Contribution of Sodium Taurocholate Co-Transporting Polypeptide to the Uptake of Its Possible Substrates Into Rat Hepatocytes1

As one of the Na-dependent transporters responsible for the hepatic uptake of ligands, sodium taurocholate (TC) co-transporting polypeptide (NTCP) has been cloned from rat liver and its substrate specificity has been clarified by examining the inhibition of TC uptake mediated by NTCP. The contribution of NTCP to the Na-dependent uptake of ligands into rat hepatocytes, however, still needs to be clarified. To determine the contribution of NTCP, we examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells, transiently expressing NTCP, and normalized the uptake of ligands with TC as a reference compound. Western Blot analysis indicated that NTCP was glycosylated much less extensively in the transfected COS-7 cells, although the expression level was comparable for the cultured hepatocytes and transfectant. Kinetic parameters for the Na-dependent uptake of TC were similar for the cultured hepatocytes and NTCPtransfected COS-7 cells (Km 5 17.7 vs. 17.4 mM; Vmax 5 1.63 vs. 1.45 nmol/min/mg protein). Glycocholic acid and cholic acid were taken up by NTCP-transfected COS-7 cells. The contribution of NTCP to the Na-dependent uptake of glycocholic acid into rat hepatocytes was approximately 80%, whereas that of cholic acid was 40%. In addition, the analysis indicated that the contribution of NTCP to the Na-dependent uptake of several ligands (ouabain, ibuprofen, glutathione-conjugate of bromosulfophthalein, glucuronideand sulfate-conjugates of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole) was negligible. Thus, this is a convenient method to determine the contribution of NTCP to the uptake of ligands into hepatocytes. It is also suggested that multiple transport mechanisms are responsible for the Na-dependent uptake of organic anions into hepatocytes. In addition to renal excretion, hepatic elimination is one of the main pathways involved in the detoxification of xenobiotics. Hepatic uptake is the initial process for the elimination of xenobiotics mediated by metabolism and/or biliary excretion. The mechanism for the hepatic uptake of ligands has been studied by kinetic analysis of the experimental data obtained in vivo, in situ with perfused liver, in vitro in isolated and/or cultured hepatocytes and isolated sinusoidal membrane vesicles (Suchy, 1993; Elferink et al., 1995; Yamazaki et al., 1996). For the hepatic uptake of bile acids, it has been established that approximately 80 and 40% of TC and CA uptake, respectively, are mediated by a Na-dependent mechanism (Yamazaki et al., 1993). In addition, based on the kinetic studies, it has been suggested that some ligands are transported across the sinusoidal membrane via mechanisms shared with bile acids. Zimmerli et al. (1989) found that the Na-dependent uptake of TC into isolated sinusoidal membrane vesicles was competitively inhibited by bile acids (such as CA, taurochenodeoxycholate and chenodeoxycholate), steroids (such as progesterone and 17-b-estradiol-3-sulfate), bumetanide, furosemide, verapamil and phalloidin, and suggested a broad substrate specificity for the Na-dependent bile acid transporter. With isolated hepatocytes, cumulative evidence has been obtained to support the hypothesis that several compounds can act as possible substrates of Na-dependent bile acid transporter (Frimmer and Ziegler, 1988). In addition, recently, Terasaki et al. (1995) and Nakamura et al. (1996) found that octreotide (a somaReceived for publication December 19, 1997. 1 This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, and the Core Research for Evolutional Sciences and Technology of Japan Sciences and Technology Corporation. We would like to thank Eizai Co., Ltd., for providing labeled E3040 glucuronide and sulfate and Dr. PJ Meier, for providing anti-rat NTCP serum. ABBREVIATIONS: NTCP, sodium taurocholate co-transporting polypeptide; OATP, organic anion transporting polypeptide; TC, taurocholate, taurocholic acid; GCA, glycocholate, glycocholic acid; CA, cholate, cholic acid; BSP, bromosulfophthalein; BSP-SG, glutathione-conjugate of bromosulfophthalein; E3040, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole; SD, Sprague-Dawley; Km, Michaelis constant; Vmax, maximum transport velocity; CLuptake, uptake clearance; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin; SSC, saline sodium citrate; SDS, sodium dodecyl sulfate; mEH, microsomal epoxide hydrolase; TBS-T, Tris-buffered saline containing 0.05% Tween 20. 0022-3565/98/2862-1043$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286:1043–1050, 1998 1043 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from tostatin analog) and a cyclic peptide (BQ-123, an endothelin antagonist) are taken up by isolated hepatocytes in a Nadependent manner and demonstrated that the uptake of the two ligands is competitively inhibited by TC. In the same manner, Blitzer et al. (1982) and Petzinger et al. (1989) provided kinetic evidence to support the hypothesis that bumetanide and TC share a common transport mechanism. Cumulative evidence suggests that the Na-independent uptake mechanism of many organic anions is shared by CA (Petzinger, 1994; Elferink et al., 1995; Meier, 1995; Yamazaki et al., 1996). To get more detailed information on the mechanism for hepatic uptake, the cDNA species for NTCP and OATP1 were isolated from rat liver based on expression cloning with Xenopus laevis oocytes (Hagenbuch et al., 1991; Jacquemin et al., 1994). Moreover, the human homologs of these transporters (NTCP and OATP) have been cloned (Hagenbuch and Meier, 1994; Kullak-Ublick et al., 1995). The sinusoidal localization of these transporters was confirmed with antibodies (Ananthanarayanan et al., 1994; Stieger et al., 1994), and in the transport properties were characterized with oocytes injected with cRNA and mammalian cells transfected with cDNA (Meier, 1995; Hagenbuch and Meier, 1996). Functional analysis of NTCP, has shown that NTCP-mediated TC uptake is inhibited by several bile acid derivatives (such as taurochenodeoxycholate, chenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate and CA), bumetanide and BSP (Hagenbuch et al., 1991; Hagenbuch and Meier, 1994; Boyer et al., 1994). However, much less information is available on whether the previously described inhibitors of TC uptake are transported via NTCP. Platte et al. (1996) reported that the transport of CA and GCA into an immortalized liver-derived cell line was not stimulated by transfection of NTCP, although the two bile acid derivatives were effective inhibitors of NTCP-mediated TC uptake in this transfected cell line. The purpose of the present study is to examine whether several organic anions which are taken up by hepatocytes via a Na-dependent mechanism can be a substrate for NTCP. In addition, we propose a convenient method to examine the contribution of NTCP to the Na-dependent uptake of ligands, because multiple systems may be responsible for their hepatic uptake. For this purpose, we examined the uptake of ligands into primary cultured hepatocytes and into COS-7 cells transiently expressing NTCP and normalized the uptake of ligands with TC as a reference compound. Materials and Methods Materials. COS-7 cells were purchased from American Type Culture Collection (Rockville, MD). [H]TC (128.4 GBq/mmol), [H]CA (906.5 GBq/mmol) and [H]ouabain (758.5 GBq/mmol) were purchased from New England Nuclear (Boston, MA). [C]GCA (2.11 GBq/mmol) and [H]ibuprofen (18.5 GBq/mmol) were purchased from Amersham International (Buckinghamshire, England). The glucuronideand sulfate-conjugates of [C]E3040 (1.85 GBq/mmol), prepared according to the method described previously (Hibi et al., 1994), were kindly donated by Eizai Co., Ltd. (Tokyo, Japan). The [H]BSP-SG was synthesized according to the method described by Saxena and Henderson (1995) with BSP (Aldrich, Milwaukee, WI) and [H]glutathione (1739 GBq/mmol; New England Nuclear). All other chemicals were commercially available and of reagent grade. Transient expression of NTCP cDNA in COS-7 Cells. Fulllength cDNA for NTCP was cloned initially by screening the rat liver cDNA library with the reported sequence according to the method described previously in detail (Ito et al., 1997). NTCP cDNA rescued into the pBluescript II S/K (2) vector (TOYOBO, Osaka, Japan) was excised with XhoI and XbaI (Takara, Tokyo, Japan) to perform the subcloning into the XhoI site in the pCAGGS vector (Niwa et al., 1991) after converting to blunt ends. For transfection, COS-7 cells were cultured in 150-mm dishes in DMEM supplemented with 5% fetal bovine serum. At 30% confluence, cells were exposed to serum-free DMEM containing plasmid (1 mg/ml) and Lipofectamine (1 mg/ml; BRL, Gaithersburg, MD). At 8 h after transfection, the plasmid-Lipofectamine solution was removed, and the medium consisting of DMEM supplemented with 5% fetal bovine serum was allowed to culture for overnight. Then, the transfected cells were treated with trypsin to seed approximately 1.6 3 10 cells onto 22-mm dishes and cultured overnight. An uptake study was performed at 48 h after transfection. Primary cultured rat hepatocytes. Rat hepatocytes were isolated from male SD rats (200 250 g, Nihon Ikagaku Dobutsu Shizai Kenkyusyo, Tokyo, Japan) after perfusion of the liver with collagenase. Cell viability was checked routinely by the trypan blue [0.4% (wt/vol)] exclusion test. After preparation, freshly isolated cells were suspended in Williams’ medium E. Approximately 5 3 10 cells were placed on collagen-coated 22-mm dishes and cultured for 4 h. Northern Blot analysis. Northern Blot analysis was performed as described previously (Ito et al., 1997). Poly(A)RNA (0.5 or 2 mg), prepared from COS-7 cells 48 h after transfection and SD rat liver, were separated on 0.8% agarose gel containing 3.7% formaldehyde and transferred to a nylon membrane before fixation by baking for 2 h at 80°C. Blots were prehybridized in medium containing 4 3 SSC, 5 3 Denhardt’s solution, 0.2% SDS, 0.1 mg/ml sonicated salmon sperm DNA and 50% formamide at 42°C for 2 h. We used 0.9 kbp NTCP cDNA (nucleic acid, 260–1186 bp) as a hybridization probe and hybridization was performed overnight in the same buffer containing 10 cpm/ml P-labeled cDNA prepared by the random primed labeling method. As a control, P-labeled cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech Laboratories, Inc., Palo Alto, CA) was used. The hybridized membrane was washed in 2 3 SSC and 0.1% SDS at 55°C for 20 min and then in 0.1 3 SSC and 0.1% SDS at 55°C for 20 min. After the membrane was exposed for 1 h to the imaging plate at room temperature, it was analyzed with Bio-Image Analyzer (Bas 2000; Fuji Film, Tokyo, Japan). Western Blot analysis. For the Western Blot analysis crude membrane fraction was prepared from COS-7 cells 48 h after transfection, rat hepatocytes cultured for 4 h and SD rat liver according to the method of Gant et al. (1991). Cells were homogenized in five volumes 0.1 M Tris-HCl buffer (pH 7.4) containing 1 mg/ml leupeptin and pepstatin A and 50 mg/ml phenylmethylsulfonyl fluoride with 20 strokes of a Dounce homogenizer. The supernatant, after centrifugation (1500 3 g for 10 min) of homogenate, was centrifuged again (100,000 3 g for 30 min). The precipitate was suspended in Tris-HCl buffer and centrifuged again (100,000 3 g for 30 min). The crude membrane fraction was resuspended in the 0.1 M Tris-HCl buffer containing the proteinase inhibitors with 5 strokes of a Dounce homogenizer and stored at 280°C before being used for Western Blot analysis. All procedures were performed at 0 to 4°C . The membrane protein concentrations were determined according to the method of Lowry et al. (1951). Fifty micrograms crude membrane was dissolved in 10 ml of 2 3 0.25 M Tris-HCl buffer containing 2% SDS, 30% glycerol and 0.01% bromophenol blue (pH 6.8) and was loaded on a 7.5% SDS-polyacrylamide gel electrophoresis plate with a 4.4% stacking gel. Molecular weight was assessed with a prestained protein marker (NEB, Beverly, MA). Proteins were transferred electrophoretically to a nitrocellulose membrane (Millipore, Bedford, MA) with a blotter (Bio-Rad Laboratories, Richmond, CA) at 15V for 1 h. The membrane was blocked with TBS-T and 5% BSA for 1 to 2 h at room temperature. After washing with TBS-T (3 3 5 min), the membrane was incubated with anti-rat NTCP serum (dilution 1044 Kouzuki et al. Vol. 286 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from 1:5000), which was kindly donated by Dr. PJ Meier (Stieger et al., 1994), in TBS-T containing 5% BSA overnight at 4°C and then washed with TBS-T (3 3 5 min). The membrane was allowed to bind to I-labeled sheep anti-rabbit Ig in TBS-T containing 5% BSA for 1 h at room temperature and then washed with TBS-T (3 3 5 min). After the membrane was exposed overnight to the imaging plate at room temperature, it was analyzed with Bio-Image Analyzer (Bas 2000; Fuji Film, Tokyo, Japan).