- Email: [email protected]
taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes
Archives of Biochemistry and Biophysics 562 (2014) 115–121
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Electrophysiological characterization of human Na+/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes Masayuki Masuda a, Yukari Ichikawa a, Kazumi Shimono a, Maki Shimizu a, Yoshio Tanaka a, Toshifumi Nara b, Seiji Miyauchi a,⇑ a b
Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan
a r t i c l e
i n f o
Article history: Received 6 June 2014 and in revised form 12 August 2014 Available online 27 August 2014 Keywords: Bile acid Electrogenicity Hepatic uptake Na+-dependent transporter NTCP SLC10A1 Xenopus laevis oocyte
a b s t r a c t The Na+/taurocholate cotransporting polypeptide (NTCP) plays a major role in Na+-dependent bile acid uptake into hepatocytes. The purpose of the present study was to establish the heterologous expression of human NTCP (hNTCP) in Xenopus laevis oocytes and to elucidate whether the transport of bile acid via hNTCP is electrogenic using electrophysiological techniques. First, we evaluated the uptake of taurocholate (TCA) by hNTCP heterologously expressed in Xenopus oocytes utilizing [3H]-labeled TCA. The uptake of 1.2 lM TCA by cRNA-injected oocytes increased more than 100-fold compared to H2O-injected oocytes, indicating that hNTCP is robustly expressed in the oocytes. hNTCP-mediated transport of TCA is saturable with a Michaelis constant of 10.5 ± 2.9 lM. The Na+-activation kinetics describing the relationship between the concentration of Na+ and the magnitude of the TCA uptake rate by hNTCP were sigmoidal with a Hill coefficient of 2.3 ± 0.4, indicating the involvement of more than one Na+ in the transport process. Ntcp in primary cultured hepatocytes from rats exhibited similar Na+-activation kinetics of TCA uptake rate with a Hill coefficient of 1.9 ± 0.1, suggesting that hNTCP could be expressed properly in the oocytes and exhibit the electrogenic property of Na+-coupled TCA transport. The transport of TCA via hNTCP was subsequently determined in the oocytes by the inward currents induced via TCA uptake under voltage (50 mV). Two hundred micromolar TCA induced significant inward currents that were entirely abolished by the substitution of Na+ with N-methyl-D-glucamine (NMDG) in the perfusate, indicating that the TCA-induced currents were obligatorily dependent on the presence of Na+. The TCAinduced currents were saturable, and the substrate concentration needed for half-maximal induction of the current was consistent with the Michaelis constant. Transportable substrates, such as rosuvastatin and fluvastatin, also induced currents. These results in the hNTCP heterologously expressed in Xenopus oocytes directly demonstrated that hNTCP is an electrogenic Na+-dependent transporter. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Bile acids are synthesized from cholesterol in the liver and secreted into the bile. They play an important role not only in the maintenance of bile flow and biliary lipid secretion but also in cholesterol homeostasis through its secretion in the form of bile acids [1,2]. Furthermore, bile acids secreted into the intestine function as solubilizing agents of various lipophilic substances [3–5]. More than 95% of the bile acids secreted are efficiently reabsorbed by the intestinal epithelial cells and returned to the liver through ⇑ Corresponding author. E-mail address: [email protected] (S. Miyauchi). http://dx.doi.org/10.1016/j.abb.2014.08.010 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.
portal blood circulation. Bile acids are also taken up by hepatocytes and resecreted into bile, thus undergoing enterohepatic circulation in which several transporters in the liver and intestine are crucial [6–8]. Among them, NTCP (SLC10A1), which is predominantly expressed at the basolateral membrane, is a major component of Na+-dependent uptake of the bile acids into the liver [1,8,9]. The Na+-dependent transport system is responsible for approximately 80% of the hepatic uptake of bile acids. Thus, NTCP is the key transporter for hepatic uptake of bile acids, and alteration in the transport activity via NTCP impacts the homeostasis of liver function, especially bile flow and enterohepatic circulation [6–8]. It is plausible that impaired uptake of bile acids via NTCP may cause a remarkable increase in the bile acid concentration in the blood,
116
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
which is associated with cytotoxicity, cellular membrane damage, mitochondrial dysfunction and alteration of cellular metabolism [10–12]. Hypercholanemia has often been associated with adverse extrahepatic effects, including the well-known effect of pruritus [13]. In addition to the important role of NTCP in the maintenance of normal liver function, another important feature is its broad substrate recognition and capability to transport a variety of amphiphilic compounds [14–17]. In addition to bile acids, NTCP also has the potential to transport some structurally unrelated drugs, such as HMG-CoA reductase inhibitors [18–20], the liver function diagnostics bromosulfophthalein and indocyanine green [21,22], as well as the antifungal drug micafungin [23]. Consequently, it is possible that various drugs in clinical use could interact with NTCP and inhibit its function. Such inhibition could be involved in NTCP-mediated drug–drug interactions and drug-induced toxicities [7]. For the sake of the identification and avoidance of NTCP inhibitors, their interactions with NTCP have been investigated extensively with human hepatocytes [20,24,25] as well as mammalian cells expressing hNTCP [15,17,19]. To date, the transport cycle of Na+ and bile acid co-transport has only been minimally studied, despite an increase in evidence with regard to NTCP-mediated drug–drug interactions and its substrate recognition [7,8,15–17]. Although substrate transport via NTCP unequivocally exhibits an obligatory Na+ dependency, the electrogenicity of the NTCP transport cycle remains controversial [26–32]. In the present study, we established heterologous expression of hNTCP in Xenopus laevis oocytes and directly demonstrated and characterized the electrogenic transport of bile acids via hNTCP. hNTCP functioned properly and robustly in X. laevis oocytes, in which TCA induced Na+-dependent inward currents associated with the transport via hNTCP. As far as we are aware, the present study is the first report to directly demonstrate the electrogenicity of hNTCP expressed in X. laevis oocytes. Materials and methods Materials [3H]-TCA (specific radioactivity, 10 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). Bile acids including TCA and its derivatives were purchased from Sigma (Tokyo, Japan). Six-week-old Sprague Dawley (SD)1 rats were purchased from Sankyo Labo Corp. Inc. (Tokyo, Japan). All other chemicals used were of the highest purity available from Wako Chemical Inc. (Osaka, Japan). Amplification of the coding region of hNTCP The cloning of the coding region of hNTCP from a human liver cDNA library (Takara, Tokyo, Japan) was performed with polymerase chain reaction (PCR) as described previously [33]. Based on the published sequence in the GenBank™ data base (accession number L21893), we designed primers introduced restriction enzyme site for cloning purposes. The sense primer, containing an initiation codon, was 50 -GAATTCGATATATAGCCATGGAGGCCCACAACGCGTC, and the antisense primer, located near the stop codon, was 50 AAGCTTCTAGGCTGTGCAAGGGGAGC. The underlined sequences in the sense and antisense primers are EcoRI and HindIII sites, respectively. PCR with these primers using human liver cDNA 1 Abbreviations used: Hepes, N-2-hydroxy-ethylpiperazine-N0 -2-ethanesulfonic acid; hNTCP, human Na+/taurocholate cotransporting polypeptide; LC–MS/MS, liquid chromatography coupled with tandem mass spectrometry; Mes, 2-(N-morpholino) ethanesulfonate; NMDG, N-methyl-D-glucamine; PCR, polymerase chain reaction; SD, Sprague Dawley; TCA, taurocholate; Tris, Tris (hydroxymethyl) aminomethane.
library as the template yielded a 1 kbp product, as expected. This product was subcloned into a TA-cloning vector, pGEM-T Easy (Promega, Madison, WI). The sequence of the subcloned PCR product was determined on both strands with the Taq DyeDeoxy terminator cycle method using Applied Biosystems 3500 (Life Technologies Japan Ltd., Tokyo). The insert containing the entire hNTCP was excised with EcoRI and HindIII and subcloned into pGH19. The pGH19 vector (kindly provided by Dr. Peter S. Aronson, Yale University School of Medicine) contains the 30 - and 50 untranslated region of the X. laevis b-globin gene down- and up-stream of the cloning site, respectively. Functional expression of hNTCP in X. laevis oocytes The amplified hNTCP cDNA was expressed heterologously in X. laevis oocytes by cRNA injection. Capped cRNA from hNTCP in the pGH19 vector was synthesized using AmpliCap™ T7 High Yield Message Maker Kits (Epicentre Biotechnologies, Madison, WI). Mature oocytes (stage V–VI) from X. laevis were isolated by treatment with 1.0 mg/ml collagenase A (Roche Diagnostics GmbH, Mannheim, Germany), manually defolliculated and incubated at 18 °C in modified Barth’s medium supplemented with 50 lg/ml gentamicin as described previously [33,34]. On the following day, each oocyte was injected with 10 ng hNTCP cRNA in a 50 nl volume and incubated for 3–5 days. The oocytes were used for uptake and electrophysiological studies 3–4 days after cRNA injection. H2Oinjected oocytes served as controls. Transport analysis of hNTCP expressed in X. laevis oocytes Uptake of [3H]-TCA into oocytes injected with hNTCP-cRNA or H2O was measured as described previously [35]. Eight to ten oocytes were pre-incubated at room temperature for 2 min in NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM N-2-hydroxy-ethylpiperazine-N0 -2-ethanesulfonic acid (Hepes), adjusted to pH 7.5 with Tris (hydroxymethyl) aminomethane (Tris). The buffer was then replaced by the NaCl buffer supplemented with TCA containing [3H]-TCA (2 lCi/ml) at the given concentrations (1–200 lM). At the given times, the oocytes were gently washed 5 times with 2 ml of ice-cold NMDGCl buffer, in which NaCl is replaced by an iso-osmolar amount of N-methyl-Dglucamine (NMDG) chloride in the NaCl buffer. Each oocyte was transferred to a scintillation vial and dissolved in a 200 lL aliquot of 1% SDS/0.2 M NaOH solution. The radioactivity was determined in a 10 mL aliquot of a scintillation counting cocktail (Clear-sol I, Nacalai Tesque Inc., Tokyo, Japan) with Tri-Carb 2910TR liquid scintillation counter (PerkinElmer Inc., Waltham, MA). The uptake data in the presence of competitors at various concentrations were normalized by the value in the absence of competitor and expressed as the percent of control. The Na+-activation kinetics of TCA uptake was analyzed by measuring the TCA uptake in the presence of increasing Na+ concentrations. The Na+ concentration was varied by iso-osmotically replacing NaCl with NMDG chloride. Electrophysiological studies were performed with the two-electrode voltage clamp (TEVC) technique as described previously [33,36]. The membrane potential was clamped at 50 mV. The oocyte was always superfused with perfusion buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM Hepes/Tris adjusted to pH 7.5). After the current stabilized, the oocyte was superfused with NaCl buffer containing 200 lM quinine. The quinine was used as K+ channel blocker to minimize outward-directed K+ leakage [31]. Up to 400 lM quinine did not have any influence on TCA uptake via hNTCP (Fig. 3). The oocyte was superfused with the same buffer additionally containing test compound. After application of the test compound, the current reached the maximum and steady state, and the test compound was washed out with NMDGCl
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
117
Fig. 1. TCA uptake via NTCP in X. laevis oocytes (A) Na+ dependency of TCA uptake via hNTCP. The uptake of 1.2 lM TCA was measured for 30 min at room temperature and pH 7.5. cRNA and H2O represent hNTCP cRNA-injected and H2O-injected oocytes, respectively. The closed and open bars represent the uptake in NaCl and NMDGCl buffers, respectively. Each bar represents the mean ± SE (n = 10). (B) Time course of TCA uptake by X. laevis oocytes expressing hNTCP. The uptake of 1.2 lM TCA by H2O-(open circles) or hNTCP cRNA-injected (filled circles) oocytes was measured at room temperature and pH 7.5. Each point represents the mean ± SE (n = 10).
buffer. The substrate-induced current at an applied voltage was calculated as the difference between the steady-state currents recorded in the absence and presence of substrate. Cl-free NaMes buffer was composed of 2 mM K gluconate, 1 mM Mg(gluconate)2, 1 mM Ca(gluconate)2, 100 mM sodium 2-(N-morpholino) ethanesulfonate (NaMes) and 10 mM Hepes/Tris adjusted to pH 7.5. Uptake measurements by primary cultured hepatocytes from rat liver Hepatocytes were isolated from the livers of 6-week-old male SD rats that were given free access to food and water. The isolation procedure was basically the same as previously reported [37,38]. Cell viability, determined by trypan blue exclusion, ranged from 95% to 98%. Cells were suspended at the density of 5 105 cells/ ml in William’s E medium supplemented with dexamethasone (1 nM), insulin (1 nM), 10% fetal calf serum, 100 U/ml penicillin and 100 lg/ml streptomycin. One milliliter aliquot cell suspension was added to each well of a 12-well plate. The culture medium was exchanged with fresh medium after 4 h following the initial seeding. Cells were cultured for an additional 16 h at 37 °C in the incubator with 5% CO2/95% air and then used for TCA uptake experiments [39]. The experimental protocol for the use of the animals was approved by the University Animal Care and Use Committee. Uptake of TCA in primary culture hepatocytes was measured at 37 °C in uptake buffer (pH 7.5) as described previously [39]. The composition of the uptake buffer was 140 mM NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 10 mM Hepes/Tris
adjusted to pH 7.5. The Na+-activation kinetics of TCA uptake was analyzed by measuring the TCA uptake in the presence of increasing Na+ concentrations. The Na+ concentration was varied by iso-osmotically replacing NaCl with NMDG chloride. After TCA uptake by hepatocytes for the designated period, the uptake was immediately terminated by washing the cells twice with ice-cold NMDGCl buffer. The cells were then dissolved in a 500 ll aliquot of 1% SDS/0.2 M NaOH solution, and the radioactivity associated with the cells was measured.
Kinetics analyses Saturation kinetics was analyzed by fitting the concentration dependency of the initial uptake rate of TCA to a Michaelis Menten-type equation with a saturable component [33]. The kinetic parameters (Vmax, the maximum uptake rate; Km, Michaelis constant) were determined by an iterative nonlinear least-squares method in Origin version 8.5 (MicroCal, Northampton, MA). The currents evoked by TCA were analyzed in a similar manner; kinetic parameters were defined by the maximum induced current, Imax (nA), and the concentration necessary for half-maximal induction of the current, K0.5 (lM), instead of Vmax and Km, respectively. The Na+-activation kinetics was analyzed by fitting the data to the Hill equation, and the Hill coefficient (h, the number of Na+ ions involved in the activation process) and K0.5 value (the concentration of Na+ necessary for half-maximal activation) were determined [35].
Fig. 2. Saturation kinetics and Na+-activation kinetics for TCA uptake via hNTCP. (A) Concentration dependency of TCA uptake by hNTCP. The uptake rates of TCA were measured over 10 min at room temperature and pH 7.5 with an increase in substrate concentrations (2–200 lM). The inset figure shows the Eadie–Hofstee plot of TCA uptake via hNTCP. Each point represents the mean ± SE (n = 7). Lines fit to the non-linear or linear forms of the Michaelis–Menten equation are shown. (B) Na+ activation of TCA uptake by hNTCP. The uptake rate of 2 lM TCA was measured for 10 min at room temperature and pH 7.5 with an increase in Na+ concentrations. TCA uptake rate in (B) represents the Na+-dependent uptake rate, which was estimated by the subtraction of TCA uptake rate in the absence of Na+ from TCA uptake rate in the presence of Na+ at various concentrations. The inset figure shows the Hill plot of the Na+ dependency of the uptake via hNTCP. Each point represents the mean ± SE (n = 7–8). Lines fit to the nonlinear or linear forms of the Hill equation are shown.
118
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
Statistical analyses All experiments were conducted at least 5 times, and results are expressed as the mean ± SE. Statistical analyses between two groups were performed with Student’s t-test. A difference between means was considered if P < 0.05. Results Functional expression of hNTCP in X. laevis oocytes Uptake of 1.2 lM TCA was measured in X. laevis oocytes injected with hNTCP cRNA, and the results are summarized in Fig. 1. In cRNA-injected oocytes, the uptake of 1.2 lM TCA increased more than 100-fold compared to that in H2O injected oocytes, indicating that hNTCP is expressed robustly in X. laevis oocytes. Removal of Na+ from the uptake buffer reduced the uptake to the levels found in H2O-injected oocytes, indicating that the enhanced uptake required Na+. The TCA uptake linearly increased over 15 min, and the initial uptake rate was determined by the uptake in 10 min. The saturation kinetics and Na+-activation kinetics for TCA transport via hNTCP were analyzed and are depicted in Fig. 2. The TCA uptake rate was saturable, and the data fit the Michaelis–Menten equation describing a single saturable process, which was also confirmed by the linearity of Eadie–Hofstee plot shown in the inset. The kinetic parameters Km (Michaelis–Menten constant) and Vmax (maximal uptake rate) were 10.5 ± 2.9 lM and 18.6 ± 1.5 nmol/oocyte/10 min, respectively. The Km value for hNTCP expressed in X. laevis oocytes was similar to that reported for hNTCP expressed in mammalian cells [15,19]. The Na+-mediated increase of the TCA uptake rate showed sigmoidal features. The relationship fit the Hill equation well, with Hill coefficient (h) and concentration necessary for half-maximal activation (K0.5) values estimated to be 1.9 ± 0.1 and 26.9 ± 2.0 mM, respectively. The h value implied that for every TCA molecule transported, at least two Na+ ions were cotransported. Because TCA is a single anion at physiological pH, a Na+:TCA stoichiometry of 2:1 could suggest an electrogenic transport mechanism via hNTCP. The h value obtained from oocytes expressing hNTCP was compatible with that in oocytes expressing rat Ntcp and the isolated rat hepatocytes [30,40,41].
The effects of various bile acids and statins on the uptake of [3H]-TCA via hNTCP were examined and are summarized in Fig. 3. All bile acids inhibited TCA uptake; 200 lM taurochenodeoxycholate (TCDCA) inhibited TCA uptake most potently, indicating that NTCP might prefer conjugated bile acids to unconjugated bile acids. In addition, statins such as rosuvastatin and fluvastatin also inhibited TCA uptake by hNTCP at levels comparable to those reported by others [18–20]. In contrast, up to 400 lM quinine, which was used as a K+ channel blocker in electrophysiological experiments, had no effect on TCA uptake (Fig. 3) [31,32]. Na+-activation kinetics of TCA uptake in primary cultured hepatocytes from rat liver To elucidate whether hNTCP was properly expressed in X. laevis oocytes, we investigated the Na+-activation kinetics of TCA uptake in primary cultured rat hepatocytes. Fig. 4 shows that the uptake of TCA by primary cultured rat hepatocytes was linear up to 2 min, and two-thirds of the TCA uptake exhibited Na+-dependency. The uptake rates were evaluated by the uptake amounts at 2 min. The Na+-activation kinetics of TCA uptake also exhibited sigmoidal kinetics, indicating the participation of more than one Na+ in the TCA transport process. The values of h and K0.5 values by rat Ntcp in primary cultured hepatocytes were essentially the same (h = 1.9 ± 0.3 and 89 ± 21, respectively) as those of hNTCP expressed in X. laevis oocytes (Fig. 2), suggesting that hNTCP functions properly irrespective of the robust expression. Substrate-induced currents associated with Na+-dependent transport via heterologously expressed hNTCP in X. laevis oocytes To investigate whether the Na+-dependent TCA transport is electrogenic or not, the transport of TCA via hNTCP was monitored by electrophysiological techniques using the two-electrode voltage-clamp method. As shown in Fig. 5, 200 lM TCA induced an inward current. H2O-injected oocytes did not exhibit TCA-induced currents. The substitution of Na+ with NMDG in the perfusate completely abolished the TCA-induced current, whereas Cl substitution with Mes in the perfusate had a minimal influence on the TCA-induced current. The effect of ion substitution indicates that the induced currents were associated with Na+-dependent TCA
Fig. 3. Inhibition of [3H]-TCA uptake via hNTCP by various bile acids (A) and statins (B). The uptake rate of 2 lM [3H]-TCA was measured for 10 min at room temperature and pH 7.5 with and without 20 or 200 lM bile acids and 20, 200 or 500 lM statins. The abbreviations of bile acids are as follows: CA, cholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; LCA, lithocholic acid. The uptake rates are normalized to those in the absence of bile acids or statins. Each bar represents the mean ± SE (n = 10). All test compounds except for 200 lM and 400 lM quinine (#) and 20 lM pravastatin (*) exhibit significantly different from control (P < 0.01). #Not significantly different from control. ⁄Significantly different from control (P < 0.05).
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
119
Fig. 4. Time course of TCA uptake (A) and Na+-activation kinetics (B) for TCA uptake by primary cultured rat hepatocytes. The uptake of 1 lM TCA in the presence (filled circles) or absence (open circles) of Na+ was measured at 37 °C and pH 7.5. Each point represents the mean ± SE (n = 6). The uptake rate of TCA in Na+-activation kinetics was measured for 2 min at 37 °C and pH 7.5. TCA uptake rate in (B) represents the Na+-dependent uptake rate, which was estimated by the subtraction of TCA uptake rate in the absence of Na+ from TCA uptake rate in the presence of Na+ at various concentrations. Each point represents the mean ± SE (n = 6).
Fig. 5. Tracers of representative 200 lM TCA induced currents in H2O-injected (A) and hNTCP cRNA-injected (B) X. laevis oocytes. Inward currents induced by 200 lM TCA were monitored in oocytes at membrane potential clamped at 50 mV. Three different perfusion buffers, NaCl buffer (+Na+), NMDGCl buffer (Na+), and NaMes buffer (–Cl), were used. The buffers used just before and during TCA superfusion contained 200 lM quinine.
Fig. 6. Saturation kinetics for TCA-induced currents. Inward currents induced by increasing concentrations of TCA were monitored at 50 mV in five different oocytes expressing hNTCP. The data were normalized for variations in cRNA expression in different oocytes by taking the maximal value for the induced current (Vmax) as 1 in each oocyte. The estimated Vmax value was 37.3 ± 3.4 nA (mean ± SE (n = 5)). The inset figure shows Eadie–Hofstee plot of TCA-induced currents via hNTCP. Each point represents the mean ± SE (n = 5). Lines fit to the non-linear or linear forms of the Michaelis–Menten equation are shown.
transport via hNTCP, but there is no Cl involvement in the transport. We also analyzed the saturation kinetics of TCA transport via hNTCP by monitoring the TCA-induced current (Fig. 6). The induced currents attributed to TCA transport were saturable, and the data fit the Michaelis–Menten equation describing a single saturable process. The substrate concentration needed for half-
maximal induction of current (K0.5) was 30 ± 6.2 lM (± calculated SD), which means that the Michaelis constant for TCA transport is comparable with those reported by others [15,19,42–44]. Furthermore, the transport of TCDCA and various statins by hNTCP were determined by studying the inward currents induced by these compounds (Fig. 7). The current induced by TCDCA was the most potent, suggesting that TCDCA is a good transportable substrate. Among statins, fluvastatin induced the most potent current followed by rosuvastatin, but pravastatin induced a minimal current. Their induced currents were completely abolished by the removal of Na+ from the perfusate. The magnitude of the currents induced by statins correlated with the transport activities of various statins by hNTCP reported by others [18–20]. This good relationship could imply that the present experimental system exhibits the potential advantage that we can easily investigate whether drugs currently in clinical use have the potential to interact with NTCP and alter transport activity via NTCP without the need for radiolabeled drugs or a precise assay system such as a liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS).
Discussion NTCP is predominantly expressed at the basolateral membrane in the liver and is mostly involved in Na+-dependent uptake of bile acids from the portal blood circulation. To date, the Na+-dependent uptake via hNTCP has been corroborated by various experimental
120
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
systems with isolated perfused liver [45], isolated hepatocytes [20,23,24,46], isolated membrane vesicles [47] and hNTCP expressed in mammalian cultured cells [15–17] or heterologously in X. laevis oocytes [22,43]. As far as we are aware, however, there is no direct evidence for the electrogenic nature of bile acid transport via hNTCP. The electrogenicity of hNTCP still remains controversial, although there are ample evidences supporting the electrogenic feature, exemplified by the Na+-bile acid coupling stoichiometry (2:1) [40,41], the depolarization potential induced by bile acid uptake via NTCP and the stimulation of Na+-dependent bile acid uptake by the hyperpolarization of the membrane potential [30–32]. In the present study, we used a conventional electrophysiological technique in X. laevis oocytes expressing hNTCP to directly measure the current induced by the bile acid transport via hNTCP. We established a heterologous hNTCP expression system in X. laevis oocytes and characterized the features of TCA transport. As shown in Fig. 3, the transport activity in hNTCPexpressing oocytes was robust compared to that of H2O-injected oocytes, and the transport function was dependent on Na+. Na+activation kinetics of hNTCP-mediated TCA uptake showed that multiple Na+ ions are involved in each transport cycle as is evident from the sigmoidal feature of the Na+ stimulation in TCA uptake (Fig. 5). The Hill coefficient for the activation process is 1.9 ± 0.1 (Fig. 2), suggesting that the involvement of more than one Na+ per transport cycle is feasible and implying an electrogenic characteristic of the hNTCP transport process. Studies using an electrophysiological approach provided unequivocal data corroborating the electrogenic feature of hNTCP transport process; Na+-dependent TCA transport via hNTCP induced significant transport currents, which exhibited dependency on Na+ as well as saturation kinetics (Fig. 6). The concentration necessary for the half maximal induced current (K0.5 = approx. 30 lM) was compatible with the Km values obtained in the saturation kinetics of TCA uptake and those reported by others [15,19,42–44]. The transportable substrates fluvastatin and rosuvastatin also induced currents (see Fig. 7), the magnitudes of which were consistent with the uptake rate obtained through direct assay of the amounts of statins [18–20]. This consistency between the two methodologies argued for the most feasible explanation, that the substrate–induced currents are attributed to their transport via hNTCP. In recent years, the crystal structures of the apical sodium-dependent transporter (ASBT) homologs were reported by Hu et al. [48] and Zhou et al. [49]. These ASBT homologs have extensive sequence and functional
Fig. 7. Inward currents induced by bile acids and statins via hNTCP. Oocytes were perfused with the NaCl buffer followed by the same buffer containing bile acids (200 lM TCA and 100 lM TCDCA) and statins (500 lM pravastatin, 500 lM rosuvastatin and 200 lM fluvastatin). Substrate-induced currents were determined by the difference in the currents in the presence and absence of substrate when clamped at 50 mV. The substrate-induced currents were normalized to 200 lM TCA-induced currents. Data represent mean ± SE (n = 6). The current induced by 200 lM TCA were 32.2 ± 1.8 nA (mean ± SE (n = 6)).
similarities to NTCP. In particular, the key amino acid residues involved in the transport function are conserved among these transporters, leading us to a plausible hypothesis that they translocate Na+ ions and bile acids by a similar molecular mechanism. Therefore, the crystal structures of ASBT homologs potently facilitate our understanding of their molecular mechanisms of substrate recognition, translocation and energy coupling with Na+ [48,49]. These structures clearly showed an inward open conformation in which two Na+ ions and TCA bound to the core domain. The Na+binding sites were located behind the crossover region between the helixes to regulate the accessibility of the solvent from the extracellular and cytoplasmic spaces. Furthermore, they demonstrated that more than one Na+ cooperatively binds to the transporter, independent of the TCA bound, with the assay of Na+ binding to the purified ASBT. They also solved another structure of the ASBT homolog in which the TCA-binding domain was largely rotated. Based on these features of the ASBT homolog, they proposed a preliminary model of the ASBT transport cycle in which the release of two Na+ ions driven by exposure to lower Na+ concentrations in the cytoplasm triggers TCA release, so two Na+ ions and one TCA molecule are simultaneously transported. The proposed model explains our data with regard to the electrogenicity of NTCP [49] (see Fig. 7). The present study provided unequivocal and direct evidence that hNTCP transports TCA electrogenically with the involvement of more than one Na+ per TCA transport cycle. However, earlier investigations of TCA transport via Ntcp with isolated hepatocytes and liver basolateral membrane vesicles yielded controversial results in favor of either an electroneutral or electrogenic transport mechanism [26–32]. These studied focused on measurements of radio isotopic flux and provided only indirect evidence for electrogenicity due to TCA uptake. An electroneutral transport mechanism has been suggested based on the flux ratio of Na+ to TCA (1:1) in isolated rat hepatocytes [26] due to the lack of stimulatory effects of valinomycin-enhanced K+-gradient and permeable anions on TCA uptake [28,29]. In contrast, an electrogenic transport mechanism has been corroborated in hepatocytes by the stoichiometry of Na+ and TCA cotransport (2:1) [30,40], the depolarization of the membrane potential caused by the Na+/TCA cotransporter and the enhancement of TCA uptake by hyperpolarization of the membrane potential induced by glucagon or valinomycin [50,51]. The reason for these controversial results does not seem to be entirely clear. One plausible explanation is that it is very difficult to precisely control the membrane potential of isolated vesicles and hepatocytes with ionophores and permeable anions. This difficulty might cause uncertainty. Another explanation might be that the Na+ and TCA fluxes via Ntcp in isolated vesicles or isolated hepatocytes induce a minimal current whose measurement is hampered by the large total membrane conductance. In other words, changes in other ion membrane conductance could compensate for small currents attributed to electrogenic fluxes of Na+ and bile acid via NTCP. In the present study, to overcome these drawbacks of using native membrane vesicles and hepatocytes, we established an hNTCP expression system in X. laevis oocytes that is able to produce a measurable current due to the transport of bile acid. However, we should carefully consider a disadvantage that the robust expression might provide a nonphysiological environment and perturb the function of the transporter as well as the membrane fluidity, thereby affecting an ion conductance. In addition, it should also be considered that bile acids are relatively good detergents and may alter membrane fluidity and the ion conductance of membranes [3]. It is thus very difficult to completely rule out the effect of conductance of any other ion on the induced currents due to bile acid transport via hNTCP in the present study. Consequently, to precisely study the relationship between the induced currents and the fluxes of Na+ and TCA, we
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
should use a complementary approach to minimize nonspecific changes in the conductance of any ions. Furthermore, we should also investigate the effect of bile acid on the alteration of ion conductance. In the present study, we established a useful experimental system in which the transport activity of bile acids and their analogs via hNTCP can be monitored directly by studying substrate-induced currents. If the radiolabeled test compounds or precise assay systems such as a LC–MS/MS are not available, this system enables us to distinguish whether compounds are transportable substrates for hNTCP or are blockers that block the uptake of bile acids by competing for the substrate-binding site in hNTCP without being transported across the membrane [34,52]. If the test compound is a transportable substrate, it should induce inward Na+-dependent currents in oocytes expressing hNTCP. If the test compound is a blocker and not a transportable substrate, no inward current associated with the transport via hNTCP would be observed; instead, the inward currents induced by bile acids would be abolished in a concentration-dependent manner. In terms of drug–drug interactions, the present experimental system exhibits the potential advantage that we can easily investigate whether drugs currently in clinical use have the potential to interact with hNTCP and alter transport activity via hNTCP. In conclusion, we established an hNTCP-expressing oocyte system and directly demonstrated that the transport mechanism of hNTCP is electrogenic. This system is a useful tool to clarify whether a compound that interacts with hNTCP is a transportable substrate or merely a blocker that is not transported across the membrane. Acknowledgment This work was supported by the grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology to Seiji Miyauchi (23590062). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
P.J. Meier, B. Stieger, Annu. Rev. Physiol. 64 (2002) 635–661. P.A. Dawson, T. Lan, A. Rao, J. Lipid Res. 50 (2009) 2340–2357. A.F. Hofmann, Gastroenterology 48 (1965) 484–494. G.M. Murphy, E. Signer, Gut 15 (1974) 151–163. K. Ebihara, B.O. Schneeman, J. Nutr. 119 (1989) 1100–1106. G.A. Kullak-Ublick, B. Stieger, P.J. Meier, Gastroenterology 126 (2004) 322–342. C. Pauli-Magnus, P.J. Meier, Hepatology 44 (2006) 778–787. B. Doring, T. Lutteke, J. Geyer, E. Petzinger, Curr. Top. Membr. 70 (2012) 105– 168. W.A. Alrefai, R.K. Gill, Pharm. Res. 24 (2007) 1803–1823. J.R. Spivey, S.F. Bronk, G.J. Gores, J. Clin. Invest. 92 (1993) 17–24. B. Bouscarel, S. Ceryak, T.W. Gettys, H. Fromm, F. Noonan, Am. J. Physiol. 268 (1995) G908–916. J.P. Meng, S. Ceryak, Z. Aratsu, L. Jones, L. Epstein, B. Bouscarel, Am. J. Physiol. Cell Physiol. 291 (2006) C546–554. E.M. Kuiper, K.J. van Erpecum, U. Beuers, B.E. Hansen, H.B. Thio, R.A. de Man, H.L. Janssen, H.R. van Buuren, Hepatology 52 (2010) 1334–1340.
121
[14] P.J. Meier, U. Eckhardt, A. Schroeder, B. Hagenbuch, B. Stieger, Hepatology 26 (1997) 1667–1677. [15] R.B. Kim, B. Leake, M. Cvetkovic, M.M. Roden, J. Nadeau, A. Walubo, G.R. Wilkinson, J. Pharmacol. Exp. Ther. 291 (1999) 1204–1209. [16] R. Greupink, S.B. Nabuurs, B. Zarzycka, V. Verweij, M. Monshouwer, M.T. Huisman, F.G. Russel, Toxicol. Sci. 129 (2012) 35–48. [17] Z. Dong, S. Ekins, J.E. Polli, Mol. Pharm. 10 (2013) 1008–1019. [18] R.H. Ho, R.G. Tirona, B.F. Leake, H. Glaeser, W. Lee, C.J. Lemke, Y. Wang, R.B. Kim, Gastroenterology 130 (2006) 1793–1806. [19] R. Greupink, L. Dillen, M. Monshouwer, M.T. Huisman, F.G. Russel, Eur. J. Pharm. Sci. 44 (2011) 487–496. [20] Y.A. Bi, X. Qiu, C.J. Rotter, E. Kimoto, M. Piotrowski, M.V. Varma, A.F. Ei-Kattan, Y. Lai, Biopharm. Drug Dispos. 34 (2013) 452–461. [21] S. Hata, P. Wang, N. Eftychiou, M. Ananthanarayanan, A. Batta, G. Salen, K.S. Pang, A.W. Wolkoff, Am. J. Physiol. Gastrointest. Liver Physiol. 285 (2003) G829–G839. [22] W. de Graaf, S. Hausler, M. Heger, T.M. van Ginhoven, G. van Cappellen, R.J. Bennink, G.A. Kullak-Ublick, R. Hesselmann, T.M. van Gulik, B. Stieger, J. Hepatol. 54 (2011) 738–745. [23] S.B. Yanni, P.F. Augustijns, D.K. Benjamin Jr., K.L. Brouwer, D.R. Thakker, P.P. Annaert, Drug Metab. Dispos. 38 (2010) 1848–1856. [24] E.I. Lepist, H. Gillies, W. Smith, J. Hao, C. Hubert, R.L. St Claire III, K.R. Brouwer, A.S. Ray, PLoS One 9 (2014) e87548. [25] S.B. Yanni, P.B. Smith, D.K. Benjamin Jr., P.F. Augustijns, D.R. Thakker, P.P. Annaert, Biopharm. Drug Dispos. 32 (2011) 222–232. [26] B.F. Scharschmidt, J.E. Stephens, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 986– 990. [27] M. Inoue, R. Kinne, T. Tran, I.M. Arias, Hepatology 2 (1982) 572–579. [28] M.C. Duffy, B.L. Blitzer, J.L. Boyer, J. Clin. Invest. 72 (1983) 1470–1481. [29] P.J. Meier, A. St, J. Biol. Chem. 259 (1984) 10614–10622. [30] S.D. Lidofsky, J.G. Fitz, R.A. Weisiger, B.F. Scharschmidt, Am. J. Physiol. 264 (1993) G478–485. [31] S.A. Weinman, R.P. Weeks, Am. J. Physiol. 265 (1993) G73–80. [32] S.A. Weinman, Yale J. Biol. Med. 70 (1997) 331–340. [33] S. Miyauchi, E. Gopal, Y.J. Fei, V. Ganapathy, J. Biol. Chem. 279 (2004) 13293– 13296. [34] S. Miyauchi, E. Gopal, E. Babu, S.R. Srinivas, Y. Kubo, N.S. Umapathy, S.V. Thakkar, V. Ganapathy, P.D. Prasad, Biochim. Biophys. Acta 1798 (2010) 1164– 1171. [35] S. Miyauchi, S.R. Srinivas, Y.J. Fei, E. Gopal, N.S. Umapathy, H. Wang, S.J. Conway, V. Ganapathy, P.D. Prasad, Placenta 27 (2006) 550–559. [36] E. Gopal, Y.J. Fei, M. Sugawara, S. Miyauchi, L. Zhuang, P. Martin, S.B. Smith, P.D. Prasad, V. Ganapathy, J. Biol. Chem. 279 (2004) 44522–44532. [37] S. Miyauchi, Y. Sawada, T. Iga, M. Hanano, Y. Sugiyama, Pharm. Res. 10 (1993) 434–440. [38] T. Iga, D.L. Eaton, C.D. Klaassen, Am. J. Physiol. 236 (1979) C9–14. [39] E. Gopal, S. Miyauchi, P.M. Martin, S. Ananth, S.R. Srinivas, S.B. Smith, P.D. Prasad, V. Ganapathy, Am. J. Physiol. Gastrointest. Liver Physiol. 292 (2007) G402–408. [40] M.S. Anwer, D. Hegner, Hoppe Seylers Z. Physiol. Chem. 359 (1978) 181–192. [41] B. Hagenbuch, P.J. Meier, Semin. Liver Dis. 16 (1996) 129–136. [42] S.A. Azer, N.H. Stacey, Biochem. Pharmacol. 46 (1993) 813–819. [43] B. Hagenbuch, P.J. Meier, J. Clin. Invest. 93 (1994) 1326–1331. [44] S. Abu-Hayyeh, P. Martinez-Becerra, S.H. Sheikh, J. Biol. Chem. 285 (2010) 16504–16512. [45] B.N. Melgert, P. Olinga, B. Weert, M.J. Slooff, D.K. Meijer, K. Poelstra, G.M. Groothuis, Drug Metab. Dispos. 29 (2001) 361–367. [46] G.W. Sandker, B. Weert, P. Olinga, H. Wolters, M.J. Slooff, D.K. Meijer, G.M. Groothuis, Biochem. Pharmacol. 47 (1994) 2193–2200. [47] D.A. Novak, F.C. Ryckman, F.J. Suchy, Hepatology 10 (1989) 447–453. [48] N.J. Hu, S. Iwata, A.D. Cameron, D. Drew, Nature 478 (2011) 408–411. [49] X. Zhou, E.J. Levin, Y. Pan, J.G. McCoy, R. Sharma, B. Kloss, R. Bruni, M. Quick, M. Zhou, Nature 505 (2014) 569–573. [50] J.W. Edmondson, B.A. Miller, L. Lumeng, Am. J. Physiol. 249 (1985) G427–433. [51] J.G. Fitz, B.F. Scharschmidt, Am. J. Physiol. 252 (1987) G56–64. [52] S. Itagaki, E. Gopal, L. Zhuang, Y.J. Fei, S. Miyauchi, P.D. Prasad, V. Ganapathy, Pharm. Res. 23 (2006) 1209–1216.
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Electrophysiological characterization of human Na+/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes Masayuki Masuda a, Yukari Ichikawa a, Kazumi Shimono a, Maki Shimizu a, Yoshio Tanaka a, Toshifumi Nara b, Seiji Miyauchi a,⇑ a b
Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime 790-8578, Japan
a r t i c l e
i n f o
Article history: Received 6 June 2014 and in revised form 12 August 2014 Available online 27 August 2014 Keywords: Bile acid Electrogenicity Hepatic uptake Na+-dependent transporter NTCP SLC10A1 Xenopus laevis oocyte
a b s t r a c t The Na+/taurocholate cotransporting polypeptide (NTCP) plays a major role in Na+-dependent bile acid uptake into hepatocytes. The purpose of the present study was to establish the heterologous expression of human NTCP (hNTCP) in Xenopus laevis oocytes and to elucidate whether the transport of bile acid via hNTCP is electrogenic using electrophysiological techniques. First, we evaluated the uptake of taurocholate (TCA) by hNTCP heterologously expressed in Xenopus oocytes utilizing [3H]-labeled TCA. The uptake of 1.2 lM TCA by cRNA-injected oocytes increased more than 100-fold compared to H2O-injected oocytes, indicating that hNTCP is robustly expressed in the oocytes. hNTCP-mediated transport of TCA is saturable with a Michaelis constant of 10.5 ± 2.9 lM. The Na+-activation kinetics describing the relationship between the concentration of Na+ and the magnitude of the TCA uptake rate by hNTCP were sigmoidal with a Hill coefficient of 2.3 ± 0.4, indicating the involvement of more than one Na+ in the transport process. Ntcp in primary cultured hepatocytes from rats exhibited similar Na+-activation kinetics of TCA uptake rate with a Hill coefficient of 1.9 ± 0.1, suggesting that hNTCP could be expressed properly in the oocytes and exhibit the electrogenic property of Na+-coupled TCA transport. The transport of TCA via hNTCP was subsequently determined in the oocytes by the inward currents induced via TCA uptake under voltage (50 mV). Two hundred micromolar TCA induced significant inward currents that were entirely abolished by the substitution of Na+ with N-methyl-D-glucamine (NMDG) in the perfusate, indicating that the TCA-induced currents were obligatorily dependent on the presence of Na+. The TCAinduced currents were saturable, and the substrate concentration needed for half-maximal induction of the current was consistent with the Michaelis constant. Transportable substrates, such as rosuvastatin and fluvastatin, also induced currents. These results in the hNTCP heterologously expressed in Xenopus oocytes directly demonstrated that hNTCP is an electrogenic Na+-dependent transporter. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Bile acids are synthesized from cholesterol in the liver and secreted into the bile. They play an important role not only in the maintenance of bile flow and biliary lipid secretion but also in cholesterol homeostasis through its secretion in the form of bile acids [1,2]. Furthermore, bile acids secreted into the intestine function as solubilizing agents of various lipophilic substances [3–5]. More than 95% of the bile acids secreted are efficiently reabsorbed by the intestinal epithelial cells and returned to the liver through ⇑ Corresponding author. E-mail address: [email protected] (S. Miyauchi). http://dx.doi.org/10.1016/j.abb.2014.08.010 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.
portal blood circulation. Bile acids are also taken up by hepatocytes and resecreted into bile, thus undergoing enterohepatic circulation in which several transporters in the liver and intestine are crucial [6–8]. Among them, NTCP (SLC10A1), which is predominantly expressed at the basolateral membrane, is a major component of Na+-dependent uptake of the bile acids into the liver [1,8,9]. The Na+-dependent transport system is responsible for approximately 80% of the hepatic uptake of bile acids. Thus, NTCP is the key transporter for hepatic uptake of bile acids, and alteration in the transport activity via NTCP impacts the homeostasis of liver function, especially bile flow and enterohepatic circulation [6–8]. It is plausible that impaired uptake of bile acids via NTCP may cause a remarkable increase in the bile acid concentration in the blood,
116
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
which is associated with cytotoxicity, cellular membrane damage, mitochondrial dysfunction and alteration of cellular metabolism [10–12]. Hypercholanemia has often been associated with adverse extrahepatic effects, including the well-known effect of pruritus [13]. In addition to the important role of NTCP in the maintenance of normal liver function, another important feature is its broad substrate recognition and capability to transport a variety of amphiphilic compounds [14–17]. In addition to bile acids, NTCP also has the potential to transport some structurally unrelated drugs, such as HMG-CoA reductase inhibitors [18–20], the liver function diagnostics bromosulfophthalein and indocyanine green [21,22], as well as the antifungal drug micafungin [23]. Consequently, it is possible that various drugs in clinical use could interact with NTCP and inhibit its function. Such inhibition could be involved in NTCP-mediated drug–drug interactions and drug-induced toxicities [7]. For the sake of the identification and avoidance of NTCP inhibitors, their interactions with NTCP have been investigated extensively with human hepatocytes [20,24,25] as well as mammalian cells expressing hNTCP [15,17,19]. To date, the transport cycle of Na+ and bile acid co-transport has only been minimally studied, despite an increase in evidence with regard to NTCP-mediated drug–drug interactions and its substrate recognition [7,8,15–17]. Although substrate transport via NTCP unequivocally exhibits an obligatory Na+ dependency, the electrogenicity of the NTCP transport cycle remains controversial [26–32]. In the present study, we established heterologous expression of hNTCP in Xenopus laevis oocytes and directly demonstrated and characterized the electrogenic transport of bile acids via hNTCP. hNTCP functioned properly and robustly in X. laevis oocytes, in which TCA induced Na+-dependent inward currents associated with the transport via hNTCP. As far as we are aware, the present study is the first report to directly demonstrate the electrogenicity of hNTCP expressed in X. laevis oocytes. Materials and methods Materials [3H]-TCA (specific radioactivity, 10 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). Bile acids including TCA and its derivatives were purchased from Sigma (Tokyo, Japan). Six-week-old Sprague Dawley (SD)1 rats were purchased from Sankyo Labo Corp. Inc. (Tokyo, Japan). All other chemicals used were of the highest purity available from Wako Chemical Inc. (Osaka, Japan). Amplification of the coding region of hNTCP The cloning of the coding region of hNTCP from a human liver cDNA library (Takara, Tokyo, Japan) was performed with polymerase chain reaction (PCR) as described previously [33]. Based on the published sequence in the GenBank™ data base (accession number L21893), we designed primers introduced restriction enzyme site for cloning purposes. The sense primer, containing an initiation codon, was 50 -GAATTCGATATATAGCCATGGAGGCCCACAACGCGTC, and the antisense primer, located near the stop codon, was 50 AAGCTTCTAGGCTGTGCAAGGGGAGC. The underlined sequences in the sense and antisense primers are EcoRI and HindIII sites, respectively. PCR with these primers using human liver cDNA 1 Abbreviations used: Hepes, N-2-hydroxy-ethylpiperazine-N0 -2-ethanesulfonic acid; hNTCP, human Na+/taurocholate cotransporting polypeptide; LC–MS/MS, liquid chromatography coupled with tandem mass spectrometry; Mes, 2-(N-morpholino) ethanesulfonate; NMDG, N-methyl-D-glucamine; PCR, polymerase chain reaction; SD, Sprague Dawley; TCA, taurocholate; Tris, Tris (hydroxymethyl) aminomethane.
library as the template yielded a 1 kbp product, as expected. This product was subcloned into a TA-cloning vector, pGEM-T Easy (Promega, Madison, WI). The sequence of the subcloned PCR product was determined on both strands with the Taq DyeDeoxy terminator cycle method using Applied Biosystems 3500 (Life Technologies Japan Ltd., Tokyo). The insert containing the entire hNTCP was excised with EcoRI and HindIII and subcloned into pGH19. The pGH19 vector (kindly provided by Dr. Peter S. Aronson, Yale University School of Medicine) contains the 30 - and 50 untranslated region of the X. laevis b-globin gene down- and up-stream of the cloning site, respectively. Functional expression of hNTCP in X. laevis oocytes The amplified hNTCP cDNA was expressed heterologously in X. laevis oocytes by cRNA injection. Capped cRNA from hNTCP in the pGH19 vector was synthesized using AmpliCap™ T7 High Yield Message Maker Kits (Epicentre Biotechnologies, Madison, WI). Mature oocytes (stage V–VI) from X. laevis were isolated by treatment with 1.0 mg/ml collagenase A (Roche Diagnostics GmbH, Mannheim, Germany), manually defolliculated and incubated at 18 °C in modified Barth’s medium supplemented with 50 lg/ml gentamicin as described previously [33,34]. On the following day, each oocyte was injected with 10 ng hNTCP cRNA in a 50 nl volume and incubated for 3–5 days. The oocytes were used for uptake and electrophysiological studies 3–4 days after cRNA injection. H2Oinjected oocytes served as controls. Transport analysis of hNTCP expressed in X. laevis oocytes Uptake of [3H]-TCA into oocytes injected with hNTCP-cRNA or H2O was measured as described previously [35]. Eight to ten oocytes were pre-incubated at room temperature for 2 min in NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM N-2-hydroxy-ethylpiperazine-N0 -2-ethanesulfonic acid (Hepes), adjusted to pH 7.5 with Tris (hydroxymethyl) aminomethane (Tris). The buffer was then replaced by the NaCl buffer supplemented with TCA containing [3H]-TCA (2 lCi/ml) at the given concentrations (1–200 lM). At the given times, the oocytes were gently washed 5 times with 2 ml of ice-cold NMDGCl buffer, in which NaCl is replaced by an iso-osmolar amount of N-methyl-Dglucamine (NMDG) chloride in the NaCl buffer. Each oocyte was transferred to a scintillation vial and dissolved in a 200 lL aliquot of 1% SDS/0.2 M NaOH solution. The radioactivity was determined in a 10 mL aliquot of a scintillation counting cocktail (Clear-sol I, Nacalai Tesque Inc., Tokyo, Japan) with Tri-Carb 2910TR liquid scintillation counter (PerkinElmer Inc., Waltham, MA). The uptake data in the presence of competitors at various concentrations were normalized by the value in the absence of competitor and expressed as the percent of control. The Na+-activation kinetics of TCA uptake was analyzed by measuring the TCA uptake in the presence of increasing Na+ concentrations. The Na+ concentration was varied by iso-osmotically replacing NaCl with NMDG chloride. Electrophysiological studies were performed with the two-electrode voltage clamp (TEVC) technique as described previously [33,36]. The membrane potential was clamped at 50 mV. The oocyte was always superfused with perfusion buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM Hepes/Tris adjusted to pH 7.5). After the current stabilized, the oocyte was superfused with NaCl buffer containing 200 lM quinine. The quinine was used as K+ channel blocker to minimize outward-directed K+ leakage [31]. Up to 400 lM quinine did not have any influence on TCA uptake via hNTCP (Fig. 3). The oocyte was superfused with the same buffer additionally containing test compound. After application of the test compound, the current reached the maximum and steady state, and the test compound was washed out with NMDGCl
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
117
Fig. 1. TCA uptake via NTCP in X. laevis oocytes (A) Na+ dependency of TCA uptake via hNTCP. The uptake of 1.2 lM TCA was measured for 30 min at room temperature and pH 7.5. cRNA and H2O represent hNTCP cRNA-injected and H2O-injected oocytes, respectively. The closed and open bars represent the uptake in NaCl and NMDGCl buffers, respectively. Each bar represents the mean ± SE (n = 10). (B) Time course of TCA uptake by X. laevis oocytes expressing hNTCP. The uptake of 1.2 lM TCA by H2O-(open circles) or hNTCP cRNA-injected (filled circles) oocytes was measured at room temperature and pH 7.5. Each point represents the mean ± SE (n = 10).
buffer. The substrate-induced current at an applied voltage was calculated as the difference between the steady-state currents recorded in the absence and presence of substrate. Cl-free NaMes buffer was composed of 2 mM K gluconate, 1 mM Mg(gluconate)2, 1 mM Ca(gluconate)2, 100 mM sodium 2-(N-morpholino) ethanesulfonate (NaMes) and 10 mM Hepes/Tris adjusted to pH 7.5. Uptake measurements by primary cultured hepatocytes from rat liver Hepatocytes were isolated from the livers of 6-week-old male SD rats that were given free access to food and water. The isolation procedure was basically the same as previously reported [37,38]. Cell viability, determined by trypan blue exclusion, ranged from 95% to 98%. Cells were suspended at the density of 5 105 cells/ ml in William’s E medium supplemented with dexamethasone (1 nM), insulin (1 nM), 10% fetal calf serum, 100 U/ml penicillin and 100 lg/ml streptomycin. One milliliter aliquot cell suspension was added to each well of a 12-well plate. The culture medium was exchanged with fresh medium after 4 h following the initial seeding. Cells were cultured for an additional 16 h at 37 °C in the incubator with 5% CO2/95% air and then used for TCA uptake experiments [39]. The experimental protocol for the use of the animals was approved by the University Animal Care and Use Committee. Uptake of TCA in primary culture hepatocytes was measured at 37 °C in uptake buffer (pH 7.5) as described previously [39]. The composition of the uptake buffer was 140 mM NaCl, 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 10 mM Hepes/Tris
adjusted to pH 7.5. The Na+-activation kinetics of TCA uptake was analyzed by measuring the TCA uptake in the presence of increasing Na+ concentrations. The Na+ concentration was varied by iso-osmotically replacing NaCl with NMDG chloride. After TCA uptake by hepatocytes for the designated period, the uptake was immediately terminated by washing the cells twice with ice-cold NMDGCl buffer. The cells were then dissolved in a 500 ll aliquot of 1% SDS/0.2 M NaOH solution, and the radioactivity associated with the cells was measured.
Kinetics analyses Saturation kinetics was analyzed by fitting the concentration dependency of the initial uptake rate of TCA to a Michaelis Menten-type equation with a saturable component [33]. The kinetic parameters (Vmax, the maximum uptake rate; Km, Michaelis constant) were determined by an iterative nonlinear least-squares method in Origin version 8.5 (MicroCal, Northampton, MA). The currents evoked by TCA were analyzed in a similar manner; kinetic parameters were defined by the maximum induced current, Imax (nA), and the concentration necessary for half-maximal induction of the current, K0.5 (lM), instead of Vmax and Km, respectively. The Na+-activation kinetics was analyzed by fitting the data to the Hill equation, and the Hill coefficient (h, the number of Na+ ions involved in the activation process) and K0.5 value (the concentration of Na+ necessary for half-maximal activation) were determined [35].
Fig. 2. Saturation kinetics and Na+-activation kinetics for TCA uptake via hNTCP. (A) Concentration dependency of TCA uptake by hNTCP. The uptake rates of TCA were measured over 10 min at room temperature and pH 7.5 with an increase in substrate concentrations (2–200 lM). The inset figure shows the Eadie–Hofstee plot of TCA uptake via hNTCP. Each point represents the mean ± SE (n = 7). Lines fit to the non-linear or linear forms of the Michaelis–Menten equation are shown. (B) Na+ activation of TCA uptake by hNTCP. The uptake rate of 2 lM TCA was measured for 10 min at room temperature and pH 7.5 with an increase in Na+ concentrations. TCA uptake rate in (B) represents the Na+-dependent uptake rate, which was estimated by the subtraction of TCA uptake rate in the absence of Na+ from TCA uptake rate in the presence of Na+ at various concentrations. The inset figure shows the Hill plot of the Na+ dependency of the uptake via hNTCP. Each point represents the mean ± SE (n = 7–8). Lines fit to the nonlinear or linear forms of the Hill equation are shown.
118
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
Statistical analyses All experiments were conducted at least 5 times, and results are expressed as the mean ± SE. Statistical analyses between two groups were performed with Student’s t-test. A difference between means was considered if P < 0.05. Results Functional expression of hNTCP in X. laevis oocytes Uptake of 1.2 lM TCA was measured in X. laevis oocytes injected with hNTCP cRNA, and the results are summarized in Fig. 1. In cRNA-injected oocytes, the uptake of 1.2 lM TCA increased more than 100-fold compared to that in H2O injected oocytes, indicating that hNTCP is expressed robustly in X. laevis oocytes. Removal of Na+ from the uptake buffer reduced the uptake to the levels found in H2O-injected oocytes, indicating that the enhanced uptake required Na+. The TCA uptake linearly increased over 15 min, and the initial uptake rate was determined by the uptake in 10 min. The saturation kinetics and Na+-activation kinetics for TCA transport via hNTCP were analyzed and are depicted in Fig. 2. The TCA uptake rate was saturable, and the data fit the Michaelis–Menten equation describing a single saturable process, which was also confirmed by the linearity of Eadie–Hofstee plot shown in the inset. The kinetic parameters Km (Michaelis–Menten constant) and Vmax (maximal uptake rate) were 10.5 ± 2.9 lM and 18.6 ± 1.5 nmol/oocyte/10 min, respectively. The Km value for hNTCP expressed in X. laevis oocytes was similar to that reported for hNTCP expressed in mammalian cells [15,19]. The Na+-mediated increase of the TCA uptake rate showed sigmoidal features. The relationship fit the Hill equation well, with Hill coefficient (h) and concentration necessary for half-maximal activation (K0.5) values estimated to be 1.9 ± 0.1 and 26.9 ± 2.0 mM, respectively. The h value implied that for every TCA molecule transported, at least two Na+ ions were cotransported. Because TCA is a single anion at physiological pH, a Na+:TCA stoichiometry of 2:1 could suggest an electrogenic transport mechanism via hNTCP. The h value obtained from oocytes expressing hNTCP was compatible with that in oocytes expressing rat Ntcp and the isolated rat hepatocytes [30,40,41].
The effects of various bile acids and statins on the uptake of [3H]-TCA via hNTCP were examined and are summarized in Fig. 3. All bile acids inhibited TCA uptake; 200 lM taurochenodeoxycholate (TCDCA) inhibited TCA uptake most potently, indicating that NTCP might prefer conjugated bile acids to unconjugated bile acids. In addition, statins such as rosuvastatin and fluvastatin also inhibited TCA uptake by hNTCP at levels comparable to those reported by others [18–20]. In contrast, up to 400 lM quinine, which was used as a K+ channel blocker in electrophysiological experiments, had no effect on TCA uptake (Fig. 3) [31,32]. Na+-activation kinetics of TCA uptake in primary cultured hepatocytes from rat liver To elucidate whether hNTCP was properly expressed in X. laevis oocytes, we investigated the Na+-activation kinetics of TCA uptake in primary cultured rat hepatocytes. Fig. 4 shows that the uptake of TCA by primary cultured rat hepatocytes was linear up to 2 min, and two-thirds of the TCA uptake exhibited Na+-dependency. The uptake rates were evaluated by the uptake amounts at 2 min. The Na+-activation kinetics of TCA uptake also exhibited sigmoidal kinetics, indicating the participation of more than one Na+ in the TCA transport process. The values of h and K0.5 values by rat Ntcp in primary cultured hepatocytes were essentially the same (h = 1.9 ± 0.3 and 89 ± 21, respectively) as those of hNTCP expressed in X. laevis oocytes (Fig. 2), suggesting that hNTCP functions properly irrespective of the robust expression. Substrate-induced currents associated with Na+-dependent transport via heterologously expressed hNTCP in X. laevis oocytes To investigate whether the Na+-dependent TCA transport is electrogenic or not, the transport of TCA via hNTCP was monitored by electrophysiological techniques using the two-electrode voltage-clamp method. As shown in Fig. 5, 200 lM TCA induced an inward current. H2O-injected oocytes did not exhibit TCA-induced currents. The substitution of Na+ with NMDG in the perfusate completely abolished the TCA-induced current, whereas Cl substitution with Mes in the perfusate had a minimal influence on the TCA-induced current. The effect of ion substitution indicates that the induced currents were associated with Na+-dependent TCA
Fig. 3. Inhibition of [3H]-TCA uptake via hNTCP by various bile acids (A) and statins (B). The uptake rate of 2 lM [3H]-TCA was measured for 10 min at room temperature and pH 7.5 with and without 20 or 200 lM bile acids and 20, 200 or 500 lM statins. The abbreviations of bile acids are as follows: CA, cholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; LCA, lithocholic acid. The uptake rates are normalized to those in the absence of bile acids or statins. Each bar represents the mean ± SE (n = 10). All test compounds except for 200 lM and 400 lM quinine (#) and 20 lM pravastatin (*) exhibit significantly different from control (P < 0.01). #Not significantly different from control. ⁄Significantly different from control (P < 0.05).
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
119
Fig. 4. Time course of TCA uptake (A) and Na+-activation kinetics (B) for TCA uptake by primary cultured rat hepatocytes. The uptake of 1 lM TCA in the presence (filled circles) or absence (open circles) of Na+ was measured at 37 °C and pH 7.5. Each point represents the mean ± SE (n = 6). The uptake rate of TCA in Na+-activation kinetics was measured for 2 min at 37 °C and pH 7.5. TCA uptake rate in (B) represents the Na+-dependent uptake rate, which was estimated by the subtraction of TCA uptake rate in the absence of Na+ from TCA uptake rate in the presence of Na+ at various concentrations. Each point represents the mean ± SE (n = 6).
Fig. 5. Tracers of representative 200 lM TCA induced currents in H2O-injected (A) and hNTCP cRNA-injected (B) X. laevis oocytes. Inward currents induced by 200 lM TCA were monitored in oocytes at membrane potential clamped at 50 mV. Three different perfusion buffers, NaCl buffer (+Na+), NMDGCl buffer (Na+), and NaMes buffer (–Cl), were used. The buffers used just before and during TCA superfusion contained 200 lM quinine.
Fig. 6. Saturation kinetics for TCA-induced currents. Inward currents induced by increasing concentrations of TCA were monitored at 50 mV in five different oocytes expressing hNTCP. The data were normalized for variations in cRNA expression in different oocytes by taking the maximal value for the induced current (Vmax) as 1 in each oocyte. The estimated Vmax value was 37.3 ± 3.4 nA (mean ± SE (n = 5)). The inset figure shows Eadie–Hofstee plot of TCA-induced currents via hNTCP. Each point represents the mean ± SE (n = 5). Lines fit to the non-linear or linear forms of the Michaelis–Menten equation are shown.
transport via hNTCP, but there is no Cl involvement in the transport. We also analyzed the saturation kinetics of TCA transport via hNTCP by monitoring the TCA-induced current (Fig. 6). The induced currents attributed to TCA transport were saturable, and the data fit the Michaelis–Menten equation describing a single saturable process. The substrate concentration needed for half-
maximal induction of current (K0.5) was 30 ± 6.2 lM (± calculated SD), which means that the Michaelis constant for TCA transport is comparable with those reported by others [15,19,42–44]. Furthermore, the transport of TCDCA and various statins by hNTCP were determined by studying the inward currents induced by these compounds (Fig. 7). The current induced by TCDCA was the most potent, suggesting that TCDCA is a good transportable substrate. Among statins, fluvastatin induced the most potent current followed by rosuvastatin, but pravastatin induced a minimal current. Their induced currents were completely abolished by the removal of Na+ from the perfusate. The magnitude of the currents induced by statins correlated with the transport activities of various statins by hNTCP reported by others [18–20]. This good relationship could imply that the present experimental system exhibits the potential advantage that we can easily investigate whether drugs currently in clinical use have the potential to interact with NTCP and alter transport activity via NTCP without the need for radiolabeled drugs or a precise assay system such as a liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS).
Discussion NTCP is predominantly expressed at the basolateral membrane in the liver and is mostly involved in Na+-dependent uptake of bile acids from the portal blood circulation. To date, the Na+-dependent uptake via hNTCP has been corroborated by various experimental
120
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
systems with isolated perfused liver [45], isolated hepatocytes [20,23,24,46], isolated membrane vesicles [47] and hNTCP expressed in mammalian cultured cells [15–17] or heterologously in X. laevis oocytes [22,43]. As far as we are aware, however, there is no direct evidence for the electrogenic nature of bile acid transport via hNTCP. The electrogenicity of hNTCP still remains controversial, although there are ample evidences supporting the electrogenic feature, exemplified by the Na+-bile acid coupling stoichiometry (2:1) [40,41], the depolarization potential induced by bile acid uptake via NTCP and the stimulation of Na+-dependent bile acid uptake by the hyperpolarization of the membrane potential [30–32]. In the present study, we used a conventional electrophysiological technique in X. laevis oocytes expressing hNTCP to directly measure the current induced by the bile acid transport via hNTCP. We established a heterologous hNTCP expression system in X. laevis oocytes and characterized the features of TCA transport. As shown in Fig. 3, the transport activity in hNTCPexpressing oocytes was robust compared to that of H2O-injected oocytes, and the transport function was dependent on Na+. Na+activation kinetics of hNTCP-mediated TCA uptake showed that multiple Na+ ions are involved in each transport cycle as is evident from the sigmoidal feature of the Na+ stimulation in TCA uptake (Fig. 5). The Hill coefficient for the activation process is 1.9 ± 0.1 (Fig. 2), suggesting that the involvement of more than one Na+ per transport cycle is feasible and implying an electrogenic characteristic of the hNTCP transport process. Studies using an electrophysiological approach provided unequivocal data corroborating the electrogenic feature of hNTCP transport process; Na+-dependent TCA transport via hNTCP induced significant transport currents, which exhibited dependency on Na+ as well as saturation kinetics (Fig. 6). The concentration necessary for the half maximal induced current (K0.5 = approx. 30 lM) was compatible with the Km values obtained in the saturation kinetics of TCA uptake and those reported by others [15,19,42–44]. The transportable substrates fluvastatin and rosuvastatin also induced currents (see Fig. 7), the magnitudes of which were consistent with the uptake rate obtained through direct assay of the amounts of statins [18–20]. This consistency between the two methodologies argued for the most feasible explanation, that the substrate–induced currents are attributed to their transport via hNTCP. In recent years, the crystal structures of the apical sodium-dependent transporter (ASBT) homologs were reported by Hu et al. [48] and Zhou et al. [49]. These ASBT homologs have extensive sequence and functional
Fig. 7. Inward currents induced by bile acids and statins via hNTCP. Oocytes were perfused with the NaCl buffer followed by the same buffer containing bile acids (200 lM TCA and 100 lM TCDCA) and statins (500 lM pravastatin, 500 lM rosuvastatin and 200 lM fluvastatin). Substrate-induced currents were determined by the difference in the currents in the presence and absence of substrate when clamped at 50 mV. The substrate-induced currents were normalized to 200 lM TCA-induced currents. Data represent mean ± SE (n = 6). The current induced by 200 lM TCA were 32.2 ± 1.8 nA (mean ± SE (n = 6)).
similarities to NTCP. In particular, the key amino acid residues involved in the transport function are conserved among these transporters, leading us to a plausible hypothesis that they translocate Na+ ions and bile acids by a similar molecular mechanism. Therefore, the crystal structures of ASBT homologs potently facilitate our understanding of their molecular mechanisms of substrate recognition, translocation and energy coupling with Na+ [48,49]. These structures clearly showed an inward open conformation in which two Na+ ions and TCA bound to the core domain. The Na+binding sites were located behind the crossover region between the helixes to regulate the accessibility of the solvent from the extracellular and cytoplasmic spaces. Furthermore, they demonstrated that more than one Na+ cooperatively binds to the transporter, independent of the TCA bound, with the assay of Na+ binding to the purified ASBT. They also solved another structure of the ASBT homolog in which the TCA-binding domain was largely rotated. Based on these features of the ASBT homolog, they proposed a preliminary model of the ASBT transport cycle in which the release of two Na+ ions driven by exposure to lower Na+ concentrations in the cytoplasm triggers TCA release, so two Na+ ions and one TCA molecule are simultaneously transported. The proposed model explains our data with regard to the electrogenicity of NTCP [49] (see Fig. 7). The present study provided unequivocal and direct evidence that hNTCP transports TCA electrogenically with the involvement of more than one Na+ per TCA transport cycle. However, earlier investigations of TCA transport via Ntcp with isolated hepatocytes and liver basolateral membrane vesicles yielded controversial results in favor of either an electroneutral or electrogenic transport mechanism [26–32]. These studied focused on measurements of radio isotopic flux and provided only indirect evidence for electrogenicity due to TCA uptake. An electroneutral transport mechanism has been suggested based on the flux ratio of Na+ to TCA (1:1) in isolated rat hepatocytes [26] due to the lack of stimulatory effects of valinomycin-enhanced K+-gradient and permeable anions on TCA uptake [28,29]. In contrast, an electrogenic transport mechanism has been corroborated in hepatocytes by the stoichiometry of Na+ and TCA cotransport (2:1) [30,40], the depolarization of the membrane potential caused by the Na+/TCA cotransporter and the enhancement of TCA uptake by hyperpolarization of the membrane potential induced by glucagon or valinomycin [50,51]. The reason for these controversial results does not seem to be entirely clear. One plausible explanation is that it is very difficult to precisely control the membrane potential of isolated vesicles and hepatocytes with ionophores and permeable anions. This difficulty might cause uncertainty. Another explanation might be that the Na+ and TCA fluxes via Ntcp in isolated vesicles or isolated hepatocytes induce a minimal current whose measurement is hampered by the large total membrane conductance. In other words, changes in other ion membrane conductance could compensate for small currents attributed to electrogenic fluxes of Na+ and bile acid via NTCP. In the present study, to overcome these drawbacks of using native membrane vesicles and hepatocytes, we established an hNTCP expression system in X. laevis oocytes that is able to produce a measurable current due to the transport of bile acid. However, we should carefully consider a disadvantage that the robust expression might provide a nonphysiological environment and perturb the function of the transporter as well as the membrane fluidity, thereby affecting an ion conductance. In addition, it should also be considered that bile acids are relatively good detergents and may alter membrane fluidity and the ion conductance of membranes [3]. It is thus very difficult to completely rule out the effect of conductance of any other ion on the induced currents due to bile acid transport via hNTCP in the present study. Consequently, to precisely study the relationship between the induced currents and the fluxes of Na+ and TCA, we
M. Masuda et al. / Archives of Biochemistry and Biophysics 562 (2014) 115–121
should use a complementary approach to minimize nonspecific changes in the conductance of any ions. Furthermore, we should also investigate the effect of bile acid on the alteration of ion conductance. In the present study, we established a useful experimental system in which the transport activity of bile acids and their analogs via hNTCP can be monitored directly by studying substrate-induced currents. If the radiolabeled test compounds or precise assay systems such as a LC–MS/MS are not available, this system enables us to distinguish whether compounds are transportable substrates for hNTCP or are blockers that block the uptake of bile acids by competing for the substrate-binding site in hNTCP without being transported across the membrane [34,52]. If the test compound is a transportable substrate, it should induce inward Na+-dependent currents in oocytes expressing hNTCP. If the test compound is a blocker and not a transportable substrate, no inward current associated with the transport via hNTCP would be observed; instead, the inward currents induced by bile acids would be abolished in a concentration-dependent manner. In terms of drug–drug interactions, the present experimental system exhibits the potential advantage that we can easily investigate whether drugs currently in clinical use have the potential to interact with hNTCP and alter transport activity via hNTCP. In conclusion, we established an hNTCP-expressing oocyte system and directly demonstrated that the transport mechanism of hNTCP is electrogenic. This system is a useful tool to clarify whether a compound that interacts with hNTCP is a transportable substrate or merely a blocker that is not transported across the membrane. Acknowledgment This work was supported by the grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology to Seiji Miyauchi (23590062). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
P.J. Meier, B. Stieger, Annu. Rev. Physiol. 64 (2002) 635–661. P.A. Dawson, T. Lan, A. Rao, J. Lipid Res. 50 (2009) 2340–2357. A.F. Hofmann, Gastroenterology 48 (1965) 484–494. G.M. Murphy, E. Signer, Gut 15 (1974) 151–163. K. Ebihara, B.O. Schneeman, J. Nutr. 119 (1989) 1100–1106. G.A. Kullak-Ublick, B. Stieger, P.J. Meier, Gastroenterology 126 (2004) 322–342. C. Pauli-Magnus, P.J. Meier, Hepatology 44 (2006) 778–787. B. Doring, T. Lutteke, J. Geyer, E. Petzinger, Curr. Top. Membr. 70 (2012) 105– 168. W.A. Alrefai, R.K. Gill, Pharm. Res. 24 (2007) 1803–1823. J.R. Spivey, S.F. Bronk, G.J. Gores, J. Clin. Invest. 92 (1993) 17–24. B. Bouscarel, S. Ceryak, T.W. Gettys, H. Fromm, F. Noonan, Am. J. Physiol. 268 (1995) G908–916. J.P. Meng, S. Ceryak, Z. Aratsu, L. Jones, L. Epstein, B. Bouscarel, Am. J. Physiol. Cell Physiol. 291 (2006) C546–554. E.M. Kuiper, K.J. van Erpecum, U. Beuers, B.E. Hansen, H.B. Thio, R.A. de Man, H.L. Janssen, H.R. van Buuren, Hepatology 52 (2010) 1334–1340.
121
[14] P.J. Meier, U. Eckhardt, A. Schroeder, B. Hagenbuch, B. Stieger, Hepatology 26 (1997) 1667–1677. [15] R.B. Kim, B. Leake, M. Cvetkovic, M.M. Roden, J. Nadeau, A. Walubo, G.R. Wilkinson, J. Pharmacol. Exp. Ther. 291 (1999) 1204–1209. [16] R. Greupink, S.B. Nabuurs, B. Zarzycka, V. Verweij, M. Monshouwer, M.T. Huisman, F.G. Russel, Toxicol. Sci. 129 (2012) 35–48. [17] Z. Dong, S. Ekins, J.E. Polli, Mol. Pharm. 10 (2013) 1008–1019. [18] R.H. Ho, R.G. Tirona, B.F. Leake, H. Glaeser, W. Lee, C.J. Lemke, Y. Wang, R.B. Kim, Gastroenterology 130 (2006) 1793–1806. [19] R. Greupink, L. Dillen, M. Monshouwer, M.T. Huisman, F.G. Russel, Eur. J. Pharm. Sci. 44 (2011) 487–496. [20] Y.A. Bi, X. Qiu, C.J. Rotter, E. Kimoto, M. Piotrowski, M.V. Varma, A.F. Ei-Kattan, Y. Lai, Biopharm. Drug Dispos. 34 (2013) 452–461. [21] S. Hata, P. Wang, N. Eftychiou, M. Ananthanarayanan, A. Batta, G. Salen, K.S. Pang, A.W. Wolkoff, Am. J. Physiol. Gastrointest. Liver Physiol. 285 (2003) G829–G839. [22] W. de Graaf, S. Hausler, M. Heger, T.M. van Ginhoven, G. van Cappellen, R.J. Bennink, G.A. Kullak-Ublick, R. Hesselmann, T.M. van Gulik, B. Stieger, J. Hepatol. 54 (2011) 738–745. [23] S.B. Yanni, P.F. Augustijns, D.K. Benjamin Jr., K.L. Brouwer, D.R. Thakker, P.P. Annaert, Drug Metab. Dispos. 38 (2010) 1848–1856. [24] E.I. Lepist, H. Gillies, W. Smith, J. Hao, C. Hubert, R.L. St Claire III, K.R. Brouwer, A.S. Ray, PLoS One 9 (2014) e87548. [25] S.B. Yanni, P.B. Smith, D.K. Benjamin Jr., P.F. Augustijns, D.R. Thakker, P.P. Annaert, Biopharm. Drug Dispos. 32 (2011) 222–232. [26] B.F. Scharschmidt, J.E. Stephens, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 986– 990. [27] M. Inoue, R. Kinne, T. Tran, I.M. Arias, Hepatology 2 (1982) 572–579. [28] M.C. Duffy, B.L. Blitzer, J.L. Boyer, J. Clin. Invest. 72 (1983) 1470–1481. [29] P.J. Meier, A. St, J. Biol. Chem. 259 (1984) 10614–10622. [30] S.D. Lidofsky, J.G. Fitz, R.A. Weisiger, B.F. Scharschmidt, Am. J. Physiol. 264 (1993) G478–485. [31] S.A. Weinman, R.P. Weeks, Am. J. Physiol. 265 (1993) G73–80. [32] S.A. Weinman, Yale J. Biol. Med. 70 (1997) 331–340. [33] S. Miyauchi, E. Gopal, Y.J. Fei, V. Ganapathy, J. Biol. Chem. 279 (2004) 13293– 13296. [34] S. Miyauchi, E. Gopal, E. Babu, S.R. Srinivas, Y. Kubo, N.S. Umapathy, S.V. Thakkar, V. Ganapathy, P.D. Prasad, Biochim. Biophys. Acta 1798 (2010) 1164– 1171. [35] S. Miyauchi, S.R. Srinivas, Y.J. Fei, E. Gopal, N.S. Umapathy, H. Wang, S.J. Conway, V. Ganapathy, P.D. Prasad, Placenta 27 (2006) 550–559. [36] E. Gopal, Y.J. Fei, M. Sugawara, S. Miyauchi, L. Zhuang, P. Martin, S.B. Smith, P.D. Prasad, V. Ganapathy, J. Biol. Chem. 279 (2004) 44522–44532. [37] S. Miyauchi, Y. Sawada, T. Iga, M. Hanano, Y. Sugiyama, Pharm. Res. 10 (1993) 434–440. [38] T. Iga, D.L. Eaton, C.D. Klaassen, Am. J. Physiol. 236 (1979) C9–14. [39] E. Gopal, S. Miyauchi, P.M. Martin, S. Ananth, S.R. Srinivas, S.B. Smith, P.D. Prasad, V. Ganapathy, Am. J. Physiol. Gastrointest. Liver Physiol. 292 (2007) G402–408. [40] M.S. Anwer, D. Hegner, Hoppe Seylers Z. Physiol. Chem. 359 (1978) 181–192. [41] B. Hagenbuch, P.J. Meier, Semin. Liver Dis. 16 (1996) 129–136. [42] S.A. Azer, N.H. Stacey, Biochem. Pharmacol. 46 (1993) 813–819. [43] B. Hagenbuch, P.J. Meier, J. Clin. Invest. 93 (1994) 1326–1331. [44] S. Abu-Hayyeh, P. Martinez-Becerra, S.H. Sheikh, J. Biol. Chem. 285 (2010) 16504–16512. [45] B.N. Melgert, P. Olinga, B. Weert, M.J. Slooff, D.K. Meijer, K. Poelstra, G.M. Groothuis, Drug Metab. Dispos. 29 (2001) 361–367. [46] G.W. Sandker, B. Weert, P. Olinga, H. Wolters, M.J. Slooff, D.K. Meijer, G.M. Groothuis, Biochem. Pharmacol. 47 (1994) 2193–2200. [47] D.A. Novak, F.C. Ryckman, F.J. Suchy, Hepatology 10 (1989) 447–453. [48] N.J. Hu, S. Iwata, A.D. Cameron, D. Drew, Nature 478 (2011) 408–411. [49] X. Zhou, E.J. Levin, Y. Pan, J.G. McCoy, R. Sharma, B. Kloss, R. Bruni, M. Quick, M. Zhou, Nature 505 (2014) 569–573. [50] J.W. Edmondson, B.A. Miller, L. Lumeng, Am. J. Physiol. 249 (1985) G427–433. [51] J.G. Fitz, B.F. Scharschmidt, Am. J. Physiol. 252 (1987) G56–64. [52] S. Itagaki, E. Gopal, L. Zhuang, Y.J. Fei, S. Miyauchi, P.D. Prasad, V. Ganapathy, Pharm. Res. 23 (2006) 1209–1216.