Introduction

Top Abstract Introduction Materials and methods Results Discussion References

The vascular endothelium releases a variety of vasoactive substances such as prostacyclin, endothelium-derived relaxing factor (nitric oxide), and endothelin (ET). Although ET-1 was originally identified as a 21-amino acid vasoconstrictor peptide in the supernatant of cultured porcine aortic endothelial cells, it has also been found in humans (Yanagisawa et al., 1989). Several studies have characterized two ET receptor subtypes, ET_A and ET_B, in mammalian systems, and considerable effort has been devoted to identifying ET receptor antagonists because such compounds may lead to useful therapeutic agents (Doherty, 1992).

BQ-123 is a cyclic pentapeptide ET_A receptor antagonist that has been isolated from Streptomyces misakiensis (Ihara et al., 1991; Kojiri et al., 1991). This compound acts as a potent and selective ET_A receptor antagonist not only in vitro but also in vivo. It is expected to be a useful therapeutic agent for the treatment of ET-related human diseases such as renal failure (Chan et al., 1994; Mino et al., 1992;). However, because the drug is rapidly eliminated in bile without any biotransformation after i.v. administration (Nakamura et al., 1996; Shin et al., 1996), longer-acting ET-receptor antagonists will be required to treat chronic diseases.

Highly efficient biliary excretion has also been found for several types of small peptides such as somatostatin analogs and renin inhibitors (Greenfield et al., 1989; Lemaire et al., 1989; Cathapermal et al., 1991). Some of these compounds are reported to be taken up by hepatocytes via active transport systems that also recognize bile acids and/or organic anions or cations (Bertrams et al., 1991; Ziegler et al., 1988, 1991; Terasaki et al., 1995). In addition, a few of them are reported to be concentratively excreted into bile via the primary active transport systems located at the bile canalicular membrane (Ziegler et al., 1994; Yamada et al., 1996; Takahashi et al., 1997). Thus, transporters on both the sinusoidal and canalicular membranes represent the major elimination mechanism for these peptides from the circulating plasma into the bile. To design longer-acting therapeutic peptides, understanding of factors affecting the specificity of such transporters has to be considered.

Although the uptake mechanism of peptides has been extensively investigated, there is only limited information on the transport mechanism at the bile canalicular membrane. In this study, we synthesized three derivatives of BQ-123, including a linear anionic peptide, BQ-485, and a zwitterionic peptide, compound A. Using isolated canalicular membrane vesicles (CMVs), we examined their biliary excretion mechanism in rats. Although we previously reported that the biliary excretion clearance of compound A is much lower than that of BQ-123 (Kato et al., 1999), their uptake by isolated rat hepatocytes is not very different (S. Akhteruzzaman, Y. Kato, H. Kouzuki, H. Suzuki, A. Hisaka, B. Stieger, P. J. Meier, and Y. Sugiyama, submitted). Therefore, it is possible that these two compounds differ in their transport across the bile canalicular membrane.

The existence of an ATP-driven primary active transport system on the bile canalicular membrane of hepatocytes has been demonstrated for many types of endogenous and exogenous compounds (Yamazaki et al., 1996; Müller et al., 1997). These include P-glycoprotein, which recognizes amphipathic cationic and neutral compounds, canalicular multispecific organic anion transporter (cMOAT), and canalicular bile acid transporter (cBAT). BQ-123 transport across the bile canalicular membrane is mainly governed by cMOAT (Shin et al., 1997); accordingly, there may be a large difference in transport activity by cMOAT between BQ-123 and compound A. The purpose of the present study was to clarify the biliary excretion mechanism for the BQ-123 derivatives. We examined the involvement of cMOAT in their biliary excretion using CMV obtained from normal Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR), which have a hereditary defect of cMOAT. The transport activity mediated by cMOAT was different for each compound and that for compound A was by far the lowest.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Chemicals and Reagents. BQ-123 (cyclo[D-Trp-D-Asp-L-Pro-D-Val-L-Leu]), BQ-485 (perhydroazepino-N-carbonyl-L-Leu-D-Trp-D-Trp), BQ-518 (cyclo[D-Trp-D-Asp-L-Pro-D-Thg-L-Leu]), compound A (cyclo[D-Trp-D-Asp-L-Hyp(L-Arg)-D-Val-L-Leu]), BQ-587 (cyclo[D-Trp-D-Asp-L-Hyp(N, N-dimethyl-L-Lys)-D-Val-L-Leu]), compound B (perhydroazepino-N-carbonyl-L-Leu-D-Trp-2-aminobutyric acid), and compound C [cyclo(D-Trp(COOMe)-D-Asp-L-Pro-D-Val-L-Leu)] were synthesized at the Tsukuba Research Institute of Banyu Pharmaceutical Co., Ltd. (Tsukuba, Japan). [prolyl-3,4(n)-³H]BQ-123 (31.0 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK) and [³H(G)]taurocholic acid ([³H]TCA) (2.0 Ci/mmol) was purchased from New England Nuclear (Boston, MA). [³H]Dinitrophenyl-glutathione ([³H]DNP-SG) was synthesized with radiolabeled glutathione (0.25 mM) and 1-chloro-2,4-dinitrobenzene (0.4 mM) in the presence of glutathione S-transferase according to the method described by Kobayashi et al. (1990). ATP, creatine phosphate, and creatine phosphokinase were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals and reagents were commercial products of analytical grade.

Animals. Male SD rats, weighing approximately 250 to 300 g, were purchased from Nisseizai (Tokyo, Japan), whereas male EHBR, weighing 250 to 300 g, were supplied by Eisai Laboratories (Gifu, Japan). This study was carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" as adopted and promulgated by the National Institutes of Health.

Preparation of CMVs. CMVs, prepared from male SD rats according to Kobayashi et al. (1990), were suspended in 50 mM Tris·HCl buffer, pH 7.4, containing 250 mM sucrose, frozen in liquid N₂, and stored at 100°C until used. Marker enzyme activities such as Mg⁺⁺-ATPase and alkaline phosphatase activities were routinely determined by the method of Schoner et al. (1967) and Yachi et al. (1989), respectively, to check the purity of the prepared CMVs. 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) after a 2-min incubation at 37°C. The concentration of protein in CMVs was determined by Bradford's method with bovine serum albumin as a standard, using the BioRad protein assay kit (Bio-Rad, Hercules, CA).

Inhibition of CMV Uptake of [³H]BQ-123 by ET Antagonists. The uptake of [³H]BQ-123 was measured by a rapid filtration technique as described previously (Niinuma et al., 1997). The transport medium (250 mM sucrose and 10 mM MgCl₂ in 10 mM Tris·HCl buffer, pH 7.4) containing 0.1 µM [³H]BQ-123 and 0 to 1000 µM concentration of each inhibitor peptide (BQ-485, BQ-518, compound A, compound B, compound C, and BQ-587) were preincubated for 3 min at 37°C in the presence of 5 mM ATP and an ATP-generating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). The reaction was started by adding the vesicle preparation (5 µl of stocked solution) to the preincubated transport medium and incubating it again for 2 min at 37°C. The total volume and the amount of CMVs in the medium were 20 µl and 10 µg of protein, respectively. The uptake reaction was stopped by the addition of 1 ml of ice-cold stop buffer that contained 100 mM NaCl, 250 mM sucrose, and 10 mM Tris·HCl (pH 7.4). A 50-µl aliquot of the reaction mixture was mixed with 5 ml of scintillation cocktail (Clearsol II; Nacali Tesque Inc., Kyoto, Japan). A 900-µl aliquot of the reaction mixture was filtered through a Millipore filter (0.45-µm HAWP; Millipore Corp., Bedford, MA). The filter was washed twice with 5 ml of ice-cold stop buffer and dissolved in the scintillation cocktail. The radioactivity retained on the filter and in the reaction mixture was determined by liquid scintillation counter (LS 6000E; Beckman Instruments). The uptake of [³H]BQ-123 was normalized with respect to both the amount of membrane vesicle and the concentration of substrate in the transport medium. ATP-dependent uptake was determined by subtracting the uptake in the absence of ATP from that in its presence.

Uptake of ET Antagonists by CMVs. The uptake of unlabeled BQ-123, BQ-485, BQ-518, and compound A by CMVs was measured by the same rapid filtration method as described above. Peptides (1-1000 µM) were incubated with CMVs in a transport medium in the presence of ATP and the ATP-generating system. The reaction was stopped by the addition of 1 ml of ice-cold stop buffer. Then, 100 µl of the mixture was mixed with an equal volume of ethanol and centrifuged. The supernatant then was assayed by high-performance liquid chromatography (HPLC) to determine the medium concentration. The compound retained on the filter was extracted by soaking the filter paper in 300 µl of a mixture of stop buffer and ethanol (1:1). After extraction, it was centrifuged, and the supernatant was assayed by HPLC to determine the uptake amount.

HPLC Analysis. HPLC analysis was performed by using a Spherisorb S3 ODS2 (4.6 × 150 mm) column (Tosoh, Japan). The mobile phase consisted of 0.1% (v/v) trifluoroacetic acid and 35% (v/v) acetonitrile for BQ-123, compound A, and BQ-518 and 55% acetonitrile for BQ-485. A flow rate of 0.8 ml/min (BQ-485) and 1.0 ml/min (BQ-123, BQ-518, and compound A) and an injection volume of 50 µl was used for all experiments. The fluorescent detector was operated at an excitation wavelength of 287 nm and an emission wavelength of 348 nm.

Determination of Kinetic Parameters for Uptake by CMVs. To estimate the inhibitory effects of the peptides on the uptake of [³H]BQ-123 by CMVs, the inhibition constant K_i values were obtained by fitting the data into the following equation:

(1)

where V_{0(+inhibitor)} and V_0(inhibitor) represent the initial rate of uptake in the presence and absence of inhibitor, respectively; I represents the inhibitor concentration, and K_i is the inhibitor concentration that produces a 50% reduction in V₀ compared with the rate in the absence of inhibitor. This equation was derived based on the assumption of competitive or noncompetitive inhibition and the fact that the [³H]BQ-123 concentration (0.1 µM) is much lower than the K_m value of BQ-123 uptake (Shin et al., 1997).

(2)

	Results

Top Abstract Introduction Materials and methods Results Discussion References

Transport by CMVs. To check the transport activity of the CMVs, the uptake of standard substrates TCA (1 µM) and DNP-SG (1 µM) by CMVs from SD rats and EHBR was initially investigated. The uptake of [³H]TCA was comparable between SD rats (62.5 ± 3.0 pmol/min/mg protein, mean ± S.E. of 16 different membrane preparations) and EHBR (70.5 ± 10.3 pmol/min/mg protein, mean ± S.E. of three determinations from one membrane preparation), whereas that of [³H]DNP-SG, a representative substrate of cMOAT, was much greater in SD rats (71.0 ± 3.5 pmol/min/mg protein) than in EHBR (2.7 ± 0.3 pmol/min/mg protein). The fold enrichment of alkaline phosphatase and Mg⁺⁺-ATPase in SD rats was 189 ± 11 and 55.9 ± 5.1, whereas the corresponding values in EHBR were 123 ± 25 and 46.9 ± 7.8, respectively.

Effects of ET Antagonists on ATP-Dependent [³H]BQ-123 Uptake by CMVs from SD Rats. All of the ET antagonists examined inhibited the ATP-dependent uptake of [³H]BQ-123 by CMVs from SD rats in a concentration-dependent manner (Fig. 1). This inhibition was almost complete, and the K_i values obtained are shown in Table 1. The K_i for compound A was the lowest, followed by that for BQ-485 (Table 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Inhibitory effect of ET antagonists on ATP-dependent [3H]BQ-123 uptake by CMVs. ATP-dependent uptake of [3H]BQ-123 for 2 min by CMVs from SD rats was determined in the presence of various concentrations of each ET antagonist. Data are the mean ± S.E. of six determinations in three different preparations. The lines represent the calculated curves obtained by fitting to eq. 1. , BQ-123; , BQ-485; , BQ-518; , compound A; , BQ-587; , compound B; , compound C.

Uptake of BQ-123, BQ-485, BQ-518, and Compound A by CMVs from SD Rats. The uptake of BQ-123, BQ-485, BQ-518, and compound A was examined in the presence or absence of ATP in the medium (Fig. 2). The uptake of all of the compounds examined was greater in the presence of ATP than in its absence (Fig. 2). An "overshoot" phenomenon was observed in the uptake of these compounds in the presence of ATP (Fig. 2). The ATP-dependent uptake, obtained by subtracting the uptake in the absence of ATP from that in its presence, of these four ET antagonists showed saturation (Fig. 3), and the K_m values are shown in Table 1. The K_m for the four compounds was almost comparable to the K_i (Table 1).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Time course for the uptake of ET antagonists by CMVs. CMVs were incubated in the presence of each ET antagonist (10 µM) with () or without () ATP and ATP-generating system. Data are the mean ± S.E. of three determinations.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Eadie-Hofstee plot for the ATP-dependent uptake of ET antagonists by CMVs. Uptake rate of each peptide was determined at 2-min incubation in the presence or absence of ATP and ATP-generating system. ATP-dependent uptake () was estimated by subtracting the uptake in the absence of ATP () from that in its presence. Data are the mean ± S.E. of four determinations in two different preparations. The lines represent the calculated curves obtained by fitting to eq. 2.

Effects of BQ-123 and DNP-SG on Uptake of BQ-485 by CMVs from SD Rats. BQ-123 inhibited the ATP-dependent uptake of BQ-485 (Fig. 4A), and a K_i of BQ-123 was comparable to the K_m for the ATP-dependent uptake of BQ-123 (Table 1; 84.1 µM versus 97.8 µM). Also, DNP-SG (600 µM) almost completely inhibited the ATP-dependent uptake of BQ-485 (Fig. 4B).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Inhibitory effect of anionic compounds on ATP-dependent BQ-485 uptake by CMVs from SD rats. A, inhibitory effect of BQ-123 on ATP-dependent uptake of BQ-485 (10 µM). The lines represent the calculated curves obtained by fitting to eq. 1. Data are mean ± S.E. of four determinations in two different preparations. B, inhibitory effect of BQ-123 and DNP-SG on ATP-dependent uptake of BQ-485 (10 µM). ATP-dependent uptake of BQ-485 for 2 min was determined in the presence or absence of each inhibitor. ATP-dependent uptake of BQ-485 at 1 mM was also determined. The uptake of BQ-485 was normalized with respect to the BQ-485 concentration in the medium. Data are mean ± S.E. of four determinations in two different preparations.

Uptake of BQ-123, BQ-485, BQ-518, and Compound A by CMVs from EHBR. The ATP-dependent uptake of BQ-123, BQ-485, BQ-518, and compound A was compared using CMVs from SD rats and EHBR (Fig. 5). Because there may be a y-intercept in the ATP-dependent uptake for some compounds (Fig. 2), the uptake rate in Fig. 5 was obtained by subtracting the uptake at 0.5 min from that at 2 min to allow more precise estimation of the transport activity and compare it in SD rats and EHBR. In the SD rats, the ATP-dependent uptake of BQ-485 was the greatest, whereas that of compound A was much less than that of the others. The ATP-dependent uptake of BQ-123, BQ-485, and BQ-518 in SD rats was much greater than that in EHBR, whereas the ATP-dependent uptake of compound A did not differ much between SD rats and EHBR (Fig. 5).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   ATP-dependent uptake of ET antagonists by CMVs from SD rat and EHBR. CMVs were incubated in the presence of 10 µM BQ-123 A, BQ-485 (B), BQ-518 (C), and compound A (D), with or without ATP and ATP-generating system. The uptake represents the ATP-dependent uptake obtained by subtracting data for 0.5 min from that for 2 min. Data are the mean ± S.E. of three different determinations. , SD rat; , EHBR.

Effect of DNP-SG on ATP-Dependent Uptake of Compound A. The ATP-dependent uptake of compound A (10 µM) was 13.7 ± 2.3, 16.4 ± 1.6, 15.4 ± 3.9, 15.7 ± 3.1, and 17.4 ± 4.7 pmol/min/mg protein in the presence of 0, 10, 100, 300, and 1000 µM DNP-SG (mean ± S.E. of three different determinations). DNP-SG did not show any inhibitory effect on the ATP-dependent uptake of compound A. In this study, the highest DNP-SG concentration (1 mM) should almost completely saturate its own transporter considering the K_m value (18 µM) for the ATP-dependent uptake of DNP-SG determined in our previous study (Niinuma et al., 1997).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

cMOAT has been shown to be responsible for the biliary excretion of a wide variety of organic anions (Yamazaki et al., 1996; Müller et al., 1997). One of its physiological functions is the secretion of xenobiotic metabolites, such as glutathione and glucuronide conjugates, into bile. Such primary active transport systems of xenobiotic metabolites have been proposed to function as a "phase III" detoxification system (Ishikawa, 1992), whereas phases I and II represent oxidation and subsequent conjugation, respectively. The other function of cMOAT is to efficiently excrete certain types of anionic therapeutic agents into the bile because biliary excretion is one of the predominant elimination pathways for such organic anions. In both in vivo experiments and in vitro CMV uptake studies, we previously demonstrated that the biliary excretion of BQ-123, an anionic cyclopentapeptide, is primarily mediated by cMOAT (Shin et al., 1997). In EHBR, the biliary excretion clearance (CL_{bile, h}), defined as the steady-state biliary excretion rate divided by the hepatic concentration during i.v. infusion, was only 30% of that in SD rats (Shin et al., 1997). In addition, the ATP-dependent uptake of BQ-123 was reduced in CMVs from EHBR compared with that in CMVs from SD rats (Shin et al.,1997). Such primary active transport of BQ-123 by cMOAT is one of the major factors responsible for reducing the level of BQ-123 in circulating plasma after its i.v. administration. In the present study, we examined the primary active transport of BQ-123 derivatives to identify those ET antagonists with different transport activity on the bile canalicular membrane. The present finding demonstrates that it is possible to design such antagonist peptides, which will undergo relatively less transport by cMOAT.

The ATP dependence (Fig. 2) and saturation (Fig. 3) of the CMV uptake of BQ-485, a linear anionic tripeptide, and BQ-518, an anionic cyclopentapeptide, indicate the involvement of a primary active transport system. These two peptides share cMOAT with BQ-123 because 1) the K_i and K_m values for BQ-485 and BQ-518 were similar (Table 1), 2) the K_i for inhibition of BQ-485 transport by BQ-123 was also similar to the K_m of BQ-123 uptake (Table 1), and 3) the ATP-dependent uptake of BQ-485 and BQ-518 in EHBR was much lower than that in SD rats (Fig. 5). The fact that BQ-485 exhibits the greatest transport activity of the four peptides (Figs. 2 and 5) indicates that cMOAT transports linear as well as cyclic peptides. The K_m for the ATP-dependent uptake of BQ-123 was approximately three times higher than that obtained in our previous report (Shin et al., 1997). This discrepancy may come from differences in the preparation of CMVs as well as in the experimental protocol, such as differences in the substrate concentration points and uptake period for assessing the initial uptake. Therefore, the absolute values need to be discussed with care. Nevertheless, it is possible to compare them for each compound because the data shown in Fig. 3 were obtained using the same CMV preparation.

In the light of the ATP-dependence and saturation in the uptake of compound A (Figs. 2 and 3), a primary active transport system is also involved in its biliary excretion. It should be noted that its ATP-dependent uptake is very similar in SD rats and EHBR (Fig. 5), suggesting that a primary active transport system other than cMOAT is responsible for the biliary excretion of this compound. Nevertheless, compound A almost completely inhibits the ATP-dependent uptake of [³H]BQ-123 (Fig. 1), with cMOAT being its major transporter. The above findings can be explained if compound A inhibits cMOAT-mediated BQ-123 transport but is not a substrate of cMOAT and it is transported by another system. The validity of this hypothesis is supported by the finding that DNP-SG cannot inhibit the ATP-dependent uptake of compound A. Interestingly, the finding that compound A has the lowest K_i (1 µM) of the ET antagonists examined (Table 1) indicates that it has the highest affinity for cMOAT. In addition, the K_m of its own uptake (3 µM) is close to the K_i (Table 1). Thus, the affinity of compound A for its own transporter is also high and very similar to that for cMOAT. Nevertheless, its transport efficiency on CMVs is much lower than that of the other compounds (Figs. 2 and 5) because of the low capacity (V_max) of its primary active transport (Table 1).

Several reports have suggested that primary active transport systems other than cMOAT are involved in the biliary excretion of small peptides. Ziegler et al. (1994) reported the ATP-dependent uptake of a cationic linear renin-inhibiting peptide EMD-51921 in CMVs from Wistar rats that was unchanged in CMVs isolated from TR rats, which have a hereditary defect in cMOAT like EHBR. Takahashi et al. (1997) found that ditekiren, a pseudohexapeptide, is taken up by rat CMVs in an ATP- and a temperature-dependent manner. This ATP-dependent uptake shows saturation and is competitively inhibited by EMD 51921 and daunomycin, but not by glutathione disulfide, a substrate of cMOAT (Takahashi et al., 1997). We also reported the ATP-dependent uptake of a cationic cyclo-octapeptide, octreotide, by CMV that was comparable between SD rats and EHBR and could be inhibited by verapamil and PSC-833 (Yamada et al., 1996). Thus, there is a major transport system other than cMOAT. Additionally, in the present study, the ATP-dependent uptake of BQ-485, BQ-518, and compound A was still observed in CMVs from EHBR (Fig. 5). This suggests that transporters other than cMOAT also mediate the biliary excretion of these ET antagonists. Thus, multiple primary active transport systems act as the biliary excretion mechanism for small peptides across the bile canalicular membrane.

We previously determined the CL_{bile, h} in vivo in rats and found that the CL_{bile, h} for BQ-485 was the greatest, followed by that of BQ-123, BQ-518, and compound A (Kato et al., 1999). Since CL_{bile, h} represents transport activity from the intracellular compartment into the bile, it is reasonable that the transport activity of these compounds in CMVs (Figs. 2 and 5) is reflected in the magnitude of CL_{bile, h}. Thus, the efficiency in the primary active transport on bile canalicular membrane determines the degree of biliary excretion of these ET antagonists in vivo. The biliary excretion of compound A is much lower than BQ-123, the biliary excretion clearance defined in terms of the plasma concentration of compound A being 13% that of BQ-123 (Kato et al., 1999). Nevertheless, the hepatic uptake clearance of compound A is not very different from BQ-123 (66% of BQ-123) (Kato et al., 1999). Thus, the primary active transport governed by cMOAT on bile canalicular membrane is one of the major factors that determines the efficiency of the net biliary excretion of these peptides. The present study demonstrates the importance of recognition by cMOAT, which governs the degree of biliary excretion of these anionic small peptides.

The present study also identified compound A as a cMOAT antagonist. The other peptides that can inhibit cMOAT but whose transport by cMOAT is minor include cyclosporin A because the ATP-dependent uptake of leukotriene C₄, a substrate of cMOAT, by rat CMV is inhibited by cyclosporin A with a K_i of 3.5 µM (Bohme et al., 1993). Thus, this compound also has a high affinity for cMOAT, similar to compound A. Although the transport of cyclosporin A by cMOAT is minor, such high affinity for cMOAT may lead us to consider the possibility of drug interactions during clinical applications. For example, Gupta et al. (1996) reported the modulation of the pharmacokinetics of irinotecan and its metabolites in rats after the coadministration of cyclosporin A. Because biliary excretion is the major elimination pathway, with cMOAT being responsible for the primary active transport (Chu et al., 1997a,b) of irinotecan and its metabolites, the drug interaction with cyclosporin A occurs during biliary excretion. Thus, it is important to consider the possibility of drug interactions for such hydrophobic compounds. Interestingly, cyclosporin A is a neutral compound, whereas compound A has a cationic moiety in their structures. Nevertheless, they both potently inhibit cMOAT, which is believed to mainly recognize anionic compounds. Thus, not only the anionic charge but also other physicochemical properties may determine the affinity for cMOAT.

Nonlinear kinetics of the ATP-independent BQ-123 uptake was found both in Fig. 3 and in our previous report (Shin et al., 1997). This possibly represents adsorption to CMVs with saturable behavior because the uptake of BQ-123 in the absence of ATP exhibits no osmolarity dependence (Shin et al., 1997).

In conclusion, a primary active transport system is responsible for the biliary excretion of BQ-485, BQ-518, and compound A. cMOAT is primarily involved in the transport of both BQ-485 and BQ-518, whereas another transporter mainly mediates the transport of compound A, which can efficiently inhibit the cMOAT-mediated active transport of BQ-123. The primary active transport of compound A is much less than that of the others due to its lower transport capacity. Thus, it is possible to design ET antagonists with different transport activities recognized by cMOAT.