Ulrike Buchholz

The multidrug resistance-associated protein (MRP) is the product of an ATP-binding cassette transporter gene overexpressed in some tumor cells resistant to antineoplastic agents. We studied the transport function of MRP in membrane vesicles prepared from HeLa cells transfected with an MRP expression vector and overexpressing this 190-kDa membrane glycoprotein. ATP-dependent primary-active transport into the vesicles was demonstrated for leukotriene C4 (LTC4), LTD4, LTE4, and S-(2,4-dinitrophenyl)glutathione with relative rates, at a substrate concentration of 50 nM, of 1.0, 0.27, 0.14, and 0.16, respectively. The endogenous glutathione conjugate LTC4 had the highest affinity for this transporter with a Km of 97 nM. The Km for ATP was 19 microM. Direct photoaffinity labeling with [3H]LTC4 labeled a 190-kDa membrane protein predominantly in the MRP-transfected HeLa cells. ATP-dependent LTC4 transport was effectively inhibited by the LTD4 receptor antagonist MK 571, whereas cyclosporin A and, particularly, its analog PSC 833 were much less potent. The respective Ki values were 0.6, 5, and 27 microM, respectively. In addition, MK 571 preferentially inhibited photoaffinity labeling of the 190-kDa protein in the MRP transfectants. Our results provide direct evidence that the MRP gene encodes a primary-active ATP-dependent export pump for conjugates of lipophilic compounds with glutathione and several other anionic residues. We conclude that the biosynthetic release of LTC4 from cells is mediated by the 190-kDa product of the MRP gene.

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 [3H]monoglucuronosyl bilirubin, [3H]bisglucuronosyl bilirubin, and [3H]sulfobromophthalein withKm values of 0.10, 0.28, and 0.14 μm, respectively. High-affinity uptake of unconjugated [3H]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 [3H]sulfobromophthalein and [3H]monoglucuronosyl bilirubin with lower affinity, withKm values of 3.3 and 0.5 μm, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a Ki 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. 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 [3H]monoglucuronosyl bilirubin, [3H]bisglucuronosyl bilirubin, and [3H]sulfobromophthalein withKm values of 0.10, 0.28, and 0.14 μm, respectively. High-affinity uptake of unconjugated [3H]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 [3H]sulfobromophthalein and [3H]monoglucuronosyl bilirubin with lower affinity, withKm values of 3.3 and 0.5 μm, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a Ki 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. sulfobromophthalein 17%-glucuronosyl estradiol human serum albumin indocyanine green organic anion-transporting polypeptide high pressure liquid chromatography Bilirubin, the main bile pigment in most mammals, is the end product of heme catabolism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar). In the blood circulation, bilirubin is bound to serum albumin, which prevents its potential toxicity thought to be caused by the free ligand (2Brodersen R. Stern L. Acta Paediatr. Scand. 1990; 79: 12-19Crossref PubMed Scopus (64) Google Scholar). Despite high-affinity binding to albumin, bilirubin is rapidly and selectively taken up into the liver (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 4Arias I.M. Johnson L. Wolfson S. Am. J. Physiol. 1961; 200: 1091-1094Crossref PubMed Scopus (82) Google Scholar), biotransformed upon conjugation with glucuronate (5Senafi S.B. Clarke D.J. Burchell B. Biochem. J. 1994; 303: 233-240Crossref PubMed Scopus (228) Google Scholar), and secreted into bile across the canalicular membrane of hepatocytes by an ATP-dependent conjugate export pump termed multidrug resistance protein 2 (transporter symbol ABCC2) (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar, 7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar). In addition to a reduction of UDP-glucuronosyl transferase activity (8Black M. Billing B.H. N. Engl. J. Med. 1969; 280: 1266-1271Crossref PubMed Scopus (233) Google Scholar), impaired bilirubin uptake from the blood circulation into liver has been suggested to contribute to a subgroup of patients with Gilbert's syndrome (9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar), which is characterized by a mild unconjugated hyperbilirubinemia. Uptake of bilirubin by hepatocytes was considered to be a process mediated by specific membrane proteins, although passive diffusion has also been proposed as a possible mechanism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar, 3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar,10Mediavilla M.G. Pascolo L. Rodriguez J.V. Guibert E.E. Ostrow J.D. Tiribelli C. FEBS Lett. 1999; 463: 143-145Crossref PubMed Scopus (21) Google Scholar). Because of its instability and low solubility in aqueous solution, hepatic uptake of bilirubin was studied predominantly by use of structurally related anionic substances like sulfobromophthalein (BSP)1 and indocyanine green (ICG) (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 11Okuda K. Ohkubo H. Musha H. Kotoda K. Abe H. Tanikawa K. Gut. 1976; 17: 588-594Crossref PubMed Scopus (24) Google Scholar, 12Pascolo L. Del Vecchio S. Koehler R.K. Bayon J.E. Webster C.C. Mukerjee P. Ostrow J.D. Tiribelli C. Biochem. J. 1996; 316: 999-1004Crossref PubMed Scopus (24) Google Scholar). A transport protein for BSP with a Michaelis-Menten constant (Km) of 1.5 μm has been cloned from rat liver (13Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar) and designated as organic anion-transporting polypeptide 1 (rat OATP1). Rat OATP1 belongs to a family of transport proteins (OATP family, symbol SLC21A) mediating the transport of organic anions including bile salts, steroid conjugates, thyroid hormones, prostaglandins, and BSP (14Meier P.J. Eckhardt U. Schroeder A. Hagenbuch B. Stieger B. Hepatology. 1997; 26: 1667-1677Crossref PubMed Scopus (322) Google Scholar). For human OATP1 (SLC21A3), which is expressed at high levels in brain and kidney and at a low level in human liver, kinetic studies revealed only a moderate affinity for BSP with a Km value of 20 μm (15Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (368) Google Scholar). In a search for additional OATP isoforms in human liver, we and other groups have recently cloned a new member of this transporter family, human OATP2 (also known as LST1 or OATP-C, gene symbol SLC21A6) (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 17Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 18Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Most recently, we cloned an additional human liver OATP isoform termed OATP8 (gene symbol SLC21A8), which shares 80% identical amino acids with human OATP2 (19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Antibodies raised against both transport proteins localized them to the basolateral membrane of human hepatocytes (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Northern blot analyses demonstrated an apparently exclusive hepatic expression of both transporters (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). The availability of cell lines stably expressing human OATP2 and OATP8 enabled us to answer the question of whether these two major human hepatic OATP family members are capable of transporting bilirubin and its conjugates from blood across the basolateral membrane into hepatocytes.DISCUSSIONWe conclude that uptake of bilirubin into human hepatocytes, the first step of its detoxification, is mediated by OATP2, a major transport protein localized to the basolateral membrane of hepatocytes, but not by the isoform OATP8 localized to the same membrane domain. Our conclusion is based on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin were high-affinity substrates for OATP2, with nanomolarKm values, whereas OATP8 transported BSP and monoglucuronosyl bilirubin with markedly lower affinity (Fig. 1 and Table I); (b) OATP2, but not OATP8, was able to extract substrates from albumin (Fig. 2) to which bilirubin binds with high affinity; (c) ICG inhibited OATP2 at nanomolar concentrations but exerted no inhibitory effect on OATP8 at concentrations up to 10 μm (Fig. 3); and (d) [3H]bilirubin uptake by OATP2 was directly demonstrated by uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination pathway of bilirubin in human liver (Fig. 5): bilirubin (B) bound to albumin is taken up across the basolateral membrane by OATP2 and conjugated in the hepatocyte by the UDP-glucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl bilirubin and bisglucuronosyl bilirubin. Bilirubin glucuronides are finally excreted into bile by the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) membrane (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar,7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar).Our results here establish a carrier-mediated uptake of bilirubin into hepatocytes. However, we do not exclude additional bilirubin uptake through passive diffusion. The differentiation between carrier-mediated and diffusional bilirubin uptake into the liver will be supported by the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the basolateral membrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contribute to the selective uptake of bilirubin from the blood circulation into liver. Bilirubin, the main bile pigment in most mammals, is the end product of heme catabolism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar). In the blood circulation, bilirubin is bound to serum albumin, which prevents its potential toxicity thought to be caused by the free ligand (2Brodersen R. Stern L. Acta Paediatr. Scand. 1990; 79: 12-19Crossref PubMed Scopus (64) Google Scholar). Despite high-affinity binding to albumin, bilirubin is rapidly and selectively taken up into the liver (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 4Arias I.M. Johnson L. Wolfson S. Am. J. Physiol. 1961; 200: 1091-1094Crossref PubMed Scopus (82) Google Scholar), biotransformed upon conjugation with glucuronate (5Senafi S.B. Clarke D.J. Burchell B. Biochem. J. 1994; 303: 233-240Crossref PubMed Scopus (228) Google Scholar), and secreted into bile across the canalicular membrane of hepatocytes by an ATP-dependent conjugate export pump termed multidrug resistance protein 2 (transporter symbol ABCC2) (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar, 7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar). In addition to a reduction of UDP-glucuronosyl transferase activity (8Black M. Billing B.H. N. Engl. J. Med. 1969; 280: 1266-1271Crossref PubMed Scopus (233) Google Scholar), impaired bilirubin uptake from the blood circulation into liver has been suggested to contribute to a subgroup of patients with Gilbert's syndrome (9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar), which is characterized by a mild unconjugated hyperbilirubinemia. Uptake of bilirubin by hepatocytes was considered to be a process mediated by specific membrane proteins, although passive diffusion has also been proposed as a possible mechanism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar, 3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar,10Mediavilla M.G. Pascolo L. Rodriguez J.V. Guibert E.E. Ostrow J.D. Tiribelli C. FEBS Lett. 1999; 463: 143-145Crossref PubMed Scopus (21) Google Scholar). Because of its instability and low solubility in aqueous solution, hepatic uptake of bilirubin was studied predominantly by use of structurally related anionic substances like sulfobromophthalein (BSP)1 and indocyanine green (ICG) (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 11Okuda K. Ohkubo H. Musha H. Kotoda K. Abe H. Tanikawa K. Gut. 1976; 17: 588-594Crossref PubMed Scopus (24) Google Scholar, 12Pascolo L. Del Vecchio S. Koehler R.K. Bayon J.E. Webster C.C. Mukerjee P. Ostrow J.D. Tiribelli C. Biochem. J. 1996; 316: 999-1004Crossref PubMed Scopus (24) Google Scholar). A transport protein for BSP with a Michaelis-Menten constant (Km) of 1.5 μm has been cloned from rat liver (13Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar) and designated as organic anion-transporting polypeptide 1 (rat OATP1). Rat OATP1 belongs to a family of transport proteins (OATP family, symbol SLC21A) mediating the transport of organic anions including bile salts, steroid conjugates, thyroid hormones, prostaglandins, and BSP (14Meier P.J. Eckhardt U. Schroeder A. Hagenbuch B. Stieger B. Hepatology. 1997; 26: 1667-1677Crossref PubMed Scopus (322) Google Scholar). For human OATP1 (SLC21A3), which is expressed at high levels in brain and kidney and at a low level in human liver, kinetic studies revealed only a moderate affinity for BSP with a Km value of 20 μm (15Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (368) Google Scholar). In a search for additional OATP isoforms in human liver, we and other groups have recently cloned a new member of this transporter family, human OATP2 (also known as LST1 or OATP-C, gene symbol SLC21A6) (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 17Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 18Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Most recently, we cloned an additional human liver OATP isoform termed OATP8 (gene symbol SLC21A8), which shares 80% identical amino acids with human OATP2 (19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Antibodies raised against both transport proteins localized them to the basolateral membrane of human hepatocytes (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Northern blot analyses demonstrated an apparently exclusive hepatic expression of both transporters (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). The availability of cell lines stably expressing human OATP2 and OATP8 enabled us to answer the question of whether these two major human hepatic OATP family members are capable of transporting bilirubin and its conjugates from blood across the basolateral membrane into hepatocytes. DISCUSSIONWe conclude that uptake of bilirubin into human hepatocytes, the first step of its detoxification, is mediated by OATP2, a major transport protein localized to the basolateral membrane of hepatocytes, but not by the isoform OATP8 localized to the same membrane domain. Our conclusion is based on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin were high-affinity substrates for OATP2, with nanomolarKm values, whereas OATP8 transported BSP and monoglucuronosyl bilirubin with markedly lower affinity (Fig. 1 and Table I); (b) OATP2, but not OATP8, was able to extract substrates from albumin (Fig. 2) to which bilirubin binds with high affinity; (c) ICG inhibited OATP2 at nanomolar concentrations but exerted no inhibitory effect on OATP8 at concentrations up to 10 μm (Fig. 3); and (d) [3H]bilirubin uptake by OATP2 was directly demonstrated by uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination pathway of bilirubin in human liver (Fig. 5): bilirubin (B) bound to albumin is taken up across the basolateral membrane by OATP2 and conjugated in the hepatocyte by the UDP-glucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl bilirubin and bisglucuronosyl bilirubin. Bilirubin glucuronides are finally excreted into bile by the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) membrane (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar,7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar).Our results here establish a carrier-mediated uptake of bilirubin into hepatocytes. However, we do not exclude additional bilirubin uptake through passive diffusion. The differentiation between carrier-mediated and diffusional bilirubin uptake into the liver will be supported by the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the basolateral membrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contribute to the selective uptake of bilirubin from the blood circulation into liver. We conclude that uptake of bilirubin into human hepatocytes, the first step of its detoxification, is mediated by OATP2, a major transport protein localized to the basolateral membrane of hepatocytes, but not by the isoform OATP8 localized to the same membrane domain. Our conclusion is based on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin were high-affinity substrates for OATP2, with nanomolarKm values, whereas OATP8 transported BSP and monoglucuronosyl bilirubin with markedly lower affinity (Fig. 1 and Table I); (b) OATP2, but not OATP8, was able to extract substrates from albumin (Fig. 2) to which bilirubin binds with high affinity; (c) ICG inhibited OATP2 at nanomolar concentrations but exerted no inhibitory effect on OATP8 at concentrations up to 10 μm (Fig. 3); and (d) [3H]bilirubin uptake by OATP2 was directly demonstrated by uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination pathway of bilirubin in human liver (Fig. 5): bilirubin (B) bound to albumin is taken up across the basolateral membrane by OATP2 and conjugated in the hepatocyte by the UDP-glucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl bilirubin and bisglucuronosyl bilirubin. Bilirubin glucuronides are finally excreted into bile by the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) membrane (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar,7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar). Our results here establish a carrier-mediated uptake of bilirubin into hepatocytes. However, we do not exclude additional bilirubin uptake through passive diffusion. The differentiation between carrier-mediated and diffusional bilirubin uptake into the liver will be supported by the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the basolateral membrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contribute to the selective uptake of bilirubin from the blood circulation into liver. We thank W. D. Lehmann (Deutsches Krebsforschungszentrum, Spectroscopy, Heidelberg, Germany) for analysis of the labeled and unlabeled BSP by nanoelectrospray mass spectrometry, J. M. Crawford (University of Florida, Department of Pathology, Gainesville, FL) and A. F. McDonagh (University of California, Division of Gastroenterology, San Francisco, CA) for advice on the preparation of [3H]bilirubin, K. Bode and M. Donner (Deutsches Krebsforschungszentrum, Division of Tumor Biochemistry, Heidelberg, Germany) for help during the biosynthesis of [3H]bilirubin, and G. Jedlitschky (Deutsches Krebsforschungszentrum, Division of Tumor Biochemistry, Heidelberg, Germany) for critical reading of the manuscript.

The ATP-dependent transport of the endogenous glutathione conjugate leukotriene C4 (LTC4) was more than 25-fold higher in membrane vesicles prepared from human leukemia cells (HL60/ADR) overexpressing the multidrug resistance-associated protein than from drug-sensitive parental HL60 cells or revertant cells. Similar results were obtained with S-(2,4-dinitrophenyl)glutathione as substrate. Photoaffinity labeling detected preferentially in the HL60/ADR membranes a 190-kilodalton protein binding [3H]LTC4 and 8-azido[alpha-32P]ATP. The [3H]LTC4-labeled 190-kilodalton protein was immunoprecipitated by an antiserum against the COOH-terminal sequence of multidrug resistance-associated protein. Our results indicate that multidrug resistance-associated protein mediates the ATP-dependent transport of LTC4 and structurally related anionic amphiphilic conjugates.

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.