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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Characterization of the Transport Properties of Organic Anion Transporting Polypeptide 1 (oatp1) and Na+/Taurocholate Cotransporting Polypeptide (Ntcp): Comparative Studies on the Inhibitory Effect of their Possible Substrates in Hepatocytes and cDNA-Transfected COS-7 Cells

Hirokazu Kouzuki, Hiroshi Suzuki, Bruno Stieger, Peter J. Meier and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics February 2000, 292 (2) 505-511;

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Abstract

In the present study, we compared the inhibitory effects of organic anions (including bile acids) on the uptake of taurocholate (TC) and estradiol 17β-d-glucuronide (E217βG), typical substrates for sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide (oatp1), respectively, using primary cultured rat hepatocytes and Ntcp- or oatp1-transfected COS-7 cells. The Na+-dependent uptake of TC was inhibited by nine bile acids and five nonbile acid organic anions in a concentration-dependent manner, and their inhibitory effects were similar in both primary cultured rat hepatocytes and Ntcp-transfected COS-7 cells. BQ-123 (1 μM) and indomethacin (10 μM), both of which exhibit no Ntcp-mediated transport, significantly inhibited the Na+-dependent uptake of TC mediated by Ntcp. In addition, the Na+-independent uptake of E217βG was inhibited by 15 organic anions in a concentration-dependent manner, and their inhibitory effects were similar between primary cultured rat hepatocytes and oatp1-transfected COS-7 cells. BQ-123 (1 μM), pravastatin (1 μM), and indomethacin (10 μM), all of which do not undergo oatp1-mediated transport, significantly inhibited the Na+-independent uptake of E217βG mediated by oatp1. These results are consistent with the hypothesis that the hepatic uptake of TC and E217βG is predominantly mediated by Ntcp and oatp1, respectively. In addition, it was clearly demonstrated that we cannot refer to the substrate specificity of transporters based on inhibition studies.

Recently, the molecular mechanism underlying the hepatocellular uptake of organic anions has been clarified. Using Xenopus laevis oocytes injected with mRNA from rat liver, sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide (oatp)1 have been cloned as transporters responsible for the Na+-dependent uptake of bile acids and Na+-independent uptake of organic anions, respectively (Meier, 1995). Oatp2 and oatp3 have been identified as homologs of oatp1 (Noé et al., 1997; Abe et al., 1998). Moreover, the hepatic expression of oat2 and 3, homologs of oat1 initially cloned from rat kidney, have been demonstrated (Sekine et al., 1997,1998; Kusuhara et al., 1999). The transport properties of these cDNA products have been investigated by examining the substrate uptake intoXenopus laevis oocytes injected with cRNA and/or into mammalian cells transfected with cDNA (Meier, 1995; Hagenbuch and Meier, 1996; Noe et al., 1997; Sekine et al., 1997, 1998; Kusuhara et al., 1999). It has been demonstrated that Ntcp accepts taurocholate (TC), cholate (CA), taurochenodeoxycholate (TCDCA), tauroursodeoxycholate, glychocholate (GCA), and estrone-3-sulfate as substrates (Meier, 1995; Hagenbuch and Meier, 1996; Kouzuki et al., 1998; Schroeder et al., 1998). In addition, oatp1 has been demonstrated to transport bromosulfophthalein (BSP), bile acids (such as TC and CA), anionic steroid conjugates (such as estrone-3-sulfate and estradiol 17β-d-glucuronide; E217βG), neutral steroids (such as ouabain, aldosterone, and cortisol), and even some amphipathic organic cations such as N-(4,4-azo-n-pentyl)-21-deoxyajmalinium, a permanently charged photolabile derivative of the antiarrhythmic drugN-propylajmaline (Meier, 1995; Hagenbuch and Meier, 1996), whereas oatp2 preferentially accepts digoxin as a substrate rather than oatp1 substrates (Noé et al., 1997). Substrates for oat2 includep-aminohippurate, salicylate, acetylsalicylate, prostaglandin E2, and dicarboxylate (Sekine et al., 1998) and those for oat3 include p-aminohippurate, ochratoxin A, estrone sulfate, and cimetidine, a cationic compound (Kusuhara et al., 1999).

Moreover, Hagenbuch et al. (1996) and our group (Kouzuki et al., 1998,1999; Akhteruzzaman et al., 1999) have determined the contribution of Ntcp and oatp1 to the hepatic uptake of their ligands. By examining the effect of simultaneous injection of specific antisense sequences against Ntcp and oatp1, Hagenbuch et al. (1996) found that >95% of TC transport is mediated by Ntcp. Our group has suggested that the uptake of E217βG by hepatocytes is predominantly mediated by oatp1, based on the agreement in theVmax value for the uptake of E217βG and the expression level of oatp1 protein between hepatocytes and transfected COS-7 cells (Kouzuki et al., 1999). Moreover, we have compared the ligand uptake into hepatocytes and into COS-7 cells transfected with Ntcp and oatp1 cDNAs and normalized the ligand uptake with standard substrates (TC and E217βG for Ntcp and oatp1, respectively) to determine the contribution of these cloned transporters (Kouzuki et al., 1998, 1999).

The details of the hepatic uptake of organic anions, however, still remain to be clarified. For example, although hepatic uptake of cyclic peptide (BQ-123; an endothelin antagonist) is mediated, at least in part, by an Na+-dependent transporter that is competitively inhibited by TC and, moreover, the Na+-dependent uptake of TC is inhibited by BQ-123 (Nakamura et al., 1996), no Ntcp-mediated transport of BQ-123 is detected if COS-7 cells are used for Ntcp cDNA transfection (Akhteruzzaman et al., 1999). This complexity of Na+-dependent transport has also been reported for Na+-dependent uptake of bumetanide (Blitzer et al., 1982; Petzinger et al., 1989, 1996). As far as oatp1 is concerned, the same paradox has been reported for BQ-123 (Nakamura et al., 1996), glucuronide-conjugates of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040; Takenaka et al., 1997), and a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor (pravastatin;Yamazaki et al., 1993). In the present study, we have characterized the nature of the transport mediated by Ntcp and oatp1, particularly focusing on the inhibitory effect of a series of bile acids and nonbile acid organic anions on the uptake of their typical substrates (TC and E217βG, respectively) by hepatocytes and cDNA-transfected COS-7 cells.

Experimental Procedures

Materials.

COS-7 cells were purchased from American Type Culture Collection (Rockville, MD). [3H]TC (128.4 GBq/mmol) and [3H]E217βG (1813 GBq/mmol) were purchased from New England Nuclear (Boston, MA). TC, CA, GCA, chenodeoxycholate (CDCA), TCDCA, litocholate (LCA), deoxycholate (DCA), ursodeoxycholate (UDCA), glycochenodeoxycholate (GCDCA), indomethacin, and probenecid were purchased from Wako Pure Chemical Industries (Osaka, Japan). E217βG, benzylpenicillin (BZP) and estrone-3-sulfate were purchased from Sigma Chemical (St. Louis, MO). Dibromosulfophthalein (DBSP) was obtained from the Societe d'Etudes et de Recherches Biologiques (Paris, France). 7,8-Dihydro-5-[(E)-[[a-(3-pyridyl)benzylidene]aminooxy]ethyl]-1-naphthyoxy]acetic acid (ONO-1301) was kindly donated by ONO Pharmaceutical Co., Ltd. (Osaka, Japan). BQ-123 and a linear peptide (BQ-485) were donated by Banyu Pharmaceutical Co., Ltd. (Ibaraki, Japan). The glucuronide and sulfate conjugates of E3040, prepared according to the method described previously (Hibi et al., 1994), were donated by Eizai Co., Ltd. (Tokyo, Japan). Pravastatin was a gift from Sankyo Co., Ltd. (Tokyo, Japan). The glutathione conjugate of BSP (BSP-SG) was synthesized by the method described by Saxena and Henderson (1995) using BSP (Aldrich, Milwaukee, WI) and glutathione (Wako, Osaka, Japan). All other chemicals were commercially available and of reagent grade.

Transient Expression of Ntcp and oatp1 cDNA in COS-7 Cells.

The pCAGGS plasmids containing Ntcp and oatp1 cDNAs were used in the present study. The structure of the plasmid construct has been described previously (Kouzuki et al., 1998, 1999).

The procedure for the transfection of plasmids into COS-7 cells has been described previously (Kouzuki et al., 1998, 1999). Briefly, COS-7 cells were cultured in 150-mm dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum. At 30% confluence, cells were exposed to serum-free DMEM containing plasmid (1 μg/ml) and Lipofectamine (1 μg/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 cultured overnight. Then, the transfected cells were treated with trypsin and approximately 1.6 × 105 cells were seeded onto 22-mm dishes and cultured overnight. An uptake study was performed at 48 h after transfection.

Primary Cultured Rat Hepatocytes.

The procedure for the preparation of primary cultured rat hepatocytes has been described previously (Kouzuki et al., 1998, 1999). Briefly, rat hepatocytes were isolated from male Sprague-Dawley rats (200–250 g, Japan Laboratory Animal Inc., Tokyo, Japan) after perfusing the liver with collagenase. Cell viability was routinely checked by the trypan blue [0.4% (w/v)] exclusion test. After preparation, freshly isolated cells were suspended in Williams' medium E. Approximately 5 × 105 cells were placed on collagen-coated 22-mm dishes and cultured for 4 h.

Inhibition Study.

Uptake of [3H]TC and [3H]E217βG (1 μM) was examined by the method described previously (Kouzuki et al., 1998,1999). Briefly, the experiments were initiated by adding the radiolabeled ligands to the medium in the absence or presence of inhibitors, after washing the cultured cells three times and preincubating with Krebs-Henseleit buffer or choline buffer at 37 for 5 min. The Krebs-Henseleit buffer consisted of 142 mM NaCl, 23.8 mM Na2CO3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM glucose and 1.53 mM CaCl2 adjusted to pH 7.3. The composition of the choline buffer was the same as that of the Krebs-Henseleit buffer except that NaCl and NaHCO3 were replaced with isotonic choline chloride and choline bicarbonate, respectively. At designated times, the reaction was terminated by adding ice-cold Krebs-Henseleit buffer. Just before the designated times, 50 μl of medium was transferred to scintillation vials. Then, cells were washed three times with 2 ml of ice-cold Krebs-Henseleit buffer and solubilized in 500 μl of 1 N NaOH. After adding 500 μl of distilled water, 800-μl aliquots were transferred to scintillation vials. The radioactivity associated with the cells and medium was determined in a liquid scintillation counter (LS 6000SE; Beckman Instruments, Inc., Fullerton, CA) after adding 8 ml of scintillation fluid (Hionic flow; Packard Instrument Co., Downers Grove, IL) to the scintillation vials. The remaining 100-μl aliquots of cell lysate were used to determine protein concentrations by the method of Lowry et al. (1951) with BSA as a standard. Ligand uptake is given as the cell-to-medium concentration ratio, determined as the amount of ligand associated with the cells divided by the medium concentration.

Because the initial velocity of the uptake of TC and E217βG was linear up to 2 and 1 min, respectively, the uptake of these ligands at respective times was used to determine the kinetic parameters.Ki values of a series of anionic compounds for the uptake of TC (1 μM) and E217βG (1 μM) were calculated assuming a competitive inhibition. Na+-dependent uptake was calculated by subtracting the Na+-independent uptake (measured in choline buffer) from the total uptake (measured in Krebs-Henseleit buffer).

Results

Inhibitory Effect of Organic Anions on the Uptake of TC.

The inhibitory effects of bile acids on the Na+-dependent uptake of TC by primary cultured rat hepatocytes and Ntcp-transfected COS-7 cells were investigated at several concentrations (1–300 μM). The uptake of TC (1 μM) was inhibited by bile acids in a concentration-dependent manner in these two cell types (Fig. 1). Among them, TCDCA and GCDCA were potent inhibitors compared with GCA, CA, and LCA (Fig. 1).

Figure 1
Figure 1

Comparison of the inhibition of [3H]TC uptake by bile acids in primary cultured rat hepatocytes (left) and Ntcp-transfected COS-7 cells (right). Uptake of [3H]TC (1 μM) was measured for 2 min in the presence or absence of Na+. Inhibitors used were 10 to 300 μM GCA (A, ▪), 1 to 300 μM TC (A, ■), 10 to 300 μM GCDCA (A, ●), 10 to 300 μM UDCA (B, ○), 10 to 300 μM DCA (B, ▴), 10 to 300 μM CDCA (B, ▵), 10 to 300 μM LCA (C, ▾), 10 to 300 μM CA (C, ▿), and 10 to 300 μM TCDCA (C, ⋄). Each symbol and vertical bar represent the mean ± S.E. of six determinations in two different preparations.

In the same manner, other nonbile acid organic anions inhibited the Na+-dependent uptake of TC by these two cell types in a concentration-dependent manner (Fig.2). Of these, ONO-1301 was a potent inhibitor compared with BQ-123, DBSP, and indomethacin (Fig. 2). For the uptake of TC, a significant correlation was observed in the inhibitory effect of bile acids and nonbile acid organic anions (100 and 300 μM) between Ntcp-transfected COS-7 cells and primary cultured hepatocytes (Figs. 3 and4).

Figure 2
Figure 2

Comparison of the inhibition of [3H]TC uptake by organic anions in primary cultured rat hepatocytes (left) and Ntcp-transfected COS-7 cells (right). Uptake of [3H]TC (1 μM) was measured for 2 min in the presence or absence of Na+. Inhibitors used were 10 to 300 μM DBSP (A, ▪), 10 to 300 μM indomethacin (A, ■), 1 to 300 μM ONO-1301 (A, ●), 10 to 300 μM BQ-123 (B, ○), and 10 to 300 μM BQ-485 (B, ▴). Each symbol and vertical bar represent the mean ± S.E. of six determinations in two different preparations.

Figure 3
Figure 3

Correlation of inhibition of [3H]TC uptake by bile acids in primary cultured rat hepatocytes and Ntcp-transfected COS-7 cells. All data were calculated using the data shown in Fig. 1. Concentrations of inhibitors were 10 (A), 100 (B), and 300 μM (C). Key: ▿, CA; ▪, GCA; ▾, LCA; ○, UDCA; ▴, DCA; ■, TC; ▵, CDCA; ●, GCDCA; ⋄, TCDCA. Each symbol and vertical bar represent the mean ± S.E. of six determinations in two different preparations.

Figure 4
Figure 4

Correlation of inhibition of [3H]TC uptake by organic anions in primary cultured rat hepatocytes and Ntcp-transfected COS-7 cells. All data were calculated using the data shown in Fig. 3. Concentrations of inhibitors were 10 (A), 100 (B), and 300 μM (C). Key: ■, indomethacin; ▪, DBSP; ○, BQ-123; ▴, BQ-485; ●, ONO-1301. Each symbol and vertical bar represent the mean ± S.E. of six determinations in two different preparations.

Inhibitory Effect of Organic Anions on the Uptake of E217βG.

The inhibitory effects of organic anions on the Na+-independent uptake of E217βG by primary cultured rat hepatocytes and oatp1-transfected COS-7 cells were investigated at several concentrations (1–300 μM). The uptake of E217βG (1 μM) was inhibited by bile acids (TC and CA) and by nonbile acid organic anions in a concentration-dependent manner in these two cell types (Fig. 5). Of these, ONO-1301, estrone-3-sulfate and BQ-485 were potent inhibitors compared with BQ-123, BSP-SG, E3040 glucuronide, probenecid, and BZP (Fig. 5). For the uptake of E217βG, a significant correlation was observed in the inhibitory effect of organic anions (10, 100, and 300 μM) between oatp1-transfected COS-7 cells and primary cultured hepatocytes (Fig.6).

Figure 5
Figure 5

Inhibition of [3H]E217βG uptake by organic anions in primary cultured rat hepatocytes (left) and oatp1-transfected COS-7 cells (right). Uptake of [3H]E217βG (1 μM) was measured for 1 min in the presence of Na+. Inhibitors used were 10 to 300 μM TC (A, ▪), 10 to 300 μM CA (A, ■), 10 to 300 μM CDCA (A, ●), 10 to 300 μM pravastatin (B, ○), 10 to 300 μM indomethacin (B, ▴), 1 to 300 μM ONO-1301 (B, ▵), 10 to 300 μM E3040 glucuronide (C, ▾), 1 to 300 μM estrone-3-sulfate (C, ▿), 1 to 300 μM E3040 sulfate (C, ♦), 10 to 300 μM probenecid (D, ⋄), 10 to 300 μM BQ-123 (D, ▪), 10 to 300 μM BQ-485 (D, ■), 10 to 300 μM BZP (E, ●), 10 to 300 μM BSP-SG (E, ○), and 1 to 300 μM DBSP (E, ▴). Each symbol and vertical bar represent the mean ± S.E. of six determinations in two different preparations.

Figure 6
Figure 6

Correlation of inhibition of [3H]E217βG uptake by organic anions in primary cultured rat hepatocytes and oatp1-transfected COS-7 cells. All data were calculated using the data shown in Fig. 5. Concentrations of inhibitors were 10 (A), 100 (B), and 300 μM (C). Key: 1, BSP-SG; 2, BZP; 3, BQ-123; 4, E3040 glucuronide; 5, indomethacin; 6, TC; 7, pravastatin; 8, probenecid; 9, CA; 10, E3040 sulfate; 11, DBSP; 12, CDCA; 13, estrone-3-sulfate; 14, BQ-485; 15, ONO-1301. Each symbol and vertical bar represent the mean ± S.E. of six determinations in two different preparations.

Discussion

In the present study, we compared the inhibitory effects on the uptake of TC and E217βG in primary cultured rat hepatocytes and Ntcp- or oatp1-transfected COS-7 cells. The Na+-dependent and/or Ntcp-mediated uptake of TC was inhibited by bile acids in a concentration-dependent manner and their inhibitory effects were similar in both primary cultured rat hepatocytes and Ntcp-transfected COS-7 cells (Figs. 1 and 3). The rank order of the Ki values was TCDCA < GCDCA < CDCA DCA UDCA GCA < CA < LCA in rat hepatocytes and Ntcp-transfected COS-7 cells (Fig. 1). These results should be compared with the previously reported kinetic parameters. With sinusoidal membrane vesicles, Zimmerli et al. (1989) found that the Na+-dependent uptake of TC was competitively inhibited by CA (Ki = 140 μM), TCDCA (Ki = 9 μM), and CDCA (Ki = 53 μM). In addition, it has been shown that the Ntcp-mediated uptake of TC is inhibited to approximately 10 to 20% by TCDCA (100 μM), to 20 to 30% by CDCA (100 μM), to 20 to 50% by UDCA (100 μM), to 30 to 50% by CA (100–200 μM), and to 60% by GCA (100 μM), whereas DCA (100 μM) does not significantly inhibit the uptake in oocytes injected with cRNA and mammalian cells transfected with cDNA (Hagenbuch et al., 1991;Boyer et al., 1994; Platte et al., 1996). The results of the present study are in good agreement with the previous reports, although we have no good reason to account for the discrepancy in the inhibitory nature of DCA between the previous study (Platte et al., 1996) and the present one (Figs. 1 and 3).

We also examined the inhibitory effects of nonbile acid organic anions on TC transport. The Na+-dependent and/or Ntcp-mediated uptake of TC was inhibited by organic anions in a concentration-dependent manner (Fig. 2), and their inhibitory effects were similar in both primary cultured rat hepatocytes and Ntcp-transfected COS-7 cells (Fig. 4). The rank order ofKi values was ONO-1301 < BQ-485 DBSP indomethacin < BQ-123 in the Ntcp-transfected COS-7 cells (Fig. 2). The inhibitory effect of ONO-1301 is consistent with our previous findings; we have reported that the Na+-dependent uptake of TC into isolated rat hepatocytes was inhibited by ONO-1301 with an IC50 of ∼10 μM (Imawaka and Sugiyama, 1998). In addition, we could demonstrate that the transfection of Ntcp cDNA into COS-7 cells stimulated the Na+-dependent uptake of ONO-1301 (H. Imawaka and Y.S., unpublished observations). The inhibitory effect of BQ-123 and indomethacin needs to be discussed. Nakamura et al. (1996) found that BQ-123 is taken up by isolated hepatocytes, at least in part, in an Na+-dependent manner (Km = 5.9 μM), which was competitively inhibited by BQ-485 (Ki= 1.6 μM) and TC (Ki = 9.1 μM). The calculated Ki value (9.1 μM) of TC for the Na+-dependent uptake of BQ-123 was comparable with the Km value for the uptake of TC itself (13.1 μM), which is consistent with the hypothesis that the Na+-dependent uptake of BQ-123 may be mediated by Ntcp (Nakamura et al., 1996). However, we found that the contribution of Ntcp to the Na+-dependent uptake of BQ-123 was negligible (Akhteruzzaman et al., 1999). Moreover, although we found that indomethacin is partly taken up by rat hepatocytes in an Na+-dependent manner, it was revealed that this drug transport was not significantly mediated by Ntcp expressed on COS-7 cells (Kouzuki et al., 2000). Thus, it appears that both BQ-123 and indomethacin reduced the Na+-dependent uptake of TC by hepatocytes and that by transfected COS-7 cells, to a comparable degree without being substrates for this transporter. These ligands may act as “antagonists” for Ntcp. Moreover, the presence of multiplicity for Na+-dependent transport of organic anions should be assumed. Although one of the candidates is microsomal epoxide hydrolase (von Dippe et al., 1996), the presence of additional transporter(s) should be hypothesized.

Furthermore, we also examined the inhibitory effects of organic anions (including bile acids) on the uptake of E217βG. The Na+-independent and/or oatp1-mediated uptake of E217βG was inhibited by organic anions in a concentration-dependent manner and their inhibitory effects were similar in both primary cultured rat hepatocytes and oatp1-transfected COS-7 cells (Figs. 5 and 6). The rank order ofKi values was ONO-1301 < estrone-3-sulfate BQ-485 CDCA DBSP E3040 sulfate < indomethacin TC CA pravastatin < E3040 glucuronide probenecid BQ-123 BZP BSP-SG in the oatp1-transfected COS-7 cells. The inhibition of oatp1-mediated E217βG transport by TC, CA, and estrone-3-sulfate is consistent with the previous results that the hepatic uptake of these organic anions shares a common mechanism (Anwer and Hegner, 1978; Brouwer et al., 1987), which were confirmed by the studies with cloned oatp1 product (Jacquemin et al., 1994; Kanai et al., 1996; Bossuyt et al., 1996). The inhibitory effect of TC, CA, estrone-3-sulfate, and E3040 sulfate is also consistent with our previous kinetic analysis, which indicated that the uptake of these substrates is mediated partly by oatp1; we found that the contribution of oatp1 to the Na+-independent uptake of TC and CA into rat hepatocytes was more than 50 to 60%, whereas the corresponding values for the estrone-3-sulfate and E3040 sulfate were 20 to 30% (Kouzuki et al., 1999). Moreover, the potency of the inhibitory effect of TC, estrone-3-sulfate, and E3040 sulfate agreed with their Km values; theKm values for TC and estrone-3-sulfate were determined as 50 and 4.5 μM in Xenopus laevis oocytes injected with oatp1 cRNA (Kullak-Ublick et al., 1994; Bossuyt et al., 1996), whereas the Km value of E3040 sulfate was 25 μM in isolated rat hepatocytes (Takenaka et al., 1997). Much less potency of inhibition of BZP may be ascribed to its low affinity to the uptake transporter (Km = 473 μM; Tsuji et al., 1986). The inhibition potency of small peptides (BQ-123 and BQ-485) is also consistent with our previous results in isolated rat hepatocytes; the Km value for the Na+-independent uptake of BQ-123 (12 μM) into rat hepatocytes was higher than the Kivalue of BQ-485 (2.5 μM) on the uptake of BQ-123 (Nakamura et al., 1996). However, the inhibitory effect of BQ-123, E3040 glucuronide, and pravastatin should be discussed in more detail, in relation to the previous findings. Nakamura et al. (1996), Takenaka et al. (1997), andYamazaki et al. (1993) found that DBSP competitively inhibits the Na+-independent uptake of BQ-123 (Km = 12 μM), E3040 glucuronide (Km = 59 μM), and pravastatin (Km = 29 μM) withKi values of 3.5, 8.4, and 6.3 μM, respectively. If we consider the facts that: 1) DBSP is taken up by isolated rat hepatocytes via an Na+-independent transport system; and 2) the Km value for the uptake of DBSP (2.1 μM; Blom et al., 1981) is comparable with the previously described Ki values, it is plausible that the uptake of BQ-123, E3040 glucuronide, and pravastatin is mediated by oatp1. However, we found that the contribution of oatp1 to the Na+-independent uptake of these three organic anions was minimal as far as COS-7 cells were used (Kouzuki et al., 1999). Moreover, we found that transporter(s) other than oatp1 may be responsible for the Na+-independent hepatic uptake of indomethacin (Kouzuki et al., 2000). Collectively, BQ-123, E3040 glucuronide, pravastatin, and indomethacin inhibit the Na+-independent uptake of E217βG by hepatocytes and that by transfected COS-7 cells to a comparable degree without being substrates for this transporter. These ligands act as “antagonists” for oatp1.

Collectively, for the uptake of both TC and E217βG, the pattern of inhibition observed in hepatocytes and transfected COS-7 cells was similar (Figs. 3, 4, and6). These results can be accounted for by assuming that the hepatic uptakes of TC and E217βG are almost predominantly mediated by Ntcp and oatp1, respectively, or alternatively, by assuming the presence of additional transporters with similar transport kinetics. Although several pieces of evidence suggest that the former hypothesis may hold (Hagenbuch et al., 1996; Kouzuki et al., 1998, 1999), we cannot exclude the latter possibility. This latter hypothesis is consistent with the observation that the inhibition of oatp1-transfected COS-7 cells exceeds that of hepatocytes (Fig. 6). However, it is difficult to clearly demonstrate the presence of multiple transport systems for E217βG, due to the difficulty in finding oatp1-specific inhibitors. Moreover, the presence of multiple transport systems for E217βG could not be demonstrated by examining its transport kinetics because saturable transport of E217βG by isolated rat hepatocytes could be described by assuming one saturable component (Kouzuki et al., 1999).

In conclusion, by comparing the inhibitory effect of a series of organic anions (including bile acids) on the uptake of TC and E217βG in primary cultured rat hepatocytes and Ntcp- or oatp1-transfected COS-7 cells, we showed that several ligands, which may not be transported to a significant degree via Ntcp or oatp1, inhibit the uptake of TC and E217βG. These results indicate that we cannot refer to the substrate specificity of transporters based on inhibition studies. Moreover, the comparability of inhibition efficiency of a series of inhibitors on the uptake of TC and E217βG in primary cultured rat hepatocytes and cDNA-transfected COS-7 cells suggests that the hepatic uptakes of TC and E217βG are mediated by Ntcp and oatp1, respectively, or by other transporter(s) with similar transport kinetics.

Acknowledgments

We thank ONO Pharmaceutical Co., Ltd. for providing ONO-1301, Banyu Pharmaceutical Co., Ltd. for providing BQ-123 and BQ-485, Eizai Co., Ltd. for providing E3040 glucuronide and E3040 sulfate, and Sankyo Co., Ltd. for providing pravastatin.

Footnotes

  • Send reprint requests to: Yuichi Sugiyama, Ph.D., Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail:sugiyama{at}seizai.f.u-tokyo.ac.jp.

  • Abbreviations:
    Ntcp
    sodium taurocholate cotransporting polypeptide
    oatp
    organic anion transporting polypeptide
    TC
    taurocholate
    E217βG
    estradiol 17β-d-glucuronide
    BSP
    bromosulfophthalein
    CA
    cholate
    TCDCA
    taurochenodeoxycholate
    CDCA
    chenodeoxycholate
    E3040
    6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole
    DBSP
    dibromosulfophthalein
    GCA
    glycocholate
    LCA
    litocholate
    DCA
    deoxycholate
    UDCA
    ursodeoxycholate
    GCDCA
    glycochenodeoxycholate
    BZP
    benzylpenicillin
    ONO-1301
    7,8-dihydro-5-[(E)-[[a-(3-pyridyl)benzylidene]aminooxy]ethyl]-1-naphthyoxy]acetic acid
    BSP-SG
    glutathione-conjugate of bromosulfophthalein
    DMEM
    Dulbecco's modified Eagle's medium
    • Received July 19, 1999.
    • Accepted September 29, 1999.

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Characterization of the Transport Properties of Organic Anion Transporting Polypeptide 1 (oatp1) and Na+/Taurocholate Cotransporting Polypeptide (Ntcp): Comparative Studies on the Inhibitory Effect of their Possible Substrates in Hepatocytes and cDNA-Transfected COS-7 Cells

Hirokazu Kouzuki, Hiroshi Suzuki, Bruno Stieger, Peter J. Meier and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics February 1, 2000, 292 (2) 505-511;
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