Abstract
Eisai hyperbilirubinemic rat (EHBR) is a mutant strain with a hereditary defect in canalicular multispecific organic anion transporter (cMOAT). We examined the uptake and mutual inhibition of S-(2,4-dinitrophenyl)-glutathione (DNP-SG), which is a typical substrate for cMOAT, and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) glucuronide (E-glu) with canalicular membrane vesicles (CMV) prepared from Sprague-Dawley (SD) and EHBR rats to investigate the multiplicity of the organic anion transporter. The ATP-dependent uptake by CMV from SD rats had an apparent Km of 17.6 μM for DNP-SG and 5.7 μM for E-glu, whereas the corresponding uptake by CMV from EHBR had an apparent Km of 44.6 μM for E-glu. The effects of E-glu, 4-methylumbelliferone glucuronide (4 MUG), E3040 sulfate (E-sul) and 4-methylumbelliferone sulfate (4 MUS) on the uptake of [3H]DNP-SG were also examined. The uptake of [3H]DNP-SG was inhibited by glucuronides (E-glu and 4 MUG) in a concentration-dependent manner, although it was enhanced by the sulfate conjugates (E-sul and 4 MUS). This enhancement was shown to be caused by an increased DNP-SG affinity for the transporter. In CMV from SD rats, although ATP-dependent uptake of [3H]DNP-SG was almost completely inhibited by E-glu, that of [14C]E-glu was only reduced to about 30% of controls by DNP-SG. On the other hand, in CMV from EHBR, the ATP-dependent uptake of [14C]E-glu was not inhibited at all by DNP-SG. Kinetic analysis indicated that E-glu inhibited DNP-SG uptake competitively. In conclusion: 1) cMOAT recognizes both DNP-SG and E-glu, and another transporter present in SD rats is also involved in E-glu transport along with cMOAT; 2) the latter transporter is kinetically similar to the E-glu transporter present in EHBR; 3) E-sul enhances the uptake of DNP-SG by increasing the affinity of glucuronide for the transporter.
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 (Kamimotoet 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) (Takenakaet 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 andvice versa.
Materials and Methods
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 and3H-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 (Takenakaet 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.
Male SD rats (250–300 g b.wt.) from Charles River Japan Inc. (Kanagawa, Japan) and male EHBR (270–360 g) from Eisai laboratories (Gifu, Japan) were used to obtain the CMVs.
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 Scharschmidtet 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:
The Ki
value was calculated by use of equation 2 from the inhibition data (see fig. 4) obtained by varying the inhibitor concentration (i) while the [3H]DNP-SG (1.0 μM) or [14C]E-glu (20 μM) concentration was kept constant
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 V o/S vs. normalized i(i app) form according to equation 7. The inhibitor concentration on the abscissa represents thei app calculated based on equation 8. The kinetic parameters obtained are listed in table 1.
Kinetic parameters of ATP-dependent uptake of DNP-SG and E-glu into CMV from SD rats and EHBR
The apparent kinetic parameters (Km
app,V
max
app andP
dif
app) for ATP-dependent uptake of DNP-SG in the presence of E-glu were also estimated by fitting the data (see fig. 7) to equation 1. The inhibition constant (Ki
) for E-glu was also calculated with the following equation, assuming competitive inhibition:
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.
Equation 1 can be represented as equation 4 for the ATP-dependent uptake of DNP-SG and E-glu:
When Km
≫ S, that is, the substrate concentration is much less than theKm
value, equation 6 becomes:
Results
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.
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 aKm value of 17.6 μM and aV 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 Km of 5.7 μM and a V max of 385 pmol/min/mg protein (fig. 2B); EHBR provided a Km of 44.6 μM and a V max of 207 pmol/min/mg protein (fig. 2C).
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 V o/S vs. V o 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).
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, theKi of E-glu for [3H]DNP-SG uptake was calculated as 3.84 μM and the Ki 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).
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.
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 Km (Km app) increased, whereas the V max showed only a minimal change (table 1), which suggests competitive inhibition. The E-glu K ifor 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 apparentKm (Km app) value fell by one third, whereas the V max fell by a half (table 1).
Discussion
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 (Takenakaet 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 Km (Km app) for the ATP-dependent uptake of [3H]DNP-SG was found to be 56.8 μM, which is higher than the Km (15.8 μM) when E-glu is not present, whereas the V maxis not very different from that in the absence of E-glu (fig. 7, table 1). The Ki 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 ownKm value, 5.7 μM (table 1). In addition, the Ki of DNP-SG for ATP-dependent [14C]E-glu uptake was calculated to be 17.1 μM (fig. 4A), which is also similar to theKm (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).
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 Km , 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 Km (Km app) value for the ATP-dependent uptake of DNP-SG by CMV fell by about one third and theV 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. TheKm 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 Km 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 Km value, it would be necessary to have a substrate concentration much lower than theKm (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 theKm 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.
Footnotes
-
Send reprint requests to: Yuichi Sugiyama, Ph.D., Professor and Chair, Faculty of Pharmaceutical Sciences, University of Tokyo, 7–3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan.
-
↵1 This study was supported in part by a Grant-in Aid for Scientific Research provided by the Ministry of Education, Science and Culture of Japan.
- Abbreviations:
- DNP-SG
- S-(2,4-dinitrophenyl)-glutathione
- EHBR
- Eisai hyperbilirubinemic rat
- cMOAT
- canalicular multispecific organic anion transporter
- CMV
- canalicular membrane vesicles
- SD rat
- Sprague-Dawley rat
- E3040
- 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole
- E-glu
- E3040-glucuronide
- E-sul
- E3040-sulfate
- 4 MU
- 4-methylumbelliferone
- 4 MUG
- 4 MU-glucuronide
- 4 MUS
- 4 MU-sulfate
- LTC4
- leukotriene C4
- BSP
- sulfobromophthalein
- DBSP
- dibromosulfophthalein
- ICG
- indocyanine green
- LG
- Liquiritigenin
- Km
- Michaelis constant
- Vmax
- maximum transport velocity
- Pdif
- nonspecific diffusion clearance
- Ki
- inhibitory constant
- TCA
- taurocholate
- NPG
- p-nitrophenyl glucuronide
- Received September 4, 1996.
- Accepted April 8, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
