Carnitine/organic cation transporter (OCTN1/SLC22A4) accepts various therapeutic agents as substrates in vitro and is expressed ubiquitously, although its function in most organs has not yet been examined. The purpose of the present study was to evaluate functional expression of OCTN1 in small intestine and liver, using octn1 gene knockout [octn1(−/−)] mice. After oral administration of [3H]ergothioneine ([3H]ERGO), a typical substrate of OCTN1, the amount of [3H]ERGO remaining in the small intestinal lumen was much higher in octn1(−/−) mice than in wild-type mice. In addition, uptake of [3H]ERGO by human embryonic kidney 293 cells heterologously expressing OCTN1 gene product and uptake of [3H]ERGO at the apical surface of intestinal everted sacs from wild-type mice were inhibited by OCTN1 substrates, tetraethylammonium and verapamil. Immunohistochemical analysis revealed that OCTN1 is localized on the apical surface of small intestine in mice and humans. These results suggest that OCTN1 is responsible for small intestinal absorption of [3H]ERGO. However, the plasma concentration of [3H]ERGO after oral administration was higher in octn1(−/−) mice than in wild-type mice, despite the lower absorption in octn1(−/−) mice. This was probably because of efficient hepatic uptake of [3H]ERGO, as revealed by integration plot analysis; the uptake clearance was close to the hepatic plasma flow rate. The uptake of [3H]ERGO by isolated hepatocytes was minimal, whereas [3H]ERGO uptake was observed in isolated nonparenchymal cells. This finding is consistent with immunostaining of OCTN1 in liver sinusoids. Thus, our results indicate that OCTN1 is functionally expressed in nonparenchymal liver cells.
Various types of transporters have been proposed to play important roles in the absorption of nutrients and xenobiotics in the small intestine. For example, hydrophilic nutrients such as saccharides, oligopeptides, and hydrophilic vitamins can only minimally penetrate through the plasma membrane by passive diffusion, and specific solute carriers (SLCs) are involved in their transport into epithelial cells across the apical membrane. On the other hand, hydrophobic compounds, such as many therapeutic agents and other xenobiotics, are thought to be absorbed by passive diffusion, but some of them are actively pumped out by ATP-binding cassette transporters, thereby hindering their oral absorption. SLC transporters localized on the apical membrane of small intestine often have relatively wide substrate specificity. Consequently, it has been proposed that these SLC transporters play an important role in the import of not only nutrients but also therapeutic agents into epithelial cells. Such SLC transporters may include oligopeptide transporter (PEPT1/SLC15A) and organic anion-transporting polypeptide 2B1 and 1A2 in humans (Groneberg et al., 2001; Kobayashi et al., 2003; Glaeser et al., 2007).
The presence of such influx transporters has an impact on drug development, because it may be possible to use them to improve the oral absorption of therapeutic agents. For example, valacyclovir, the l-valyl ester of acyclovir, was developed as a prodrug of acyclovir that is recognized as a substrate by PEPT1 (Balimane et al., 1998). This is considered to be the reason for the greater bioavailability of valacyclovir than acyclovir (Soul-Lawton et al., 1995). The recognition of various types of therapeutic agents by PEPT1 has led to use of this transporter in rational drug design (Li et al., 2008; Brandsch, 2009). However, it is also important to consider influx transporter-mediated drug interactions (Banfield et al., 2002; Lilja et al., 2005a,b). Lilja et al. (2005a,b) have reported decreased plasma concentrations of several β-blockers after ingestion of citrus juice. Subsequent studies in experimental animals confirmed the presence of small intestinal influx transporters for β-blockers (Kato et al., 2009; Shirasaka et al., 2009). However, because of the presence of other mechanism(s) also affecting oral drug absorption, the contribution of influx transporters to the overall absorption of therapeutic agents remains to be fully clarified. In addition, possible involvement of influx transporters has been demonstrated only for relatively few therapeutic agents so far, and the mechanism of membrane permeation for most orally administered drugs has not yet been clarified.
The carnitine/organic cation transporter OCTN1 (SLC22A4) was first identified in human fetal liver (Tamai et al., 1997). It is ubiquitously distributed, being particularly highly expressed in the kidney, and is localized on the apical membrane of renal proximal tubular epithelial cells (Tamai et al., 2000, 2004). OCTN1 transports several cationic compounds, including tetraethylammonium (TEA), pyrilamine, quinidine, verapamil, donepezil, betonicine, ergothioneine (ERGO), and stachydrine (Yabuuchi et al., 1999; Gründemann et al., 2005). Gründemann et al. (2005) reported that ERGO was most efficiently transported by OCTN1 heterologously transfected in a mammalian cell line in vitro. On the other hand, we have recently developed octn1 gene knockout [octn1(−/−)] mice and identified ERGO as a substrate of OCTN1 in vivo by metabolome analysis: ERGO, presumably derived from the diet, is present at micromolar to millimolar levels in blood and all organs of wild-type mice, whereas its concentration was below the detection limit in octn1(−/−) mice, apparently because of the abrogation of OCTN1-mediated renal tubular reabsorption (Kato et al., 2010). Accordingly, isotope-labeled ERGO may be a useful probe compound to analyze functional expression of OCTN1 in the body, because ERGO is not biosynthesized or metabolized in mammals (Mayumi et al., 1978). However, limited information is available on the pharmacokinetics of ERGO. Furthermore, the role of OCTN1 in most organs, except kidney, is not yet known (Urban et al., 2008).
Long-term analysis (∼14 days) of the plasma concentration profile of [3H]ERGO in both wild-type and octn1(−/−) mice has already been reported (Kato et al., 2010), but the detailed mechanism underlying the difference in the plasma profile between the two strains has not yet been clarified. In the present study, we performed pharmacokinetic analysis of [3H]ERGO in both wild-type and octn1(−/−) mice, with the aim of clarifying the role of OCTN1 in small intestinal absorption. Because ERGO was found to be subject to predominant first-pass extraction after oral absorption, we also examined its hepatic disposition. The present study has provided the first evidence for functional expression of OCTN1 in both small intestine and liver.
Materials and Methods
Reagents and Animals.
[3H]ERGO (293 Ci/mol) and [14C]inulin (2.8 μCi/mg) were obtained from Moravek Biochemicals (Brea, CA) and American Radiolabeled Chemicals Inc. (St. Louis, MO), respectively. Sulfobromophthalein (BSP) was purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were commercial products of reagent grade. The octn1(−/−) mice were generated according to the previous report (Kato et al., 2010). Animals were maintained, and experiments were performed according to the Guideline for the Care and Use of Laboratory Animals in Kanazawa University.
Pharmacokinetic Studies in Mice.
Male mice (6–8 weeks old) were fasted overnight with access to water and anesthetized by intraperitoneal injection of pentobarbital. [3H]ERGO at 100 or 330 μg/kg with [14C]inulin (5 mg/kg) was intravenously or orally administered, respectively. Blood samples were collected at the designated time intervals from the right jugular vein of each mouse and centrifuged to obtain plasma. At 4 h after the oral administration, the mice were decapitated, and intestinal tissue was excised and divided into three parts. The radioactivity remaining inside the lumen was recovered by manually washing the luminal side of each segment with 5 ml of ice-cold saline using a syringe and summing up the radioactivity recovered from the three segments. At 4 h after the intravenous injection, the mice were decapitated, and tissues were excised immediately, rinsed with approximately 50 ml of ice-cold saline, blotted dry, weighed, and solubilized with Solene-350 (PerkinElmer Life and Analytical Sciences, Waltham, MA) at 50°C for 3 h. The solution was treated with hydrogen peroxide and neutralized with 5 M HCl. The associated radioactivity was measured with a liquid scintillation counter (LSC-5100; Aloka, Tokyo, Japan), with Clearsol I (Nacalai Tesque, Kyoto, Japan) as the scintillation fluid.
Integration Plot Analysis.
Mice were anesthetized with pentobarbital, and [3H]ERGO (100 μg/kg) was injected via the left jugular vein. Blood was withdrawn from the right jugular vein at designated times, and plasma was separated by centrifugation. After 1, 2, 3, or 5 min, the mice were sacrificed, and liver was obtained and solubilized. The solubilized liver and plasma were each mixed with scintillation liquid, and the associated radioactivity was measured with a liquid scintillation counter as described above. Efflux from the liver soon after intravenous administration was assumed to be negligible, and the tissue uptake clearance (CLuptake) was calculated with the following equation: where XT(t) and CP(t) are the tissue amount and the plasma concentration at time t, respectively. AUC is the area under the plasma concentration-time curve. V0 is the volume of distribution, within which a rapid equilibrium with the plasma compartment is assumed. When [XT(t)/CP(t)] is plotted against [AUC/CP(t)], the slope represents the value of CLuptake.
Uptake of [3H]ERGO by Everted Intestinal Sac.
The experimental procedure was as described previously (Nakashima et al., 1984). The freshly isolated small intestine was equally divided into upper, middle, and lower segments. Each intestinal segment was everted with polyethylene tubing, and the everted sacs were washed with the buffer solution. After a 5-min preincubation, the sacs were incubated with [3H]ERGO (40 nM) and an extracellular marker, [14C]inulin, for 10 min. Then they were washed with ice-cold buffer solution, blotted dry, weighed, and solubilized. The associated radioactivity was measured as described above. The buffer solution was composed of 123 mM NaCl, 5.1 mM KCl, 1.4 mM CaCl2, 1.3 mM MgSO4, 21 mM NaHCO3, 1.3 mM KH2PO4, and 5 mM d-glucose, adjusted to pH 6.0, maintained at 37°C, and gassed with O2 before and during the experiment.
Transport Studies in HEK293/OCTN1 Cells.
Plasmid DNA encoding mouse OCTN1 was transiently transfected into HEK293 cells according to the calcium phosphate precipitation method (Tamai et al., 2000). At 48 h after transfection, the HEK293/OCTN1 cells were harvested and suspended in the transport buffer (125 mM NaCl, 4.8 mM KCl, 5.6 mM d-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES, pH 7.4). The uptake experiment was then performed according to the silicone oil layer method (Tamai et al., 2000).
Isolation and Transport Experiments in Nonparenchymal Liver Cells.
The separation of liver parenchymal and nonparenchymal cells was performed by the collagenase perfusion method (Berry and Friend, 1969; Horiuchi et al., 1985) with some modifications. In brief, mice were anesthetized with pentobarbital, and their body temperature was maintained at 37°C during the experiment. The liver was perfused first with Ca2+, Mg2+-free buffer from the inferior vena cava for 3 min, followed by perfusion buffer containing 5 mM CaCl2, 0.03% collagenase, and 0.005% trypsin inhibitor for 10 min. As soon as the perfusion was started, the portal vein was cut, and the perfusion rate was maintained at 4 ml/min. The dispersed liver cells were separated into parenchymal (PC) and nonparenchymal cells (NPC) by differential centrifugation. The isolated cells were resuspended in ice-cold transport buffer. After incubation with [3H]ERGO (40 nM), the mixture was 10-fold diluted with ice-cold transport buffer and then centrifuged for 1 min, and the supernatant was removed. The cells were washed three times with ice-cold buffer, followed by solubilization with NaOH. After solubilization, the cell lysate was neutralized with HCl. The associated radioactivity was measured as described above. Cellular protein content was determined with a protein assay kit (Bio-Rad Laboratories, Hercules, CA). BSP was quantified by high-performance liquid chromatography (LC-10A series; Shimadzu, Kyoto, Japan). The reverse-phase column (COSMOSIL 5C18-AR-II, 4.6 × 150-mm; Nacalai Tesque) was maintained at 40°C in a column oven. The mobile phase was a mixture of 20 mM potassium phosphate buffer (pH 6.0) and acetonitrile (70:30). The UV detector was set at 230 nm. The flow rate was 1.0 ml/min.
Immunohistochemical Studies and Western Blot Analysis.
Mice were anesthetized and transcardially perfused with 4% paraformaldehyde. The liver and small intestine were removed and sequentially immersed in 4% paraformaldehyde, 10, 20, and 30% sucrose-phosphate-buffered saline. The tissue was then frozen and sectioned with a cryostat, and the sections (10 μm) were mounted on glass slides. Slides of 4-μm sections of formalin-fixed, paraffin-embedded normal human small intestinal tissue were purchased from COSMO BIO Co., Ltd. (Tokyo, Japan). The slides were then incubated with a mixture of first antibodies at 4°C overnight and further incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rat, and Alexa Fluor 594 anti-rabbit IgG conjugates) (Invitrogen, Carlsbad, CA) for 30 min at room temperature. Finally, they were mounted in VECTASHIELD mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) to fix the sample and to stain nuclei. The specimens were examined with a confocal laser scanning fluorescence microscope (LSM 510; Carl Zeiss, Jena, Germany). Mouse intestinal brush-border membrane vesicles (BBMVs) were prepared according to the previous study (Sugiura et al., 2008). Hepatic NPC, PC, and HEK293/OCTN1 cells were obtained as described above. These samples were solubilized in RIPA-Y buffer containing 1% Nonidet P-40, 75 mM NaCl, 50 mM Tris-HCl, pH 7.5, and protease inhibitors. Solubilized samples were analyzed by SDS-polyacrylamide gel electrophoresis, followed by immunoblotting with antisera against OCTN1. Western blot analysis was performed as described previously (Kato et al., 2005).
Statistical analyses were performed by means of Student's t test or analysis of variance with Tukey's post hoc comparison test for single and multiple comparisons, respectively. Differences were considered statistically significant at p < 0.05.
Absorption and Uptake from Apical Side of [3H]ERGO in Small Intestine.
To investigate the role of OCTN1 in the small intestine, [3H]ERGO was first administered orally to wild-type and octn1(−/−) mice, and radioactivity remaining inside the small intestinal lumen was measured to estimate gastrointestinal absorption. Less than 3% of the dose was recovered in the gastrointestinal tract of wild-type mice, whereas approximately 30% of the dose remained in octn1(−/−) mice (Fig. 1A). On the other hand, the recovery of [14C]inulin was not very different between the two strains [39.6 ± 2.3 and 52.7 ± 2.8% of the dose in wild-type and octn1(−/−) mice, respectively]. These results suggest that gastrointestinal absorption of [3H]ERGO was reduced in octn1(−/−) mice.
To further compare the small intestinal membrane permeability of [3H]ERGO in wild-type and octn1(−/−) mice, uptake of [3H]ERGO from the apical side of the small intestine was measured by means of the everted sac method. [3H]ERGO uptake was observed in the upper, middle, and lower parts of the small intestine of wild-type mice, whereas that in octn1(−/−) mice was much lower (Fig. 2A). The uptake of [3H]ERGO by everted sacs of wild-type mice was reduced in the presence of unlabeled ERGO in a concentration-dependent manner (Fig. 2B). Furthermore, the [3H]ERGO uptake in wild-type mice was reduced in the presence of TEA or verapamil, both of which are substrates for OCTN1 (Fig. 3A).
Characterization of the Uptake of [3H]ERGO by OCTN1.
The uptake of [3H]ERGO by mouse OCTN1 was characterized using HEK293/OCTN1 cells. The OCTN1-mediated uptake of [3H]ERGO, obtained by subtracting the uptake in vector-transfected HEK293 cells from that in HEK293/OCTN1 cells, was reduced in the presence of TEA and verapamil (Fig. 3B). Such an inhibition profile is similar to that in the case of [3H]ERGO uptake in mouse small intestine (Fig. 3, A and B).
Expression and Localization of OCTN1 in Small Intestine.
Localization of OCTN1 in the small intestine was next investigated by immunohistochemistry (Fig. 4, A–J). OCTN1 was detected on the apical region of epithelial cells in wild-type mice (Fig. 4, A and E) but not in octn1(−/−) mice (Fig. 4C). OCTN1 was also detected in the intestinal crypts (Fig. 4A). Anti-Na+/K+-ATPase antibody was used as a basolateral membrane marker (Fig. 4F), and the OCTN1 signal was distinct from that of Na+/K+-ATPase, as revealed by double staining of the two membrane proteins (Fig. 4G). In addition, to confirm apical localization of OCTN1, colocalization with PDZK1, an apical membrane scaffold protein, was further examined. Double-labeled immunohistochemical analysis with antisera against OCTN1 and PDZK1 yielded merged signals, indicating apical localization of OCTN1 (Fig. 4, H, I, and J). This finding is further supported by the detection of OCTN1 gene product in the small intestinal BBMVs in Western blot analysis (Fig. 4K). Expression of OCTN1 was also examined in human small intestine and was again detected on the apical membrane (Fig. 5).
First-Pass Uptake of [3H]ERGO in Liver.
Based on the data shown in Figs. 1A, 2, and 3, the plasma concentration of [3H]ERGO in octn1(−/−) mice after oral administration was expected to be lower than that in wild-type mice. In our previous experiments, however, we found that the plasma concentration of [3H]ERGO after oral administration was transiently higher in octn1(−/−) mice compared with that in wild-type mice (Kato et al., 2010). Because the oral absorption profile was not further examined during the short time period covered in the previous analysis, we next examined the plasma concentration profile of [3H]ERGO until 4 h after oral administration. The plasma concentration of [3H]ERGO in octn1(−/−) mice was higher than that in wild-type mice immediately after oral administration (Fig. 1B), confirming our previous observation (Kato et al., 2010). A possible explanation is efficient hepatic first-pass extraction of [3H]ERGO in wild-type but not in octn1(−/−) mice. To examine this hypothesis, [3H]ERGO was injected intravenously, and its distribution to the liver was compared in wild-type and octn1(−/−) mice. After the intravenous injection, plasma [3H]ERGO initially fell rapidly in wild-type mice and then declined with a longer half-life, whereas that in octn1(−/−) mice showed a gradual and continuous decrease (Fig. 6A). The more rapid elimination phase in octn1(−/−) mice (Fig. 6A) was compatible with much higher urinary excretion in octn1(−/−) mice (Fig. 6B). The hepatic uptake process of [3H]ERGO was then investigated by integration plot analysis (Fig. 7). The integration plot for hepatic uptake of [3H]ERGO showed an almost linear increase in wild-type mice, but that in octn1(−/−) mice showed only a minimal increase (Fig. 7). CLuptake of [3H]ERGO in wild-type mice was estimated to be 1.07 ml/min/g of tissue, whereas that in octn1(−/−) mice was 0.0145 ml/min/g of tissue. Efficient hepatic uptake was also supported by the fact that the highest value of the tissue/plasma concentration ratio (Kp) of [3H]ERGO was measured at 4 h after intravenous administration (Table 1). In addition to the liver, the kidney, spleen, lung, heart, small intestine, large intestine, testis, pancreas, thymus, and bone marrow of octn1(−/−) mice had significantly lower Kp values of [3H]ERGO than those in wild-type mice (Table 1).
Uptake of [3H]ERGO, Expression, and Localization of OCTN1 in NPC.
To evaluate which fraction of the liver cells contributes to the hepatic uptake of [3H]ERGO, we next performed an uptake study using freshly isolated hepatic PC and NPC. In PC of wild-type mice, uptake of [3H]ERGO was almost negligible, being similar to that of [14C]inulin, whereas strong uptake of BSP was observed (Fig. 8A). On the other hand, [3H]ERGO was taken up into NPC isolated from wild-type mice in a time-dependent manner, but uptake into NPC of octn1(−/−) mice was much lower and showed minimal time dependence (Fig. 8B). The localization of OCTN1 in the liver was then investigated by immunohistochemical analysis (Fig. 9). A fluorescence signal corresponding to OCTN1 was observed in sinusoidal vessels but not on plasma membranes of hepatocytes (Fig. 9, A and D). Anti-CD31 antibody was used as an endothelial cell marker (Fig. 9, B and E), and its signal at least partially overlapped with that of OCTN1 in wild-type mice (Fig. 9, C, F, and G). Furthermore, Western blot analysis detected the band of OCTN1 gene product in the hepatic NPC but not in PC (Fig. 4K), supporting the higher transport activity of [3H]ERGO in NPC compared with that in PC of wild-type mice.
ERGO is a natural thiol compound present in mushrooms and mammalian tissues (Ey et al., 2007). Biosynthesis of ERGO occurs only in certain microorganisms, and so ERGO is presumably absorbed from daily foods in mammals. The membrane permeability of ERGO is expected to be limited because of its hydrophilicity; therefore, it is reasonable to speculate that a transporter is involved in its gastrointestinal absorption. However, there has been no report on the oral absorption mechanism of this compound. Here, we found that when [3H]ERGO was administered orally to wild-type and octn1(−/−) mice, a greater amount of [3H]ERGO remained in the gastrointestinal tract of octn1(−/−) mice, and the amount remaining in wild-type mice was quite small (Fig. 1A). These observations indicate the involvement of OCTN1 in absorption of ERGO from the luminal side of the small intestine. This conclusion was further supported by uptake studies of [3H]ERGO using the everted sac method (Figs. 2 and 3): [3H]ERGO was efficiently taken up by intestinal tissue in wild-type mice, but the uptake in octn1(−/−) mice was negligible (Fig. 2A). The uptake of [3H]ERGO by everted sacs of wild-type mice was reduced in the presence of unlabeled ERGO (Fig. 2B), and this concentration dependence was consistent with the reported Km value of mouse OCTN1 for ERGO (∼5 μM) (Kato et al., 2010). TEA and verapamil, both of which are substrates of OCTN1, also inhibited the uptake of [3H]ERGO by everted sacs, and the inhibition pattern is similar to that found in HEK293/OCTN1 cells (Fig. 3). Finally, immunostaining for OCTN1 was detected on the small intestinal apical membrane of wild-type mice (Fig. 4, A–J) and humans (Fig. 5). This apical localization of OCTN1 was further confirmed by Western blot analysis of intestinal BBMVs (Fig. 4K). All of these findings indicate that OCTN1 is functionally expressed on the apical membrane of small intestinal epithelial cells in vivo.
The OCTN1-mediated ERGO uptake at the small intestinal apical membrane was inhibited by a therapeutic agent, verapamil (Fig. 3). In view of the wide substrate specificity of OCTN1 (Yabuuchi et al., 1999; Gründemann et al., 2005), the apical localization of this transporter both in humans and mice may require further consideration of the contribution of OCTN1 to oral absorption of other therapeutic agents; indeed, this transporter might be a novel target for rational design of orally administrable drugs. OCTN1 can act as an exchanger for H+ and organic cations in OCTN1 gene-transfected HEK293 cells (Tamai et al., 2004). On the other hand, some cationic compounds, such as cimetidine and guanidine, were reported to be transported via H+/cation exchange mechanisms across the brush-border membranes in small intestine, but the transport properties seem not to be fully consistent with those of OCTN1 (Piyapolrungroj et al., 1999).
The plasma concentration profile of [3H]ERGO in octn1(−/−) mice until 4 h after oral administration was unexpectedly higher than that in wild-type mice (Fig. 1B), despite the lower intestinal absorption of [3H]ERGO in octn1(−/−) mice (Fig. 1A). To elucidate this apparent inconsistency, we examined hepatic extraction of [3H]ERGO. Integration plot analysis (Fig. 7) revealed continuous uptake of [3H]ERGO in the liver of wild-type mice, and the CLuptake was estimated to be ∼1 ml/min g of tissue, which is close to the hepatic plasma flow rate (Davies and Morris, 1993). Thus, the hepatic uptake of ERGO in wild-type mice seems to be blood flow-limited. On the other hand, the integration plot for octn1(−/−) mice revealed almost negligible uptake (Fig. 7). Therefore, lower hepatic extraction in octn1(−/−) mice (Fig. 7) may overcompensate for the lower oral absorption (Fig. 1A), leading to a higher concentration of [3H]ERGO in the systemic circulation of octn1(−/−) mice (Fig. 1B). Furthermore, hepatic NPC, but not PC, seem to play a crucial role in the efficient uptake of [3H]ERGO in the liver (Fig. 8). This observation was supported by the overlap of immunostainings for OCTN1 and an endothelial cell marker CD31, whereas hepatocytes were not stained for OCTN1 (Fig. 9). The detection of OCTN1 by Western blot analysis in NPC but not in PC of wild-type mice (Fig. 4K) was also consistent with the transport activity of [3H]ERGO by NPC (Fig. 8). It seems be noteworthy that NPC account for only one-third of total liver cells but are responsible for a more than 200 times higher concentration of [3H]ERGO in the liver (Kp ∼238) compared with the systemic circulation, meaning that ERGO is highly concentrated inside NPC. Although the physiological role of ERGO in NPC should be clarified by further analyses, ERGO is a very stable antioxidant and is capable of scavenging reactive oxygen species (Melville, 1958; Hartman, 1990). Liver sinusoidal endothelial cells are the primary sites of phagocyte attachment and play an important role in defense against phagocyte-derived reactive oxygen species (Spolarics, 1998). Dysfunction of the liver sinusoidal cells has been observed during arsenic-induced oxidative stress, leading to vascular disease atherosclerosis (States et al., 2009). Bedirli et al. (2004) have demonstrated that exogenous administration of ERGO improves survival in animals with liver injury induced by ischemia and reperfusion. Thus, ERGO may function as one of the defense mechanisms acting under such pathological conditions. However, the present findings do not rule out the possible existence of OCTN1 in NPC other than endothelial cells. More detailed studies are required to understand the role of OCTN1.
A substantial amount of [3H]ERGO was orally absorbed even in octn1(−/−) mice (Fig. 1B), indicating a contribution of other membrane permeation mechanism(s) at the apical membrane of the small intestine. Considering the hydrophilicity of ERGO, passive diffusion through lipid bilayers is unlikely to be significant. Although OCTN2 is also expressed on the apical membrane of small intestinal epithelial cells (Kato et al., 2006), [3H]ERGO was not transported by HEK293 cells transfected with mouse OCTN2 (Kato et al., 2010). OCTN3 is another member of the OCTN family but is expressed on the basolateral membrane in the small intestine (Durán et al., 2005). Accordingly, some other transporter(s) localized on the apical membrane of small intestine is likely to be involved in intestinal absorption of ERGO. The presence of other influx transporters for ERGO was also supported by the present finding in everted sacs that a part of the uptake of [3H]ERGO is not inhibited even in the presence of 100 μM unlabeled ERGO (Fig. 2B). Thus, the present findings indicate the presence of multiple influx transporters for ERGO on small intestinal apical membrane.
After intravenous administration to wild-type mice, [3H]ERGO rapidly disappeared from the circulation in the initial phase (Fig. 6A). Such rapid elimination can probably be explained by the large tissue distribution of [3H]ERGO (Table 1). For example, hepatic distribution (Kp ∼ 240 ml/g of tissue) (Table 1) can account for the distribution volume of ∼10 l/kg in wild-type mice but was negligible in octn1(−/−) mice (Fig. 7; Table 1). This may lead to relatively rapid elimination in the terminal phase in octn1(−/−) mice (Fig. 6A). Such an OCTN1-mediated pharmacokinetic profile of ERGO would be quite similar to the OCTN2-mediated disposition of carnitine, which was clarified by the use of wild-type and octn2 mutant mice (Yokogawa et al., 1999): OCTN2 contributes to intestinal absorption, tissue distribution, and renal tubular reabsorption of carnitine (Yokogawa et al., 1999; Kato et al., 2006), as seen in the case of ERGO and OCTN1.
We thank Lica Ishida for technical assistance. We also thank Kazuki Matsubara and Takayuki Taguchi for isolation of liver cells and uptake experiments, respectively.
This study was supported by the Ministry of Education, Science and Culture of Japan [Grant-in-Aid for Scientific Research]; and the Japan Research Foundation for Clinical Pharmacology.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
- solute carrier
- oligopeptide transporter
- carnitine/organic cation transporter 1
- human embryonic kidney
- parenchymal cells
- nonparenchymal cells
- brush-border membrane vesicle.
- Received February 16, 2010.
- Accepted July 2, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics