Hepatic Uptake of Bilirubin and Its Conjugates by the Human Organic Anion Transporter SLC21A6*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 13, Issue of March 30, pp , y The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hepatic Uptake of Biliruin and Its Conjugates y the Human Organic Anion Transporter SLC21A6* Received for pulication, June 8, 2000, and in revised form, Decemer 12, 2000 Pulished, JBC Papers in Press, Decemer 27, 2000, DOI /jc.M Yunhai Cui, Jörg König, Inka Leier, Ulrike Buchholz, and Dietrich Keppler From the Division of Tumor Biochemistry, Deutsches Kresforschungszentrum, D Heidelerg, Germany Biliruin, the end product of heme cataolism, is taken up from the lood circulation into the liver. This work identifies a high-affinity transport protein mediating the uptake of iliruin and its conjugates into human hepatocytes. Human emryonic kidney cells (HEK293) permanently expressing the recominant organic anion-transporting polypeptide 2 (human OATP2, also known as LST-1 or OATP-C; symol SLC21A6) showed uptake of [ 3 H]monoglucuronosyl iliruin, [ 3 H]isglucuronosyl iliruin, and [ 3 H]sulforomophthalein with K m values of 0.10, 0.28, and 0.14 M, respectively. High-affinity uptake of unconjugated [ 3 H]iliruin y OATP2 occurred in the presence of alumin and was not mediated y another asolateral hepatic uptake transporter, human OATP8 (symol SLC21A8). OATP2 and OATP8 differed y their capacity to extract sustrates from alumin efore transport. In comparison to the high-affinity transport y OATP2, OATP8 transported [ 3 H]sulforomophthalein and [ 3 H]monoglucuronosyl iliruin with lower affinity, with K m values of 3.3 and 0.5 M, respectively. The organic anion indocyanine green potently inhiited transport mediated y OATP2, with a K i value of 112 nm, ut did not inhiit transport mediated y OATP8. Human OATP2 may play a key role in the prevention of hyperiliruinemia y facilitating the selective entry of unconjugated iliruin and its glucuronate conjugates into human hepatocytes. Biliruin, the main ile pigment in most mammals, is the end product of heme cataolism (1). In the lood circulation, iliruin is ound to serum alumin, which prevents its potential toxicity thought to e caused y the free ligand (2). Despite high-affinity inding to alumin, iliruin is rapidly and selectively taken up into the liver (3, 4), iotransformed upon conjugation with glucuronate (5), and secreted into ile across the canalicular memrane of hepatocytes y an ATP-dependent conjugate export pump termed multidrug resistance protein 2 (transporter symol ABCC2) (6, 7). In addition to a reduction of UDP-glucuronosyl transferase activity (8), impaired iliruin uptake from the lood circulation into liver has een suggested to contriute to a sugroup of patients with Gilert s syndrome (9), which is characterized y a mild unconjugated hyperiliruinemia. Uptake of iliruin y hepatocytes was considered to * The work was supported in part y Deutsche Forschungsgemeinschaft through SFB601 and the Fonds der Chemischen Industrie. The costs of pulication of this article were defrayed in part y the payment of page charges. This article must therefore e herey marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should e addressed: Division of Tumor Biochemistry, Deutsches Kresforschungszentrum, Im Neuenheimer Feld 280, D Heidelerg, Germany. Tel.: ; Fax: ; y.cui@dkfz-heidelerg.de e a process mediated y specific memrane proteins, although passive diffusion has also een proposed as a possile mechanism (1, 3, 10). Because of its instaility and low soluility in aqueous solution, hepatic uptake of iliruin was studied predominantly y use of structurally related anionic sustances like sulforomophthalein (BSP) 1 and indocyanine green (ICG) (3, 9, 11, 12). A transport protein for BSP with a Michaelis- Menten constant (K m )of1.5 M has een cloned from rat liver (13) and designated as organic anion-transporting polypeptide 1 (rat OATP1). Rat OATP1 elongs to a family of transport proteins (OATP family, symol SLC21A) mediating the transport of organic anions including ile salts, steroid conjugates, thyroid hormones, prostaglandins, and BSP (14). For human OATP1 (SLC21A3), which is expressed at high levels in rain and kidney and at a low level in human liver, kinetic studies revealed only a moderate affinity for BSP with a K m value of 20 M (15). In a search for additional OATP isoforms in human liver, we and other groups have recently cloned a new memer of this transporter family, human OATP2 (also known as LST1 or OATP-C, gene symol SLC21A6) (16 18). Most recently, we cloned an additional human liver OATP isoform termed OATP8 (gene symol SLC21A8), which shares 80% identical amino acids with human OATP2 (19). Antiodies raised against oth transport proteins localized them to the asolateral memrane of human hepatocytes (16, 19). Northern lot analyses demonstrated an apparently exclusive hepatic expression of oth transporters (16, 19). The availaility of cell lines staly expressing human OATP2 and OATP8 enaled us to answer the question of whether these two major human hepatic OATP family memers are capale of transporting iliruin and its conjugates from lood across the asolateral memrane into hepatocytes. EXPERIMENTAL PROCEDURES Cell Culture and Cell Lines HEK293 cells were cultured in minimum essential medium (Sigma) supplemented with 10% fetal ovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37 C, 95% humidity, and 5% CO 2 as descried recently (16, 19). HEK-OATP2 cells (16) and HEK-OATP8 cells (19) permanently expressed high levels of human recominant OATP2 and OATP8, respectively. The Gen- Bank TM /European Molecular Biology Laoratory accession numers for the sequences of OATP2 and OATP8 are AJ and AJ251506, respectively. Biosynthesis of [ 3 H]Biliruin [ 3 H]Biliruin was otained iosynthetically in rats in a procedure similar to the one descried y Crawford et al. (20). Two Harlan Sprague-Dawley rats were given an intravenous or intraportal injection of 500 Ci of [3,5-3 H]deltaaminolevulinic acid (25.9 GBq/mmol; NEN Life Sciences, Boston, MA) at a dose of 83 and 42 MBq/kg ody weight, respectively. [ 3 H]Biliruin was isolated from ile y hydrolysis of its glucuronides and extraction 1 The areviations used are: BSP, sulforomophthalein; E 2 17 G, 17 -glucuronosyl estradiol; HSA, human serum alumin; ICG, indocyanine green; OATP, organic anion-transporting polypeptide; HPLC, high pressure liquid chromatography. This paper is availale on line at

2 Biliruin Transport y Human OATP with chloroform (20). The purity of the [ 3 H]iliruin was confirmed y HPLC, and the specific radioactivity was 120, ,000 dpm/nmol ( GBq/mmol) for the material otained from oth rats. In all experiments, [ 3 H]iliruin was protected from exposure to light. Synthesis of [ 3 H]Monoglucuronosyl Biliruin and [ 3 H]Bisglucuronosyl Biliruin Biliruin glucuronides were prepared using recominant UDP-glucuronosyl transferase 1A1 and UDP[1-3 H]glucuronic acid (0.56 TBq/mmol; Biotrend, Köln, Germany) together with unlaeled iliruin as descried previously (6, 7) and purified using radio-hplc. Uptake Studies For uptake assays, cells were seeded in 6-well plates (coated with 0.1 mg/ml poly-d-lysine) at a density of cells/well and cultured with 10 mm sodium utyrate for 24 h. Before the uptake experiments, cells were washed with uptake uffer (142 mm NaCl, 5 mm KCl, 1 mm KH 2 PO 4, 1.2 mm MgSO 4, 1.5 mm CaCl 2,5mM glucose, and 12.5 mm HEPES, ph 7.3). The transport assay was started y the addition of 1 ml of uptake uffer containing 3 H-laeled sustrate ( MBq/ml) to the cells. 3 H-laeled sustrates were otained from NEN Life Sciences. [ 3 H]BSP (0.5 TBq/mmol) was otained y custom synthesis (Hartmann Analytic, Köln, Germany); its purity ( 98%) was confirmed y reverse-phase HPLC analysis on a C 18 Hypersil column (5- m particles; Shandon, Runcorn, United Kingdom) using two different systems. The isocratic elution was performed with 45% methanol/55% water containing 50 mm NaH 2 PO 4 and5mm Na 2 SO 4 at ph 2.8. The linear gradient elution was performed from 100% uffer A (45% methanol/55% water containing 2 mm tetrautylammonium hydroxide at ph 6.0) to 100% uffer B (90% methanol/10% water containing 2 mm tetrautylammonium hydroxide at ph 6.0). The specific radioactivity of [ 3 H]BSP did not change during repeated HPLC analyses, indicating that 3 H exchange was elow detectaility. [ 3 H]BSP strictly co-chromatographed with unlaeled BSP during HPLC. Moreover, the unlaeled BSP and the 3 H- laeled BSP were analyzed y nanoelectrospray mass spectrometry as descried y Lehmann and Kaspersen (21). The isotopic pattern of molecular ions of unlaeled BSP and [ 3 H]BSP was very similar, and quantitative evaluation indicated the following relative amounts: unlaeled BSP, 63.1%; singly laeled [ 3 H]BSP 27.4%; and douly laeled [ 3 H]BSP, 9.5%. These data correspond to a specific radioactivity of 0.5 TBq/mmol. For inhiition studies, inhiitors were included in the uptake uffer. After incuation at 37 C, transport was stopped at different time points y the addition of 1 ml of cold uptake uffer. Cells were susequently washed three times with uptake uffer and lysed with 1 ml of 0.2% SDS in water. Aliquots (250 l) of the lysate were counted for radioactivity. Protein content was determined y the Lowry method using 100 l of lysate. Uptake Studies with [ 3 H]Biliruin Due to high ackground inding of [ 3 H]iliruin to the poly-d-lysine-coated plastic dishes, uptake of [ 3 H]iliruin into transfected cells was measured in cell suspension. Cells were cultured with 10 mm utyrate for 24 h as descried previously (16). For uptake assays, cells were detached from culture flasks y knocking, washed twice with uptake uffer, and resuspended in uptake uffer at a density of cells/ml. [ 3 H]Biliruin was diluted with human serum alumin (HSA; Sigma; fatty acid-free) in uptake uffer (75, ,000 dpm/ml). Unlaeled iliruin was added to give the desired final concentrations. Uptake was started y mixing 1 ml of cell suspension with 1 ml of iliruin/alumin solution to give a final radioactivity of 37,500 50,000 dpm/ml and stopped at different time points y centrifugation of the mixture at 13,000 rpm for 10 s. Cell pellets were washed twice with 1 ml of uptake uffer containing HSA and lysed in 2 ml of 0.2% SDS in water. Aliquots (300 l) of the lysate were counted for radioactivity. To determine the nonspecific inding of [ 3 H]iliruin, cells were incuated with [ 3 H]iliruin in the presence of HSA for 1 min at 4 C. Cell-associated radioactivity measured under this condition was used as a lank and sutracted from all other values. No differences etween this method and the method using adherent cells were oserved for other sustrates like BSP. Immunolot Analysis Preparation of crude memrane fractions from transfected cells and immunolot analysis were performed as descried previously (16, 19). The polyclonal antiody ESL (16) was used to detect recominant human OATP2. RESULTS Sulforomophthalein, Monoglucuronosyl Biliruin, and Bisglucuronosyl Biliruin Are High-affinity Sustrates for Human OATP2 BSP, a widely used anionic model compound for studies on uptake into the liver, is a high-affinity sustrate for OATP2 with a K m value of 140 nm (Fig. 1). In comparison with FIG. 1. BSP uptake mediated y human OATP2. A, [ 3 H]BSP transport into cells was measured at a concentration of 1 M using HEK293 cells transfected with human OATP2 (HEK-OATP2) or with vector alone (HEK-Co). B, concentration dependence of [ 3 H]BSP uptake. Uptake of [ 3 H]BSP into HEK-OATP2 cells (f) and HEK-Co cells ( ) was measured at concentrations etween 10 and 210 nm. The net uptake rates ( ) were calculated y sutracting values otained with HEK-Co cells from those otained with HEK-OATP2 cells. Data are the means S.D. from two triplicate experiments. OATP8, OATP2 showed a 24-fold higher affinity for BSP (Tale I). In addition, monoglucuronosyl iliruin and isglucuronosyl iliruin were identified as high-affinity sustrates for OATP2 with nanomolar K m values. Monoglucuronosyl iliruin was also a good sustrate for human OATP8 (Tale I). Kinetic properties of additional sustrates for OATP2 and OATP8 are summarized in Tale I. Despite the remarkale difference in their affinities for BSP, human OATP2 and OATP8 showed similar affinities for 17 -glucuronosyl estradiol (E 2 17 G). OATP2, ut not OATP8, Transports Sustrates in the Presence of Alumin An important aspect with regard to BSP uptake mediated y oth transporters is the differential influence of HSA. As shown in Fig. 2A, HSA, in a 20-fold molar excess, did not significantly affect OATP2-mediated BSP uptake ut aolished OATP8-mediated BSP uptake. One-third of the respective K m value (50 nm for OATP2 and 1 M for OATP8) was chosen as the BSP concentration in these experiments. A similar influence of HSA was oserved when BSP uptake was measured for oth transporters at a constant BSP concentration (1 M) with varying HSA concentrations (Fig. 2B). HSA caused only a minor decrease of OATP2-mediated BSP uptake. Uptake assays using 1 M E 2 17 G as sustrate showed that 5 M HSA did not change the uptake rate with OATP2. The OATP2-mediated uptake was not chloride-dependent in the presence or asence of HSA ecause replacement of chloride in uptake uffer y gluconate had no effect on the rate of uptake. It is well known that many organic anions ind to HSA in the lood circulation, and a particular role of HSA in the hepatic uptake of organic anions has een proposed (22). It is therefore conceivale that a hepatic transport protein like OATP2 has the aility to extract sustrates from HSA. For OATP8, the interaction with HSA is different (Fig. 2). We have identified four lipophilic organic anions (monoglucuronosyl iliruin, BSP, E 2 17 G, and dehydroepiandrosterone 3-sulfate) as 3 H- laeled sustrates for OATP8. These experiments, however, were performed without HSA in the uffer (Tale I). The fact that OATP8 is not capale of extracting sustrates from HSA raises the question of the physiological function of OATP8 in the asolateral hepatocyte memrane. From the data otained thus far, it is feasile that OATP8 functions predominantly under circumstances where the inding capacity of alumin for lipophilic organic anions is exceeded. Indocyanine Green Inhiits Transport Mediated y OATP2 ut not Transport Mediated y OATP8 We tested a numer of organic anions for their aility to inhiit uptake y human OATP2 and OATP8. ICG did not affect OATP8-mediated E 2 17 G transport at concentrations up to 10 M (Fig. 3A) ut

3 9628 Biliruin Transport y Human OATP2 TABLE I Kinetic constants for sustrate transport mediated y OATP2 and OATP8 Transport studies with 3 H-laeled sustrates were performed in the asence of HSA using HEK293 cells staly expressing recominant human OATP2 or human OATP8 (GenBank /European Molecular Biology Laoratory data ank accession numers AJ and AJ251506, respectively). Transport into control vector-transfected cells served as a lank. Michaelis-Menten constants (K m ) are given as means S.D. (n 6). K m ( M) Sustrate OATP2 OATP8 Monoglucuronosyl iliruin Bisglucuronosyl iliruin a Sulforomophthalein Glucuronosyl estradiol Cholyl taurine Cholate Estrone 3-sulfate Dehydroepiandrosterone 3-sulfate c a No significant transport was detected at a concentration of 1 M. No significant transport was detected at a concentration of 5 M. c No saturation up to a concentration of 30 M. FIG. 2.Effect of HSA on BSP uptake mediated y OATP2 and OATP8. A, uptake of [ 3 H]BSP was measured at 50 nm for OATP2 and 1 M for OATP8 in the presence of 1 M (OATP2) or 20 M (OATP8) HSA (BSP:HSA 1: 20 in oth cases). No significant effect of alumin on [ 3 H]BSP uptake was oserved for OATP2, whereas OATP8-mediated [ 3 H]BSP uptake was completely aolished y the addition of alumin. B, uptake of 1 M [ 3 H]BSP measured with OATP2 and OATP8 in the presence of increasing concentrations of HSA ranging from 0.2 to 5 M. HSA had a much greater effect on the OATP8-mediated [ 3 H]BSP uptake than on the OATP2-mediated uptake (p 0.01). Data are the means S.D. (n 6). inhiited OATP2-mediated uptake in a competitive manner with a K i value of 112 nm. Although BSP and ICG were thought to e taken up into hepatocytes y the same mechanism (3), patients with apparently normal hepatic BSP uptake ut delayed ICG clearance have een reported (11). Our studies raise the possiility that such patients may have a deficiency in OATP2, leading to delayed ICG clearance, ut normal OATP8, which may function in BSP uptake under this condition. Unlike ICG, the drugs pravastatin, rifamycin SV, and rifampicin inhiited oth OATP2- and OATP8-mediated uptake of [ 3 H]E 2 17 G (Fig. 3B). The HMG-CoA reductase inhiitor pravastatin was a competitive inhiitor of OATP2-mediated transport of E 2 17 G with an inhiition constant of 53 M. This is in line with a recent study showing that pravastatin is a sustrate for human OATP2 with a K m value of aout 30 M (17). Unconjugated Biliruin Is Transported y OATP2 in the Presence of Alumin The fact that BSP, monoglucuronosyl iliruin, and isglucuronosyl iliruin are high-affinity sustrates for human OATP2 and the fact that ICG inhiits OATP2-mediated uptake competitively at nanomolar concentrations suggested that OATP2 might e the long-sought uptake transporter for unconjugated iliruin in the asolateral hepatocyte memrane. We therefore measured [ 3 H]iliruin uptake y OATP2-transfected HEK293 cells (HEK-OATP2) at a concentration of 1 M in the presence of 2 M HSA. The FIG. 3.Inhiition of OATP2- and OATP8-mediated transport. Uptake of 17 -glucuronosyl [ 3 H]estradiol (E 2 17 G; 2 M) y HEK293 cells transfected with OATP2 or OATP8 was measured in the presence of different inhiitors and in the asence of HSA. A, uptake of [ 3 H]E 2 17 G mediated y OATP2 was potently inhiited y ICG, whereas ICG exerted no significant inhiition on OATP8-mediated uptake. B, oth OATP2 and OATP8 were inhiited y pravastatin (25 M), rifamycin SV (50 M), and rifampicin (75 M). Data are the means S.D. from two triplicate experiments. calculated free iliruin concentration under this condition, using the dissociation constant for HSA and iliruin (12), is aout 25 nm. As shown in Fig. 4A, [ 3 H]iliruin was taken up y HEK-OATP2 cells time-dependently at 37 C. The uptake rate into HEK-OATP2 cells (8.5 pmol min 1 mg protein 1 ) differed from that into vector-transfected HEK293 cells (1.5 pmol min 1 mg protein 1 ) y a factor of 5.7 (p 0.01). When the uptake was determined at 4 C, no significant difference in uptake rates was detected etween OATP2-expressing cells and control cells. Uptake of iliruin mediated y recominant human OATP2 was concentration-dependent as shown in Fig. 4B. In these experiments, the HSA concentration was kept constant at 20 M so that the iliruin:hsa ratio did not exceed 0.5. Because of the uncertainty of calculated free iliruin concentrations (12), the K m value for free unconjugated iliruin could only e estimated and was aout 160 nm. For comparison, we investigated whether OATP8 would also transport unconjugated iliruin. At the same [ 3 H]iliruin and HSA concentrations and under the same conditions used for OATP2 (Fig. 4), no significant uptake of [ 3 H]iliruin was detected with HEK293 cells expressing human OATP8 (data not shown). Biliruin, which was examined as a complex with HSA, inhiited the uptake of E 2 17 G and BSP y human OATP2, with 50% inhiition at 5 M with E 2 17 G as sustrate and at 20 M with BSP as sustrate. Uptake of [ 3 H]iliruin into HEK-OATP2 cells was strongly inhiited y BSP (Fig. 4C), demonstrating mutual inhiition of BSP and iliruin uptake. DISCUSSION We conclude that uptake of iliruin into human hepatocytes, the first step of its detoxification, is mediated y OATP2, a major transport protein localized to the asolateral memrane of hepatocytes, ut not y the isoform OATP8 localized to the same memrane domain. Our conclusion is ased on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl iliruin, and isglucuronosyl iliruin were highaffinity sustrates for OATP2, with nanomolar K m values, whereas OATP8 transported BSP and monoglucuronosyl iliruin with markedly lower affinity (Fig. 1 and Tale I); () OATP2, ut not OATP8, was ale to extract sustrates from alumin (Fig. 2) to which iliruin inds with high affinity; (c) ICG inhiited OATP2 at nanomolar concentrations ut exerted no inhiitory effect on OATP8 at concentrations up to 10 M (Fig. 3); and (d) [ 3 H]iliruin uptake y OATP2 was directly demonstrated y uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination

4 Biliruin Transport y Human OATP FIG. 5.Biliruin uptake and conjugate export y human hepatocytes. Biliruin (B) is taken up across the asolateral memrane y human OATP2 (SLC21A6; Fig. 4A) and conjugated with glucuronate (GA) y UDP-glucuronosyl transferase 1A1 (UGT1A1), resulting in monoglucuronosyl iliruin (BGA) and isglucuronosyl iliruin (B(GA) 2 ) (5). The excretion of BGA and B(GA) 2 is mediated y the apical ATP-dependent conjugate export pump, multidrug resistance protein 2 (symol ABCC2) (6, 7). pathway of iliruin in human liver (Fig. 5): iliruin (B) ound to alumin is taken up across the asolateral memrane y OATP2 and conjugated in the hepatocyte y the UDPglucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl iliruin and isglucuronosyl iliruin. Biliruin glucuronides are finally excreted into ile y the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) memrane (6, 7). Our results here estalish a carrier-mediated uptake of iliruin into hepatocytes. However, we do not exclude additional iliruin uptake through passive diffusion. The differentiation etween carrier-mediated and diffusional iliruin uptake into the liver will e supported y the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the asolateral memrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contriute to the selective uptake of iliruin from the lood circulation into liver. Acknowledgments We thank W. D. Lehmann (Deutsches Kresforschungszentrum, Spectroscopy, Heidelerg, Germany) for analysis of the laeled and unlaeled BSP y 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 [ 3 H]iliruin, K. Bode and M. Donner (Deutsches Kresforschungszentrum, Division of Tumor Biochemistry, Heidelerg, Germany) for help during the iosynthesis of [ 3 H]iliruin, and G. Jedlitschky (Deutsches Kresforschungszentrum, Division of Tumor Biochemistry, Heidelerg, Germany) for critical reading of the manuscript. FIG. 4.Biliruin as a sustrate for human OATP2. A, uptake of [ 3 H]iliruin was measured at 1 M iliruin in the presence of 2 M HSA. Cell-associated radioactivity after incuation of cells with [ 3 H]iliruin for 1 min at 4 C was used as a lank and sutracted from all other values. The uptake rate into HEK-OATP2 cells was 8.5 pmol min 1 mg protein 1 (n 10) and differed significantly from the uptake rate into vector-transfected HEK293 cells (1.5 pmol min 1 mg protein 1 ; n 5). B, concentration dependence of iliruin uptake. Uptake of [ 3 H]iliruin at different concentrations was measured at a constant HSA concentration of 20 M. The estimated free iliruin concentration ranged from 5 to 25 nm. Measurements of [ 3 H]iliruin uptake at each concentration were performed at 1 min (lank) and 10 min; the rate of uptake was calculated from the difference etween these two measurements (n 5). C, inhiition of [ 3 H]iliruin uptake y BSP. Uptake of 10 M [ 3 H]iliruin was measured in the presence of 20 M HSA (n 5), and the uptake was calculated as descried in B; for inhiition studies, 5 M BSP was included into the uptake uffer. All experiments were reproduced at least twice. Data are the means S.D. Asterisks indicate a significant difference compared with controls (p 0.01). Inset in A, immunolot with the polyclonal antiody ESL (16) of REFERENCES 1. Chowdhury, J. R., Chowdhury, N. R., Wolkoff, A. W., and Arias, I. M. (1994) in The Liver: Biology and Pathoiology (I. M. Anas et al., eds.) 3rd Ed., pp , Raven Press, New York 2. Brodersen, R., and Stern, L. (1990) Acta Paediatr. Scand. 79, Scharschmidt, B. F., Waggoner, J. G., and Berk, P. D. (1975) J. Clin. Invest. 56, Arias, I. M., Johnson, L., and Wolfson, S. (1961) Am. J. Physiol. 200, Senafi, S. B., Clarke, D. J., and Burchell, B. (1994) Biochem. J. 303, Kamisako, T., Leier, I., Cui, Y., König, J., Buchholz, U., Hummel-Eiseneiss, J., and Keppler, D. (1999) Hepatology 30, Jedlitschky, G., Leier, I., Buchholz, U., Hummel-Eiseneiss, J., Burchell, B., and Keppler, D. (1997) Biochem. J. 327, Black, M., and Billing, B. H. (1969) N. Engl. J. Med. 280, Martin, J. F., Vierling, J. M., Wolkoff, A. W., Scharschmidt, B. F., Vergalla, J., Waggoner, J. G., and Berk, P. D. (1976) Gastroenterology 70, Mediavilla, M. G., Pascolo, L., Rodriguez, J. V., Guiert, E. E., Ostrow, J. D., and Tirielli, C. (1999) FEBS Lett. 463, Okuda, K., Ohkuo, H., Musha, H., Kotoda, K., Ae, H., and Tanikawa, K. (1976) Gut 17, memranes (20 g of protein) memranes (20 g of protein) from HEK-OATP2 cells (left lane) or HEK-Co cells (right lane). The arrow points to the fully glycosylated OATP2 at 90 kda.

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