences.
SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000
plifying one of several conditions under which MRP3
and MRP2 are inversely regulated in liver and hepatocyte-derived cells.68
INTERACTION OF THE APICAL AND
BASOLATERAL CONJUGATE EXPORT
PUMPS: MRP2 AND MRP3
The release on anionic conjugates from hepatocytes
into sinusoidal blood under conditions of extrahepatic
cholestasis or MRP2 deficiency can be mediated by the
basolateral export pump MRP3 (Fig. 2). This is indicated by the basolateral localization of MRP3 in human
and rat hepatocytes20,21 and by the substrate preference
of MRP3 for anionic conjugates formed inside hepatocytes both under normal conditions and during cholestasis or MRP2 deficiency.42,78,79 Substrates for MRP3 include glucuronosyl bilirubin,78 17-glucuronosyl
estradiol,42,79 and sulfated bile salts such as sulfatolithocholyl taurine and sulfatochenodeoxycholyl taurine,42,79
whereas glutathione conjugates are relatively poor substrates for MRP3 when compared with MRP1 and
MRP2.79 The pronounced expression of MRP3 in the
basolateral membrane of hepatocytes from patients with
Dubin-Johnson syndrome20 serves as a compensatory
transport pathway for conjugates that cannot be secreted
into bile and must therefore be released into blood followed by renal excretion. The conjugated hyperbilirubinemia in Dubin-Johnson syndrome suggests that the
release of bilirubin glucuronides from hepatocytes via
MRP3 is more rapid than renal excretion. The compensatory role of MRP3 in MRP2 deficiency and in extrahepatic cholestasis is also consistent with the upregulation of MRP3 in rat liver after bile duct ligation and in
rats with hereditary MRP2 deficiency.80 The noninvasive assessment of hepatobiliary and renal secretion of
anionic conjugates in rats under such conditions has indicated a prolonged time period for intracellular storage
and metabolism and an approximately threefold extension of hepatic excretion half-times followed by the
complete renal elimination of substances that are eliminated under normal conditions via MRP2 into bile.17
The molecular mechanisms leading to the upregulation of MRP3 have not been sufficiently elucidated.42,68,79,80,81 Characterization of the 5-flanking region of human MRP3 demonstrates that MRP3 is under
the control of a TATA-less promoter and that Sp1 binding sites may be involved in the regulation of its expression.81 Evidence has not been obtained supporting a direct action of bilirubin as an inducer.81 The basal
promoter activity of human MRP3 is only 4 % of that
measured for MRP268; however, MRP3 promoter activity, mRNA, and protein are markedly increased after
disruption of microtubules by nocadazol.68 This treatment leads, on the other hand, to a downregulation of
promoter activity, mRNA, and protein of MRP2, exem-
ABBREVIATIONS
ABC
ABCC
kbp
MRP
ATP-binding cassette
ATP-binding cassette transporter subfamily C
kilo-base pair
multidrug resistance protein
REFERENCES
1. Kobayashi K, Sogame Y, Hayashi K, et al. ATP stimulates the
uptake of S-dinitrophenylglutathione by rat liver plasma membrane vesicles. FEBS Lett 1988;240:55–58
2. Kitamura T, Jansen P, Hardenbrook C, et al. Defective ATPdependent bile canalicular transport of organic anions in mutant
(TR-) rats with conjugated hyperbilirubinemia. Proc Natl Acad
Sci USA 1990;87:3557–3561
3. Ishikawa T, Müller M, Klünemann C, et al. ATP-dependent primary active transport of cysteinyl leukotrienes across liver
canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates. J Biol Chem 1990;265:
19279–19286
4. Akerboom TPM, Narayanaswami V, Kunst M, et al. ATPdependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver. J Biol Chem
1991;266:13147–13152
5. Fernández-Checa JC, Takikawa H, Horie T, et al. Canalicular
transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. J Biol Chem 1992;267:1667–1673
6. Oude Elferink RPJ, Meijer DKF, Kuipers F, et al. Hepatobiliary
secretion of organic compounds: Molecular mechanisms of
membrane transport. Biochim Biophys Acta 1995;1241:
215–268
7. Mayer R, Kartenbeck J, Büchler M, et al. Expression of the MRP
gene-encoded conjugate export pump in liver and its selective
absence from the canalicular membrane in transport-deficient
mutant hepatocytes. J Cell Biol 1995;131:137–150
8. Paulusma CC, Bosma PJ, Zaman GJR, et al. Congenital jaundice
in rats with a mutation in a multidrug resistance-associated protein gene. Science 1996;271:1126–1128
9. Büchler M, König J, Brom M, et al. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein,
cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J Biol Chem 1996;271:15091–15098
10. Jedlitschky G, Leier I, Buchholz U, et al. ATP-dependent transport of glutathione S-conjugates by the multidrug resistanceassociated protein. Cancer Res 1994;54:4833–4836
11. Leier I, Jedlitschky G, Buchholz U, et al. The MRP gene encodes
an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 1994;269:27807–27810
12. Müller M, Meijer C, Zaman G, et al. Overexpression of the gene
encoding the multidrug resistance-associated protein results in
increased ATP-dependent glutathione S-conjugate transport.
Proc Natl Acad Sci USA 1994;91:13033–13037
13. Jedlitschky G, Leier I, Buchholz U, et al. Transport of
glutathione, glucuronate, and sulfate conjugates by the MRP
gene-encoded conjugate export pump. Cancer Res 1996;56:
988–994
14. Jansen PLM, Peters WHM, et al. Hereditary chronic conjugated
271
HEPATIC SECRETION—KEPPLER, KÖNIG
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
hyperbilirubinemia in mutant rats caused by defective hepatic
anion transport. Hepatology 1985;5:573–579
Huber M, Guhlmann A, Jansen PLM, et al. Hereditary defect of
hepatobiliary cysteinyl leukotriene elimination in mutant rats
with defective hepatic anion excretion. Hepatology 1987;7:
224–228
Ito K, Suzuki H, Hirohashi T, et al. Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR.
Am J Physiol 1997;272:G16–G22.
Guhlmann A, Krauss K, Oberdorfer F, et al. Noninvasive assessment of hepatobiliary and renal elimination of cysteinyl leukotrienes by positron emission tomography. Hepatology 1995;21:
1568–1575
Cui Y, König J, Buchholz U, et al. Drug resistance and ATPdependent conjugate transport mediated by the apical multidrug
resistance protein, MRP2, permanently expressed in human and
canine cells. Mol Pharmacol 1999;55:929–937
Keppler D, Kartenbeck J. The canalicular conjugate export
pump encoded by the cmrp/cmoat gene. In: Boyer JL, Ockner
RK (eds). Progress in liver diseases. Vol. 14. Philadelphia: W.B.
Saunders, 1996:55–67
König J, Rost D, Cui Y, et al. Characterization of the human
multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 1999;29:1156–1163
Kool M, van der Linden M, de Haas M, et al. MRP3, an organic
anion transporter able to transport anti-cancer drugs. Proc Natl
Acad Sci USA 1999;96:6914–6919
Loo TW, Clarke DM. Merck Frosst Award Lecture 1998. Molecular dissection of the human multidrug resistance Pglycoprotein.
Biochem Cell Biol 1999;77:11–23
Ambudkar SV, Dey S, Hrycyna CA, et al. Biochemical, cellular,
and pharmacological aspects of the multidrug transporter. Annu
Rev Pharmacol Toxicol 1999;39:361–398.
Tusnady, GE, Bakos E, Varadi A, et al. Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters. FEBS Lett 1997;402:1–3
Bakos E, Hegedus T, Hollo Z, et al. Membrane topology and
glycosylation of the human multidrug resistance-associated protein. J Biol Chem 1996;271:12322–12326.
Hipfner DR, Almquist KC, Stride BD, et al. Location of a
protease-hypersensitive region in the multidrug resistance protein (MRP) by mapping of the epitope of MRP-specific monoclonal antibody QCRL-1. Cancer Res 1996;56:3307–3314
Kast C, Gros P. Epitope insertion favors a six transmembrane
domain model for the carboxy-terminal portion of the multidrug
resistance-associated protein. Biochemistry 1998;37:2305–
2313
Kast C, Gros P. Topology mapping of the amino-terminal half of
multidrug resistance-associated protein by epitope insertion and
immunofluorescence. J Biol Chem 1997;272:26479–26487
Hipfner DR, Almquist KC, Leslie EM, et al. Membrane topology of the multidrug resistance protein (MRP). A study of
glycosylation-site mutants reveals an extracytosolic NH2 terminus. J Biol Chem 1997;272:23623–23630
König J, Nies AT, Cui Y, et al. Conjugate export pumps of the
multidrug resistance protein (MRP) family: Localization, substrate specificity, and MRP2-mediated drug resistance. Biochim
Biophys Acta 1999;1461:377–394
Cole SP, Bhardwaj G, Gerlach JH, et al. Overexpression of a
transporter gene in a multidrug-resistant human lung cancer cell
line. Science 1992;258:1650–1654
Grant CE, Kurz EU, Cole SP, et al. Analysis of the intron-exon
organization of the human multidrug-resistance protein gene
(MRP) and alternative splicing of its mRNA. Genomics 1997;
45:368–378
Taniguchi K, Wada M, Kohno K, et al. A human canalicular multispecific organic anion transporter (cMOAT) gene is overex-
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
pressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation. Cancer Res 1996;56:4124–4129
Paulusma CC, Kool M, Bosma PJ, et al. A mutation in the human
canalicular multispecific organic anion transporter gene causes
the Dubin-Johnson syndrome. Hepatology 1997;25:1539–1542
Keppler D, König J. Hepatic canalicular membrane 5: Expression and localization of the conjugate export pump encoded by
the MRP2 (cMRP/cMOAT) gene in liver. FASEB J 1997;11:
509–516
Evers R, Kool M, van Deemter L, et al. Drug export activity of
the human canalicular multispecific organic anion transporter in
polarized kidney MDCK cells expressing cMOAT (MRP2)
cDNA. J Clin Invest 1998;101:1310–1319
Tsujii H, König J, Rost D, et al. Exon-intron organization of the
human multidrug-resistance protein 2 (MRP2) gene mutated in
Dubin-Johnson syndrome. Gastroenterology 1999;117:653–660
Toh S, Wada M, Uchiumi T, et al. Genomic structure of the
canalicular multispecific organic anion-transporter gene
(MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome. Am J Hum Genet 1999;
64:739–746
Allikmets R, Gerrard B, Hutchinson A, et al. Characterization of
the human ABC superfamily: Isolation and mapping of 21 new
genes using the expressed sequence tags database. Hum Mol
Genet 1996;5:1649–1655
Kool M, de Haas M, Scheffer GL, et al. Analysis of expression
of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of
the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res 1997;57:3537–3547
Kiuchi Y, Suzuki H, Hirohashi T, et al. cDNA cloning and inducible expression of human multidrug resistance associated
protein 3 (MRP3). FEBS Lett 1998;433:149–152
Hirohashi T, Suzuki H, Takikawa H, et al. ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3
(Mrp3). J Biol Chem 2000;275:2905–2910
Ogawa K, Suzuki H, Hirohashi T, et al. Characterization of inducible nature of MRP3 in rat liver. Am J Physiol Gastrointest
Liver Physiol 2000;278:G438-G446
Kamisako T, Leier I, Cui Y, et al. Transport of monoglucuronosyl and bisglucuronosyl bilirubin by recombinant human and rat
multidrug resistance protein 2. Hepatology 1999;30:485–490
Keppler D, König J, Büchler M. The canalicular multidrug resistance protein, cMRP/MRP2, a novel conjugate export pump expressed in the apical membrane of hepatocytes. Adv Enzyme
Regul 1997;37:321–333
van Aubel RA, van Kuijck MA, Koenderink JB, et al. Adenosine
triphosphate-dependent transport of anionic conjugates by the
rabbit multidrug resistance-associated protein Mrp2 expressed
in insect cells. Mol Pharmacol 1998;53:1062–1067.
Schaub TP, Kartenbeck J, König J, et al. Expression of the conjugate export pump encoded by the mrp2 gene in the apical
membrane of kidney proximal tubules. J Am Soc Nephrol
1997;8:1213–1221
Schaub TP, Kartenbeck J, König J, et al. Expression of the MRP2
gene-encoded conjugate export pump in human kidney proximal
tubules and in renal cell carcinoma. J Am Soc Nephrol 1999;10:
1159–1169
Nies AT, Cantz T, Broim M, et al. Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line,
WIF-B. Hepatology 1998;28:1332–1340
Walle UK, Galijatovic A, Walle T. Transport of the flavonoid
chrysin and its conjugated metabolites by the human intestinal
cell line Caco-2. Biochem Pharmacol 1999;58:431–438
Bock KW, Eckle T, Ouzzine M, et al. Coordinate induction by
antioxidants of UDP-glucuronosyltransferase UGT1A6 and the
apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells. Biochem Pharmacol 2000;59:467–470
272
52. Cantz T, Nies AT, Brom M, et al. MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles
of Hep G2 cells. Am J Physiol Gastrointest Liver Physiol
2000;278:G522-G531
53. Kubitz R, D’Urso D, Keppler D, et al. Osmodependent dynamic
localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane. Gastroenterology 1997;113:
1438–1442
54. Roelofsen H, Soroka CJ, Keppler D, et al. Cyclic AMP stimulates sorting of the canalicular organic anion transporter
(Mrp2/cMoat) to the apical domain in hepatocyte couplets. J
Cell Sci 1998;111:1137–1145
55. Trauner M, Arrese M, Soroka CJ, et al. The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic
and obstructive cholestasis. Gastroenterology 1997;113:
255–264
56. Vos TA, Hooiveld GJ, Koning H, et al. Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation
of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver. Hepatology 1998;28:
1637–1644
57. Kubitz R, Wettstein M, Warskulat U, et al. Regulation of the
multidrug resistance protein 2 in the rat liver by lipopolysaccharide and dexamethasone. Gastroenterology 1999;116:401–410
58. Hagmann W, Denzlinger C, Keppler D. Role of peptide leukotrienes and their hepatobiliary elimination in endotoxin action.
Circ Shock 1984;14:223–235
59. Keppler D, Hagmann W, Rapp S, et al. The relation of leukotrienes to liver injury. Hepatology 1985;5:883–891
60. Takikawa H, Sano N, Narita T, et al. Biliary excretion of bile
acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley
rat. Hepatology 1991;14:352–360
61. Suzuki H, Sugiyama Y. Transporters for bile acids and organic
anions. In: Amidon GL, Sadée W (eds). Membrane transporters
as drug targets. New York: Kluwer Academic/Plenum, 1999:
387–439
62. Leier I, Hummel-Eisenbeiss J, Cui Y, et al. ATP-dependent paraaminohippurate transport by apical multidrug resistance protein
MRP2. Kidney Int 2000;57:1636–1642
63. Norman BH. Inhibitors of MRP1-mediated multidrug resistance.
Drugs Future 1998;23:1001–1013
64. Evers R, Zaman GJ, van Deemter L, et al. Basolateral localization and export activity of the human multidrug resistanceassociated protein in polarized pig kidney cells. J Clin Invest
1996;97:1211–1218
65. Borst P, Evers R, Kool M, et al. The multidrug resistance protein
family. Biochim Biophys Acta 1999;1461:347–357
66. Kool M, van der Linden M, de Haas, et al. Expression of human
MRP6, a homologue of the multidrug resistance protein gene
MRP1, in tissues and cancer cells. Cancer Res 1999;59:175–182
67. Madon J, Hagenbuch B, Landmann L, et al. Transport function
and hepatocellular localization of mrp6 in rat liver. Mol Pharmacol 2000;57:634–641
SEMINARS IN LIVER DISEASE—VOL. 20, NO. 3, 2000
68. Stöckel B, König, J, Nies AT, et al. Characterization of the 5’flanking region of the human multidrug resistance protein 2
(MRP2) gene and its regulation in comparison with the multidrug resistance protein 3 (MRP3) gene. Eur J Biochem 2000;
267:1347–1358
69. Hirohashi T, Suzuki H Chu XY, et al. Function and expression of
multidrug resistance-associated protein family in human colon
adenocarcinoma cells (Caco-2). J Pharmacol Exp Ther 2000;
292:265–270
70. Wada M, Toh S, Taniguchi K, et al. Mutations in the canalicular
multispecific organic anion transporter (cMOAT) gene, a novel
ABC transporter, in patients with hyperbilirubinemia II/DubinJohnson syndrome. Hum Mol Genet 1998;7:203–207
71. Dubin IN, Johnson FB. Chronic idiopathic jaundice with unidentified pigment in liver cells: A new clinicopathologic entity with
report of 12 cases. Medicine 1954;33:155–179
72. Sprinz H, Nelson RS. Persistent nonhemolytic hyperbilirubinemia associated with lipochrome-like pigment in liver cells: Report of four cases. Ann Intern Med 1954;41:952–962
73. Roy Chowdhury J, Roy Chowdhury N, Wolkoff AW, et al. Heme
and bile pigment metabolism. In: Arias IM, Boyer JL, Fausto N,
et al. (eds). The liver: Biology and pathobiology. New York:
Raven Press, 1994:471–504
74. Kartenbeck J, Leuschner U, Mayer R, et al. Absence of the
canalicular isoform of the MRP gene-encoded conjugate export
pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology 1996;23:1061–1066
75. Kajihara S, Hisatomi A, Mizuta T, et al. A splice mutation in the
human canalicular multispecific organic anion transporter gene
causes Dubin-Johnson syndrome. Biochem Biophys Res Commun 1998;253:454–457
76. Mor-Cohen R, Zivelin A, Rosenberg N, et al. Identification of a
common ile1173phe mutation in the canalicular multispecific
organic anion transporter gene in patients with Dubin-Johnson
syndrome of Iranian-Jewish origin. Am J Human Genetics 1999;
65(Suppl):581
77. Thermann R, Neu-Yilik G, Deters A, et al. Binary specification
of nonsense codons by splicing and cytoplasmic translation.
EMBO J 1998;17:3484–3494
78. Keppler D, Kamisako T, Leier I, et al. Localization, substrate
specificity, and drug resistance conferred by conjugate export
pumps of the MRP family. Adv Enzyme Regul 2000;40:339–349
79. Hirohashi T, Suzuki H, Sugiyama Y. Characterization of the
transport properties of cloned rat multidrug resistanceassociated protein 3 (MRP3). J Biol Chem 1999;274:
15181–15185
80. Hirohashi T, Suzuki H, Ito K, et al. Hepatic expression of multidrug resistance-associated protein-like proteins maintained in
Eisai hyperbilirubinemic rats. Mol Pharmacol 1998;53:
1068–1075
81. Takada T, Suzuki H, Sugiyama Y. Characterization of 5-flanking region of human MRP3. Biochem Biophys Res Commun
2000;270:728–732