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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Functional Characterization of pH-Sensitive Organic Anion Transporting Polypeptide OATP-B in Human

Takashi Nozawa, Kozue Imai, Jun-Ichi Nezu, Akira Tsuji and Ikumi Tamai
Journal of Pharmacology and Experimental Therapeutics February 2004, 308 (2) 438-445; DOI: https://doi.org/10.1124/jpet.103.060194

Abstract

The pH-sensitive activity of human organic anion transporting polypeptide OATP-B, which is expressed at the apical membrane of human small intestinal epithelial cells, was functionally characterized. When initial uptake of estrone-3-sulfate, a typical substrate of OATP, was studied kinetically, we observed an increase in Vmax with decrease of pH from 7.4 to 5.0, whereas the change in Km was negligible. OATP-B-mediated uptake of estrone-3-sulfate was independent of sodium, chloride, bicarbonate, or glutathione, whereas the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone exhibited a pH-dependent inhibitory effect, suggesting that a proton gradient is a driving force for OATP-B. When OATP-B was expressed in human embryonic kidney 293 cells, uptake activities for anionic compounds showed various kinds of pH sensitivity. Dehydroepiandrosterone-sulfate, estrone-3-sulfate, and fexofenadine were transported by OATP-B at both neutral and acidic pH, whereas estradiol-17β-glucuronide, acetic acid, and lactic acid were not transported at all. Transport of taurocholic acid and pravastatin by OATP-B was observed only at acidic pH, demonstrating a pH-sensitive substrate specificity of OATP-B. Because the physiological pH close to the surface of intestinal epithelial cells is acidic, the roles of OATP-B in the small intestine might be different from those in other tissues, such as liver basolateral membrane. Although the driving force for OATP-B has not been fully established, the clarification of factors, such as pH, that affect the OATP-B-activity is essential for an understanding of the physiological and pharmacological relevance of the transporter in the small intestine.

Since the discovery of rat oatp1 (Jacquemin et al., 1994), the number of known organic anion transporting polypeptides (OATP: SLCO) has been increasing. OATPs are in general characterized by multispecificity, i.e., the ability to transport clinically used drugs as well as physiologically important compounds, and by the variety of tissue expression profiles among OATP members (Hagenbuch and Meier, 2003). Previous OATP studies have mainly focused on the hepatic transport of bile acids, bilirubin, and xenobiotics such as clinically used drugs and their metabolites, and it was found that OATP molecules expressed at the human liver basolateral membranes are OATP-B, OATP-C, and OATP8 (König et al., 2000a,b; Kullak-Ublick et al., 2001). Among liver-expressed human OATPs, OATP-C is likely a key transporter for the hepatic uptake of organic anions, based on its specific and abundant expression in liver, its multispecificity, and its localization at the liver basolateral membrane (Tirona and Kim, 2002). Other human OATPs are expressed in a variety of tissues other than liver and presumably have distinct physiological roles (Tamai et al., 2000a). At present, OATP-B, OATP-D, OATP-E, and OATP-F are known to act as transporters of sulfated steroid in the placenta, prostaglandin in several tissues, thyroid hormone in several tissues, and thyroid hormone in brain and testis, respectively (Fujiwara et al., 2001; Pizzagalli et al., 2002; St-Pierre et al., 2002; Adachi et al., 2003). Thus, the roles of each human OATP have been at least partially established.

Among human OATPs, OATP-B and OATP-C are commonly expressed in the liver, although their substrate specificities are different, with OATP-B being more selective than OATP-C (Kullak-Ublick et al., 2001; Tamai et al., 2001). Furthermore, OATP-B is expressed in several tissues, including placenta, small intestine, and others, in marked contrast to the case of OATP-C. Very recently, we reported that OATP-B in small intestine exhibited localized expression at the apical membrane of small intestinal epithelial cells and showed pH-sensitive transport with higher activity at acidic pH than that at neutral pH (Kobayashi et al., 2003). Organic weak acids are usually thought to be absorbed through passive diffusion according to the pH-partition hypothesis because of the presence of an acidic microclimate pH around the intestinal epithelial cells (Shiau et al., 1985). However, there are reports that suggest the involvement of pH-dependent and carrier-mediated absorption for anionic compounds, including short-chain fatty acids (Stein et al., 2000), the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin (Tamai et al., 1995), lactic acid (Tamai et al., 2000b), and other anionic compounds (Tamai et al., 1996). Based on these observations, OATP-B may function as a pH-dependent organic anion transporter in the small intestine and be involved in the intestinal absorption of anionic drugs as well as physiological compounds. Furthermore, we found genetic polymorphisms of OATP-B, and one of the single nucleotide polymorphisms of OATP-B, which has an allelic frequency of about 10% in the Japanese population, showed decreased activity in an in vitro transport assay using transfected cells (Nozawa et al., 2002). Accordingly, it is important to characterize OATP-B in detail to establish its importance for small intestinal function.

Although the previous experiments had demonstrated that estrone-3-sulfate and pravastatin are transported by OATP-B in a pH-sensitive manner, more precise studies on pH-sensitive transport by OATP-B, including the substrate selectivity and specificity of increased transport at acidic pH, as well as the mechanism of the increase of activity at acidic pH, are now needed. Accordingly, the purpose of the present study is to characterize further the transport activity of OATP-B at acidic pH, by means of kinetic analysis using a variety of test compounds.

Materials and Methods

Materials. [3H]Estrone-3-sulfate, ammonium salt (1702.0 GBq/mmol), [3H]dehydroepiandrosterone-sulfate (2926.7 GBq/mmol), [3H]taurocholic acid (74.0 GBq/mmol), [3H]estrodiol-17β-glucuronide (1665.0 GBq/mmol), and [14C]lactic acid (4847.0 MBq/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). [14C]Acetic acid (2.02 GBq/mmol) and [14C]inulin carboxyl (92.5 MBq/g) were obtained from American Radiolabeled Chemicals (St. Louis, MO). Pravastatin and [14C]pravastatin (529.1 MBq/mmol) were kindly supplied by Sankyo Co. (Tokyo, Japan). pcDNA3 and pcDNA5 vectors were obtained from Invitrogen (Carlsbad, CA). Fexofenadine (terfenadine carboxylate) was from Salford Ultrafine Chemical and Research (Manchester, UK). HEK293 cells were obtained from Health Science Research Resources Bank (Tokyo, Japan). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and Wako Pure Chemicals (Osaka, Japan).

Transport Experiments. For the transport experiments using HEK293 cells, the construct pcDNA3 including OATP-B- or ASBT (SLC10A2)-cDNA was used to transfect HEK293 according to the calcium phosphate precipitation method. HEK293 cells were routinely grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin, and streptomycin in a humidified incubator at 37°C under 5% CO2. After cultivation of HEK293 cells for 24 h in 15-cm dishes, pcDNA3/OATP-B, pcDNA5/ASBT, or vector pcDNA3 or pcDNA5 alone was transfected by adding 20 μg of the plasmid DNA per dish. At 40 to 48 h after transfection, the cells were harvested and suspended in suspension medium containing 125 mM NaCl, 4.8 mM KCl, 5.6 mM d-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 12 mM MgSO4, and 25 mM HEPES, adjusted to pH 7.4. To avoid the variability of the expressed levels of OATP-B in HEK293 cells, the set of experiments was done using the same batches of HEK293 cells transfected at the same time. The cell suspension was preincubated at 37°C for 20 min in the suspension medium (pH 7.4), and then centrifuged, and the resultant cell pellets were mixed with the uptake medium (pH 5.0 to 7.4) containing a test compound to initiate uptake. Uptake medium contained the same constituents as the suspension medium except for 25 mM 2-(N-morpholino)ethane sulfonic acid instead of HEPES, adjusted to pH 5.0 to 6.0 with HEPES, or adjusted to pH 6.5 to 7.4 with tris(hydroxymethyl)aminomethane. In Na+-free and Cl–-free medium, NaCl was replaced with choline chloride and Na-gluconate, respectively. At appropriate times, aliquots of the mixture were withdrawn, and the cells were separated from the uptake medium by centrifugal filtration through a layer of a mixture of silicone oil (SH550; Toray Dow Corning Co., Tokyo, Japan) and liquid paraffin (Wako Pure Chemicals) with a density of 1.03. Each cell pellet was solubilized in 3 N KOH and then neutralized with HCl. The cell-associated radioactivity was measured with a liquid scintillation counter using Cleasol-1 as a liquid scintillation fluid (Nacalai Tesque, Kyoto, Japan). HEK293 cells transfected with pcDNA3 vector alone were used to obtain the background activity (termed mock). In the case of fexofenadine, the cell pellet sedimented in 3 N KCl instead of 3 N KOH was solubilized by sonication, mixed with methanol to be 50% final concentration, and deproteinized by centrifugation for 5 min at 15,000 rpm. The resultant supernatant was evaporated to be dryness and dissolved by the mobile phase for high-performance liquid chromatography.

Analytical Methods. Cellular protein content was determined according to the method of Bradford (1976) by using a Bio-Rad protein assay kit (Hercules, CA) with bovine serum albumin as the standard. Fexofenadine was quantified by means of high-performance liquid chromatography using an ODS 80Ts analytical column (4.9 × 150 mm; Tosoh, Tokyo, Japan) with acetonitrile/methanol/12 mM ammonium acetate (19:29:52) as the mobile phase at a flow rate of 1.0 ml/min. The analytical column was kept at 40°C, and the eluent was monitored with a fluorescence detector (model FS-8010; Tosoh) at excitation and emission wavelengths of 240 and 290 nm, respectively; the retention time of fexofenadine was approximately 11 min. All the uptake values were corrected for nonspecific adsorption of test compounds on the cell surface based on the apparent uptake of [14C]inulin, which is considered not to permeate across the cell membrane. The mean values of nonspecific adsorption space were 10.7 and 10.6 μl/mg protein for mock-transfected and OATP-B-expressing HEK293 cells, respectively.

All data were expressed as means ± S.E.M., and statistical analysis was performed by the use of Student's t test with p < 0.05 as the criterion of significance. Cell-to-medium ratio was obtained by dividing the cellular uptake amount by the concentration of test compound in the uptake medium.

Results

Characterization of Estrone-3-Sulfate Transport by OATP-B at Acidic pH. Although we have already reported that the transport activity of OATP-B was increased at acidic pH compared with that at neutral pH, the precise transport characteristics remained to be established (Kobayashi et al., 2003). In the present study, the time course of [3H]estrone-3-sulfate uptake by HEK293 cells transiently transfected with OATP-B cDNA was measured to estimate the appropriate time for measurement of the initial uptake rate. Transport of [3H]estrone-3-sulfate was evaluated at pH 5.0 and 7.4. The value of OATP-B-mediated uptake of [3H]estrone-3-sulfate after corrected with that of mock cells was significantly higher at pH 5.0 than that at pH 7.4 (Fig. 1). Because the uptake increased linearly for over 10 min at pH 7.4 and 2 min at pH 5.0, the initial uptake was evaluated in terms of the uptakes for 5 min and 1 min at pH 7.4 and pH 5.0, respectively, to obtain the kinetic parameters for the OATP-B-mediated uptake of estrone-3-sulfate. Initial uptake rates of estrone-3-sulfate at concentrations ranging from 9.2 nM to 20 μM exhibited saturation at both pH 5.0 and 7.4 (Fig. 2A). Because Eadie-Hofstee plots showed a single straight line at both pHs (Fig. 2B), the kinetic parameters were evaluated by nonlinear least-squares regression analysis using the MULTI program (Yamaoka et al., 1981). The Km and Vmax values (mean ± S.E.) at pH 7.4 were 8.09 ± 1.67 μM and 300.5 ± 46.0 pmol/mg protein/min, respectively, and those at pH 5.0 were 13.1 ± 3.2 μM and 2135.8 ± 410.3 pmol/mg protein/min, respectively. Although the Km values were comparable under both conditions, the Vmax value at pH 5.0 was 7 times that at pH 7.4. Accordingly, the increase of apparent transport activity for estrone-3-sulfate at acidic extracellular pH can be ascribed to increased maximum transport rate rather than to increased affinity for OATP-B transporter.

Fig. 1.
Fig. 1.

Time course of uptake of [3H]estrone-3-sulfate by HEK293 cells expressing OATP-B. Uptake of [3H]estrone-3-sulfate (4.6 nM) by OATP-B-cDNA-transfected or mock cells over 30 min was measured at 37°C in the uptake medium at pH 5.0 or pH 7.4 after preincubation for 20 min at 37°C in the suspension medium (pH 7.4). Closed and open circles represent OATP-B-specific uptake of [3H]estrone-3-sulfate obtained by subtracting the uptake by mock cells from that by OATP-B-cDNA-transfected cells at pH 5.0 and pH 7.4, respectively. Each result represents the mean ± S.E.M. (n = 3 or 4).

Fig. 2.
Fig. 2.

Concentration dependence of estrone-3-sulfate uptake by HEK293 cells expressing OATP-B, shown by concentration-rate plot (A) and Eadie-Hofstee plot (B). A, OATP-B-specific uptake of estrone-3-sulfate, obtained by subtracting the uptake by mock cells from that by OATP-B-cDNA-transfected cells, was measured for 1 or 5 min at various concentrations from 9.2 nM to 20 μM at pH 5.0 (closed circles) and 7.4 (open circles), respectively. B, OATP-B-specific uptake of estrone-3-sulfate at various concentrations at pH 5.0 (closed circles) and 7.4 (open circles) is shown as an Eadie-Hofstee plot. Solid lines represent the calculated values using the kinetic parameters obtained by nonlinear least-squares analysis. Each result represents the mean ± S.E.M. (n = 3 or 4).

To elucidate the mechanism of the pH sensitivity, the effect of extracellular ions and compounds on OATP-B-mediated uptake of [3H]estrone-3-sulfate was studied at pH 7.4 and 5.0 (Table 1). The replacement of Na+ with choline (Na+-free) or that of Cl– with gluconate (Cl–-free) caused no change in the transport of [3H]estrone-3-sulfate. Because the exchange transport of organic anions with Math or glutathione (GSH) was suggested in the case of rat oatp (Satlin et al., 1997; Li et al., 1998, 2000), the effect of addition of extracellular Math or GSH to dissipate the outward gradients was examined. No significant change in the uptake of [3H]estrone-3-sulfate was caused by Math or GSH. In contrast, carbonylcyanide p-trifluoromethoxyphenyl hydrazone (FCCP), which is a proton ionophore, significantly decreased the uptake only in the presence of an inwardly directed proton gradient (pH 5.0) in a concentration-dependent manner, whereas the uptake by mock-transfected cells was not affected by FCCP. Although FCCP also caused some reduction in the uptake of estrone-3-sulfate in the absence of a proton gradient (pH 7.4), this change was not statistically significant. The observations suggest that the apparent pH-sensitive transport of estrone-3-sulfate by OATP-B may be ascribed to the effect of the proton gradient as a driving force of transport by OATP-B.

View this table:
TABLE 1

Effects of extracellular compounds on uptakes of [3H]estrone-3-sulfate at pH 5.0 and pH 7.4 by OATP-B

OATP-B-cDNA-transfected or mock cells were preincubated for 20 min at 37°C in the suspension medium (pH 7.4). Uptake of [3H]estrone-3-sulfate (5.07 nM) over 1 or 5 min was measured at 37°C by incubating cells in the uptake medium at pH 5.0 or 7.4, respectively, and the results are shown as percentage of control uptake measured in the uptake medium (pH 5.0 or 7.4) after correcting for the uptake by mock cells. Each value represents the mean ± S.E.M. (n = 3 or 4). The control study on the effect of FCCP includes the ethanol (0.5 %) used to dissolve FCCP.

Substrate Specificity of pH-Sensitive Transport by OATP-B. The substrate specificity of the pH-sensitive transport activity of OATP-B was examined by using dehydroepiandrosterone-sulfate and taurocholic acid as test compounds (Figs. 3 and 4). It has been reported that dehydroepiandrosterone-sulfate is transported by OATP-B at neutral pH, whereas taurocholic acid is not (Kullak-Ublick et al., 2001). In line with previous study, the uptake of [3H]dehydroepiandrosterone-sulfate mediated by OATP-B at pH 7.4 was significantly increased compared with that by mock-transfected HEK293 cells (Fig. 3). Furthermore, the uptake of [3H]dehydroepiandrosterone-sulfate was pH-sensitive, with increased activity at acidic pH. As shown in Fig. 4, at pH 7.4 there was no significant difference in the uptake of [3H]taurocholic acid by HEK293 cells between OATP-B-transfected and mock-transfected cells. However, OATP-B-mediated uptake of taurocholic acid increased with decrease of pH from 7.4 to 5.0, with a statistically significant increase under pH 6.5 (Fig. 4). Kinetic analysis of taurocholic acid uptake by OATP-B was examined at pH 5.0 in the concentration range from 0.2 to 100 μM (Fig. 5). The obtained kinetic parameters for Km and Vmax (mean ± S.E.) were 71.8 ± 12.2 μM and 1093 ± 162 pmol/2 min/mg protein, respectively.

Fig. 3.
Fig. 3.

Effect of extracellular pH on the uptake of [3H]dehydroepiandrosterone-sulfate by HEK293 cells expressing OATP-B. Uptake of [3H]dehydroepiandrosterone-sulfate (2.98 nM) by OATP-B-cDNA-transfected cells (closed circles) or mock cells (open circles) over 5 min was measured at various extracellular pH values in the range from 5.0 to 7.4. The cells were preincubated for 20 min at 37°C in the suspension medium (pH 7.4). Squares represent OATP-B-specific uptake of [3H]dehydroepiandrosterone-sulfate obtained by subtracting the uptake by mock cells from that by OATP-B-cDNA-transfected cells. Each result represents the mean ± S.E.M. (n = 3 or 4), and * indicates a significant difference from the uptake by mock cells (p < 0.05).

Fig. 4.
Fig. 4.

Effect of extracellular pH on the uptake of [3H]taurocholic acid by HEK293 cells expressing OATP-B. Uptake of [3H]taurocholic acid (0.144 μM) by OATP-B-cDNA-transfected cells (closed circles) or mock cells (open circles) over 5 min was measured at various extracellular pH values in the range from 5.0 to 7.4. The cells were preincubated for 20 min at 37°C in the suspension medium (pH 7.4). Squares represent OATP-B-specific uptake of [3H]taurocholic acid obtained by subtracting the uptake by mock cells from that by OATP-B-cDNA-transfected cells. Each result represents the mean ± S.E.M. (n = 3 or 4), and * indicates a significant difference from the uptake by mock cells (p < 0.05).

Fig. 5.
Fig. 5.

Concentration dependence of taurocholic acid uptake by HEK293 cells expressing OATP-B, shown by concentration-rate plot (A) and Eadie-Hofstee plot (B). A, OATP-B-specific uptake of taurocholic acid, obtained by subtracting the uptake by mock cells from that by OATP-B-cDNA-transfected cells, was measured for 2 min at various concentrations from 0.2 nM to 100 μM at pH 5.0. OATP-B-specific uptake of taurocholic acid at various concentrations at pH 5.0 is shown as an Eadie-Hofstee plot (B). The line represents the calculated values using the kinetic parameters obtained by nonlinear least-squares analysis. Each result represents the mean ± S.E.M. (n = 3 or 4).

Table 2 shows the transport of various anionic compounds at pH 7.4 and 5.0, including the clinically used drugs pravastatin and fexofenadine, nutritious compounds acetic acid and lactic acid, and the conjugated metabolites of steroid hormones, estrone-3-sulfate, dehydroepiandrosterone-sulfate, and estradiol-17β-glucuronide. The results can be divided to three groups. The first is the case of estrone-3-sulfate, dehydroepiandrosterone-sulfate, and fexofenadine, which were transported by OATP-B at both pH 7.4 and 5.0, although higher activities were observed at acidic pH. The second is the case of taurocholic acid and pravastatin, which were transported only at the acidic pH. The last is the case of estradiol-17β-glucuronide, acetic acid and lactic acid, which were not transported at either the acidic or neutral pH. Among these compounds, we chose pravastatin for a kinetic study, because the intestinal absorption mechanism of pravastatin remains to be fully clarified, despite the suggestion of the involvement of a carrier-mediated transport mechanism (Tamai et al., 1995) and previous observation of pH-sensitive pravastatin uptake by OATP-B (Kobayashi et al., 2003). Figure 6 shows the concentration dependence of the initial uptake of pravastatin by OATP-B at pH 5.0 in the concentration range from 29 μM to 10 mM. The Eadie-Hofstee plot exhibited the presence of a single saturable component (Fig. 6B), and the evaluated Km and Vmax values (mean ± S.E.) were 2.25 ± 0.94 mM and 41.6 ± 6.4 nmol/mg protein/10 min, respectively. These results suggest that OATP-B exhibits broader substrate selectivity at acidic pH than at neutral pH. Although the substrate specificity of OATP-B at acidic pH is not yet clear, the fact that pravastatin and fexofenadine are transported by OATP-B at acidic pH suggests that OATP-B is involved in the intestinal absorption of acidic drugs.

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TABLE 2

Effect of extracellular pH on the uptake of various compounds by OATP-B

Uptake of [3H]E13S, [3H]DHEAS, fexofenadine, and [3H]TCA were measured for 1, 5, 30, and 2 min, respectively, and other compounds were measured for 10 min. Concentration used for each test compound was shown in the respective parenthesis. OATP-B-specific was obtained by subtracting the uptake by mock-transfected cells from the OATP-B cDNA-transfected cells. Each value represents the mean ± S.E.M. (n = 3 or 4). For the uptake of [3H]TCA, the uptake medium including 0.5% BSA was used to decrease the nonspecific binding.

Fig. 6.
Fig. 6.

Concentration dependence of pravastatin uptake by HEK293 cells expressing OATP-B shown by concentration-rate plot (A) and Eadie-Hofstee plot (B). A, OATP-B-specific uptake of pravastatin over 10 min, obtained by subtracting the uptake by mock cells from that by OATP-B-cDNA-transfected cells, was measured at various concentrations from 29.4 nM to 10 mM at pH 5.0. B, OATP-B-specific uptake of pravastatin at various concentrations at pH 5.0 is shown as an Eadie-Hofstee plot. The line represents the calculated values using the kinetic parameters obtained by nonlinear least-squares analysis. Each result represents the mean ± S.E.M. (n = 3 or 4).

To confirm the pH-sensitive transport activity of OATP-B, the effect of pH on the uptake of [3H]taurocholic acid, which was transported by OATP-B only at acidic pH, by the human ileal sodium-dependent bile acid cotransporter ASBT (Wong et al., 1995), was examined. As shown in Table 3, the uptake of [3H]taurocholic acid by HEK293 cells expressing ASBT was increased compared with that by mock-transfected cells in the presence of Na+, whereas no significant increase was observed in the absence of Na+. The ASBT-mediated uptake at pH 5.0 was 20.1 ± 2.4 μl/mg protein, which was lower than that at pH 7.4 (29.0 ± 1.9 μl/mg protein). Accordingly, pH-sensitive transport of taurocholic acid is specific to OATP-B and cannot be ascribed to some nonspecific artifact.

View this table:
TABLE 3

Effect of extracellular pH on uptake of [3H]taurocholic acid by HEK293 cells expressing ASBT

NT, not tested.

Discussion

We have recently demonstrated that OATP-B (SLC2B1) is expressed at the apical membrane of human intestinal epithelial cells (Kobayashi et al., 2003). Because the physiological microclimate pH of the intestinal epithelial cells is acidic, the environment of OATP-B in the intestine is different in terms of pH from that of OATP-B in the liver or in other tissues (Shiau et al., 1985; Kullak-Ublick et al., 2001). There are transporters expressed in the intestinal epithelial cells that exhibit pH dependence. For those transporters, the proton gradient across the apical membrane can be a driving force, as demonstrated for the oligopeptide transporter PEPT1 (Fei et al., 1994) and monocarboxylic acid transporter MCT1 (Tamai et al., 1999). Because these pH-sensitive transporters often lose their transport activities at neutral pH, it is not unusual that OATP-B has differential activity between pH 7.4 and 5.0, as observed in the present and previous studies (Kobayashi et al., 2003). Accordingly, it is essential to characterize the functionality of OATP-B at acidic pH to understand the physiological and pharmacological relevance of the transporter in the small intestine.

When OATP-B activity was examined in in vitro-transfected cells, estrone-3-sulfate uptake at pH 5.0 was significantly higher than that at pH 7.4 (Fig. 1). Kinetic analysis showed that the increase was due to an increase in Vmax, rather than a change in the affinity (Km) for OATP-B (Fig. 2). The proton-gradient-dependent peptide transporter in the small intestine exhibits a decrease of Km without change in Vmax when the pH is lowered to the acidic region (Tsuji et al., 1987; Kottra and Daniel, 2001). Accordingly, the apparent change of OATP-B activity by pH may not be ascribed to the same mechanism as that of the peptide transporter. In contrast, there is a report that an acidic intracellular pH decreased Vmax without changing Km in glycocholate transport by rat oatp1 (Marin et al., 2003). Kanai et al. (1996) also observed increased uptake of sulfobromophthalein by rat oatp1 when the pH was lowered from 8.0 to 6.5. Although the underlying mechanism remains unclear, the possibilities of proton cotransport (alternatively, exchange with hydroxy ion), the presence of a pH-sensitive active site on the transporters and/or the increase of turnover rate of the active sites across the membrane were suggested. When we examined factors that might affect the OATP-B activity in terms of driving force, Na+ or Cl– had no effect (Table 1). Furthermore, previously suggested driving forces of rat oatp such as outward bicarbonate ion and GSH gradients (Satlin et al., 1997; Li et al., 2000) were unlikely to contribute to the estrone-3-sulfate uptake by OATP-B (Table 1). The proton ionophore FCCP caused a significant reduction in the estrone-3-sulfate uptake by OATP-B only at acidic pH. These results suggested that a proton gradient could be a driving force for the estrone-3-sulfate transport by OATP-B, although further studies, such as direct measurement of proton movement, are needed to confirm this and to establish the precise mechanism.

It is important to know the specificity and selectivity of the pH-sensitive transport by OATP-B. We have already reported that estrone-3-sulfate and pravastatin exhibited pH-sensitive transport by OATP-B (Kobayashi et al., 2003). In this study, various anionic compounds were examined, including previously known OATP-B substrates and nonsubstrates at neutral pH, clinically used drugs, and nutrients. At neutral pH, dehydroepiandrosterone-sulfate was a substrate of OATP-B, whereas taurocholic acid and estradiol-17β-glucuronide were not transported by OATP-B (Kullak-Ublick et al., 2001). Interestingly, the results for these three compounds in this study were variable (Table 2). First, dehydroepiandrosterone-sulfate was transported by OATP-B at both pH 5.0 and 7.4 with significantly higher uptake at pH 5.0 than that at pH 7.4. Second, estradiol-17β-glucuronide was not a substrate of OATP-B at either pH 7.4 or 5.0. Last, taurocholic acid was transported by OATP-B at pH 5.0, but not at pH 7.4. Acetic acid and lactic acid were not transported by OATP-B, whereas significant uptake of fexofenadine was observed at pH 5.0 and 7.4. These results may suggest that low-molecular-weight compounds, acetic acid (mol. wt. 60) and lactic acid (mol. wt. 90), are poor substrates, being transported instead by another pH-dependent transporter, MCT1, which seems not to transport anionic compounds of larger molecular weight (Tamai et al., 1999).

The apparent pH sensitivity of OATP-B might not be specific and might reflect the ionization state of the compounds. However, when we evaluated the taurocholic acid transport by ASBT in HEK293 cells, we observed sodium ion dependence but not pH dependence (Table 3). Accordingly, the observation in this study is OATP-B-specific, and not due to a nonspecific effect of pH on the apparent transport activity of organic anions. Accordingly, OATP-B shows pH-sensitive substrate specificity; thus, care is needed in identifying possible substrates of OATP-B, because compounds such as taurocholic acid can be substrates at acidic pH, but not at neutral pH. In the case of bile acids, they are absorbed by ASBT from the ileum. In rats, oatp3 is localized at the apical membrane of intestinal epithelial cells and was suggested to be involved in the sodium-independent absorption of bile acids (Amelsberg et al., 1999; Walters et al., 2000). Those authors proposed a bile acids-anion antiport mechanism, and oatp was considered as a possible transporter molecule. The Km of taurocholic acid (71.7 μM) for OATP-B at pH 5.0 is close to the reported Km of rat oatp3 (30 μM; Walters et al., 2000). However, the amino acid sequences of rat oatp3 exhibited higher similarity to human OATP-A, whereas human OATP-B is closely related to rat oatp9 (Hagenbuch and Meier, 2003). Rat oatp9 has a Km value for taurocholic acid of 17.6 μM (Nishio et al., 2000), which is close to the Km value of OATP-B, but no precise information is available on the pH sensitivity or expression at the intestinal apical membrane for oatp9. Accordingly, it is not easy to establish which rat oatp corresponds to human OATP-B in the small intestine. However, this study has demonstrated for the first time that the human OATP transporter expressed in the intestinal apical membrane could play specific physiological roles by showing differential substrate selectivity from that in other tissues, based on the studies of the pH sensitivity of the substrate specificity of OATP-B. However, because OATP-B can transport some substrates, including estrone-3-sulfate, dehydroepiandrosterone sulfate, and sulfobromophthalein at neutral pH (Tamai et al., 2000a, 2001; Kullak-Ublick et al., 2001), OATP-B in other tissues may play a role in the disposition of specific substrates, which may be partly different from intestine.

As regards drug absorption, the observed transport of the clinically used drugs pravastatin and fexofenadine is interesting. We previously proposed that pravastatin might be absorbed by a pH-sensitive transporter across the rabbit intestinal apical membrane based on studies in isolated intestinal apical membrane vesicles (Tamai et al., 1995). The Km value of pravastatin for OATP-B obtained in this study at pH 5.0 (2.25 mM) is close to the IC50 value (5.5 mM), which was obtained for the inhibitory effect of pravastatin on the estrone-3-sulfate uptake by OATP-B at pH 5.5 in a previous study (Kobayashi et al., 2003). A study using rabbit intestinal brush-border membrane vesicles gave a Km value of pravastain of about 15 mM at pH 5.5. Considering the species difference and pH examined, it is possible that pravastatin is transported across the intestinal epithelial apical membrane by the OATP-B-like transporter with a Km value of the order of millimolar concentrations. Fexofenadine was suggested to be absorbed by an OATP transporter, because rat oatp1, 2, and 3 and human OATP-A transported fexofenadine and fruit juice constituents reduced both the bioavailability of fexofenadine administered orally in human and the in vitro uptake activity of rat oatps and human OATP-A in cultured cells (Cvetkovic et al., 1999; Dresser et al., 2002). However, OATP-A is not expressed in human small intestine, and some other OATP might participate in the absorption of fexofenadine. The pH-sensitive transport activity of fexofenadine by OATP-B observed in this study might, at least in part, explain the mechanism of fexofenadine absorption in the small intestine.

In conclusion, it was clearly demonstrated that OATP-B exhibits pH-sensitive transport activity for various organic anions such as estrone-3-sulfate, dehydroepiandrosteronesulfate, taurocholic acid, pravastatin, and fexofenadine. The increased transport activity at lower pH seemed to be due to an increase of Vmax, but not the affinity. Because OATP-B is expressed at the apical membrane of intestinal epithelial cells, it is important to take the physiological pH in the intestinal lumen into consideration in assessing functionality. OATP is generally believed to be a multispecific transporter for various drugs as well as physiological compounds, and OATP-B exhibits a broader substrate specificity at acidic pH than that found at neutral pH in previous studies. There is a possibility that OATP-B is involved in the intestinal absorption of some clinically used acidic drugs, as well as physiological compounds. Further studies on the driving force, substrate specificity, regulatory mechanisms, and orthologous molecules in animals would be useful to clarify the significance of OATP-B as a transporter in humans.

Footnotes

  • This investigation was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by grants from The Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Novartis Foundation for the Promotion of Science.

  • ABBREVIATIONS: OATP, organic anion transporting polypeptide; HEK, human embryonic kidney; GSH, glutathione; FCCP, carbonylcyanide p-trifluoromethoxyphenyl hydrazone; ASBT, apical sodium-dependent bile acid transporter.

  • DOI: 10.1124/jpet.103.060194.

    • Received September 17, 2003.
    • Accepted November 5, 2003.

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Functional Characterization of pH-Sensitive Organic Anion Transporting Polypeptide OATP-B in Human

Takashi Nozawa, Kozue Imai, Jun-Ichi Nezu, Akira Tsuji and Ikumi Tamai
Journal of Pharmacology and Experimental Therapeutics February 1, 2004, 308 (2) 438-445; DOI: https://doi.org/10.1124/jpet.103.060194
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