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Good news on the conjugate export pump cMRP in human liver and the absence of cMRP or cMoat in canalicular membranes of TR- TR-rat hepatocytes
HEPATOLOGY Elsewhere T. JAKE LIANG, EDITOR
ADVISORY COMMITTEE BRUCE R. BACON, St. Louis, MO HENRY C. BODENHEIMER, New York, NY JAMES M. CRAWFORD, Boston, MA NORMAN D. GRACE, Boston, MA SANJEEV GUPTA, Bronx, NY JOEL LAVINE, San Diego, CA RICHARD H. MOSELEY, Ann Arbor, MI
Gastrointestinal Unit GRJ-724 Massachusetts General Hospital 32 Fruit St Boston, MA 02114
GOOD NEWS ON THE CONJUGATE EXPORT PUMP cMRP IN HUMAN LIVER AND THE ABSENCE OF cMrp OR cMoat IN CANALICULAR MEMBRANES OF TR0 TRRAT HEPATOCYTES
ABSTRACT
Mayer R, Kartenbeck J, Bu¨chler M, Jedlitschky G, Leier I, Keppler D. 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. ABSTRACT
We have previously shown that the multidrug-resistance protein (MRP) mediates the adenosine triphosphate (ATP)-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. We demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse-transcription polymerase chain reaction (RT-PCR) of human and normal rat liver messenger RNA resulted in two expected complementary DNA (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 homologue 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 TR0 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 TR0 mutant rats localized Mrp only to the lateral membrane 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. Paulusma CC, Bosma PJ, Zaman GJR, Bakker CTM, Otter M, Scheffer GL, Scheper RJ, et al. Congenital jaundice in rats with a mutation in a multidrug resistance–associated protein gene. Science 1996;271:1126-1128.
The human Dubin-Johnson syndrome and its animal model, the TR0 rat, are characterized by a chronic conjugated hyperbilirubinemia. TR0 rats are defective in the canalicular multispecific organic anion transporter (cMOAT), which mediates hepatobiliary excretion of numerous organic anions. The complementary DNA for rat cmoat, a homologue of the human multidrug-resistance gene (hMRP1), was isolated and shown to be expressed in the canalicular membrane of hepatocytes. In the TR0 rat, a single-nucleotide deletion in this gene resulted in a reduced messenger RNA level and absence of the protein. It is likely that this mutation accounts for the TR0 phenotype. COMMENTS
Excretion into bile is a major pathway for the elimination of endogenous and xenobiotic lipophilic compounds from the mammalian organism. Hepatocytes are the primary secretory units of bile formation. They are equipped with different carrier proteins of the basolateral and canalicular membrane.1 Some of these carrier proteins have a wide substrate specificity and transport endogenous and exogenous compounds. For the mamalian organism, toxic lipophilic compounds are metabolized by transferases into amphiphilic anionic conjugates with glucuronate, glutathione, or sulfate. Excretion of these conjugates across the canalicular membrane into bile is mediated by a primary-active ATP-dependent transport system, which has been characterized functionally and termed non–bile acid organic anion transporter,2 multispecific organic anion transporter cMOAT,3 glutathione S-conjugate export pump,4 or, leukotriene export pump.5 This last term refers to the endogenous glutathione S-conjugate leukotriene C4 (LTC4 ), which is the substrate with the highest affinity for this transporter.5-7 Neither the protein nor the gene encoding this ATP-dependent transporter in the canalicular membrane of human hepatocytes were previously identified. Recently, a new transport protein has been cloned and characterized, MRP, which mediates multidrug-resistance against chemotherapeutic drugs in tumor cells of the lung.8,9 Overexpression of this membrane glycophosphoprotein with a molecular mass ranging from 170 to 190 kd results in the resistance of human tumor cells to a number of cytotoxic drugs, including anthracycline, vinca alkaloids and epidophyilotoxins.10-12 MRP bears a limited amino acid similarity to MDR-1-p-glycoprotein (gp170), MDR-2-p-glycoprotein, and the cystic fibrosis transmembrane conductance regulator (CFTR). Although all are members of the superfamily of the ATP-binding cassette membrane transport proteins (ABC transporters), these transporters represent different functions in cell metabolism.8 In 1994, the function of MRP was identified in several human nonhepatic cell lines, which overexpress MRP.7,13 The glutathione conjugate LCT4 and other
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glutathione conjugates and structurally related anionic amphiphilic conjugates are high-affinity endogenous substrates of this ATP-binding transporter pump. Because substrate specifities of the conjugate export pump in rat hepatocytes and the human MRP gene–encoded export pump are very similar,7,13 Mayer et al. and Paulusma et al. concluded that the human MRP (hMRP) is possibly a candidate gene for the hepatic conjugate export pump or cMoat. High levels of MRP mRNA have been found in different tumor cells and in normal human tissue. Prior results have indicated only very small insignificant amounts of MRP mRNA in human liver.8,11 Mayer et al. have now established the expression of MRP by RT-PCR and sequencing in normal liver of humans and rats. In contrast to the preceding analyses, the authors used a high-sensitivity RT-PCR technique and avoided RNA degradation by immediate freezing of the human liver tissue before RNA extraction. Previous investigations also did not include immunodetection of human MRP in liver by four different polyclonal antibodies as well as immunofluorescence microscopy. Double immunofluorescence microscopy and confocal laser scanning microscopy confirmed that both human MRP and rat Mrp are present in canalicular as well as in the lateral membrane domains of hepatocytes. The localization of MRP in different hepatocyte membrane fractions of human and rat liver was further confirmed by comparison with specific proteins of the canalicular and lateral membranes, DPPIV and desmoplakin. DPPIV as a plasma membrane protein is highly concentrated in the canalicular structures, and desmoplakin as a component of the cytoplasmic structures of desmosomes in the lateral hepatocellular membrane.14,15 The functional characterization of the canalicular conjugate export pump and its excretion of organic anions into bile was further promoted by the finding that a group of mutant rats (TR0 or GY/TR0 rats) had a defect in the canalicular transport of amphiphilic substances.1 The process of excretion across the canalicular membrane has been recognized as an ATP-dependent primary-active transport, which is deficient in the liver of these TR0 rats, as well as in Eisai hyperbilirubinemic mutant rats.6,21 Furthermore, this functional defect in the TR0 rats seems to be very similar to the canalicular transport defect in patients with the inherited autosomalrecessive defect called Dubin-Johnson syndrome. It is clinically featured by chronic conjugated hyperbilirubinemia and a hyperpigmented grossly black liver. Therefore, the TR0 rat with its related phenotype is considered to be a suitable experimental model and has given impetus to the characterization of the canalicular organic anion transport system. As in patients with Dubin-Johnson syndrome, these rats have impaired biliary excretion of bilirubin glucuronides and many other organic anions.19 Because the substrate specifities of the conjugate export pump in rat hepatocytes and the human MRP gene–encoded transport pump are very similar,6,7,13,16,20 it has been suggested that an altered expression of rat Mrp or an isoform of Mrp in the canalicular membrane of the rat hepatocyte causes the hereditary defect in hepatobiliary excretion of anionic conjugates in these GY/TR0 rats. Very recently, Paulusma et al. have isolated the complementary DNA for the rat cMoat as a homologue of the human MRP gene product (hMRP1) expressed in the canalicular membrane of hepatocytes. After amplifying the rat homologue, sequence by PCR with nested degenerated primers based on the highly conserved sequence of the first ATPbinding cassette of hMRP1 in rat lung cDNA, this PCR fragment (rmrp1) hybridized on Northern blot with a single 9.5kilobase (kb) transcript in all tissues of TR0 rats with high expression in lung and testes but no detectable expression in liver. Analyzing two rat liver cDNA libraries using the rmrp1 as probe and on the basis of similarity searches, the full-
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length cDNA and the protein were isolated with a single open-reading frame of 1,541 amino acids. It is of interest that Mayer et al. cloned and sequenced a 347–base pair (bp) fragment termed seq1 from rat liver that was absent in the TR0 mutant rat and which is part of the full-length cDNA now described by Paulusma et al. The abundance of three different transcripts of this cDNA, ranging from approximately 6.5 to 9.5 kb, was strongly decreased in liver and other tissues of the TR0 rat, which suggests that these transcripts were related to the defect in the TR0 rat. Paulusma et al. further produced a monoclonal antibody to a bacterial fusion protein containing the 202-amino terminal end of the sequence and detected a protein of 200 kd in the canalicular plasma membrane fraction of the Wistar rat liver, in good agreement with the predicted molecular size of the cMoat protein. In the canalicular membrane fraction of TR0 rat liver, this 200-kd protein was completely absent, and after sequencing the obtained complete cDNA revealed a 1bp deletion at amino acid position 393, resulting in a frame shift and subsequent introduction of a stop codon at position 401. Thus, taken together with the functional studies, the exclusive canalicular localization of the protein of the cmoat gene in Wistar rats and the absence of the truncated, nonviable gene product from the canalicular membrane in TR0 rat livers imply that this 1-bp deleted protein is responsible for the impaired transport of organic compounds from liver to bile in the TR0 rats. It may also be concluded that this cMoat (canalicular) and not the differentially localized Mrp (lateral) is involved in the biliary organic anion transport. This is in concordance with Mayer et al. with the localization of Mrp in the lateral membrane of TR0 rats shown by double immunofluorescence microscopy of TR0 mutant rat liver. The decreased secretion rate of conjugates into the extracellular medium of isolated TR0 mutant hepatocytes has also been shown to occur predominantly across the basolateral membrane domain.3,18 It has been shown that the excretion of glutathione conjugates into the perfusion medium of isolated perfused TR0 rat liver is reduced to less than 20% of normal.18 Because the Mrp-encoded conjugate export pump at the lateral hepatocyte membrane could still be functionally active, its low specific activity could be responsible for this excretion rate. Thus, these two papers provide definitive molecular identification and localization of basolateral and canalicular proteins responsible for the transport of lipophilic organic anions from plasma to bile. Both results are important milestones and will promote further investigations in this exciting field of hepatology. At least two upcoming questions with direct clinical implications may be answered in the near future. First, is the genetic defect of the Dubin-Johnson Syndrome due to another homologue of this newly identified cmoat gene or an isoform of the human MRP yet to be identified? Second, might the overexpression of cMoat or cMRP also confer resistance of cancer cells against cytotoxic drugs or their metabolites in rat and human liver tumor tissue? MARKUS MO¨HLER, M.D. WOLFGANG STREMMEL, M.D. Department of Internal Medicine IV University of Heidelberg Bergheimer Straße 58 69115 Heidelberg, Germany REFERENCES 1. Oude Elferink RPJ, Meijer DKF, Kuipers F, Jansen PLM, Groen AK, Groothuis GMM. Hepatobiliary secetion of organic compounds; molecular mechanisms of membrane transport. Biochim Biophys Acta 1995;1241: 215-268.
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2. Arias IM, Che M, Gatmaitan Z, Leveille C, Nishida T, Pierre MST. The biology of the bile canaliculus. HEPATOLOGY 1993;17:318-329. 3. Oude Elferink RPJ, Jansen PLM. The role of the canalicular multispecific organic anion transporter in the disposal of endo- and xenobiotics. Pharmacol Ther 1994;64:77-97. 4. Ishikawa T. The ATP-dependent glutathione S-conjugate export pump. Trends Biochem Soc 1992;17:463-468. 5. Keppler D, Mu¨ller M, Klu¨nemann C, Guhlmann A, Krauss K, Mu¨ller U, Berger U, et al. Transport and in vivo elimination of cysteinyl leukotrienes. Adv Enzyme Regul 1992;32:107-116. 6. Ishikawa T, Mu¨ller M, Klu¨nemann C, Schaub T, Keppler D. ATP-dependent primary active transport of cystineyl leukotrienes across liver canalicular membrane. J Biol Chem 1990;365:19279-19286. 7. Leier I, Jedlitschky G, Buchholz U, Cole SPC, Deeley RG, Keppler D. The MRP-gene encodes an ATP-dependent export pump for Leukotriene C4 and structurally related conjugates. J Biol Chem 1994;269:27807-27810. 8. Cole SPC, Bhardwaj JH, Gerlach JE, Mackie CE, Grant CE, Wilson GM, Almquist KC, et al. Overexpression of a transporter gene in a multidrugresistant human lung cancer cell line. Science 1992;258:1650-1654. 9. Almquist K, Loe DW, Hipfner DR, Mackie JE, Cole SPC, Deeley RG. Characterization of the Mr 190,000 Multidrug Resistance Protein (MRP) in drug-selected and transfected human tumor cells. Cancer Res 1995;55: 102-110. 10. Krishnamachary N, Ma L, Zheng L, Safa AR, Center MS. Analysis of MRP gene expression and function in HL60 cells isolated for resistance to Adriamycin. Oncol Res 1994;6:119-127. 11. Zaman GJR, Versantvoort CHM, Smit JJM, Eijdems HM, de Haas M, Smith AJ, Broxterman HJ, et al. Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines. Cancer Res 1993;53:1747-1750. 12. Cole SPC, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM, Deeley RG. Pharmacological characterization of Multidrug Resistant MRPtransfected human tumor cells. Cancer Res 1994;54:5902-5910. 13. Jedlitschky G, Leier I, Buchholz U, Center M, Keppler D. ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein. Cancer Res 1994;54:4833-4836. 14. Kartenbeck J, Koch PJ, Franke WW. In: Kreis T, Vale R, eds. Guidebook to the extracellular matrix and adhesions proteins. Desmogllein. Oxford, England: Oxford University, 1993:133-135. 15. Hubbard AL, Barr VA, Scott LJ. Hepatocyte surface polarity. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter D, Shafritz DA, eds. The liver: biology and pathobiology. New York: Raven, 1994:189-213. 16. Akerboom TPM, Narayanaswami V, Kunst M, Sies H. The ATP-dependent S-(2-4-dinitrophenyl) glutathione transport in canalicular plasma membrane vesicles from rat liver. J Biol Chem 1991;266:13147-13152. 17. Leier I, Jedlitschky G, Buchholz U, Keppler D. Characterization of the ATP-dependent Leukotriene C4 export carrier in mastocytoma cells. Eur J Biochem 1994;220:599-606. 18. Oude Elferink RPJ, Ottonhoff R, Liefting W, de Haan J, Jansen PLM. Hepatobiliary transport of glutathione and glutathione conjugates in rats with hereditary hyperbilirubinimia. J Clin Invest 1989;84:476-483. 19. Jansen PLM, Peters HM, Lamers WH. Hereditary chronic conjugated hyperbilirubinimia in mutant rats caused by defective hepatic anion transport. HEPATOLOGY 1985;5:573-579. 20. Jedlitschky G, Leier I, Buchholz U, Barnouin K, Kurz G, Keppler D. Transport of glutathione, glucuronate, and sulfate conjugates by the MRP geneencoded conjugate export pump. Cancer Res 1996;56:988-994. 21. Fernandez-Checa JC, Takikawa H, Horie T, Ookhtens M, Kaplowitz N. Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. J Biol Chem 267:1667-1673.
THE SOURCE OF THE BILE: THE LEGACY ¨ KEL OF MATHIAS MO
Crawford JM, Mo¨ckel G-M, Crawford AR, Hagen SJ, Hatch VC, Barnes S, Godleski JJ, Carey MC. Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane. J Lipid Res 1995;36:2147-2163. ABSTRACT
Physical-chemical and biological studies of hepatic bile suggest that biliary phospholipid molecules are secreted as unilamellar vesicles. Systematic ultrastructural studies of bile canaliculi were undertaken to visualize this event. Liver tissue was obtained from normal adult male rats (control), from bile salt–depleted rats (by overnight biliary diversion), and from depleted rats infused intravenously with a hydrophilic-hydrophobic congener series of common taurine-conjugated bile
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salts. Livers were fixed in situ either by modified chemical methods or by ultrarapid cryofixation. In control rats, chemical fixation revealed unilamellar vesicles 63 { 17 ({SD) nm in diameter, mostly free within canalicular lumena. Vesicles were infrequent in canaliculi of bile salt-depleted rats, but were present in canaliculi of rats infused with taurocholate. In cryofixed liver tissue, vesicles 67 { 13 nm in diameter were observed in canaliculi of control rats and bile salt–depleted rats infused with common bile salts. The majority of these vesicles were affixed to the luminal side of the canalicular membrane. The average number of vesicles per bile canaliculus was in agreement with that estimated on the basis of biliary phospholipid secretion rates, mean vesicle size, and area of close-packed phosphatidylcholine molecules. By immunoelectron microscopy, canalicular vesicles were free of actin and of a 100-kd canalicular membrane protein. It is concluded that biliary phospholipid molecules are secreted from hepatocytes into bile canalicular lumena as unilamellar vesicles Ç63 to 67 nm in average diameter. We postulate that this secretion mechanism involves lumenal bile salt–induced vesiculation of lipid microdomains in the exoplasmic hemileaflet of the canalicular membrane. Mo¨ckel G-M, Gorti S, Tandon RK, Tanaka T, Carey MC. Microscope laser light-scattering spectroscopy of vesicles within canaliculi of rat hepatocyte couplets. Am J Physiol 1995;269:G73-G84. ABSTRACT
Using microscope laser light-scattering spectroscopy, the authors investigated ‘‘primary’’ bile secretion into canalicular spaces of rat hepatocyte couplets in monolayer culture. Time-dependent scattered light intensities were fitted by bi-exponential decays. The ‘‘slow’’ decay was attributed to an undulating canalicular membrane motion, whereas the ‘‘fast’’ decay was consistent with rapidly diffusing intracanalicular vesicles with mean ˚ . After addition hydrodynamic radii ({SD) of 479 { 53 A of micromolar concentrations of common bile salts, increases in the amplitude of the fast component facilitated a quantitative estimate of vesicle secretion rates. A dose-response relationship with 0.1 to 200 mmol/L sodium taurocholate was characterized by an initial concentration-dependent increase and then a decrease in the amplitude of the fast canalicular component. Because these taurocholate concentrations are nontoxic to cultured hepatocytes, the maximum in vesicle-sized particles at 10 mmol/L taurocholate suggested that its critical micellar concentration of Ç5 mmol/L was attained within the canalicular spaces. Sodium taurolithocholate resulted in time- and dose-dependent diminution in vesicle secretion rates, which after 2 hours was followed by spontaneous canalicular recovery. This suggested that acute bicellular ‘‘cholestasis’’ was followed by oxidative metabolism and detoxification of the monohydroxy bile salt. Microscope laser light-scattering spectroscopy should facilitate further physical-chemical and pathophysiological studies of bile secretion at the cellular level. COMMENTS
The process by which phosphatidylcholine and cholesterol are secreted into bile is one of the great mysteries of hepatology. Biliary secretion of cholesterol accounts for over half of cholesterol elimination in normal humans, and secretion of
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ADVISORY COMMITTEE BRUCE R. BACON, St. Louis, MO HENRY C. BODENHEIMER, New York, NY JAMES M. CRAWFORD, Boston, MA NORMAN D. GRACE, Boston, MA SANJEEV GUPTA, Bronx, NY JOEL LAVINE, San Diego, CA RICHARD H. MOSELEY, Ann Arbor, MI
Gastrointestinal Unit GRJ-724 Massachusetts General Hospital 32 Fruit St Boston, MA 02114
GOOD NEWS ON THE CONJUGATE EXPORT PUMP cMRP IN HUMAN LIVER AND THE ABSENCE OF cMrp OR cMoat IN CANALICULAR MEMBRANES OF TR0 TRRAT HEPATOCYTES
ABSTRACT
Mayer R, Kartenbeck J, Bu¨chler M, Jedlitschky G, Leier I, Keppler D. 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. ABSTRACT
We have previously shown that the multidrug-resistance protein (MRP) mediates the adenosine triphosphate (ATP)-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. We demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse-transcription polymerase chain reaction (RT-PCR) of human and normal rat liver messenger RNA resulted in two expected complementary DNA (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 homologue 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 TR0 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 TR0 mutant rats localized Mrp only to the lateral membrane 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. Paulusma CC, Bosma PJ, Zaman GJR, Bakker CTM, Otter M, Scheffer GL, Scheper RJ, et al. Congenital jaundice in rats with a mutation in a multidrug resistance–associated protein gene. Science 1996;271:1126-1128.
The human Dubin-Johnson syndrome and its animal model, the TR0 rat, are characterized by a chronic conjugated hyperbilirubinemia. TR0 rats are defective in the canalicular multispecific organic anion transporter (cMOAT), which mediates hepatobiliary excretion of numerous organic anions. The complementary DNA for rat cmoat, a homologue of the human multidrug-resistance gene (hMRP1), was isolated and shown to be expressed in the canalicular membrane of hepatocytes. In the TR0 rat, a single-nucleotide deletion in this gene resulted in a reduced messenger RNA level and absence of the protein. It is likely that this mutation accounts for the TR0 phenotype. COMMENTS
Excretion into bile is a major pathway for the elimination of endogenous and xenobiotic lipophilic compounds from the mammalian organism. Hepatocytes are the primary secretory units of bile formation. They are equipped with different carrier proteins of the basolateral and canalicular membrane.1 Some of these carrier proteins have a wide substrate specificity and transport endogenous and exogenous compounds. For the mamalian organism, toxic lipophilic compounds are metabolized by transferases into amphiphilic anionic conjugates with glucuronate, glutathione, or sulfate. Excretion of these conjugates across the canalicular membrane into bile is mediated by a primary-active ATP-dependent transport system, which has been characterized functionally and termed non–bile acid organic anion transporter,2 multispecific organic anion transporter cMOAT,3 glutathione S-conjugate export pump,4 or, leukotriene export pump.5 This last term refers to the endogenous glutathione S-conjugate leukotriene C4 (LTC4 ), which is the substrate with the highest affinity for this transporter.5-7 Neither the protein nor the gene encoding this ATP-dependent transporter in the canalicular membrane of human hepatocytes were previously identified. Recently, a new transport protein has been cloned and characterized, MRP, which mediates multidrug-resistance against chemotherapeutic drugs in tumor cells of the lung.8,9 Overexpression of this membrane glycophosphoprotein with a molecular mass ranging from 170 to 190 kd results in the resistance of human tumor cells to a number of cytotoxic drugs, including anthracycline, vinca alkaloids and epidophyilotoxins.10-12 MRP bears a limited amino acid similarity to MDR-1-p-glycoprotein (gp170), MDR-2-p-glycoprotein, and the cystic fibrosis transmembrane conductance regulator (CFTR). Although all are members of the superfamily of the ATP-binding cassette membrane transport proteins (ABC transporters), these transporters represent different functions in cell metabolism.8 In 1994, the function of MRP was identified in several human nonhepatic cell lines, which overexpress MRP.7,13 The glutathione conjugate LCT4 and other
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glutathione conjugates and structurally related anionic amphiphilic conjugates are high-affinity endogenous substrates of this ATP-binding transporter pump. Because substrate specifities of the conjugate export pump in rat hepatocytes and the human MRP gene–encoded export pump are very similar,7,13 Mayer et al. and Paulusma et al. concluded that the human MRP (hMRP) is possibly a candidate gene for the hepatic conjugate export pump or cMoat. High levels of MRP mRNA have been found in different tumor cells and in normal human tissue. Prior results have indicated only very small insignificant amounts of MRP mRNA in human liver.8,11 Mayer et al. have now established the expression of MRP by RT-PCR and sequencing in normal liver of humans and rats. In contrast to the preceding analyses, the authors used a high-sensitivity RT-PCR technique and avoided RNA degradation by immediate freezing of the human liver tissue before RNA extraction. Previous investigations also did not include immunodetection of human MRP in liver by four different polyclonal antibodies as well as immunofluorescence microscopy. Double immunofluorescence microscopy and confocal laser scanning microscopy confirmed that both human MRP and rat Mrp are present in canalicular as well as in the lateral membrane domains of hepatocytes. The localization of MRP in different hepatocyte membrane fractions of human and rat liver was further confirmed by comparison with specific proteins of the canalicular and lateral membranes, DPPIV and desmoplakin. DPPIV as a plasma membrane protein is highly concentrated in the canalicular structures, and desmoplakin as a component of the cytoplasmic structures of desmosomes in the lateral hepatocellular membrane.14,15 The functional characterization of the canalicular conjugate export pump and its excretion of organic anions into bile was further promoted by the finding that a group of mutant rats (TR0 or GY/TR0 rats) had a defect in the canalicular transport of amphiphilic substances.1 The process of excretion across the canalicular membrane has been recognized as an ATP-dependent primary-active transport, which is deficient in the liver of these TR0 rats, as well as in Eisai hyperbilirubinemic mutant rats.6,21 Furthermore, this functional defect in the TR0 rats seems to be very similar to the canalicular transport defect in patients with the inherited autosomalrecessive defect called Dubin-Johnson syndrome. It is clinically featured by chronic conjugated hyperbilirubinemia and a hyperpigmented grossly black liver. Therefore, the TR0 rat with its related phenotype is considered to be a suitable experimental model and has given impetus to the characterization of the canalicular organic anion transport system. As in patients with Dubin-Johnson syndrome, these rats have impaired biliary excretion of bilirubin glucuronides and many other organic anions.19 Because the substrate specifities of the conjugate export pump in rat hepatocytes and the human MRP gene–encoded transport pump are very similar,6,7,13,16,20 it has been suggested that an altered expression of rat Mrp or an isoform of Mrp in the canalicular membrane of the rat hepatocyte causes the hereditary defect in hepatobiliary excretion of anionic conjugates in these GY/TR0 rats. Very recently, Paulusma et al. have isolated the complementary DNA for the rat cMoat as a homologue of the human MRP gene product (hMRP1) expressed in the canalicular membrane of hepatocytes. After amplifying the rat homologue, sequence by PCR with nested degenerated primers based on the highly conserved sequence of the first ATPbinding cassette of hMRP1 in rat lung cDNA, this PCR fragment (rmrp1) hybridized on Northern blot with a single 9.5kilobase (kb) transcript in all tissues of TR0 rats with high expression in lung and testes but no detectable expression in liver. Analyzing two rat liver cDNA libraries using the rmrp1 as probe and on the basis of similarity searches, the full-
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length cDNA and the protein were isolated with a single open-reading frame of 1,541 amino acids. It is of interest that Mayer et al. cloned and sequenced a 347–base pair (bp) fragment termed seq1 from rat liver that was absent in the TR0 mutant rat and which is part of the full-length cDNA now described by Paulusma et al. The abundance of three different transcripts of this cDNA, ranging from approximately 6.5 to 9.5 kb, was strongly decreased in liver and other tissues of the TR0 rat, which suggests that these transcripts were related to the defect in the TR0 rat. Paulusma et al. further produced a monoclonal antibody to a bacterial fusion protein containing the 202-amino terminal end of the sequence and detected a protein of 200 kd in the canalicular plasma membrane fraction of the Wistar rat liver, in good agreement with the predicted molecular size of the cMoat protein. In the canalicular membrane fraction of TR0 rat liver, this 200-kd protein was completely absent, and after sequencing the obtained complete cDNA revealed a 1bp deletion at amino acid position 393, resulting in a frame shift and subsequent introduction of a stop codon at position 401. Thus, taken together with the functional studies, the exclusive canalicular localization of the protein of the cmoat gene in Wistar rats and the absence of the truncated, nonviable gene product from the canalicular membrane in TR0 rat livers imply that this 1-bp deleted protein is responsible for the impaired transport of organic compounds from liver to bile in the TR0 rats. It may also be concluded that this cMoat (canalicular) and not the differentially localized Mrp (lateral) is involved in the biliary organic anion transport. This is in concordance with Mayer et al. with the localization of Mrp in the lateral membrane of TR0 rats shown by double immunofluorescence microscopy of TR0 mutant rat liver. The decreased secretion rate of conjugates into the extracellular medium of isolated TR0 mutant hepatocytes has also been shown to occur predominantly across the basolateral membrane domain.3,18 It has been shown that the excretion of glutathione conjugates into the perfusion medium of isolated perfused TR0 rat liver is reduced to less than 20% of normal.18 Because the Mrp-encoded conjugate export pump at the lateral hepatocyte membrane could still be functionally active, its low specific activity could be responsible for this excretion rate. Thus, these two papers provide definitive molecular identification and localization of basolateral and canalicular proteins responsible for the transport of lipophilic organic anions from plasma to bile. Both results are important milestones and will promote further investigations in this exciting field of hepatology. At least two upcoming questions with direct clinical implications may be answered in the near future. First, is the genetic defect of the Dubin-Johnson Syndrome due to another homologue of this newly identified cmoat gene or an isoform of the human MRP yet to be identified? Second, might the overexpression of cMoat or cMRP also confer resistance of cancer cells against cytotoxic drugs or their metabolites in rat and human liver tumor tissue? MARKUS MO¨HLER, M.D. WOLFGANG STREMMEL, M.D. Department of Internal Medicine IV University of Heidelberg Bergheimer Straße 58 69115 Heidelberg, Germany REFERENCES 1. Oude Elferink RPJ, Meijer DKF, Kuipers F, Jansen PLM, Groen AK, Groothuis GMM. Hepatobiliary secetion of organic compounds; molecular mechanisms of membrane transport. Biochim Biophys Acta 1995;1241: 215-268.
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HEPATOLOGY Elsewhere
HEPATOLOGY July 1996
2. Arias IM, Che M, Gatmaitan Z, Leveille C, Nishida T, Pierre MST. The biology of the bile canaliculus. HEPATOLOGY 1993;17:318-329. 3. Oude Elferink RPJ, Jansen PLM. The role of the canalicular multispecific organic anion transporter in the disposal of endo- and xenobiotics. Pharmacol Ther 1994;64:77-97. 4. Ishikawa T. The ATP-dependent glutathione S-conjugate export pump. Trends Biochem Soc 1992;17:463-468. 5. Keppler D, Mu¨ller M, Klu¨nemann C, Guhlmann A, Krauss K, Mu¨ller U, Berger U, et al. Transport and in vivo elimination of cysteinyl leukotrienes. Adv Enzyme Regul 1992;32:107-116. 6. Ishikawa T, Mu¨ller M, Klu¨nemann C, Schaub T, Keppler D. ATP-dependent primary active transport of cystineyl leukotrienes across liver canalicular membrane. J Biol Chem 1990;365:19279-19286. 7. Leier I, Jedlitschky G, Buchholz U, Cole SPC, Deeley RG, Keppler D. The MRP-gene encodes an ATP-dependent export pump for Leukotriene C4 and structurally related conjugates. J Biol Chem 1994;269:27807-27810. 8. Cole SPC, Bhardwaj JH, Gerlach JE, Mackie CE, Grant CE, Wilson GM, Almquist KC, et al. Overexpression of a transporter gene in a multidrugresistant human lung cancer cell line. Science 1992;258:1650-1654. 9. Almquist K, Loe DW, Hipfner DR, Mackie JE, Cole SPC, Deeley RG. Characterization of the Mr 190,000 Multidrug Resistance Protein (MRP) in drug-selected and transfected human tumor cells. Cancer Res 1995;55: 102-110. 10. Krishnamachary N, Ma L, Zheng L, Safa AR, Center MS. Analysis of MRP gene expression and function in HL60 cells isolated for resistance to Adriamycin. Oncol Res 1994;6:119-127. 11. Zaman GJR, Versantvoort CHM, Smit JJM, Eijdems HM, de Haas M, Smith AJ, Broxterman HJ, et al. Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines. Cancer Res 1993;53:1747-1750. 12. Cole SPC, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM, Deeley RG. Pharmacological characterization of Multidrug Resistant MRPtransfected human tumor cells. Cancer Res 1994;54:5902-5910. 13. Jedlitschky G, Leier I, Buchholz U, Center M, Keppler D. ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein. Cancer Res 1994;54:4833-4836. 14. Kartenbeck J, Koch PJ, Franke WW. In: Kreis T, Vale R, eds. Guidebook to the extracellular matrix and adhesions proteins. Desmogllein. Oxford, England: Oxford University, 1993:133-135. 15. Hubbard AL, Barr VA, Scott LJ. Hepatocyte surface polarity. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter D, Shafritz DA, eds. The liver: biology and pathobiology. New York: Raven, 1994:189-213. 16. Akerboom TPM, Narayanaswami V, Kunst M, Sies H. The ATP-dependent S-(2-4-dinitrophenyl) glutathione transport in canalicular plasma membrane vesicles from rat liver. J Biol Chem 1991;266:13147-13152. 17. Leier I, Jedlitschky G, Buchholz U, Keppler D. Characterization of the ATP-dependent Leukotriene C4 export carrier in mastocytoma cells. Eur J Biochem 1994;220:599-606. 18. Oude Elferink RPJ, Ottonhoff R, Liefting W, de Haan J, Jansen PLM. Hepatobiliary transport of glutathione and glutathione conjugates in rats with hereditary hyperbilirubinimia. J Clin Invest 1989;84:476-483. 19. Jansen PLM, Peters HM, Lamers WH. Hereditary chronic conjugated hyperbilirubinimia in mutant rats caused by defective hepatic anion transport. HEPATOLOGY 1985;5:573-579. 20. Jedlitschky G, Leier I, Buchholz U, Barnouin K, Kurz G, Keppler D. Transport of glutathione, glucuronate, and sulfate conjugates by the MRP geneencoded conjugate export pump. Cancer Res 1996;56:988-994. 21. Fernandez-Checa JC, Takikawa H, Horie T, Ookhtens M, Kaplowitz N. Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats. J Biol Chem 267:1667-1673.
THE SOURCE OF THE BILE: THE LEGACY ¨ KEL OF MATHIAS MO
Crawford JM, Mo¨ckel G-M, Crawford AR, Hagen SJ, Hatch VC, Barnes S, Godleski JJ, Carey MC. Imaging biliary lipid secretion in the rat: ultrastructural evidence for vesiculation of the hepatocyte canalicular membrane. J Lipid Res 1995;36:2147-2163. ABSTRACT
Physical-chemical and biological studies of hepatic bile suggest that biliary phospholipid molecules are secreted as unilamellar vesicles. Systematic ultrastructural studies of bile canaliculi were undertaken to visualize this event. Liver tissue was obtained from normal adult male rats (control), from bile salt–depleted rats (by overnight biliary diversion), and from depleted rats infused intravenously with a hydrophilic-hydrophobic congener series of common taurine-conjugated bile
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salts. Livers were fixed in situ either by modified chemical methods or by ultrarapid cryofixation. In control rats, chemical fixation revealed unilamellar vesicles 63 { 17 ({SD) nm in diameter, mostly free within canalicular lumena. Vesicles were infrequent in canaliculi of bile salt-depleted rats, but were present in canaliculi of rats infused with taurocholate. In cryofixed liver tissue, vesicles 67 { 13 nm in diameter were observed in canaliculi of control rats and bile salt–depleted rats infused with common bile salts. The majority of these vesicles were affixed to the luminal side of the canalicular membrane. The average number of vesicles per bile canaliculus was in agreement with that estimated on the basis of biliary phospholipid secretion rates, mean vesicle size, and area of close-packed phosphatidylcholine molecules. By immunoelectron microscopy, canalicular vesicles were free of actin and of a 100-kd canalicular membrane protein. It is concluded that biliary phospholipid molecules are secreted from hepatocytes into bile canalicular lumena as unilamellar vesicles Ç63 to 67 nm in average diameter. We postulate that this secretion mechanism involves lumenal bile salt–induced vesiculation of lipid microdomains in the exoplasmic hemileaflet of the canalicular membrane. Mo¨ckel G-M, Gorti S, Tandon RK, Tanaka T, Carey MC. Microscope laser light-scattering spectroscopy of vesicles within canaliculi of rat hepatocyte couplets. Am J Physiol 1995;269:G73-G84. ABSTRACT
Using microscope laser light-scattering spectroscopy, the authors investigated ‘‘primary’’ bile secretion into canalicular spaces of rat hepatocyte couplets in monolayer culture. Time-dependent scattered light intensities were fitted by bi-exponential decays. The ‘‘slow’’ decay was attributed to an undulating canalicular membrane motion, whereas the ‘‘fast’’ decay was consistent with rapidly diffusing intracanalicular vesicles with mean ˚ . After addition hydrodynamic radii ({SD) of 479 { 53 A of micromolar concentrations of common bile salts, increases in the amplitude of the fast component facilitated a quantitative estimate of vesicle secretion rates. A dose-response relationship with 0.1 to 200 mmol/L sodium taurocholate was characterized by an initial concentration-dependent increase and then a decrease in the amplitude of the fast canalicular component. Because these taurocholate concentrations are nontoxic to cultured hepatocytes, the maximum in vesicle-sized particles at 10 mmol/L taurocholate suggested that its critical micellar concentration of Ç5 mmol/L was attained within the canalicular spaces. Sodium taurolithocholate resulted in time- and dose-dependent diminution in vesicle secretion rates, which after 2 hours was followed by spontaneous canalicular recovery. This suggested that acute bicellular ‘‘cholestasis’’ was followed by oxidative metabolism and detoxification of the monohydroxy bile salt. Microscope laser light-scattering spectroscopy should facilitate further physical-chemical and pathophysiological studies of bile secretion at the cellular level. COMMENTS
The process by which phosphatidylcholine and cholesterol are secreted into bile is one of the great mysteries of hepatology. Biliary secretion of cholesterol accounts for over half of cholesterol elimination in normal humans, and secretion of
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WBS: Hepatology