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J. Biol. Chem., Vol. 278, Issue 26, 23529-23537, June 27, 2003

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Differential Modulation of the Human Liver Conjugate Transporters MRP2 and MRP3 by Bile Acids and Organic Anions^*

Adrienn Bodó  , Éva Bakos , Flóra Szeri , András Váradi  and Balázs Sarkadi  ¶

From the National Medical Center, Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences and the Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, 1113 Hungary

Received for publication, April 4, 2003 , and in revised form, April 17, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The multidrug resistance proteins MRP2 (ABCC2) and MRP3 (ABCC3) are key primary active transporters involved in anionic conjugate and drug extrusion from the human liver. The major physiological role of MRP2 is to transport conjugated metabolites into the bile canaliculus, whereas MRP3 is localized in the basolateral membrane of the hepatocytes and transports similar metabolites back to the bloodstream. Both proteins were shown to interact with a large variety of transported substrates, and earlier studies suggested that MRPs may work as co-transporters for different molecules. In the present study we expressed the human MRP2 and MRP3 proteins in insect cells and examined their transport and ATPase characteristics in isolated, inside-out membrane vesicles. We found that the primary active transport of estradiol-17--D-glucuronide (E₂17G), a major product of human steroid metabolism, was differently modulated by bile acids and organic anions in the case of human MRP2 and MRP3. Active E₂17G transport by MRP2 was significantly stimulated by the organic anions indomethacin, furosemide, and probenecid and by several conjugated bile acids. In contrast, all of these agents inhibited E₂17G transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E₂17G, and the results indicated an allosteric cross-stimulation, probably a co-transport of bile acids and glucuronate conjugates through this protein. There was no such stimulation of bile acid transport by MRP3. In conclusion, the different transport modulation of MRPs by bile acids and anionic drugs could play a major role in regulating physiological and pathological metabolite fluxes in the human liver.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The homologous multidrug resistance ABC¹ transporter proteins MRP2 and MRP3 seem to be key players in the transport of organic anionic conjugated compounds in the liver and kidney (1–7). Unlike the selective "classical" transport proteins, multidrug transporters recognize and handle a wide range of substrates. The members of the MRP family are transporting hydrophobic anionic conjugates but may also extrude hydrophobic uncharged drugs. In this latter case drug transport by MRPs has been shown to be linked to the co-transport or allosteric effect of cellular reduced glutathione, GSH (2, 3, 6–12).

MRP2 in polarized cells is localized in the apical (luminal) membrane surface, predominantly in the canalicular membrane of hepatocytes but also in the apical membranes of kidney-proximal tubules (1–3, 6, 13). In contrast, MRP3 expression in polarized cells is restricted to the basolateral membrane (4). The lack of functional MRP2 causes the human disease Dubin-Johnson syndrome, which is associated with a large increase of conjugated bilirubin and other conjugated metabolites in the bloodstream. Several animal models are available for modeling this disease condition (14, 15), and there are known mutations/polymorphisms, reducing human MRP2 activity and leading to disorders of conjugate metabolism (16–18).

Liver cells synthesize primary bile acids from cholesterol and then conjugate these compounds predominantly with taurine or glycine. The ABC transporter ABCB11 (also referred to as sister P-glycoprotein or bile salt export pump (BSEP)) is localized in the canalicular membrane and considered to be the major bile salt transporter (19, 20). However, MRP2 (7, 13, 21) and MRP3 (4, 5, 9) may also secrete these amphipathic compounds into the bile or the bloodstream, respectively. In the enterohepatic cycle a major part, about 95% of the secreted bile salts is reabsorbed in the intestine, whereas the rest is excreted into the feces after bacterial degradation. The relative role of the ABC transporters in this enterohepatic circulation is currently under study.

Elevated levels of MRP3 expression have been detected in human hepatocellular carcinoma (22) and in Dubin-Johnson patients, when in the absence of a functional MRP2, MRP3 seems to have a compensatory transport function (4, 7, 13). In this case several compounds, normally extruded into the bile, are transported by MRP3 into the sinusoidal blood. MRP3 is also up-regulated under cholestatic conditions and agents (23–25). Thus the co-regulated function of MRP2 and MRP3 may have a major effect on the conjugate metabolism and bile acid secretion in the human liver.

In the present paper we provide data for the interactions of MRP2 and MRP3 with estradiol-17--D-glucuronide and bile acids, as well as with some pharmacologically important organic anions. A major metabolite of human estrogen metabolism, estradiol-17--D-glucuronide (E₂17G), has been shown to be transported by both MRP2 (7, 13, 26, 27) and MRP3 (5, 28). This metabolite, with a significantly increased level during pregnancy and hormone replacement therapy, is secreted into the bile mainly by MRP2 (21, 29). Estrogen metabolites and other steroid glucuronides show hepatotoxic effects (30) and mutually protect against cholestasis (31). In pregnancy and in hormone replacement therapy, taurocholate decreases E₂17G uptake in isolated rat hepatocytes (32), and certain MRP3 substrates induce MRP3 overexpression in cholestatic conditions (23–25). All of these data suggest an interrelated transport of bile acids and glucuronide-conjugated metabolites in the liver cells.

To explore these relationships we examined the transport properties of human MRP2 and MRP3, expressed at similar high levels in Sf9 cells. In isolated Sf9 membrane vesicles we measured both human MRP2- and MRP3-dependent direct vesicular uptake of labeled compounds, as well as the effects of these compounds on the MRP ATPase activity. Our data suggest that both MRP2 and MRP3 play important physiological roles in the transport of glucuronide conjugates and bile salts and that MRP2 performs a co-transport of glucuronide conjugates and bile salts into the bile canaliculi. In contrast, glucuronide transport into the bloodstream by MRP3 is inhibited by bile salts. We also demonstrate a differential modulation of these transport pathways by pharmacologically active organic anions. These results may help to understand the molecular basis of the complex interactions of metabolite and drug transport in the human liver and intestine.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—E₂17G, glycocholate (GC), glycochenodeoxycholate (GCDC), taurodeoxycholate (TDC), and taurochenodeoxycholate (TCDC) were obtained from Sigma. Labeled [³H]E₂17G was obtained from PerkinElmer Life Sciences, and [¹⁴C]GC was from Moravek Biochemicals.

Expression of MRPs in Insect Cells—Recombinant baculoviruses containing the MRP cDNAs were prepared as described in Refs. 33 and 34. Sf9 (Spodoptera frugiperda) cells were cultured and infected with a baculovirus as described in Ref. 35. MRP2 and MRP3 cDNAs were obtained from Prof. Piet Borst and inserted into a baculovirus vector as described in Ref. 36.

Membrane Preparation and Immunoblotting—Virus-infected Sf9 cells were harvested, their membranes were isolated and stored, and the membrane protein concentrations were determined as described in Ref. 37. Gel electrophoresis and immunoblot detection were performed, and protein-antibody interaction was determined using the enhanced chemiluminescence technique as described in Ref. 36.

Membrane ATPase Measurements—ATPase activity was measured basically as described in Ref. 37, by determining the liberation of inorganic phosphate from ATP with a colorimetric reaction. The incubation medium contained 10 mM MgCl₂, 40 mM MOPS-Tris (pH 7.0), 50 mM KCl, 5 mM dithiothreitol, 0.1 mM EGTA, 4 mM sodium azide, 1 mM ouabain, and 4 mM ATP. Membrane ATPase activity was measured for 60 min at 37 °C in the presence of 4 mM ATP (control points), plus or minus 1 mM sodium orthovanadate (difference of the two values means the vanadate-sensitive component), and various concentrations of additional compounds, as indicated in the figures.

Transport Assay in Isolated Inside-out Membrane Vesicles—The membrane vesicles were incubated in the presence of 4 mM ATP in a buffer containing 10 mM MgCl₂, 40 mM MOPS-Tris (pH 7.0), and 50 mM KCl at 37 °C (34). Aliquots of the membrane suspensions were added to excess cold transport buffer and then rapidly filtered through nitrocellulose membranes (pore size, 0.45 µm). After washing the filters with 10 ml of ice-cold washing buffer, the radioactivity associated with the filters was measured by liquid scintillation counting. ATP-dependent transport was calculated by subtracting the values obtained in the presence of AMP from those in the presence of ATP. The figures present mean values obtained in three independent experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of Human MRP1, MRP2, and MRP3 in Insect Cells—Fig. 1A shows a Coomassie-stained blot of the proteins of isolated membranes obtained from Sf9 cells and separated by SDS gel electrophoresis. The Sf9 cells were infected with the recombinant baculoviruses inducing human MRP1, MRP2, or MRP3 expression. As documented, all three MRPs were successfully expressed at high levels (with an apparent molecular mass of about 160 kDa) in the Sf9 insect cells. The comparable amount of the expression of the three different human MRPs (about 5–7% of the total membrane proteins) allowed the direct comparison of the transport activities of these proteins in the following experiments. Immunoblotting by specific antibodies clearly identified the respective human MRP proteins expressed (Fig. 1B).

View larger version (39K): [in this window] [in a new window]   FIG. 1. A, detection of human MRP1, MRP2 and MRP3 by SDS-polyacrylamide gel electrophoresis and by Coomassie staining in isolated membranes of Sf9 cells. Mr, molecular mass marker; lane A, isolated membranes of Sf9 cells expressing human MRP1; lane B, isolated membranes of Sf9 cells expressing human MRP2; lane C, isolated membranes of Sf9 cells expressing human MRP3; lane D, isolated membranes of Sf9 cells expressing -galactosidase as control. B, detection of human MRP1, MRP2, and MRP3 on immunoblots by specific monoclonal antibodies in isolated membranes of Sf9 cells. Top panel, detection by the monoclonal anti-MRP1, M6; middle panel, detection by the monoclonal anti-MRP2, M2-III/6; bottom panel, detection by the monoclonal anti-MRP3, M3-II/9. First lane, isolated membranes of Sf9 cells expressing human MRP1; second lane, isolated membranes of Sf9 cells expressing human MRP3; third 3, isolated membranes of Sf9 cells expressing human MRP2.

In the heterologous Sf9 expression system these human proteins were produced in an underglycosylated form that has been demonstrated not to have any effect on their transport functions (10, 33–37). In the following experiments we used isolated membranes, forming inside-out membrane vesicles from these human MRP-expressing Sf9 cells.

Vesicular Transport of E₂17G by Human MRPs—In the following experiments we studied the transport of E₂17G, a typical glucuronide conjugate, which may be a physiologically relevant model substrate of these transporters. This compound has been indicated to be transported substrate for both MRP2 and MRP3 (5, 7, 13, 26–28). By using isolated, inverted Sf9 membrane vesicles, we have directly examined the vesicular transport of E₂17G by the three different MRPs and examined the modulation of this transport by bile acids, bile salt conjugates, and organic anions.

Fig. 2 documents the ATP-dependent uptake of radiolabeled E₂17G in isolated Sf9 cell membrane vesicles expressing MRP3 (panel A) and MRP2 (panel B). The uptake values were obtained by subtracting the values obtained in the presence of AMP (which was low in all experiments). Also, as a control, we used vesicles obtained from Sf9 cells expressing -galactosidase. In these latter vesicles ATP-dependent tracer uptake was negligible. In all of these experiments the linear phase of the tracer uptake was determined (2 min for E₂17G), and this period was used for studying the concentration dependence of the uptake.

View larger version (15K): [in this window] [in a new window]   FIG. 2. ATP-dependent uptake of [3H]E217G in Sf9 cell membrane vesicles expressing human MRP3 (A) or MRP2 (B). E217G concentration dependence is shown. Membrane vesicle preparations were incubated with different concentrations of E217G, including the labeled compound, at 37 °C for 2 min (see "Experimental Procedures"). B, inset, concentration dependence of E217G uptake below 100 µM E217G concentration. The mean values ± S.E. are shown.

As documented in Fig. 2A, MgATP-energized E₂17G uptake in human MRP3-containing membrane vesicles was a saturable function of the E₂17G concentration, with a calculated maximum uptake rate of about 1.3 nmol/mg membrane protein/min, and an apparent K_m value of about 25–30 µM.

When we measured E₂17G uptake in Sf9 membrane vesicles containing comparable amounts of human MRP1, the concentration dependence of E₂17G uptake gave a K_m value of about 5–8 µM, but the maximum transport rate was significantly lower (about 0.1 nmol/mg membrane protein/min) than that in the case of MRP3 (38).

In contrast to MRP3, as shown in Fig. 2B, membrane vesicles prepared from human MRP2-expressing Sf9 cells had an entirely different concentration dependence of E₂17G uptake. The rate of E₂17G uptake at low E₂17G concentrations (see inset in Fig. 2B) had an S-shaped curve, and saturation of the E₂17G uptake was only achieved above 1 mM E₂17G concentrations. Moreover, the maximum uptake rate of E₂17G by MRP2 was about 10 times higher (12 nmol/mg membrane protein/min) than that for MRP3.

These data indicate that MRP3 (and also MRP1) is a relatively high affinity but low capacity transporter for E₂17G, whereas MRP2 has a much lower affinity but significantly higher transport capacity for this glucuronate conjugate. The S-shaped curve of concentration dependence seen in the case of MRP2 indicates a complex interaction of this transporter protein with E₂17G.

Effect of E₂17G on the Membrane ATPase Activity of Human MRPs—In the following experiments we have studied the vanadate-sensitive membrane ATPase activity in isolated membranes of Sf9 cells, containing comparable amounts of the MRP proteins (Fig. 1). As documented earlier (33–37), this ATPase activity is closely related to the transport activity of the ABC transporter proteins, and substrate stimulation of the ATPase reflects the interaction of the respective transporter with its transported substrate(s).

The E₂17G concentration dependence of this vanadate-sensitive ATPase activity for MRP2 and MRP3 is shown in Fig. 3. As documented, we found only a slight (although significant) stimulation of the MRP3 ATPase activity at E₂17G concentrations between 20 and 100 µM. In the case of MRP1 only a moderate increase in the ATPase activity was seen (38), as compared with that in -galactosidase-expressing Sf9 cell membranes. In contrast, in membranes containing comparable amounts of human MRP2, there was a large increase in the membrane ATPase activity at E₂17G concentrations above 100 µM. This ATPase activity increased up to 8–10 nmol/mg membrane protein/min at about 0.5 mM E₂17G and did not reach a maximum level even at 1 mM of E₂17G concentration (higher concentrations could not be properly applied under the present experimental conditions). It is important to note that the E₂17G uptake rate by MRP2, as shown in Fig. 2B, closely correlates with these ATPase measurements, reinforcing that the vanadate-sensitive ATPase reflects transport-associated ATP hydrolysis by MRP2. These membrane ATPase experiments support the conclusions obtained from direct E₂17G transport experiments, suggesting that MRP3 (and MRP1) is a higher affinity but a much lower capacity transporter for E₂17G than MRP2.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Effect of E217G on vanadate-sensitive ATPase activity of human MRP3- and MRP2-expressing Sf9 cell membranes. The membrane preparations were incubated with different concentrations of E217G at 37 °C for 60 min in the presence of 4 mM MgATP (see "Experimental Procedures"). Vanadate-sensitive ATPase activity in the control, -galactosidase expressing Sf9 cell membranes was about 3 nmol/mg membrane protein/min and not modulated by E217G (not shown). The mean values ± S.E. are shown.

Modulation of the MRP3- and MRP2-dependent Vesicular Transport of E₂17G by Organic Anions and Bile Salts—In the following experiments we examined the effects of the organic anions, furosemide, probenecid, and indomethacin (IM) on the direct, vesicular uptake of labeled E₂17G in MRP3-containing (Fig. 4A) and MRP2-containing (Fig. 4B) membranes, respectively. These experiments were carried out at two fixed E₂17G concentrations (1 and 13 µM) for both MRP3 and MRP2, to study these modulatory effects at E₂17G concentrations below the respective K_m values. We expected that both the inhibitory or the possible allosteric stimulatory effects could be optimally studied under these conditions.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Modulation of ATP-dependent [3H] E217G transport in human MRP3-expressing (A) or MRP2-expressing (B) Sf9 cell membrane vesicles by the organic anions, furosemide (FR), probenecid (PR), and IM. The membrane vesicles were incubated at 37 °C for 2 min with 1 µM E217G in case of MRP3 and with 13 µM E217G in case of MRP2 (see "Experimental Procedures"). The transport rates are expressed as percentages of the tracer uptake measured in the absence of any additional compounds. The mean values ± S.E. are shown.

We found that in the case of MRP3, E₂17G transport was inhibited by all of the three organic anions. The approximate K_i values were 350 µM for furosemide, 400 µM for probenecid, and 60 µM for indomethacin. In the case of MRP3, a slight 20–25% stimulation of E₂17G uptake was observed by low concentrations (5–10 µM) of indomethacin. Fig. 4A shows E₂17G uptake data measured at 1 µM E₂17G concentration, but similar results were obtained at higher (13 µM) E₂17G concentrations as well. We have already described (38) that both indomethacin and furosemide significantly stimulate MRP3 ATPase activity; thus both of these anions are most probably transported substrates of MRP3. Still, their predominant effect on E₂17G uptake was inhibitory.

As shown in Fig. 4B, in the case of MRP2, the effects of these organic anions were entirely different; furosemide and probenecid, between a wide concentration range of 50–500 µM significantly stimulated the ATP-dependent E₂17G uptake by MRP2, and this stimulation reached about 150% of the transport rate measured without these organic anions. Moreover, indomethacin in concentrations between 50 and 100 µM induced a 6–6.5-fold stimulation of E₂17G transport activity by MRP2, and a 5-fold stimulation of this transport was still observed at 500 µM indomethacin. Fig. 4B shows the data measured at 13 µM E₂17G concentration, but similar results were obtained at lower (1 µM) E₂17G concentrations as well.

In the following experiments we have studied the effect various bile salt conjugates on the vesicular uptake of labeled E₂17G by MRP3 (Fig. 5A) and MRP2 (Fig. 5B), respectively. We have examined the effects of GC, GCDC, TDC, and TCDC, all potential physiological intrahepatic bile salts in humans. Again, these experiments were carried out at two fixed E₂17G concentrations (1 and 13 µM) for both MRP3 and MRP2.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Modulation of ATP-dependent [3H]E217G transport in human MRP3-expressing (A) or MRP2-expressing (B) Sf9 cell membrane vesicles by different concentrations of the bile salt conjugates, GC, GCDC, TDC, and TCDC. The membrane vesicles were incubated at 37 °C for 2 min with 1 µM E217G in case of MRP3 and with 13 µM E217G in case of MRP2 (see "Experimental Procedures"). Transport rates are expressed as percentages of the tracer uptake measured in the absence of any additional compounds. The mean values ± S.E. are shown.

As shown in Fig. 5A, in the case of MRP3, E₂17G transport (measured at 1 µM E₂17G) was inhibited by all bile salts examined. In the case of GC this inhibition was more pronounced at about 100 µM, whereas the other bile salt conjugates strongly inhibited E₂17G already at 10 µM concentrations. When E₂17G uptake was measured at higher (13 µM) E₂17G concentrations, all bile salts were inhibitory as well (data not shown).

Fig. 5B documents that in the case of MRP2, all bile salts examined significantly stimulated ATP-dependent E₂17G uptake (measured here at 1 µM E₂17G). This stimulatory effect increased up to 100 µM of bile salt concentrations and reached about 180–200% in the case of GC, TCDC, and GCDC, whereas TDC was somewhat less effective in this stimulation.

All of these data indicate that the ATP-dependent active E₂17G uptake, carried out by MRP2, is allosterically modulated by various organic anions and bile salt conjugates. To better characterize these interactions, we performed detailed E₂17G concentration dependence studies by examining the effects of IM and that of the most abundant physiological bile salt conjugate in humans, GC in MRP2-containing membrane vesicles. We examined fixed concentrations (100 µM) of IM and GC, respectively, at an E₂17G concentration range (10–100 µM), in which an S-shaped concentration dependence of E₂17G uptake was observed (Fig. 2B).

As shown in Fig. 6, both IM and GC significantly stimulated the rate of E₂17G uptake in this whole E₂17G concentration range. Moreover, in the case of IM, a significant change in the shape of the curve was observed; the stimulation of E₂17G uptake was more pronounced at lower E₂17G concentrations.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Modulation of ATP-dependent [3H]E217G transport in human MRP2 expressing Sf9 cell membrane vesicles by the organic anion, IM, and by the bile salt, GC. MRP2-expressing Sf9 cell membrane vesicles were incubated with different concentrations of E217G at 37 °C for 2 min. Additional compounds were 100 µM IM or 100 µM GC. The mean values ± S.E. are shown.

Vesicular Transport of Labeled Glycocholate by Human MRPs—In the following experiments, to clarify the relationship between the transport of E₂17G and its modulation by bile salts, we have directly measured labeled GC uptake in membrane vesicles containing MRP3 and MRP2, respectively. Labeled GC uptake was measured in a GC concentration range of 20–500 µM, and the effect of various concentrations of E₂17G was examined.

Fig. 7A shows the magnitude of ATP-dependent labeled GC uptake by MRP3- or MRP2-containing membrane vesicles (and the same transport in -galactosidase-containing membrane vesicles, as control), and the effect of 100 µM E₂17G on this active transport process. As shown, both MRP3 and MRP2 containing membranes show a well measurable and comparable rate of GC uptake, with a tendency of saturation at about 300 µM GC concentration. The addition of E₂17G in the case of MRP3 produced a slight inhibition of the GC uptake (at lower GC concentration this inhibition, because of the technical limitations, was not studied in detail). However, in the case of MRP2, in the entire GC concentration range studied, the addition of E₂17G significantly increased (between 100 and 300 µM of GC, approximately doubled) the rate of GC uptake.

View larger version (30K): [in this window] [in a new window]   FIG. 7. A, concentration dependence of ATP-dependent GC uptake in human MRP3- or MRP2-expressing Sf9 membrane vesicles, measured by following [14C]glycocholate uptake. Modulation of GC uptake by the glucuronide conjugate E217G is shown. The membrane vesicles were incubated with different concentrations of GC at 37 °C for 5 min. For studying the concentration dependence of the uptake the linear phase of the tracer uptake was determined (5 min for GC uptake). Modulation of GC uptake was examined at 100 µM concentration of E217G. -Galactosidase-expressing Sf9 cell membranes were used as controls. The mean values ± S.E. are shown. B, synergistic effect of GC and E217G on the combined, ATP-dependent, and vanadate-sensitive uptake of GC and E217G in human MRP2-containing membrane vesicles. MRP2-expressing Sf9 cell membrane vesicles were incubated with 100 µM GC and/or 20 µM concentrations of E217Gat37 °C for 2 min. The numbers on the columns indicate the mean values (three independent experiments) of combined vesicular substrate transport rates (in pmol/mg membrane protein/min). The magnitudes of standard errors are indicated on the columns.

Fig. 7B demonstrates the combined transport rates of E₂17G and GC in MRP2-containing vesicles under the above described experimental conditions. As documented, the addition of 100 µM GC in the presence of 20 µM E₂17G results in a synergistic increase in the total ATP-dependent and vanadate-sensitive vesicular substrate uptake, significantly exceeding an additive effect for these two compounds. These data, in combination with the respective cross-stimulation of the MRP2-dependent vesicular transport of GC and E₂17G (Figs. 6 and 7A), indicate a co-transport for these molecules by MRP2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The MRP subfamily of the ABC proteins contains several ATP-dependent active transporters, which have important physiological functions in various organs, predominantly in the liver (2–7, 13). In the present study we focused on the comparative investigation of the in vitro ATPase and transport properties of human MRP2 and MRP3, both key players in liver metabolite transport.

We expressed these proteins in Sf9 baculovirus expression system, because the heterologous expression in insect cells produces high and comparable expression levels of various human ABC proteins. These proteins are correctly folded and inserted into the membrane environment, although in an underglycosylated form, which has been shown not to alter the transport activity of many multidrug resistance proteins (33–37).

The present experiments demonstrated major differences in the transport properties and inhibitor sensitivities of human MRP2 and MRP3. As documented earlier (2, 3, 7, 10), MRP2 is an efficient transporter for glutathione conjugates, e.g. NEM-GS and LTC₄, whereas the transport of these compounds by MRP3 in the Sf9 vesicles was negligible (4, 5, 11, 38), and none of the MRP3 substrates examined here had any stimulatory effect in this regard (38). Membrane ATPase activity measurements revealed a similar substrate dependence; in the case of MRP2, vanadate-sensitive ATPase activity is significantly stimulated by GS conjugates (36), whereas we found no such stimulation in the case of MRP3 (38).

When we examined the vesicular uptake of a previously documented MRP2 and MRP3 substrate, E₂17G (5, 7, 13, 26–28), we found a well measurable transport of this compound by both of these human proteins. In these experiments MRP3 showed a significantly lower capacity but higher affinity for transporting E₂17G than MRP2. In the case of MRP3, the approximate K_m value for E₂17G transport was 25–30 µM, and the V_max was 1.3 nmol/mg membrane protein/min. In the case of MRP2, the K_m value could not be exactly determined, because the concentration dependence of E₂17G showed a sigmoidal curve (see below), and the maximum uptake rate could be reached only above 1 mM E₂17G concentration. However, the V_max in the case of MRP2 was about 10 times greater than for MRP3, reaching 12 nmol/mg membrane protein/min.

In the membrane ATPase measurements we found a similar phenomenon; in the MRP3-containing isolated membranes the ATPase activity was stimulated by relatively low concentrations (10–100 µM) of E₂17G, but this stimulation was much smaller than that found in the case of MRP2-containing membrane preparations. Moreover, E₂17G stimulation of MRP2 ATPase activity was continuously increasing up to 1 mM of E₂17G concentrations.

These data are in contrast to the high affinity E₂17G transport found in earlier studies (13, 26), using isolated membranes of cells expressing human or rat MRP2. However, our data correlate closely to the findings reported by Zelcer et al. (39) and may suggest that a relatively low MRP2 expression level in mammalian cell membranes and/or a complex modulation of MRP2 transport (see below) may have masked this phenomenon (for a more detailed discussion, see the accompanying article (39)).

In the following experiments we have studied the modulation of the E₂17G transport in MRP2- and MRP3-containing membranes by organic anions, which have been shown to exert significantly different effects on different MRPs. It has been reported earlier that furosemide, an anionic diuretic, has no major effect on MRP1 but strongly stimulates both MRP2 (36) and MRP3 ATPase activities (38). Probenecid, an inhibitor of MRP1, and indomethacin, a nonsteroid anti-inflammatory agent, were both found to stimulate MRP2 (36), and MRP3 ATPase activities (38). Benzbromarone, a strong inhibitor of MRP1 and MRP2, and MK571, a leukotriene receptor antagonist inhibitor of MRP1, also stimulated MRP3 ATPase activity in low concentrations (38).

When studying the direct modulatory effects of the above anionic compounds on E₂17G uptake in isolated MRP3-containing inside-out membrane vesicles, we found a relatively weak but consistent inhibition by furosemide and probenecid, whereas indomethacin exerted a slight stimulatory effect at low (10–20 µM) concentration and a significant inhibitory effect at higher concentrations (Fig. 4A).

In MRP2-containing membrane vesicles the effects of these organic anions on E₂17G uptake were entirely different; furosemide and probenecid induced an 1.5-fold activation, whereas indomethacin produced a 6-fold increase in E₂17G uptake (Fig. 4B). Thus a strong allosteric activation of the conjugate transport was observed in the case of MRP2. In the accompanying article, Zelcer et al. report major stimulatory effects of various organic anions on MRP2-dependent E₂17G uptake. In this study the most effective stimulating agent was sulfanitran, but a large variety of anionic agents had similar effects.

We found a major difference between the modulation of MRP3 and MRP2 transport activities by bile salts as well. Again, E₂17G uptake in isolated MRP3-containing inside-out vesicles was inhibited by all of the bile salts examined (GC, GCDC, TDC, and TCDC), whereas E₂17G uptake in MRP2-containing vesicles was significantly stimulated by all of these conjugated bile salts (GCDC being the most effective, reaching a 2.5-fold stimulation), in a physiologically relevant 10–100 µM concentration range (Fig. 5).

Because the E₂17G transport by MRP2 showed a sigmoidal substrate concentration dependence between 10 and 100 µM (Fig. 2B), we examined the possible allosteric modulation of this transport at these low E₂17G concentrations, by the addition of 100 µM indomethacin and GC, respectively. As shown in Fig. 6, in this concentration range a major stimulation was observed by both agents, although with somewhat different kinetics.

The demonstration of a different modulation of E₂17G transport by MRP2 and MRP3 and the allosteric effects of several compounds in the case of MRP2 prompted us to directly examine the modulation of labeled GC uptake in isolated membrane vesicles. We could demonstrate an active, concentration-dependent, saturable GC uptake by both MRP2 and MRP3, with similar V_max (100 and 250 pmol/mg membrane protein/min, respectively) and K_m (150–200 µM) values. However, although GC uptake by MRP3 was slightly inhibited by the addition of 100 µM E₂17G, in the case of MRP2 an about 2-fold activation of GC uptake was observed by 100 µM E₂17G (Fig. 7).

The relative contribution of MRP2 to the GC extrusion in hepatocytes is difficult to estimate, because the key bile acid transporter in the human liver is most probably BSEP (ABCB11) (19, 20). The recent work by Noe et al. (20) suggests that BSEP has a high capacity and a high affinity (in the 5–15 µmolar range) for various bile acids. Still, the co-stimulation of E₂17G and GC transport in MRP2 may allow this transporter to become a significant contributor to bile salt transport under certain metabolic conditions.

The well measurable cross-stimulation of active E₂17G and glycocholate transport in the case of MRP2 demonstrates a positive allosteric modulation and the presence of at least two substrate binding sites in this protein. This cross-stimulation suggests a co-transport activity of MRP2 for these compounds, reinforced by the finding that the combined transport rate for the glucuronide-conjugate E₂17G and the bile acid GC is significantly greater than that expected from an additive effect of these two substrates (Fig. 7B).

Our data indicate that under physiological conditions the secretion of bile salts and glucuronide conjugates by MRP2 is mutually facilitated by the accumulation of these compounds within the hepatocytes. Also, several anionic drugs, especially indomethacin, may greatly stimulate such a secretion process, yielding a better clearance for these cytotoxic metabolites. In contrast to MRP2, in the case of MRP3, only a cross-inhibition of the E₂17G and glycocholate transport could be observed, indicating a competition of these compounds on the transporter. This is not likely to be due to the absence of multiple binding sites in MRP3, as testified by the effects of some anionic compounds, which facilitate E₂17G transport by MRP3 (38). The inhibitory (competitive) interaction of metabolites found here may have an important physiological consequence in preventing a rapid secretion of conjugates and bile acids back into the bloodstream.

Collectively, our results indicate that despite the overlapping specificities of MRP2 and MRP3, the different transport capacities and affinities for their common substrates resulted in significant differences for their actual transport properties. Our data suggest that MRP2 plays an important role not only in glutathione and glucuronide conjugates but also in bile salt conjugate extrusion in the liver. Moreover, the different modulatory effects of transported substrates may result in a trans-cellular cross-talk, involving MRP2 and MRP3, which reside in opposite membrane compartments.

In the case of MRP2, a high V_max, a relatively high K_m (low affinity), and a cross-stimulation of the transported substrates with a simultaneous increase in substrate affinity assure an efficient, rapidly adaptable function of this protein. Increases of cellular conjugated metabolite levels are properly handled by this large capacity, low affinity pump system, actively extruding these metabolites into the bile. In contrast, in the case of MRP3, a low V_max, a low K_m (higher affinity), and a competitive cross-inhibition of substrates result in a relatively low level function of this protein under normal conditions, extruding only a limited amount of selected metabolites into venous blood. The different expression levels of these two transport proteins in the respective liver cell membranes may also contribute to the physiological direction of metabolite transport. However, under long term stress conditions, e.g. during cholestasis, MRP3-dependent conjugate export provides an important rescue mechanism for the hepatocytes, based on the relatively slow process of up-regulation of MRP3 expression (23–25).

The present results, describing the substrate interactions of MRP2 and MRP3, may facilitate the understanding of conjugated metabolite transport in hepatocytes. They may also support the design and application of new agents modulating the function of MRP2 and MRP3 in transport-dependent metabolic processes.

	FOOTNOTES

 
^* This work was supported by Grants T31952 [GenBank] , T35926 [GenBank] , and T38337 [GenBank] from OTKA and by funds from ETT (Hungary). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a Howard Hughes International Scholarship. To whom correspondence should be addressed: National Medical Center, Institute of Haematology and Immunology, 1113 Budapest, Diószegi u. 64, Hungary. Tel./Fax: 36-1-372-4353; E-mail: sarkadi{at}biomembrane.hu.

¹ The abbreviations used are: ABC, ATP-binding cassette; BSEP, bile salt export pump; E₂17G, estradiol-17--D-glucuronide; GC, glycocholate; GCDC, glycochenodeoxycholate; MRP, human multidrug resistance protein; TDC, taurodeoxycholate; TCDC, taurochenodeoxycholate; MOPS, 4-morpholinepropanesulfonic acid; IM, indomethacin.

	ACKNOWLEDGMENTS

 
The technical help by Katalin Petrovics, Judit Kis, and Ilona Zombori is gratefully acknowledged. We thank Prof. Piet Borst for the MRP2 and MRP3 cDNAs. We appreciate that Zelcer et al. (39) allowed us to read their paper and critically reviewed our manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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