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

Artigo Acesso aberto Revisado por pares

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

2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês

10.1074/jbc.m303515200

ISSN

1083-351X

Autores

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

Tópico(s)

Pregnancy and Medication Impact

Resumo

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-β-d-glucuronide (E217βG), 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 E217βG 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 E217βG transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E217βG, 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. 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-β-d-glucuronide (E217βG), 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 E217βG 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 E217βG transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E217βG, 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. The homologous multidrug resistance ABC 1The abbreviations used are: ABC, ATP-binding cassette; BSEP, bile salt export pump; E217βG, estradiol-17-β-d-glucuronide; GC, glycocholate; GCDC, glycochenodeoxycholate; MRP, human multidrug resistance protein; TDC, taurodeoxycholate; TCDC, taurochenodeoxycholate; MOPS, 4-morpholinepropanesulfonic acid; IM, indomethacin. 1The abbreviations used are: ABC, ATP-binding cassette; BSEP, bile salt export pump; E217βG, estradiol-17-β-d-glucuronide; GC, glycocholate; GCDC, glycochenodeoxycholate; MRP, human multidrug resistance protein; TDC, taurodeoxycholate; TCDC, taurochenodeoxycholate; MOPS, 4-morpholinepropanesulfonic acid; IM, indomethacin. transporter proteins MRP2 and MRP3 seem to be key players in the transport of organic anionic conjugated compounds in the liver and kidney (1Borst P. Kool M. Evers R. Semin. Cancer. Biol. 1997; 8: 3-8Google Scholar, 2Borst P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Google Scholar, 3Deeley R.G. Cole S.P.C. Semin. Cancer Biol. 1997; 8: 193-204Google Scholar, 4König J. Rost D. Cui Y. Keppler D. Hepatology. 1999; 29: 1156-1163Google Scholar, 5Hirohashi T. Suzuki H. Sugiyama Y. J. Biol. Chem. 1999; 274: 15181-15185Google Scholar, 6Keppler D. Leier I. Jedlitschky G. J. Biol. Chem. 1997; 378: 787-791Google Scholar, 7König J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar). 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 (2Borst P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Google Scholar, 3Deeley R.G. Cole S.P.C. Semin. Cancer Biol. 1997; 8: 193-204Google Scholar, 6Keppler D. Leier I. Jedlitschky G. J. Biol. Chem. 1997; 378: 787-791Google Scholar, 7König J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar, 8Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5136Google Scholar, 9Kool M. van der Linden M. de Haas M. Scheffer G.L. De Vree J.M. Smith A.J. Jansen G. Peters G.J. Ponne N. Scheper R.J. Elferink L.P. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6914-6919Google Scholar, 10van Aubel R.A. van Kuijck M.A. Koenderink J.B. Deen P.M. van Os C.H. Russel F.G. Mol. Pharmacol. 1999; 53: 1062-1067Google Scholar, 11Zeng H. Bain L.J. Belinsky M.J. Kruh G.D. Cancer Res. 1999; 59: 5964-5967Google Scholar, 12Kruh G. Zeng H. Rea P. Liu G. Chen Z. Lee K. Belinsky M. J. Bioenerg. Biomembr. 2001; 33: 493-501Google Scholar). 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 (1Borst P. Kool M. Evers R. Semin. Cancer. Biol. 1997; 8: 3-8Google Scholar, 2Borst P. Evers R. Kool M. Wijnholds J. Biochim. Biophys. Acta. 1999; 1461: 347-357Google Scholar, 3Deeley R.G. Cole S.P.C. Semin. Cancer Biol. 1997; 8: 193-204Google Scholar, 6Keppler D. Leier I. Jedlitschky G. J. Biol. Chem. 1997; 378: 787-791Google Scholar, 13Cui Y. König J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937Google Scholar). In contrast, MRP3 expression in polarized cells is restricted to the basolateral membrane (4König J. Rost D. Cui Y. Keppler D. Hepatology. 1999; 29: 1156-1163Google Scholar). 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 (14Kitamura T. Jansen P. Hardenbrook C. Kamimoto Y. Gatmaitan Z. Arias I.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3557-3561Google Scholar, 15Iyanagi T. Emi Y. Ikushiro S. Biochim. Biophys. Acta. 1998; 1407: 173-184Google Scholar), and there are known mutations/polymorphisms, reducing human MRP2 activity and leading to disorders of conjugate metabolism (16Kartenbeck J. Lenschner U. Mayer R. Keppler D. Hepatology. 1996; 23: 1061-1066Google Scholar, 17Paulusma C.C. Kool M. Bosma P.J. Scheffer G.L. ter Borg F. Scheper R.J. Tytgat G.N. Borst P. Baas F. Oude Elferink R.P. Hepatology. 1997; 25: 1539-1542Google Scholar, 18Kikuchi S. Hata M. Fukumoto K. Yamane Y. Matsui T. Tamura A. Yonemura S. Yamagishi H. Keppler D. Tsukita S. Nat. Genet. 2002; 31: 320-325Google Scholar). 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 (19Gerloff T. Stieger T. Hagenbuck B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. J. Biol. Chem. 1998; 273: 10046-10050Google Scholar, 20Noe J. Stieger B. Meier P.J. Gastroenterology. 2002; 123: 1733-1735Google Scholar). However, MRP2 (7König J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar, 13Cui Y. König J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937Google Scholar, 21Vore M. Lin Y. Huang L. Drug. Metab. Rev. 1997; 29: 183-203Google Scholar) and MRP3 (4König J. Rost D. Cui Y. Keppler D. Hepatology. 1999; 29: 1156-1163Google Scholar, 5Hirohashi T. Suzuki H. Sugiyama Y. J. Biol. Chem. 1999; 274: 15181-15185Google Scholar, 9Kool M. van der Linden M. de Haas M. Scheffer G.L. De Vree J.M. Smith A.J. Jansen G. Peters G.J. Ponne N. Scheper R.J. Elferink L.P. Baas F. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6914-6919Google Scholar) 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 (22Nies A.T. Konig J. Pfannschmidt M. Klar E. Hofmann W.J. Keppler D. Int. J. Cancer. 2001; 94: 492-499Google Scholar) and in Dubin-Johnson patients, when in the absence of a functional MRP2, MRP3 seems to have a compensatory transport function (4König J. Rost D. Cui Y. Keppler D. Hepatology. 1999; 29: 1156-1163Google Scholar, 7König J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar, 13Cui Y. König J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937Google Scholar). 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 (23Donner M.G. Keppler D. Hepatology. 2001; 34: 351-359Google Scholar, 24Hitzl M. Klein K. Zanger U.M. Fritz P. Nüssler A.K. Neuhaus P. Fromm M.F. Pharmacology. 2003; 304: 524-530Google Scholar, 25Scheffer G.L. Kool M. de Haas M. de Vree J.M. Pijnenborg A.C. Bosman D.K. Elferink R.P. van der Valk P. Borst P. Scheper R.J. Lab. Invest. 2002; 82: 193-201Google Scholar). 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-β-d-glucuronide and bile acids, as well as with some pharmacologically important organic anions. A major metabolite of human estrogen metabolism, estradiol-17-β-d-glucuronide (E217βG), has been shown to be transported by both MRP2 (7König J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar, 13Cui Y. König J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937Google Scholar, 26Ito K. Suzuki H. Sugiyama Y. Am. J. Gastroint. Liver Phys. 2001; 281: 1034-1043Google Scholar, 27Ito K. Oleschuk C.J. Westlake C. Vasa M.Z. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 38108-38114Google Scholar) and MRP3 (5Hirohashi T. Suzuki H. Sugiyama Y. J. Biol. Chem. 1999; 274: 15181-15185Google Scholar, 28Zelcer N. Saeki T. Reid G. Beijnen G.H. Borst P. J. Biol. Chem. 2001; 276: 46400-46407Google Scholar). This metabolite, with a significantly increased level during pregnancy and hormone replacement therapy, is secreted into the bile mainly by MRP2 (21Vore M. Lin Y. Huang L. Drug. Metab. Rev. 1997; 29: 183-203Google Scholar, 29Takikawa H. Yamazaki R. Sano M. Yamanaka M. Hepatology. 1996; 23: 607-613Google Scholar). Estrogen metabolites and other steroid glucuronides show hepatotoxic effects (30Slikker W.J.R. Vore M. Bailey J.R. Meyers M. Montgomery C. J. Pharmacol. Exp. Ther. 1983; 225: 138-143Google Scholar) and mutually protect against cholestasis (31Durham S. Vore M. J. Pharmacol. Exp. Ther. 1986; 237: 490-495Google Scholar). In pregnancy and in hormone replacement therapy, taurocholate decreases E217βG uptake in isolated rat hepatocytes (32Brock W.J. Vore M. Drug. Metab. Dispos. 1984; 12: 713-716Google Scholar), and certain MRP3 substrates induce MRP3 overexpression in cholestatic conditions (23Donner M.G. Keppler D. Hepatology. 2001; 34: 351-359Google Scholar, 24Hitzl M. Klein K. Zanger U.M. Fritz P. Nüssler A.K. Neuhaus P. Fromm M.F. Pharmacology. 2003; 304: 524-530Google Scholar, 25Scheffer G.L. Kool M. de Haas M. de Vree J.M. Pijnenborg A.C. Bosman D.K. Elferink R.P. van der Valk P. Borst P. Scheper R.J. Lab. Invest. 2002; 82: 193-201Google Scholar). 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. Materials—E217βG, glycocholate (GC), glycochenodeoxycholate (GCDC), taurodeoxycholate (TDC), and taurochenodeoxycholate (TCDC) were obtained from Sigma. Labeled [3H]E217βG 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. 33Bakos É. Hegedűs T. Holló Z.S. Welker E. Tusnády G.E. Zaman G.J. Flens M.J. Váradi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Google Scholar and 34Bakos É. Evers R. Szakács G. Tusnády G.E. Welker E. Szabó K. de Haas M. van Deemter L. Borst P. Váradi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Google Scholar. Sf9 (Spodoptera frugiperda) cells were cultured and infected with a baculovirus as described in Ref. 35Müller M. Bakos É. Welker E. Váradi A. Germann U.A. Gottesman M.M. Morse B.S. Roninson I.B. Sarkadi B. J. Biol. Chem. 1996; 271: 1877-1883Google Scholar. MRP2 and MRP3 cDNAs were obtained from Prof. Piet Borst and inserted into a baculovirus vector as described in Ref. 36Bakos É. Evers R. Sinkó E. Váradi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar. 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. 37Sarkadi B. Price E.M. Boucher R.C. Germann U.A. Scarborough G.A. J. Biol. Chem. 1992; 267: 4854-4858Google Scholar. Gel electrophoresis and immunoblot detection were performed, and protein-antibody interaction was determined using the enhanced chemiluminescence technique as described in Ref. 36Bakos É. Evers R. Sinkó E. Váradi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar. Membrane ATPase Measurements—ATPase activity was measured basically as described in Ref. 37Sarkadi B. Price E.M. Boucher R.C. Germann U.A. Scarborough G.A. J. Biol. Chem. 1992; 267: 4854-4858Google Scholar, 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 (34Bakos É. Evers R. Szakács G. Tusnády G.E. Welker E. Szabó K. de Haas M. van Deemter L. Borst P. Váradi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Google Scholar). 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. 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). 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 (10van Aubel R.A. van Kuijck M.A. Koenderink J.B. Deen P.M. van Os C.H. Russel F.G. Mol. Pharmacol. 1999; 53: 1062-1067Google Scholar, 33Bakos É. Hegedűs T. Holló Z.S. Welker E. Tusnády G.E. Zaman G.J. Flens M.J. Váradi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Google Scholar, 34Bakos É. Evers R. Szakács G. Tusnády G.E. Welker E. Szabó K. de Haas M. van Deemter L. Borst P. Váradi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Google Scholar, 35Müller M. Bakos É. Welker E. Váradi A. Germann U.A. Gottesman M.M. Morse B.S. Roninson I.B. Sarkadi B. J. Biol. Chem. 1996; 271: 1877-1883Google Scholar, 36Bakos É. Evers R. Sinkó E. Váradi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar, 37Sarkadi B. Price E.M. Boucher R.C. Germann U.A. Scarborough G.A. J. Biol. Chem. 1992; 267: 4854-4858Google Scholar). In the following experiments we used isolated membranes, forming inside-out membrane vesicles from these human MRP-expressing Sf9 cells. Vesicular Transport of E217βG by Human MRPs—In the following experiments we studied the transport of E217βG, 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 (5Hirohashi T. Suzuki H. Sugiyama Y. J. Biol. Chem. 1999; 274: 15181-15185Google Scholar, 7König J. Nies A.T. Cui Y. Leier I. Keppler D. Biochim. Biophys. Acta. 1999; 1461: 377-394Google Scholar, 13Cui Y. König J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937Google Scholar, 26Ito K. Suzuki H. Sugiyama Y. Am. J. Gastroint. Liver Phys. 2001; 281: 1034-1043Google Scholar, 27Ito K. Oleschuk C.J. Westlake C. Vasa M.Z. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2001; 276: 38108-38114Google Scholar, 28Zelcer N. Saeki T. Reid G. Beijnen G.H. Borst P. J. Biol. Chem. 2001; 276: 46400-46407Google Scholar). By using isolated, inverted Sf9 membrane vesicles, we have directly examined the vesicular transport of E217βG 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 E217βG 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 β-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 E217βG), and this period was used for studying the concentration dependence of the uptake. As documented in Fig. 2A, MgATP-energized E217βG uptake in human MRP3-containing membrane vesicles was a saturable function of the E217βG concentration, with a calculated maximum uptake rate of about 1.3 nmol/mg membrane protein/min, and an apparent Km value of about 25–30 μm. When we measured E217βG uptake in Sf9 membrane vesicles containing comparable amounts of human MRP1, the concentration dependence of E217βG uptake gave a Km 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 (38Bodó A. Bakos E.́. Szeri F. Váradi A. Sarkadi B. Toxicology Let. 2003; 140–141C: 133-143Google Scholar). 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 E217βG uptake. The rate of E217βG uptake at low E217βG concentrations (see inset in Fig. 2B) had an S-shaped curve, and saturation of the E217βG uptake was only achieved above 1 mm E217βG concentrations. Moreover, the maximum uptake rate of E217βG 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 E217βG, 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 E217βG. Effect of E217βG 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 (33Bakos É. Hegedűs T. Holló Z.S. Welker E. Tusnády G.E. Zaman G.J. Flens M.J. Váradi A. Sarkadi B. J. Biol. Chem. 1996; 271: 12322-12326Google Scholar, 34Bakos É. Evers R. Szakács G. Tusnády G.E. Welker E. Szabó K. de Haas M. van Deemter L. Borst P. Váradi A. Sarkadi B. J. Biol. Chem. 1998; 273: 32167-32175Google Scholar, 35Müller M. Bakos É. Welker E. Váradi A. Germann U.A. Gottesman M.M. Morse B.S. Roninson I.B. Sarkadi B. J. Biol. Chem. 1996; 271: 1877-1883Google Scholar, 36Bakos É. Evers R. Sinkó E. Váradi A. Borst P. Sarkadi B. Mol. Pharmacol. 2000; 57: 760-768Google Scholar, 37Sarkadi B. Price E.M. Boucher R.C. Germann U.A. Scarborough G.A. J. Biol. Chem. 1992; 267: 4854-4858Google Scholar), 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 E217βG 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 E217βG concentrations between 20 and 100 μm. In the case of MRP1 only a moderate increase in the ATPase activity was seen (38Bodó A. Bakos E.́. Szeri F. Váradi A. Sarkadi B. Toxicology Let. 2003; 140–141C: 133-143Google Scholar), as compared with that in β-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 E217βG concentrations above 100 μm. This ATPase activity increased up to 8–10 nmol/mg membrane protein/min at about 0.5 mm E217βG and did not reach a maximum level even at 1 mm of E217βG concentration (higher concentrations could not be properly applied under the present experimental conditions). It is important to note that the E217βG 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 E217βG transport experiments, suggesting that MRP3 (and MRP1) is a higher affinity but a much lower capacity transporter for E217βG than MRP2. Modulation of the MRP3- and MRP2-dependent Vesicular Transport of E217βG 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 E217βG in MRP3-containing (Fig. 4A) and MRP2-containing (Fig. 4B) membranes, respectively. These experiments were carried out at two fixed E217βG concentrations (1 and 13 μm) for both MRP3 and MRP2, to study these modulatory effects at E217βG concentrations below the respective Km values. We expected that both the inhibitory or the possible allosteric stimulatory effects could be optimally studied under these conditions. We found that in the case of MRP3, E217βG transport was inhibited by all of the three organic anions. The approximate Ki 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 E217βG uptake was observed by low concentrations (5–10 μm) of indomethacin. Fig. 4A shows E217βG uptake data measured at 1 μm E217βG concentration, but similar results were obtained at higher (13 μm) E217βG concentrations as well. We have already described (38Bodó A. Bakos E.́. Szeri F. Váradi A. Sarkadi B. Toxicology Let. 2003; 140–141C: 133-143Google Scholar) 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 E217βG 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 E217βG 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 E217βG 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 E217βG concentration, but similar results were obtained at lower (1 μm) E217βG concentrations as well. In the following experiments we have studied the effect various bile salt conjugates on the vesicular uptake of labeled E217βG 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 E217βG concentrations (1 and 13 μm) for both MRP3 and MRP2. As shown in Fig. 5A, in the case of MRP3, E217βG transport (measured at 1 μm E217βG) 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 E217βG already at 10 μm concentrations. When E217βG uptake was measured at higher (13 μm) E217βG 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 E217βG uptake (measured here at 1 μm E217βG). 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 E217βG uptake, carried out by MRP2, is allosterically modulated by various organic anions and bile salt conjugates. To better characterize these interactions, we performed detailed E217βG 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 E217βG concentration range (10–100 μm), in which an S-shaped concentration dependence of E217βG uptake was observed (F

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