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Research ArticleArticle

Carrier-Mediated Hepatic Uptake of Peptidic Endothelin Antagonists in Rats

Sharif Akhteruzzaman, Yukio Kato, Hirokazu Kouzuki, Hiroshi Suzuki, Akihiro Hisaka, Bruno Stieger, Peter J. Meier and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics September 1999, 290 (3) 1107-1115;

Abstract

The endothelin antagonist BQ-123, an anionic cyclopentapeptide, is taken up by rat hepatocytes through active transport systems. Here, we have examined the hepatocellular uptake mechanism for several BQ-123 derivatives with anionic charges using isolated rat hepatocytes. BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, were taken up by hepatocytes in a concentration-dependent manner. The uptake of BQ-485 was most efficient, whereas compound A showed comparable uptake with BQ-123. The uptake of these peptides was Na+- and energy-dependent, suggesting that active transport mechanisms are involved in their uptake into hepatocytes. BQ-485, BQ-518, and compound A can almost completely inhibit both the Na+-dependent and -independent uptake of [3H]BQ-123, with inhibition constants (Ki) that are comparable to the Michaelis-Menten constants (Km) for their Na+-dependent and -independent uptake, respectively. Inhibition by BQ-485 was competitive, and the uptake of BQ-485 can be inhibited by BQ-123, with Ki values that are comparable with the Km values for BQ-123 uptake. The uptake of BQ-123 by COS-7 cells transfected with either Na+-dependent taurocholate-cotransporting polypeptide (Ntcp) or Na+-independent basolateral organic anion-transporting polypeptide (oatp1) was minimal. Thus, these three peptides share the transporters that also recognize BQ-123 but appear to differ from Ntcp and oatp1.

BQ-123, initially developed as a selective receptor antagonist for endothelin (ET) receptor subtype ETA is a cyclic pentapeptide with a single carboxyl group (Mino et al., 1992; Itoh et al., 1993). BQ-123 exhibits a variety of biological and therapeutic activities, including an antihypertensive effect as well as a protective effect against proximal tubular necrosis in acute renal failure due to ischemia in rats (Mino et al., 1992; Itoh et al., 1993). It is also stable to enzymatic degradation in the gastrointestinal tract and the liver. In spite of its therapeutic potential and stability, the compound is quickly eliminated from the systemic circulation with a short half-life because of rapid transport from the circulating plasma into the bile (Shin et al., 1997). Almost 90% of an i.v. dose is recovered in the bile unchanged in rats (Nakamura et al., 1996). This short plasma half-life limits its usefulness as a therapeutic agent.

Studies have recently reported, as mechanisms for the hepatic uptake of peptides, specific carrier-mediated transport systems on the sinusoidal membrane of hepatocytes for some peptides, e.g., octreotide (Terasaki et al., 1995; Yamada et al., 1996), cholecystokinin (Gores et al., 1986; Gores et al., 1989), renin inhibitors (Bertrams and Ziegler, 1991a,b), and cyclosomatostatin (Ziegler et al., 1988, 1991). A hepatic uptake mechanism of BQ-123 has also been studied recently (Nakamura et al., 1996). The compound is taken up extensively by isolated rat hepatocytes in both a Na+-dependent and Na+-independent manner. The Na+-dependent system transports BQ-123 with high affinity and low capacity, whereas the Na+-independent system has low affinity and high capacity (Nakamura et al., 1996). Both of these uptake systems were found to be active transport systems and could be inhibited by bile acid (taurocholate) and an organic anion (dibromosulfophthalein) (Nakamura et al., 1996). The existence of these transport systems may explain the large hepatic distribution and short half-life of this compound in plasma (Nakamura et al., 1996; Shin et al., 1996).

To find a long-acting ET receptor antagonist, we synthesized a series of linear and cyclic derivatives of BQ-123. All of these compounds showed high ET antagonist activity (ETA-binding affinity: BQ-123, 22.0 nM; BQ-485, 3.4 nM; BQ-518, 3.0 nM; compound A, 13 nM; BQ-587, 20 nM; compound B, 15.5 nM) (Itoh et al., 1993; Fukami et al., 1995, 1996). In the present study, the efficiency of hepatic uptake of these BQ-123 derivatives was studied in isolated rat hepatocytes. Additionally, to clarify the hepatic uptake mechanism, we performed inhibition studies in isolated hepatocytes and found that these derivatives share the same transport systems as BQ-123.

Recently, several transporter genes have been identified for the uptake of bile acids and organic anions across the sinusoidal membrane both in rats and humans (Hagenbuch et al., 1991; Dippe et al., 1993, 1996;Hagenbuch and Meier, 1994; Jacquemin et al., 1994; Kanai et al., 1996). Peptidic compounds with anionic charges may be substrates for these transporters. Eckhardt et al. (1996) reported that a peptide-based thrombin inhibitor CRC-220 is a substrate for the Na+-independent basolateral organic anion-transporting polypeptide (oatp), whereas Ziegler et al. (1996)found that neither oatp1 nor Na+-dependent taurocholate-cotransporting polypeptide (Ntcp) is responsible for the uptake of the linear hydrophilic renin inhibitor EMD-56133. However, information about the transporter for small peptides is still limited. In the present study, we also used the transfectant system expressing these transporters to help identify the transporters for BQ-123 and its derivatives.

Materials and Methods

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(α),N(ε)-dimethyl-l-Lys)-d-Val-l-Leu), and compound B (perhydroazepino-N-carbonyl-l-Leu-d-Trp-2-aminobutyric acid) were synthesized at the Tsukuba Research Institute of Banyu Pharmaceutical Co., Ltd. (Tsukuba, Japan). Their chemical structures are shown in Fig. 1. [Prolyl-3,4(n)-3H]BQ-123 (31.0 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK), [3H(G)]taurocholic acid ([3H]TCA, 2.0 Ci/mmol) and [3H]estradiol 17β-d-glucuronide ([3H]E2–17G, 51 Ci/mmol) was purchased from New England Nuclear (Boston, MA), trypsine inhibitor, TCA, rotenone and carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) were purchased from Sigma Chemical Co. (St. Louis, MO). Dibromosulfopthalein (DBSP) was obtained from the Socite d’Etudes et de Researches Biologiques (Paris, France). Collagenase was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All other chemicals and reagents were commercial products of analytical grade. COS-7 cells were purchased from American Type Culture Collection (Rockville, MD).

Figure 1
Figure 1

Structures of peptidic ET antagonists.

Animals.

Male Sprague-Dawley rats, weighing approximately 200 to 300 g, were purchased from Nisseizai (Tokyo, 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 Isolated Hepatocytes.

Hepatocytes were isolated from rats by the procedure described (Yamazaki et al., 1993b). After isolation, the hepatocytes were suspended at 4°C in albumin-free Krebs-Henseleit buffer [5 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4 · 7H2O, 12 mM HEPES, 5 mM glucose, 2 mM CaCl2, 118 mM NaCl, 24 mM NaHCO3, pH 7.3]. The cells were then divided into two aliquots and washed separately three times with (+)Na+ buffer (Krebs-Henseleit buffer as described above) and (−) Na+ buffer (Krebs-Henseleit buffer in which the NaCl and NaHCO3 were replaced isotonically with choline chloride and choline bicarbonate), respectively. Cell viability was routinely checked by the trypan blue (0.4% w/v) exclusion test. Isolated hepatocytes with a viability of >85% were routinely used.

Uptake of [3H]BQ-123 in the Presence of ET Antagonists by Isolated Hepatocytes.

The uptake of [3H]BQ-123 (0.1 μM) was initiated by adding the ligand and the inhibitor peptides (BQ-485, BQ-518, compound A, BQ-587, and compound B) (0–1000 μM) to the preincubated (3 min at 37°C) cell suspension (6 × 106 cells/ml). At designated times, the reaction was terminated by separating the cells from the medium by using a centrifugal filtration technique described previously (Yamazaki et al., 1993b). Briefly, 200 μl of 2 N NaOH was covered with a 100-μl mixture of silicone and mineral oil (density 1.015). The sample (200 μl of the incubation mixture) was put onto the oil layer and then centrifuged for 10 s in a tabletop centrifuge (Microfuge E; Beckman Instrument, Fullerton, CA). The centrifugation pelleted the hepatocytes into the NaOH solution through the oil layer. After the cells were dissolved into the alkaline solution, the tube was sliced with a razor blade, and the medium side was transferred into scintillation vials. The bottom side was neutralized with 50 μl of 2 N HCl. Five milliliters of scintillation cocktail (Clearsol I; Nacali Tesque Inc., Kyoto, Japan) was then added into each vial, and radioactivity was determined by a liquid scintillation spectrophotometer (LS 6000SE; Beckman Instruments, Fullerton, CA). The uptake in the presence of (+)Na+ was designated as the total uptake. To estimate the Na+-independent uptake, the uptake study was performed in the absence of external Na+. For this experimental condition, a (−)Na+ buffer was used as the incubation buffer. The Na+-dependent uptake was calculated by subtracting the Na+-independent uptake from the total uptake. The initial velocity for [3H]BQ-123 uptake was estimated by subtracting the uptake at 30 s from that at 90 s.

Uptake of ET Antagonists by Isolated Hepatocytes.

The uptake of BQ-485, BQ-518, and compound A was investigated by a slight modification of the above rapid filtration technique. Peptides were added to preincubated (3 min, at 37°C) cell suspension (6 × 106 cells/ml). At designated times, the reaction was terminated by separating the cells from the medium using the above-mentioned centrifugal filtration technique. This method differs from the above one in that the centrifuge tube contained 50 μl of 250 mM sucrose instead of 2 N NaOH covered with a 100-μl mixture of silicone and mineral oil. After overnight preservation at −20°C, 100 μl of medium and 40 μl of cell suspension were transferred to an Eppendorf tube, mixed with four volumes of ethanol, and centrifuged. The supernatants were then assayed by HPLC to determine the intracellular and medium concentrations. The initial velocity of the uptake of each peptide was estimated by subtracting the uptake at 30 s from that at 90 s.

Experiments with Metabolic Inhibitors.

Peptides were added to hepatocytes (6 × 106 cells/ml) preincubated for 3 min at 37°C with 30 μM rotenone or 2 μM FCCP that had been prepared as stock solutions by dissolving in dimethyl sulfoxide and diluting with the preincubation mixture 1000 times.

HPLC Analysis.

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

Uptake Study in Primary Cultured Rat Hepatocytes.

Isolated rat hepatocytes were suspended in Williams’ medium E, and 5 × 105 cells were placed on collagen-coated 22-mm dishes and cultured in 5% CO2 at 37°C for 4 h (Yamada et al., 1997; Kouzuki et al., 1998). Uptake was initiated by adding the ligand to the medium after the culture dishes had been washed 3 times and preincubated with (+)Na+ buffer or (−)Na+buffer at 37°C for 5 min. At designated times, the reaction was terminated by adding ice-cold Krebs-Henseleit buffer. Immediately before each designated time, 50 μl of medium was transferred to a scintillation vial. Cells were then 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 each scintillation vial. The radioactivity in both cells and medium was determined in a liquid scintillation spectrophotometer after adding 8 ml of scintillation fluid (Hionic-fluor, Packard Instrument Co., Downers Grove, IL) to the vials. The remaining 100 μl of each aliquot was used to determine the protein concentration according to the method of Lowry (Lowry et al., 1951) with BSA as a standard. [3H]BQ-123 was used at a final concentration of 1 μM.

Transient Expression of Ntcp cDNA in COS-7 Cells.

The insert of the Ntcp clone was ligated into the pCAGGS vector and transfected in COS-7 cell-expression systems as described (Kouzuki et al., 1998). At 8 h after transfection, the medium was replaced with Dulbecco’s modified Eagle’s medium plus 5% fetal bovine serum and cultured overnight in 5% CO2 atmosphere at 37°C. Approximately 1.6 × 105 cells were then placed on 22-mm dishes and cultured overnight in 5% CO2 at 37°C. At 48 h after transfection, an uptake study was performed as described for primary cultured hepatocytes. Ntcp-mediated uptake was calculated by subtracting the uptake into COS-7 cells with the pCAGGS without the Ntcp insert from the uptake into COS-7 cells with the pCAGGS with the Ntcp insert in the presence of Na+.

Transient Expression of oatp1 cDNA in COS-7 Cells.

The insert of the oatp1 cDNA was ligated into the pCAGGS vector and transfected in COS-7 cells as described (Kouzuki et al., 1999).

Determination of Kinetic Parameters.

To estimate the inhibitory effects of the peptides on the uptake of [3H]BQ-123, the inhibition constant (Ki) was obtained by fitting the data into the equationV0(+inhibitor)V0(−inhibitor)=11+IKi Equation 1where V0(+inhibitor) andV0(−inhibitor) represent the initial uptake rate of [3H]BQ-123 in the presence and absence of inhibitor at a concentration I.Ki equals the inhibitor concentration that produces a 50% reduction in V0as compared to V0(−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 Km value of BQ-123 uptake (see Results).

The kinetic parameters for the uptake of BQ-123, BQ-485, BQ-518, and compound A were determined according to the equationV0=Vmax·SKm+S+Pdif·S Equation 2where V0 is the initial uptake rate of the ligand, S is the concentration of the ligand,Km is the Michaelis-Menten constant,Vmax is the maximum uptake velocity, and Pdif is the nonspecific uptake clearance. The uptake data were fitted to eq. 2 using the iterative nonlinear least-squares method. The algorithm used for the fitting was the Dumping Gauss-Newton method (Yamaoka et al., 1981).

To determine inhibition type, the initial velocity of the BQ-123 uptake (V0) in the presence and the absence of a constant concentration of an inhibitor peptide was fitted to the following equations in which competitive (eq. 3) or noncompetitive (eq.4) inhibition was assumed:V0=Vmax·SKm1+IKi+S+Pdif·S Equation 3V0=Vmax1+IKi·SKm+S+Pdif·S Equation 4The Km,Vmax,Pdif, andKi values were simultaneously obtained using this analysis. The type of inhibition was determined by the goodness of fit considering Akaike’s information criteria (Yamaoka et al., 1981).

Statistical Analysis.

For the analysis of differences between two data sets, the test for equal variance (F test) and a subsequent Student’s t test were performed on the two means of the unpaired data. A p value < 0.05 was considered to be statistically significant.

Results

Effect of ET Antagonists on [3H]BQ-123 Uptake.

The inhibitory effect of several ET antagonists on [3H]BQ-123 uptake was examined in isolated rat hepatocytes (Fig. 2). All of these compounds showed concentration-dependent inhibition (Fig. 2). TheKi obtained using eq. 1 is shown in Table. 1. TheKi of BQ-485 and compound B, both being linear peptides, was <10 μM and lower than for other compounds (Table 1).

Figure 2
Figure 2

Inhibitory effect of ET antagonists on the Na+-independent and Na+-dependent [3H]BQ-123 uptake by isolated rat hepatocytes. Initial velocity of Na+-independent (A) and Na+-dependent (B) [3H]BQ-123 (0.1 μM) uptake by isolated rat hepatocytes were measured in the presence of various concentrations of ET antagonists. Data are the mean ± S.E. of six determinations in three different preparations. ■, BQ-123; ○, BQ-485; ▵, BQ-518; ▪, BQ-587; ●, compound A; ▴, compound B.

View this table:
Table 1

Kinetic parameters for the uptake of endothelin antagonists by isolated rat hepatocytes

Uptake of ET Antagonists by Isolated Rat Hepatocytes.

The uptake of [3H]BQ-123, BQ-485, BQ-518, and compound A was examined with or without Na+ in the medium (Fig. 3). All four compounds exhibited both Na+-dependent and Na+-independent uptake (Fig. 3). The latter was higher than the former for all compounds (Fig. 3). The absolute value of the hepatocellular uptake was highest for BQ-485, followed by BQ-518 (Fig. 3). The time profiles of both Na+-dependent and Na+-independent uptake of all compounds in the presence of either rotenone (30 μM) or FCCP (2 μM) differed from those in their absence (Fig. 4). To differentiate between the effect of these ATP depletors on the initial uptake velocity and on the initial binding of the peptides, the initial slope of the uptake was calculated by subtracting the uptake at 30 s from that at 90 s. The mean value for the slope of both Na+-independent and -dependent uptake of all compounds was decreased in the presence of FCCP or rotenone compared to the control. A significant difference was observed 1) between the initial slope for Na+-independent uptake of BQ-123, BQ-485, and compound A in the control and in the presence of FCCP; 2) between the initial slope for Na+-independent uptake of BQ-123 and compound A in the control and in the presence of rotenone; 3) between the initial slope for Na+-dependent uptake of BQ-123, BQ-518, and compound A in the control and in the presence of FCCP; and 4) between the initial slope for Na+-dependent uptake of BQ-123 in the control and in the presence of rotenone. Thus, the effect of FCCP on the initial slope was relatively marked compared to that of rotenone. According to our previous report, the reduction in ATP content in isolated hepatocytes in our experimental system was more rapid in the presence of FCCP (2 μM) than in the presence of rotenone (30 μM) (Yamazaki et al., 1993a). The ATP content after a 3-min incubation with rotenone was approximately 25% of the control, whereas that after a 3-min incubation with FCCP was 8% of the control. Therefore, it appears that the effect of FCCP is more marked than rotenone. Both the Na+-dependent and Na+-independent uptake of all compounds showed saturation (Fig. 5). TheKm values obtained are shown in Table1, and these were comparable with theKi values for the inhibitory effect of the respective compounds on [3H]BQ-123 uptake. BQ-123, BQ-485, and BQ-518 are metabolically stable (Kato et al., 1999), and therefore, the radioactivity associated with BQ-123 uptake should represent the parent compound. Although we found that compound A is hydrolyzed in the physiological buffer, this compound should be also stable during the incubation period in the present study because the half-life of the hydrolysis was about 180 min (Kato et al., 1999).

Figure 3
Figure 3

Na+ dependence for the uptake of ET antagonists by isolated rat hepatocytes. Both the Na+-independent (●) and Na+-dependent (○) uptake of BQ-123 (A), BQ-485 (B), BQ-518 (C), and compound A (D) at 3 μM were measured in isolated rat hepatocytes. Each value represents the mean ± S.E. of six determinations in two different preparations.

Figure 4
Figure 4

Effects of metabolic inhibitors on the uptake of ET antagonists by isolated rat hepatocytes. The Na+-independent (A–D) and Na+-dependent (E–H) uptake of BQ-123 (A and E), BQ-485 (B and F), BQ-518 (C and G), and compound A (D and H) at 3 μM were determined alone (■) or in the presence of 30 μM rotenone (▵) and 2 μM FCCP (○). Each value represents six determinations in two different preparations.

Figure 5
Figure 5

Eadie-Hofstee plot for the uptake of ET antagonists by isolated rat hepatocytes. Initial velocity for the Na+-independent (●) and the Na+-dependent (○) uptake of BQ-123 (A), BQ-485 (B), BQ-518 (C), and compound A (D) was determined by subtracting the uptake at 30 s from that at 90 s at various concentrations of each substrate. Each value represents the mean ± S.E. of six determinations in two different preparations.

Effects of BQ-123, TCA, and DBSP on the Uptake of BQ-485.

BQ-123 inhibited both the Na+-dependent and Na+-independent uptake of BQ-485 (Fig.6A), withKi values shown in Table 1. Both TCA and DBSP also inhibited the Na+-dependent and Na+-independent uptake of BQ-485 (Fig. 6B).

Figure 6
Figure 6

Inhibitory effect of several anionic compounds on BQ-485 uptake by isolated rat hepatocytes. Initial velocity for BQ-485 uptake was determined by subtracting the uptake at 30 s from that at 90 s in the presence and absence of each compound. A, inhibitory effects of BQ-123 on Na+-independent (●) and Na+-dependent (○) uptake of BQ-485 (1 μM) by isolated hepatocytes. B, inhibitory effects of BQ-123, TCA, and DBSP on Na+-independent (▪) and Na+-dependent (■) BQ-485 uptake (1 μM). The initial uptake velocity of BQ-485 was normalized by the BQ-485 concentration in the medium to examine the saturation of the uptake in the presence of 100 μM BQ-485. ∗, significantly different from control (p < .05).

Competitive Inhibition of BQ-123 Uptake by BQ-485.

Although BQ-485 had a minimal effect on the apparent maximum velocity of BQ-123, it changed the apparent Km value (Fig.7). The Akaike’s information criteria obtained by fitting based on eqs. 3 and 4 were −28.3 and 5.65 for the Na+-independent uptake and −10.5 and 2.78 for the Na+-dependent uptake, respectively. Thus, competitive inhibition of BQ-123 uptake by BQ-485 was observed (Fig.7), with a Ki shown in Table 1.

Figure 7
Figure 7

Concentration-dependent uptake of BQ-123 in the presence and absence of BQ-485 by isolated hepatocytes. Initial uptake of various concentrations of BQ-123 by isolated rat hepatocytes was determined by subtracting the uptake at 30 s from that at 90 s in the presence and absence of BQ-485 (5 μM). The uptake of BQ-123 in the absence of BQ-485 is also shown in Fig. 5 and is shown as broken lines here. The obtained parameters are shown in Table 1. A, Na+-independent; B, Na+-dependent.

Uptake of [3H]BQ-123 by Primary Cultured Rat Hepatocytes and COS-7 Cells Transfected with cDNA for oatp1 or Ntcp.

Uptake of [3H]BQ-123 was examined in COS-7 cells transfected with cDNA for oatp1 or Ntcp. Because cultured monolayer cells were used in those studies, rat hepatocyte monolayers in primary culture were also used in a control experiment. Na+-independent uptake of [3H]BQ-123 was observed in cultured hepatocytes (Fig. 8A), whereas its uptake was minimal in COS-7 cells transfected with oatp1 (Fig. 8B). Na+-independent uptake of [3H]E2–17G was observed in both cases (Fig. 8, A and B). Na+-dependent uptake of [3H]BQ-123 was observed in cultured hepatocytes (Fig. 9A), whereas its uptake was minimal in COS-7 transfectant (Fig. 9B). Na+-dependent uptake of [3H]TCA was observed in both cases (Fig. 9, A and B).

Figure 8
Figure 8

Na+-independent uptake of [3H]E2–17G and [3H]BQ-123 by primary cultured hepatocytes (A) and oatp1-transfected COS-7 cells (B). The oatp1-mediated uptake represents the difference in uptake between oatp1 and control plasmid-transfected cells at the substrate concentration of 1 μM.

Figure 9
Figure 9

Na+-dependent uptake of [3H]TCA and [3H]BQ-123 by primary cultured rat hepatocytes (A) and Ntcp-transfected COS-7 cells (B). Ntcp-mediated uptake represents the difference in uptake between Ntcp and control plasmid-transfected cells at the substrate concentration of 1 μM. Each value represents the mean ± S.E. of six determinations in two different preparations.

Discussion

Small peptides that are resistant to metabolism by peptidase are known to be eliminated primarily through biliary excretion (Bertrams and Ziegler, 1991a; Nakamura et al., 1996; Takahashi et al., 1997;Yamada et al., 1997). We previously reported that both Na+-dependent and Na+-independent active transport systems are involved in the hepatocellular uptake of BQ-123 in rats (Nakamura et al., 1996). In the present study, the uptake of BQ-485, BQ-518, and compound A by isolated rat hepatocytes also shows Na+ dependence (Fig. 3), energy dependence (Fig.4), and saturation (Fig. 5), suggesting that active transport systems are involved in the uptake of these three peptides as well as BQ-123. In addition, all three of these compounds inhibited both the Na+-dependent and Na+-independent uptake of [3H]BQ-123 (Fig. 2), withKi values comparable to theKm values of their own Na+-dependent and Na+-independent uptakes, respectively (Table 1). Inhibition of BQ-123 uptake by BQ-485 was competitive (Fig. 7), with aKi comparable to theKm of BQ-485 (Table 1). TheKi values for the inhibition of BQ-485 uptake by BQ-123 were also comparable to theKm values for the uptake of BQ-123 itself (Table 1). Both the Na+-dependent and Na+-independent uptake of BQ-123 can be competitively inhibited by TCA and DBSP (Nakamura et al., 1996). The uptake of BQ-485 can also be inhibited by TCA and DBSP (Fig. 6B). These results suggest that the uptake systems for BQ-485, BQ-518, and compound A are the same as for BQ-123 and that they also recognize certain types of bile acids and organic anions.

Among these four compounds, only BQ-485 has a linear structure, whereas the others are cyclic. Thus, linear as well as cyclic peptides can be a substrate for such active-transport systems. The uptake of BQ-485 showed the highest absolute values, with the highestVmax and the lowestKm values among these ET antagonists (Figs. 3-5; Table 1). This result suggests that the efficiency of transport via the uptake systems is highest for BQ-485. TheKi of this compound as well as for compound B, which is also a linear peptide with a similar structure to BQ-485, was also the lowest (Table 1). Thus, these two linear peptides have higher affinity for the transporters. On the other hand, both compound A and BQ-587 have a cationic moiety. The uptake of compound A was comparable to that of BQ-123 (Figs. 3-5). Thus, active transport systems are involved in the hepatocellular uptake of a variety of peptides, including cyclic and linear types with anionic and zwitterionic charges, with a different affinity for each.

The fact that compound A and BQ-587 have a slightly lower affinity (higher Ki) for the Na+-independent uptake system of [3H]BQ-123, compared with BQ-123 itself, is compatible with our previous finding that the total body clearance of compound A (Kato et al., 1999) and BQ-587 (Nakamura et al., 1996) is much lower than that of BQ-123. We also observed that the biliary excretion clearance of compound A is approximately 8 times lower than that of BQ-123 (Kato et al., 1999). However, the difference between the uptake of BQ-123 and compound A was minimal (Figs. 3-5). Thus, the difference in the uptake process of these two compounds does not fully explain the difference in their biliary excretion clearance. The difference in transport activity across the bile canalicular membrane might be a much more critical factor in determining the degree of net biliary excretion of these compounds. We observed that an ATP-dependent uptake of compound A by isolated rat canalicular membrane vesicles was much lower than BQ-123 (Akhteruzzaman et al., 1999).

All of the peptides examined have an anionic moiety, whereas only compound A also has a cationic charge and a slightly lower affinity for the Na+-independent [3H]BQ-123 transporter (Table 1). Thus, this transporter might specifically recognize organic anions. Several researchers have suggested that an Na+-independent active transport system, which also recognizes bile acids and organic anions, is involved in the uptake of small peptides. Gores et al. (1986, 1989) showed that the hepatocellular uptake of a linear octapeptide cholecystokinin-8 is inhibited either by TCA or DBSP in a liver perfusion system and in isolated rat hepatocytes. Bertrams and Ziegler (1991b) reported that the uptake of EMD-51921, a linear hydrophobic renin inhibitor, by isolated rat hepatocytes is energy-dependent and is competitively inhibited either by TCA or cholate. Ziegler et al. (1988) found that cyclohexapeptide 008 is also competitively inhibited by TCA, cholate, and bromosulfophthalein. Na+-independent uptake of BQ-123 is also competitively inhibited by TCA and DBSP (Nakamura et al., 1996). The above findings may be explained if these small peptides share the same multispecific transporter with both bile acids and organic anions as substrates. Jacquemin et al. (1994) has succeeded in the molecular cloning of oatp1 with TCA, cholate, E2–17G, and bromosulfophthalein as its substrates (see also Kanai et al., 1996). Therefore, one possible Na+-independent transport system for these small peptides is oatp1. In fact, CRC-220 (Eckhardt et al., 1996), a modified dipeptide, and the mycotoxin ochratoxin A (Kontaxi et al., 1996), which consists of a dihydroisocoumarin moiety linked to a l-β-phenylalanine molecule by an amide bond, are reported to be substrates of oatp1. The oatp1-mediated uptake of BQ-123 was minimal (Fig. 8), suggesting that the major transporter for BQ-123 is different from oatp1. It should be also noted, however, that from this result alone, we cannot conclude that the contribution of oatp1 to Na+-independent BQ-123 uptake by rat hepatocytes is negligible. This is because, first, the assay system we used may not have been enough to detect the small transporting activities, and, second, the configuration of oatp1 expressed in the present COS-7 transfectant system may not have been suitable for accepting BQ-123 as a substrate. Although additional studies should be performed to estimate the exact contribution of oatp1 to BQ-123 uptake, the present result suggests the existence of transporters other than oatp1 for the Na+-independent uptake of BQ-123. Ziegler et al. (1996) demonstrated that uptake of EMD-56133 by Xenopus laevis oocytes is not stimulated by preinjection of oatp1 cRNA. Thus, although oatp1 recognizes certain types of small peptides, other transporters might also be expressed on the sinusoidal membrane for these types of small peptides. Recently, gene expression of other oatp family members, oatp2 and oatp3, which transport the several organic anions, has been identified in the liver (Noe et al., 1997; Abe et al., 1998). Thus, there appears to be a large family of oatp transporters that could play an important role in peptide transport.

Several small peptides, such as octreotide, which is a cationic cyclooctapeptide, and azidobenzamido-008 (a008), which is a cyclohexapeptide, are also known to be taken up by hepatocytes in an Na+-dependent manner (Ziegler et al., 1988;Terasaki et al., 1995). We found that uptake of BQ-485, BQ-518, and compound A, as well as BQ-123, is at least partially Na+-dependent (Fig. 3). Also, a008 competitively inhibits Na+-dependent TCA uptake by isolated rat hepatocytes, with a Ki value similar to the Km of its own uptake (Ziegler et al., 1988). The Na+-dependent uptake of both octreotide and BQ-123 can be competitively inhibited by TCA, with aKi that is comparable to theKm of TCA uptake (Nakamura et al., 1996; Yamada et al., 1996). Hagenbuch et al. (1991) have cloned Ntcp. Minimal Na+-dependent uptake of [3H]BQ-123 could be observed in COS-7 cells transfected with Ntcp (Fig. 9). Also in this case, from this result alone, we cannot conclude that the contribution of Ntcp to Na+-dependent BQ-123 transport in rat hepatocytes is negligible because of the two reasons as described above. Although further studies should be performed to estimate the exact contribution of Ntcp to BQ-123 uptake, the present result suggests the existence of transporters other than Ntcp for the Na+-dependent uptake of BQ-123 by hepatocytes. Several reports have also shown that Ntcp is not responsible for the hepatocellular uptake of other peptidic compounds such as EMD-56133 (Ziegler et al., 1996) or CRC-220 (Eckhardt et al., 1996). Thus, transport systems other than Ntcp might be involved in the Na+-dependent uptake of these small peptides in hepatocytes. Recently, Dippe et al. (1993; 1996) found Na+-dependent uptake of TCA and cholate by Madin-Darby canine kidney cells transfected with the cDNA for microsomal epoxide hydrolase. This transport system might be one of the candidiates involved in the Na+-dependent uptake of small peptides.

Footnotes

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

  • ↵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 and in part by Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation.

  • Abbreviations:
    ET
    endothelin
    Ntcp
    Na+-dependent taurocholate-cotransporting polypeptide
    oatp
    Na+-independent basolateral organic anion-transporting polypeptide
    DBSP
    dibromosulfophthalein
    FCCP
    carbonyl cyanidep-trifluoromethoxy-phenylhydrazone
    TCA
    taurocholic acid
    E2–17G
    estradiol 17β-d-glucuronide
    a008
    azidobenzamido-008
    • Received October 6, 1998.
    • Accepted May 17, 1999.

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Carrier-Mediated Hepatic Uptake of Peptidic Endothelin Antagonists in Rats

Sharif Akhteruzzaman, Yukio Kato, Hirokazu Kouzuki, Hiroshi Suzuki, Akihiro Hisaka, Bruno Stieger, Peter J. Meier and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics September 1, 1999, 290 (3) 1107-1115;
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