Introduction

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The primary biliary active excretion process for several organic anions after conjugation has been designated as "Phase-3" detoxification (Ishikawa, 1992) and is one of the hepatic elimination mechanisms for xenobiotics. Currently, at least three different types of transporters are considered to be present on the bile canalicular membrane and to transport xenobiotics and endogenous compounds by primary active transport driven by cellular ATP hydrolysis. Of these, 1) the first is selective for bile acids, such as TCA (Adachi et al., 1991; Nishida et al., 1991) which is the main constituent of bile acid; 2) the second transporter (cMOAT; canalicular multispecific organic anion transporter) transports organic anions including several conjugates, such as LTC₄ (Ishikawa et al., 1990; Sathirakul et al., 1994), an endogenous compound, and DNP-SG (Kobayashi et al., 1990), a glutathione-conjugate; 3) the third transporter is selective for amphipathic organic cations including anticancer drugs (Kamimoto et al., 1989), called P-glycoprotein, which is the product of the mdr gene(s) (multidrug resistance). Recent progress in this area of research has been partly due to the development and use of bile CMV (Inoue et al., 1983; Meier et al., 1984), and by the discovery of the mutant rats such as the TR strain (Jansen et al., 1985) and the EHBR strain (Mikami et al., 1986) which have an inherited deficiency in their biliary excretion of organic anions. With use of such animals, several organic anions and their conjugates have been reported to be excreted into the bile via the primary active transporter which is deficient in mutant rats (Jansen et al., 1985; Fernandez-Checa et al., 1992).

However, our own recent in vivo studies suggest that there are multiple biliary excretion mechanisms for organic anions (Sathirakul et al., 1994; Shimamura et al., 1994; Takenaka et al., 1995b). For example, in EHBR, the biliary excretion of LG glucuronides was markedly reduced, whereas that of LG-sulfate was similar to that in normal rats (Shimamura et al., 1994). Furthermore, the biliary excretion of E-glu, measured by liver perfusion, was severely impaired in EHBR, whereas no significant reduction was seen for the sulfate (E-sul) (Takenaka et al., 1995b). This result was confirmed by an in vitro uptake study with use of CMV prepared from SD rats and EHBR. The ATP-dependent uptake of DNP-SG, a typical substrate for cMOAT, was markedly reduced in EHBR, whereas that of E-glu in EHBR remained at about 30% that in SD rats (Takenaka et al., 1995a). In contrast, ATP did not stimulate the uptake of E-sul into CMV and the uptake of E-sul was similar for SD rats and EHBR (Takenaka et al., 1995a). These findings suggest that there is also an ATP-dependent primary active E-glu transporter in EHBR.

To demonstrate directly the presence of multiple biliary excretion mechanisms for conjugates, we examined the kinetics of the inhibitory effect of E-glu and E-sul on the ATP-dependent uptake of DNP-SG and vice versa.

	Materials and Methods

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Materials. Unlabeled and ¹⁴C-labeled E3040 ([2-¹⁴C]6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole dihydrochloride) (specific activity, 50.9 µCi/µmol) were supplied by Eisai Co., Ltd. (Tsukuba, Japan). Unlabeled and ³H-labeled DNP-SG (50.0 µCi/nmol) was synthesized as described previously (Kobayashi et al., 1990). [³H]TCA (3.47 µCi/nmol) and [³H]glutathione (50.0 µCi/nmol) were purchased from New England Nuclear (Boston, MA). The glucuronide and sulfate of E3040 were prepared by incubating E3040 with rat liver microsomes and cytosol, respectively, as described previously (Takenaka et al., 1995b). ATP, creatine phosphate and creatine phosphokinase were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were commercially available and were reagent grade.

Preparation of CMV. CMV were prepared from male SD rats and EHBR liver as described previously (Kobayashi et al., 1990). After suspending them in 50 mM Tris buffer (pH 7.4) containing 250 mM sucrose, the membrane vesicles were frozen in liquid N₂ and stored at 100°C until used. To check the purity of the prepared CMV, the activity of Na⁺/K⁺- and Mg⁺⁺-ATPases, alkaline phosphatase and leucine aminopeptidase activities was determined by the method of Scharschmidt et al. (1979), Yachi et al. (1989) and an leucine aminopeptidase assay kit (Wako Pure Chemical Industries, Osaka, Japan), respectively. The activity of the CMV was also checked by measuring the ATP-dependent uptake of standard substrates, [³H]TCA (1 µM) and [³H] DNP-SG (1 µM) for a 2-min incubation performed at 37°C. Protein concentrations were determined as described previously (Bradford, 1976), with the Bio-Rad protein assay kit with bovine serum albumin as a standard.

Uptake study of [³H]DNP-SG, [¹⁴C]E-glu and [³H]TCA by CMV. The uptake study of [³H]DNP-SG (1 µM; labeled, 0.1 µM; unlabeled, 0.9 µM), [¹⁴C]E-glu (20 µM) and [³H]TCA (1 µM) was performed as reported previously (Sathirakul et al., 1994; Takenaka et al., 1995a). The transport medium (10 mM Tris, 250 mM sucrose and 10 mM MgCl₂·6H₂O, pH 7.4) contained the ligands, 5 mM ATP and an ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml of creatine phosphokinase). An aliquot of transport medium (17-18 µl) was mixed rapidly with the vesicle suspension (10 µg protein in 2-3 µl). The transport reaction was stopped by the addition of 1 ml ice-cold buffer containing 250 mM sucrose, 0.1 M NaCl and 10 mM Tris-HCl (pH 7.4). The stopped reaction mixture was filtered through a 0.45-µm HA filter (Millipore Corp., Bedford, MA), and then was washed twice with 5 ml of the stop solution. The radioactivity retained on the filter and reaction mixture was combined with scintillation cocktail (Clear-sol I, Nacarai Tesque, Tokyo, Japan) and measured with a liquid scintillation counter (LS 6000SE, Beckman Instruments, Fullerton, CA). Ligand uptake was normalized in terms of the amount of membrane protein.

Determination of kinetic parameters. The kinetic parameters for ATP-dependent uptake, obtained by subtracting the value in the absence of ATP from the value in its presence of [³H]DNP-SG, [¹⁴C]E-glu and [³H]TCA were estimated according to the following equation:

(1)

where V_o is the initial uptake rate of ligand by CMV (pmol/min/mg protein), S is the ligand concentration in the medium (µM), K_m is the Michaelis constant (µM), V_max is the maximum uptake rate (pmol/min/mg protein) and P_dif is the nonspecific uptake clearance (µl/min/mg protein). Equation 1 was fitted to the uptake data by an iterative nonlinear least-squares method by use of a MULTI program (Yamaoka et al., 1981) to obtain estimates of kinetic parameters. The input data were weighted as the reciprocal of the square of the observed values, and the algorithm used for the fitting was the Damping Gauss Newton Method (Yamaoka et al., 1981).

(2)

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of DNP-SG (A) and E-glu (B) on the ATP-dependent uptake of [3H]DNP-SG (1.0 µM) () and [14C]E-glu (20 µM) () by CMV prepared from SD rats. CMV were incubated for 2 min ([3H]DNP-SG) or 1 min ([14C]E-glu) in the presence of DNP-SG (A) or E-glu (B). Each point and vertical bar represent the mean ± S.E. of three determinations of one preparation. Solid lines were obtained as follows. At first data were fitted to equation 2 and then the fitted line was converted to the Vo/S vs. normalized i (iapp) form according to equation 7. The inhibitor concentration on the abscissa represents the iapp calculated based on equation 8. The kinetic parameters obtained are listed in table 1.

(3)

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Eadie-Hofstee plots of [3H]DNP-SG uptake by CMV prepared from SD rats. CMV were incubated for 2 min with or without ATP and the ATP-regenerating system in the medium in the absence () and presence of E-glu (30 µM) () or E-sul (100 µM) (). The ATP-dependent uptake was obtained by subtracting the value in the absence of ATP from that in its presence. Each point and vertical bar represents the mean ± S.E. of five determinations of two preparations. Solid lines were obtained as described in figure 2. The kinetic parameters obtained are listed in table 1.

(4)

(5)

(6)

(7)

(8)

(9)

	Results

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ATP-Dependent Uptake of DNP-SG and E-glu

Time profiles were examined for the uptake of [³H]DNP-SG and [¹⁴C]E-glu by CMV prepared from SD rats. Marked ATP-dependence and overshoot phenomena were observed in the uptake of both compounds (fig. 1). Because uptake in the presence of 5 mM ATP and the ATP-regenerating system increased linearly up to 2 min for [³H]DNP-SG and up to 1 min for [¹⁴C]E-glu (fig. 1), in the following analyses the initial uptake rates were calculated with data at 2 min ([³H]DNP-SG) and 1 min ([¹⁴C]E-glu), respectively, after the reaction started.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Time-dependent uptake of (A) [3H]DNP-SG and (B) [14C]E-glu by CMV prepared from SD rats. After 3 min preincubation, the reaction was started by adding CMV (10 µg of protein). Reaction mixtures were incubated at 37°C with () or without () ATP (5 mM) and the ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml of creatine phosphokinase) in the medium. The concentrations of [3H]DNP-SG and [14C]E-glu were 1.0 µM and 20 µM, respectively. Each point and vertical bar represent the mean ± S.E. of three different experiments with ATP, and the mean of two different experiments without ATP.

The initial uptake of [³H]DNP-SG and [¹⁴C]E-glu into CMV in the presence and absence of ATP was measured with various ligand concentrations and the data were converted into Eadie-Hofstee plots (fig. 2). ATP-dependent uptake (obtained by subtracting the value in the absence of ATP from that in its presence) of [³H]DNP-SG by CMV from SD rats provided a K_m value of 17.6 µM and a V_max estimate of 1150 pmol/min/mg protein (fig. 2A). In [¹⁴C]E-glu with CMV from SD rats, the corresponding data consisted of a K_m of 5.7 µM and a V_max of 385 pmol/min/mg protein (fig. 2B); EHBR provided a K_m of 44.6 µM and a V_max of 207 pmol/min/mg protein (fig. 2C).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Eadie-Hofstee plots of DNP-SG initial uptake by CMV prepared from SD rats (A) and E-glu uptake by CMV prepared from SD rats (B) and EHBR (C). CMV were incubated at 37°C for 2 min (A) or 1 min (B), (C) with () or without () ATP and the ATP-regenerating system in the medium. ATP-dependent uptake () was obtained by subtracting the value in the absence of ATP from that in its presence. Each point and vertical bar represent the mean ± S.E. of five (DNP-SG), six (E-glu, SD rats) and three determinations (E-glu, EHBR) of one preparation. The solid lines and dotted line were obtained as follows. At first data were fitted to equation 1 and then the fitted line was converted into the Vo/S vs. Vo form. The kinetic parameters for ATP-dependent uptake obtained are listed in table 1.

Mutual Effect of the Uptake of DNP-SG and Glucuronide and Sulfate Conjugates

We investigated the effect of the glucuronide and sulfate conjugates of E3040 (E-glu, E-sul) and 4-methylumbelliferone (4 MUG, 4 MUS) on the uptake of [³H]DNP-SG, a typical substrate for cMOAT, using CMV from SD rats. Glucuronides (E-glu, 4-MUG) inhibited the uptake of [³H]DNP-SG in a concentration-dependent manner, whereas sulfates (E-sul, 4-MUS) did not, although enhancement of [³H]DNP-SG uptake was observed (fig. 3).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Effect of various conjugates on the initial uptake of [3H]DNP-SG (1.0 µM) by CMV prepared from SD rats. CMV were incubated at 37°C for 2 min with (open bars) or without (closed bars) ATP and the ATP-regenerating system in the medium. Each bar represents the mean ± S.E. of two to four different experiments.

In addition, we examined the mutual inhibition of the ATP-dependent DNP-SG and E-glu uptake by CMV from SD rats (fig. 4). Although E-glu also inhibited the ATP-dependent uptake of [14C]E-glu and [³H]DNP-SG in a concentration-dependent manner (fig. 4B), DNP-SG inhibited ATP-dependent [¹⁴C]E-glu uptake by only 70% even at 300 µM of the normalized concentration according to Eq. 8, while it inhibited its own uptake almost completely (fig. 4A). From equation 2, the K_i of E-glu for [³H]DNP-SG uptake was calculated as 3.84 µM and the K_i of DNP-SG for [¹⁴C]E-glu uptake was 17.1 µM.

The effect of DNP-SG on the ATP-dependent uptake of [¹⁴C]E-glu by CMV from EHBR was also examined. No inhibitory effect was observed by DNP-SG on the ATP-dependent uptake of [¹⁴C]E-glu (fig. 5). Furthermore, TCA, a bile acid, had no inhibitory effect on the ATP-dependent uptake of [¹⁴C]E-glu by CMV from either SD rats or EHBR (fig. 6). TCA rather increased the ATP-dependent uptake of [¹⁴C]E-glu by CMV from SD rats (fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of DNP-SG on the ATP-dependent uptake of [14C]E-glu (20 µM) by CMV prepared from EHBR. CMV were incubated for 1 min in the presence of DNP-SG. Each point and vertical bar represents the mean ± S.E. of six determinations of one preparation.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of TCA on the ATP-dependent uptake of [3H]TCA (1.0 µM) (), [3H]DNP-SG (1.0 µM) () and [14C]E-glu (20 µM) () by CMV prepared from SD rats (A) and EHBR (B). CMV were incubated for 1 min ([3H]TCA, [14C]E-glu) or 2 min ([3H]DNP-SG) in the presence of TCA. Each point and vertical bar represents the mean ± S.E. of three different experiments.

Because glucuronides and sulfates have different effects on the uptake of [³H]DNP-SG by CMV from SD rats (fig. 3), a kinetic analysis of these effects was performed. The concentration-dependence of ATP-dependent [³H]DNP-SG uptake in the presence and absence of E-glu (30 µM) or E-sul (100 µM) was investigated (fig. 7). In the presence of E-glu, the apparent K_m (K_m^app) increased, whereas the V_max showed only a minimal change (table 1), which suggests competitive inhibition. The E-glu K_i for ATP-dependent uptake of [³H]DNP-SG was calculated to be 11.6 µM by equation 3. By contrast, in the presence of E-sul, the apparent K_m (K_m^app) value fell by one third, whereas the V_max fell by a half (table 1).

	Discussion

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Biliary excretion is an important route for the elimination of organic anions, and mutant rat strains with conjugated hyperbilirubinemia (TR, EHBR) exhibit impaired biliary excretion involving cMOAT and its non-bile acid organic anion substrates. In these mutant rats, P-glycoprotein and the primary active transport system for bile acids remain normal (Jansen et al., 1987a, b; Kitamura et al., 1990; Nishida et al., 1992a, b). In vivo studies with the Wistar-derived transport mutant rat strain TR show that the biliary excretion of organic anions, such as LTC₄, is dramatically reduced after intravenous administration (Huber et al., 1987). The mutant SD rat strain (EHBR) also exhibits a reduced biliary excretion of organic anions (Fernandez-Checa et al., 1992; Sathirakul et al., 1993). Furthermore, the transport of organic anions across the bile canalicular membrane has been characterized in vitro with CMV and the uptake of non-bile acid organic anions, such as BSP (Kitamura et al., 1990) and LTC₄ (Ishikawa et al., 1990; Sathirakul et al., 1994), into CMV from normal rats has been found to be stimulated by ATP, whereas no such enhancement is observed in CMV from mutant rats (TR, EHBR). These studies directly show that both TR and EHBR lack the primary active transport system for organic anions which is directly coupled with ATP-hydrolysis, and also that both mutant rat strains possess similar characteristics in terms of the hepatobiliary excretion of ligands. Various organic anions have been recognized by cMOAT (Fernandez-Checa et al., 1992; Sathirakul et al., 1993; Ishikawa et al., 1990; Sathirakul et al., 1994; Yamazaki et al., 1996).

E-glu is considered to be transported predominantly by cMOAT, because its biliary excretion is also severely impaired in EHBR (Takenaka et al., 1995b). To examine this notion, we performed comparative studies of the uptake of [¹⁴C]E-glu and [³H]DNP-SG, a typical substrate for cMOAT, into CMV prepared from SD rats and EHBR. The uptake of both [³H]DNP-SG and [¹⁴C]E-glu into CMV from SD rats was stimulated by ATP (fig. 1), which suggests the contribution of a primary active transporter to the biliary excretion of these ligands. In addition, because the stimulatory effect of ATP on the uptake of [¹⁴C]E-glu into CMV from EHBR was minimal, the recognition of E-glu by cMOAT was confirmed (Takenaka et al., 1995a). However, the existence of an ATP-dependent transporter other than cMOAT was suggested for E-glu transport because a slight but significant transport stimulation by ATP was also observed in EHBR (Takenaka et al., 1995a). In the present study, we examined the mutual inhibition of DNP-SG and E-glu with CMV prepared from SD rats and EHBR to investigate these results in more detail.

[³H]DNP-SG and [¹⁴C]E-glu uptake by CMV from SD rats and [¹⁴C]E-glu uptake by CMV from EHBR were stimulated by ATP and exhibited saturation (fig. 2), which confirms that both compounds are excreted into bile by primary active transporters.

In the presence of a fixed concentration of E-glu (30 µM), the apparent K_m (K_m^app) for the ATP-dependent uptake of [³H]DNP-SG was found to be 56.8 µM, which is higher than the K_m (15.8 µM) when E-glu is not present, whereas the V_max is not very different from that in the absence of E-glu (fig. 7, table 1). The K_i of E-glu for ATP-dependent [³H]DNP-SG uptake calculated from these data (equation 3) is 11.6 µM, which is similar to its own K_m value, 5.7 µM (table 1). In addition, the K_i of DNP-SG for ATP-dependent [¹⁴C]E-glu uptake was calculated to be 17.1 µM (fig. 4A), which is also similar to the K_m (17.6 µM) for ATP-dependent [³H]DNP-SG uptake (table 1), which indicates that both DNP-SG and E-glu may act mutually and competitively to inhibit the uptake of [³H]DNP-SG and [¹⁴C]E-glu; in other words, both compounds share the common transporter at least partially.

The possibility that both these compounds share the transporter is clearly demonstrated when the data are summarized as in figure 4. Although E-glu inhibits the ATP-dependent uptake of [¹⁴C]E-glu and [³H]DNP-SG in the same manner and almost completely inhibits their uptake at its normalized concentration of 300 to 500 µM (fig. 4B), DNP-SG does not inhibit ATP-dependent [¹⁴C]E-glu uptake completely and about one-third of the uptake remained even at a normalized DNP-SG concentration of 300 µM, whereas the ATP-dependent uptake of [³H]DNP-SG itself was almost completely inhibited (fig. 4A). These mutual inhibition studies suggest that DNP-SG inhibits the ATP-dependent uptake of E-glu only partially. In other words, DNP-SG shares part of the transporter(s) for E-glu and another transporter also contributes to E-glu transport (fig. 8).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Proposed mechanism for the ATP-dependent or independent transport carriers for conjugates (DNP-SG, E-glu and E-sul) on the canalicular membrane involved in their biliary excretion.

The unidentified transporter for E-glu, which is also present in EHBR, is considered to be different from that for DNP-SG, because DNP-SG does not inhibit the ATP-dependent uptake of [¹⁴C]E-glu by CMV from EHBR (fig. 5). This is in agreement with the fact that the ATP-dependent uptake of [³H]DNP-SG by CMV from EHBR is negligible (Takenaka et al., 1995a). Moreover, this transporter for E-glu is different from the bile acid transporter, because the ATP-dependent uptake of [¹⁴C]E-glu by CMV from EHBR is not inhibited by TCA at a concentration of 300 µM (more than 10 times higher than its own K_m, 6.5 µM) (fig. 6).

Previously, we reported that the biliary excretion of E-sul is similar in both SD rats and EHBR (Takenaka et al., 1995b). Moreover, the uptake of E-sul into CMV is similar in SD rats and EHBR and is not stimulated by ATP (Takenaka et al., 1995a). Consequently, we examined the effect of E-sul on the uptake of DNP-SG by CMV. The ATP-dependent uptake of DNP-SG by CMV was enhanced by E-sul (fig. 3). This enhancement was also observed with 4 MUS (fig. 3) and was common to both sulfates. Furthermore, in the presence of E-sul (100 µM), the apparent K_m (K_m^app) value for the ATP-dependent uptake of DNP-SG by CMV fell by about one third and the V_max fell by about one half (fig. 6), which suggests that the enhancement of DNP-SG uptake by sulfates mainly is a result of increased affinity for the transporter, possibly involving a conformational change.

Based on these observations, we can propose a hypothesis as shown in figure 8 where in cMOAT, which is present in SD rats and is deficient in EHBR, recognizes both DNP-SG and E-glu as substrates. In addition, another transporter, which also recognizes only E-glu, is present. A transporter which recognizes E-glu in EHBR also exists, but at present, it is not clear whether this is the same as that in SD rats. The K_m value for ATP-dependent E-glu uptake by CMV from EHBR was about 44.6 µM, which is approximately 8-fold higher than that from SD rats (table 1). It should be noted that the standard deviation of the K_m for ATP-dependent E-glu uptake by CMV from SD rats was high, because the lowest practical substrate concentration used was 7.5 µM because of the low specific activity of [¹⁴C]E-glu. To more precisely determine the K_m value, it would be necessary to have a substrate concentration much lower than the K_m (5.7 µM). However, it may be judged from the Eadie-Hofstee plot (fig. 2C) that there is also a saturable primary active transport system in EHBR. This transporter with lower affinity may also be present in SD rats. But in the present kinetic analysis, this low-affinity component could not be detected, possibly because it may be hidden by the high-affinity component.

Although many organic anions including conjugates have been shown to be excreted into bile by cMOAT, some recent results have suggested the existence of a multiplicity of organic anion transporters. For example, we have performed a kinetic analysis of the effects of DBSP and ICG on the biliary excretion of LTC₄ and found that the predominant transport system for DBSP was different from that for ICG based on the fact that the biliary excretion of LTC₄ in SD rats was inhibited by infusion of DBSP, but not by ICG (Sathirakul et al., 1994). The biliary excretion of LG glucuronides (LG-monoglucuronide and LG-glucuronide-sulfate) is reduced in EHBR, and coadministration of glycyrrhizin (containing a glucuronide moiety) and DBSP in SD rats significantly reduces their biliary excretion, but has no effect on the biliary excretion of the disulfate (Shimamura et al., 1994). Moreover, as mentioned above, one of our studies involving the biliary excretion of E-glu and E-sul also suggests different biliary excretion mechanisms for glucuronides and sulfates (Takenaka et al., 1995b). Kobayashi et al. (1991) reported that DNP-SG and LTC₄ completely inhibit the ATP-dependent uptake of NPG by CMV, whereas NPG only inhibits DNP-SG uptake by 80% even at a concentration of 200 µM (10 times the K_m value of NPG itself).

Support for the presence of several related transporters is emerging from studies at the molecular level. For example, Paulusma et al. (1996) succeeded in cloning the cDNA of cMOAT from Wistar rat liver, and the amino acid sequence deduced was the same as that cloned by us in SD rats (Ito et al., 1997). Northern blot analysis of poly(A)⁺ RNA from the liver with the entire open reading frame of cMOAT as a probe was defective in EHBR. On the other hand, most recently, we have found the cDNA fragment that was amplified by use of primers based on the conserved sequence of the carboxy-terminal ATP-binding cassette region of human multidrug resistance-associated protein by polymerase chain reaction, and also hybridized to poly(A)⁺ RNA from the liver of EHBR (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T. and Sugiyama, Y., unpublished observation).

In conclusion, the present findings suggest the presence of multiple systems for the primary active transport of organic anions across the bile canalicular membrane; one is the predominant transporter for DNP-SG, the other is a transporter that does not recognize DNP-SG but recognizes E-glu as a substrate and is also present in EHBR.