Introduction

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To date, two major families of ATP-independent organic anion transporters (OATs) have been identified. The OAT family includes Oat1 (or Roat1), Oat2, and Oat3 (Sekine et al., 1997; Sweet et al., 1997; Wolff et al., 1997; Hosoyamada et al., 1999; Sweet and Pritchard, 1999; Tojo et al., 1999). Cellular uptake of organic anions on Oat1 was recently demonstrated to be directly coupled to efflux of -ketoglutarate (Sekine et al., 1997; Sweet et al., 1997), whereas Oat2 and Oat3 do not appear to be anion exchangers (Sekine et al., 1998; Kusuhara et al., 1999). The transport mechanisms for Oat2 and Oat3 have not yet been defined.

The second family consists of the OATP-related transporters and includes rat Oatp1 (Jacquemin et al., 1994), Oatp2 (Noe et al., 1997), Oatp3 (Abe et al., 1998), Oat-k1 (Saito et al., 1996), Oat-k2 (Masuda et al., 1999a), human OATP-A (Kullak-Ublick et al., 1995; Meier et al., 1997), OATP-C/LST1 (Abe et al., 1999), and the prostaglandin transporter Pgt (Kanai et al., 1995; Lu et al., 1996; Lu and Schuster, 1998). The transporters in this family exhibit relatively high amino acid identity, although there are major differences in substrate specificity. For example, Oatp1 and Oat-k1 share 72% amino acid identity, yet Oat-k1 is unable to transport either taurocholate or leukotriene (LT)C₄ (Saito et al., 1996), whereas Oatp1 accepts both as substrates (Li et al., 1998). The driving force for uptake on this family of transporters has not been identified, although Oatp1 is believed to function as an anion exchanger. Recent evidence suggests that Oatp1 is a reduced glutathione (GSH) exchanger (Li et al., 1998), although a role for bicarbonate has also been proposed (Satlin et al., 1997).

The present study examined whether Oatp2 also mediates organic anion exchange and, if so, whether it might be influenced by the GSH electrochemical gradient. Oatp1 and Oatp2 share many common features: they exhibit 77% predicted amino acid identity (Noe et al., 1997), are both localized to the basolateral membrane of hepatocytes (Kakyo et al., 1999; Reichel et al., 1999), and share many common substrates (Kullak-Ublick, 1999). However, these two transporters exhibit differences in their tissue distribution, overall substrate specificity, and regulation by physiological and pharmacological agents (Kullak-Ublick, 1999). For example, Oatp1 is distributed homogeneously across the liver acinus, whereas Oatp2 is expressed predominantly in perivenous hepatocytes. In the choroid plexus, Oatp1 is localized exclusively to the apical plasma membrane (Angeletti et al., 1997), whereas Oatp2 is localized to the basolateral membrane (Gao et al., 1999). The functional significance of this polarized distribution in the choroid plexus is unknown. Oatp1 and Oatp2 genes also appear to have different regulatory elements, based on differential changes in expression in response to certain stimuli. For example, in acute models of cholestasis, there is significant down-regulation of Oatp1 expression, whereas Oatp2 expression is unaffected (Kullak-Ublick, 1999). The down-regulation of Oatp1 may limit hepatic uptake of potentially toxic bile salts and other compounds that accumulate as a result of the impaired biliary excretion, whereas the preserved expression of Oatp2 during cholestasis may function to facilitate export of these compounds from hepatocytes into blood plasma.

The present results support this suggestion by demonstrating that Oatp2 can mediate either net uptake or efflux of organic solutes, depending on the imposed electrochemical gradient. Our results also indicate that GSH is an intracellular substrate for Oatp2 and a stimulator of organic anion uptake, although the precise mechanism by which it acts remains to be identified.

	Experimental Procedures

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Materials and Animals. [glycine-2-³H]Glutathione (50 Ci/mmol), [³H(G)]taurocholic acid (3.47 Ci/mmol), [³H(G)]digoxin (19 Ci/mmol), and [14,15,19,20-³H(N)]LTC₄ (165 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). [³H]DNP-SG was synthesized enzymatically from [³H]GSH and 1-chloro-2,4-dinitrobenzene as previously described (Ballatori and Truong, 1995). Unlabeled S-(2,4-dinitrophenyl)-glutathione (DNP-SG) and S-sulfobromophthalein-glutathione (BSP-SG) were synthesized and purified as described previously (Whelan et al., 1970; Hinchman et al., 1991). Other chemicals and reagents were obtained from Sigma Chemical Co. (St. Louis, MO), Aldrich Chemical Co. (Milwaukee, WI), or J. T. Baker (Philipsburg, NJ). Mature Xenopus laevis were purchased from Nasco (Fort Atkinson, WI). Animals were maintained under a constant light cycle at a room temperature of 18°C.

Synthesis of Capped cRNA. Oatp1 and Oatp2 cDNAs were prepared as previously described (Noe et al., 1997; Li et al., 1998). Capped cRNA was transcribed in vitro with T3 RNA polymerase (Ambion, Austin, TX); the cRNA was precipitated with lithium chloride, and resuspended in RNase-free water for oocyte injection.

X. laevis Oocyte Preparation and Microinjection. Isolation of X. laevis oocytes was performed as described by Goldin (1992) and previously used in our laboratory (Ballatori et al., 1996; Li et al., 1998). Frogs were anesthetized by immersion for 15 min in ice-cold water containing 0.3% tricaine (Sigma Chemical Co., St. Louis, MO). Oocytes were removed from the ovary and washed with Ca²⁺-free OR-2 solution (containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES-Tris, pH 7.5) and incubated at room temperature with gentle shaking for 90 min in OR-2 solution supplemented with 2 mg/ml of collagenase (Sigma Type IA). Oocytes were transferred to fresh collagenase solution after the first 45 min of incubation. Collagenase was removed by extensive washing in OR-2 solution at room temperature. Stage V and VI defolliculated oocytes were selected and incubated at 18°C in modified Barth's solution [containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, and 20 mM HEPES-Tris, pH 7.5] supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). After 3 to 4 h of incubation, healthy oocytes were injected with 50 nl of either Oatp1 or Oatp2 cRNA (0.5-10 ng/oocyte). Control oocytes were injected with a corresponding volume of sterile H₂O. Injected oocytes were cultured at 18°C with a daily change of modified Barth's medium. Healthy oocytes with a clean brown animal half and a distinct equator line were selected for experiments.

Uptake of DNP-SG and LTC₄ into Oocytes. Uptake studies were performed 3 days postmicroinjection of cRNA. Oocytes were pretreated with 0.5 mM acivicin for 30 min at room temperature to inhibit -glutamyl transpeptidase activity. For uptake measurements, from six to eight oocytes were incubated at 25°C for 1 h in 100 µl of modified Barth's solution in the presence of 1 µCi of [³H]DNP-SG or [³H]LTC₄ and 0.5 mM acivicin (Ballatori et al., 1996; Li et al., 1998). The uptake was stopped by adding 2.5 ml of ice-cold modified Barth's solution and oocytes washed three times each with 2.5 ml of ice-cold modified Barth's solution. Two oocytes each were dissolved in a polypropylene scintillation vial with 0.2 ml of 10% sodium dodecyl sulfate and counted in a Packard model 4530 scintillation spectrometer after addition of 5 ml of Opti-Fluor (Packard Instruments, Downers Grove, IL).

Taurocholate and Digoxin Uptake into Oocytes. Uptake of 1 and 10 µM taurocholate or 0.57 µM digoxin into oocytes injected with either 5 ng of Oatp1 or Oatp2 cRNA, or water for controls, was determined at 25°C in 100 µl of modified Barth's solution supplemented with either 0.35 µCi of [³H]taurocholate or 1.1 µCi of [³H]digoxin. Uptake was terminated by the addition of ice-cold stop solution, and samples were processed as described obove for other uptake studies. The stop solution for the taurocholate experiments contained 1 mM unlabeled taurocholate to reduce unspecific binding of tracer taurocholate.

GSH and DNP-SG Efflux in Oocytes. Oocytes injected with either water or Oatp2 cRNA were reinjected with 50 nl of [³H]GSH or [³H]DNP-SG (0.5-1 µCi/µl) and allowed to recover for 30 min in modified Barth's solution with 0.5 mM acivicin. Oocytes were washed twice with 2.5 ml of modified Barth's solution before efflux studies. Efflux was measured at 25°C in 200 µl of modified Barth's solution in the presence or absence of various compounds in the extracellular medium. Efflux was terminated after 15 min by removing the medium and counting it separately from the oocytes.

Altering Intracellular GSH Concentration in Oocytes. The endogenous GSH concentration in oocytes is approximately 2.5 mM (Ballatori et al., 1996). To increase GSH, oocytes were injected with 50 nl of different GSH stock solutions (e.g., 220 mM GSH stock, for an increase of ~20 mM in the oocytes). After injection, oocytes were incubated at 25°C for approximately 30 min before they were used in the experiment. To decrease GSH, oocytes were incubated in modified Barth's solution containing 2 mM hydrogen peroxide for 1 h; they were washed with modified Barth's solution and incubated at 25°C for 30 min before they were used in the experiment. GSH content of the oocyte was measured as previously described (Griffith, 1980).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Oatp1 and Oatp2 Mediate Bidirectional Solute Transport. To test the possibility of bidirectional transport on Oatp2, X. laevis oocytes expressing either Oatp2 or Oatp1 were loaded with 0.2 mM unlabeled taurocholate by microinjection, and uptake of [³H]taurocholate was measured for 15 min at 25°C (Fig. 1). Although uptake of [³H]taurocholate was accelerated by unlabeled taurocholate in both Oatp1- and Oatp2-expressing oocytes, the trans-stimulatory effect was especially high in Oatp2-expressing oocytes, indicating bidirectional transport.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Trans-stimulation of 10 µM [3H]taurocholate uptake in Oatp1- or Oatp2-expressing oocytes by intracellular taurocholate. Oocytes were injected with 5 ng of Oatp1 or Oatp2 cRNA and cultured for 3 days. To load with 0.2 mM taurocholate, oocytes were injected with 50 nl of a 2.2 mM taurocholate stock solution at 30 min before uptake measurements. Oocytes were washed in modified Barth's solution, and uptake of 10 µM [3H]taurocholate was measured at 25°C for 15 min. Values are mean ± S.E. (n = 5-7).

Role of GSH in Oatp2-Mediated Uptake of Taurocholate and Digoxin. To test whether Oatp2-mediated uptake is driven by the outwardly directed GSH electrochemical gradient, initial studies measured uptake of [³H]taurocholate and [³H]digoxin under conditions where the physiologic GSH gradient was gradually diminished by the addition of 1 to 20 mM GSH to the extracellular medium. However, extracellular GSH had only minimal effects on [³H]taurocholate and [³H]digoxin uptake even at a concentration of 20 mM (Table 1), indicating that transport is not directly coupled to GSH efflux. Taurocholate uptake in Oatp2-expressing oocytes was cis-inhibited by ouabain, as expected (Noe et al., 1997; Reichel et al., 1999), and by bromosulfophthalein and DNP-SG (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Cis-inhibition of 10 µM [3H]taurocholate and 0.57 µM [3H]digoxin uptake by GSH and some organic anions in Oatp2-expressing oocytes Oocytes injected with Oatp2 cRNA (5 ng/oocyte) were incubated with either 10 µM [3H]taurocholate or 0.57 µM [3H]digoxin for 10 min at 25°C. Control uptakes were 127 ± 36 and 193 ± 46 fmol · oocyte1 · 10 min1 for [3H]taurocholate and [3H]digoxin, respectively. Values are mean ± S.D. (n = 4-6).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Intracellular GSH concentration dependence of 1 µM [3H]taurocholate and 0.57 µM [3H]digoxin uptake in Oatp2-expressing oocytes. Oocytes were injected with 5 ng of Oatp2 cRNA. Intracellular GSH was raised or lowered as described in Experimental Procedures. [3H]Taurocholate and [3H]digoxin uptake was measured at 25°C for 15 min in the indicated intracellular GSH concentrations. The normal (endogenous) GSH concentration is approximately 2.5 mM. Uptake measurements were started 30 min after the addition of 0.5 mM acivicin to the culture medium. Values are mean ± S.E. (n = 4-6).

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Oatp2-Mediated [³H]Taurocholate Uptake Is Also Stimulated by High Intracellular Concentrations of Glutathione S-Conjugates and a GSH Analog. Interestingly, taurocholate uptake into Oatp2-expressing oocytes was also stimulated in oocytes that were preloaded with ophthalmic acid (a GSH analog) and the glutathione S-conjugates S-methylglutathione, DNP-SG, and BSP-SG but not by N-acetylcysteine or glutarate (Fig. 3), suggesting that some GSH derivatives may also be substrates for Oatp2. DNP-SG had no effect on Oatp2-mediated [³H]taurocholate uptake at an intracellular concentration of 0.1 mM, but uptake was gradually increased at higher DNP-SG concentrations, reaching a maximum value at 0.5 mM DNP-SG (Fig. 4). Taurocholate uptake was nearly doubled in Oatp2-expressing oocytes loaded with 0.5 mM DNP-SG. In contrast to the effects of DNP-SG on Oatp2-mediated uptake, this glutathione S-conjugate did not affect Oatp1-mediated taurocholate uptake, at concentrations up to 1 mM (Figs. 3 and 4).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of intracellular GSH, glutathione S-conjugates, and other compounds on [3H]taurocholate uptake in Oatp1- or Oatp2-expressing oocytes. Oatp1- and Oatp2-expressing oocytes were injected with 50 nl of either 110 mM or 220 mM stock solutions of GSH, S-methylglutathione, ophthalmic acid, N-acetylcysteine (NAC), or glutarate or 5.5 mM stock solutions of DNP-SG or BSP-SG. Oocytes were allowed to recover for 30 min in modified Barth's solution, and 1 µM [3H]taurocholate uptake was measured at 25°C for 15 min. Values are mean ± S.E. (n = 3-6).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Relation between intracellular DNP-SG concentration and 1 µM [3H]taurocholate uptake in Oatp1- or Oatp2-expressing oocytes. Intracellular DNP-SG was raised by injecting 50 nl of DNP-SG stock solutions, and oocytes were allowed to recover for 30 min in modified Barth's solution with 0.5 mM acivicin. Uptake of 1 µM [3H]taurocholate was measured at 25°C for 15 min. Values are mean ± S.E. (n = 4).

Accelerated [³H]DNP-SG Efflux in Oatp2-Expressing Oocytes. To directly test the hypothesis that DNP-SG is an intracellular substrate for Oatp2, oocytes were microinjected with 0.5 mM [³H]DNP-SG and efflux was measured in control (water-injected), and Oatp1- and Oatp2-expressing oocytes. Because oocytes have an endogenous ATP-dependent DNP-SG efflux mechanism (Ballatori et al., 1996), the background rate of efflux is quite high (22.7 ± 0.6 pmol · oocyte¹ · 15 min¹). Nevertheless, Oatp2-expressing oocytes exported [³H]DNP-SG at a higher rate than control oocytes (Fig. 5), indicating that DNP-SG is an intracellular substrate for Oatp2. In contrast, DNP-SG efflux was unaffected in Oatp1-expressing oocytes (Fig. 5). The Oatp2-stimulated [³H]DNP-SG efflux rate was 2.3 pmol · oocyte¹ · 15 min¹, a value comparable to that measured for [³H]GSH efflux in Oatp2-expressing oocytes (2.6 pmol · oocyte¹ · 15 min¹).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Efflux of [3H]DNP-SG in Oatp1- or Oatp2-expressing oocytes. Oatp1- or Oatp2-expressing oocytes were reinjected with 50 nl of [3H]DNP-SG to achieve an intracellular concentration of 0.5 mM DNP-SG. Oocytes were allowed to recover for 30 min, and efflux was measured for 15 min in 200 µl of modified Barth's solution. The control rate of DNP-SG efflux was 22.7 ± 0.6 pmol · oocyte1 · 15 min1. Values are mean ± S.D. (n = 6). *, significantly different from control, P < .05 by Student's t test.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Several ATP-independent organic solute transporters have been identified at the molecular level; however, the functional characterization of these remains incomplete. A major unresolved question relates to the driving force for transport. For example, although there is good evidence that rat kidney Oat1 functions as an uptake transporter that is energized by exchange with intracellular -ketoglutarate (Sekine et al. 1997; Sweet et al. 1997), the transport mechanism for other members of the OAT family appears to be different. Recent studies indicate that in contrast to Oat1, the Oat2 and Oat3 transporters do not function as -ketoglutarate exchangers (Sekine et al., 1998; Kusuhara et al., 1999).

The transport mechanism for the OATP transporters also remains undefined. Data on driving forces have been reported for only one member of this family, Oatp1, but these data are discordant. One study indicates that Oatp1 may function as a GSH exchanger (Li et al., 1998), whereas another study suggests a role for bicarbonate (Satlin et al., 1997). The present study tested the hypothesis that another member of the OATP family, Oatp2, functions as a GSH exchanger.

To directly test this hypothesis, initial experiments examined [³H]taurocholate uptake in oocytes incubated in medium containing high GSH concentrations to dissipate the GSH gradient. However, the addition of GSH to the extracellular medium had minimal effects on taurocholate uptake, indicating that Oatp2 is not an obligate GSH exchanger. These results, however, do not exclude the possibilities that transport on Oatp2 either is highly asymmetric or is stimulated indirectly by intracellular GSH. For example, GSH may stimulate transport by a kinetic rather than a catalytic mechanism, as discussed further later. Alternatively, if GSH binding affinity and/or transport rate was high from the intracellular surface of the protein but low on the extracellar surface, this would allow for net outward movement of GSH even with a minimal GSH electrochemical gradient. Conversely, substrate (e.g., taurocholate) binding affinity may be high on the extracellular surface and low in the cytosol, favoring net uptake. If both GSH and extracellular substrate displayed such asymmetric transport kinetics, this may allow for net transport despite seemingly unfavorable chemical gradients. The issue of asymmetric transport kinetics is difficult to evaluate in most experimental systems, especially in intact cells, because intracellular concentrations of substrates are not readily altered or measured. A second limitation relates to the difficulty in distinguishing substrate-induced GSH transport activity from the large basal GSH transport that is observed in all cells that have been examined, including X. laevis oocytes (Ballatori et al., 1996; Ballatori and Rebbeor, 1998).

Because of these limitations, additional experiments were performed to further explore the role of intracellular GSH in Oatp2-mediated transport in X. laevis oocytes. As previously reported for Oatp1 (Li et al., 1998), there was a positive correlation between intracellular GSH concentration and organic anion uptake in Oatp2-expressing oocytes (Fig. 2), consistent with a role for GSH in organic anion uptake. Oatp2-expressing oocytes also demonstrated higher [³H]GSH efflux compared with water-injected oocytes, but GSH efflux was not further stimulated by extracellular substrates of Oatp2 (data not shown). As described in Results, this lack of effect of extracellular substrates may be explained by the relatively slow rate at which Oatp2 mediates uptake of organic anions compared with the much larger Oatp2-stimulated GSH efflux rate.

One interesting difference between Oatp1 and Oatp2 relates to their interaction with glutathione S-conjugates. [³H]DNP-SG and [³H]LTC₄ are taken up by Oatp1-, but not Oatp2-, expressing oocytes, indicating differences in extracellular substrate specificity. Conversely, DNP-SG trans-stimulates [³H]taurocholate uptake into Oatp2-, but not Oatp1-, expressing oocytes, indicating differences in intracellular substrate specificity. The demonstration of enhanced [³H]DNP-SG efflux in Oatp2-expressing oocytes provides further strong evidence that this glutathione S-conjugate is an intracellular substrate and supports the suggestion that Oatp2-mediated transport is asymmetric. Additional studies in a well-defined (polarized) isolated membrane system are needed to examine this possibility.

Additional studies are also needed to elucidate the precise role of GSH in Oatp2-mediated transport. Our data indicate that GSH is an intracellular substrate for Oatp2, as evidenced both by the ability of GSH to trans-stimulate uptake of organic anions and the ability of Oatp2 to stimulate [³H]GSH efflux. However, in contrast to Oatp1, extracellular GSH failed to cis-inhibit organic anion uptake, indicating that GSH may facilitate uptake by a kinetic rather than a catalytic mechanism. That is, GSH efflux on Oatp2 may accelerate the transition of the carrier from an inward-facing to an outward-facing mode, which in turn could stimulate uptake of extracellular substrate. Because transport is also stimulated by glutathione S-conjugates, the effects of GSH are probably not due to a redox-type reaction.

Taken together, the present findings indicate that Oatp2 can mediate bidirectional transport of organic anions and suggest that under certain conditions, Oatp2 may function primarily as an export carrier. The direction of transport across the cell membrane is most likely determined by the electrochemical gradients and the relative binding affinities of individual substrates. This interpretation is consistent with the previous suggestion that Oatp2 may function as an efflux transporter in cholestasis (Kullak-Ublick, 1999). Oatp1 and Ntcp are down-regulated in cholestasis, as might be expected for uptake transporters, whereas Oatp2 expression is unaltered, indicating that it may not function as an uptake mechanism for cholephilic compounds. Additional evidence that Oatp2 may function for efflux comes from the polarized distribution of Oatp1 and Oatp2 in the choroid plexus: Oatp1 is localized to the apical membrane, whereas Oatp2 is localized to the basolateral membrane (Angeletti et al., 1997; Gao et al., 1999). Organic anions are eliminated from cerebrospinal fluid (CSF) after i.c.v. administration or during ventriculocisternal perfusion in animals (Suzuki et al., 1997; Nishino et al., 1999), but the transporters involved have not been identified. Our observations that Oatp1 functions a GSH exchanger (Li et al., 1998) and that Oatp2 mediates bidirectional transport suggest a possible mechanism by which these carriers may work in tandem to transport compounds from the CSF into blood. That is, the apically localized Oatp1 may facilitate uptake of organic solutes from the CSF in exchange for intracellular GSH, and basolateral Oatp2 would export the same compounds down their electrochemical gradients into blood plasma. Because both Oatp1 and Oatp2 transport bidirectionally, they may also produce net transport in the opposite direction, under appropriate transepithelial gradient conditions. Additional studies are needed to evaluate these possibilities. Interestingly, another member of the OATP family, Oat-k1, was recently shown to function bidirectionally (Masuda et al., 1999b), lending support to the suggestion that some OATP transporters may function for efflux of organic solutes from cells.