Characterization of 5(6)-Carboxy-2,′7′-Dichlorofluorescein Transport by MRP2 and Utilization of this Substrate as a Fluorescent Surrogate for LTC4

Abstract

MRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug—endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties. (Journal of Biomolecular Screening 2008:295-301)

Keywords

MRP2 vesicular transport fluorescence LTC4

References

Glavinas H. , Krajcsi P. , Cserepes J. , Sarkadi B. : The role of ABC transporter in drug resistance, metabolism and toxicity . Curr Drug Deliv 2004;1:27-42.

Ambudkar SV , Kim IW , Sauna ZE : The power of the pump: mechanisms of action of P-glycoprotein (ABCB1) . Eur J Pharm Sci 2006;27: 392-400.

Locher K. : Structure and mechanism of ABC transporters. Curr Opin Struct Biol 2004;14:426-431.

Keppler D. , Konig J. : Hepatic canalicular membrane 5: expression and localization of the conjugate export pump encoded by the MRP2 (cMRP/cMOAT) gene in liver. FASEB J 1997;11:509-516.

Buchler M. , Konig J. , Brom M. , Kartenbeck J. , Spring H. , Horie T. , et al: cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J Biol Chem 1996;271:15091-15098.

Mottino AD , Hoffman T. , Jennes L. , Vore M. : Expression and localization of multidrug resistant protein mrp2 in rat small intestine. J Pharmacol Exp Ther 2000;293:717-723.

Van Aubel RA , Masereeuw R. , Russel FG : Molecular pharmacology of renal organic anion transporters. Am J Physiol Renal Physiol 2000 ;279: F216-F232.

Jedlitschky G. , Leier I. , Buchholz U. , Barnouin G. , Keppler D. : Transport of glutathione, glucuronide and sulfate conjugates by the MRP gene-encoded conjugate export pump. Cancer Res 1996;56:988-994.

Dubin IN , Johnson FB : Chronic idiopathic jaundice with unidentified pigment in liver cells: a new clinicopathologic entity with report of 12 cases. Medicine 1954;33:155-179.

10.

Kartenbeck J. , Leuschner U. , Mayer R. , Keppler D. : Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology 1996;23:1061-1066.

11.

Nezasa K. , Tian X. , Zamek-Gliszczynski MJ , Patel NJ , Raub TJ , Brouwer KL : Altered hepatobiliary disposition of 5 (and 6)-carboxy-2,′7′dichlorofluorescein in Abcg2 (Bcrp1) and Abcc2 (Mrp2) knockout mice. Drug Metab Dispos 2006;34:718-723.

12.

Donner MG , Keppler D. : Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 2001;34:351-359.

13.

Nies AT , König J. , Pfannschmidt M. , Klar E. , Hofmann WJ , Keppler D. : Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma. Int J Cancer 2001 ;94:492-499.

14.

Lengyel G. , Veres Z. , Tugyi R. , Vereczkey L. , Molnár T. , Glavinas H. , et al: Modulation of sinusoidal and canalicular elimination of bilirubinglucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes . Hepatology Res 2008;38:300-309.

15.

Bodo A. , Bakos E. , Szeri F. , Varadi A. , Sarkadi B. : Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions. J Biol Chem 2003;278:23529-23537.

16.

Cui Y. , Konig J. , Keppler D. : Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2. Mol Pharmacol 2001;60:934-943.

17.

Ito K. , Suzuki H. , Sugiyama Y. : Charged amino acids in the transmembrane domains are involved in the determination of the substrate specificity of rat Mrp2. Mol Pharmacol 2001;59:1077-1085.

18.

Ninomiya M. , Ito K. , Hiramatsu R. , Horie T. : Functional analysis of mouse and monkey multidrug resistance-associated protein 2 (Mrp2). Drug Metab Dispos 2006;34:2056-2063.

19.

El-Sheikh AA , van den Heuvel JJ , Koenderink JB , Russel FG : Interaction of nonsteroidal anti-inflammatory drugs with multidrug resistance protein (MRP) 2/ABCC2- and MRP4/ABCC4-mediated methotrexate transport. J Pharmacol Exp Ther 2007;320:229-235.

20.

Chen ZS , Kawabe T. , Ono M. , Aoki S. , Sumizawa T. , Furukawa T. , et al: Effect of multidrug resistance-reversing agents on transporting activity of human canalicular multispecific organic anion transporter. Mol Pharmacol 1999;56:1219-1228.

21.

Gao M. , Yamazaki M. , Loe DW , Westlake CJ , Grant CE , Cole SP , et al: Multidrug resistance protein: identification of regions required for active transport of leukotriene C4. J Biol Chem 1998;273:10733-10740.

22.

Zelcer N. , Huisman MT , Reid G. , Wielinga P. , Breedveld P. , Kuil A. , et al: Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J Biol Chem 2003;278: 23538-23544.

23.

Huber M. , Guhlmann A. , Jansen PLM , Keppler D. : Hereditary defect of hepatobiliary cysteinyl leukotriene elimination in mutant rats with defective hepatic anion excretion. Hepatology 1997;7:224-228.

24.

Zamek-Glyszczyinsky MJ , Xiong H. , Patel NJ , Turncliff RZ , Pollack GM , Brouwer KL : Pharmacokinetics of 5 (and 6)-Carboxy-2,′7′dichlorofluorescein and its diacetate promoiety in the liver. J Pharm Exp Ther 2003;304:801-809.

25.

Pratt S. , Chen V. , Perry WI III , Starling JJ , Dantzig AH : Kinetic validation of the use of carboxydichlorofluorescein as a drug surrogate for MRP5-mediated transport. Eur J Pharm Sci 2006;27:524-532.

26.

Xiong H. , Suzuki H. , Sugiyama Y. , Meier PJ , Pollack GM , Brouwer KL : Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment. Drug Metab Dispos 2002;30:962-969.

27.

Bakos E. , Evers R. , Sinko E. , Varadi A. , Borst P. , Sarkadi B. : Interaction of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol 2000;57:760-768.

28.

Szentpetery Z. , Kern A. , Liliom K. , Sarkadi B. , Varadi A. , Bakos E. : The role of the conserved glycerin of ATP-binding cassette signature motifs of MRP1 in the communication between the substrate-binding site and the catalytic centers . J Biol Chem 2004;279:41670-41678.

29.

Muller M. , Bakos E. , Welker E. , Varadi A. , Germann UA , Gottesman MM , et al: Altered drug-simulated ATPase activity in mutants of the human multidrug resistance protein. J Biol Chem 1996;271:1877-1883.

30.

Sarkadi B. , Price EM , Boucher RC , Germann UA , Scarborough GA : Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J Biol Chem 1992;267:4854-4858.

31.

Cheng Y. , Prusoff WH : Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction . Biochem Pharmacol 1973;22:3099-3108.

32.

Akao T. , Hanada M. , Sakashita Y. , Sato K. , Morita M. , Imanaka T. : Efflux of baicalin, a flavone glucuronide of Scutellariae Radix, on Caco-2 cells through multidrug resistance-associated protein 2. J Pharm Pharmacol 2007;59:87-93.

33.

Horikawa M. , Kato Y. , Tyson CA , Sugiyama Y. : The potential for an interaction between MRP2 (ABCC2) and various therapeutic agents: probenecid as a candidate inhibitor of the biliary excretion of irinotecan metabolites . Drug Metab Pharmacokinet 2002;17:23-33.

34.

Payen L. , Delugin L. , Courtois A. , Trinquart Y. , Guillouzo A. , Fardel O. : The sulphonylurea glibenclamide inhibits multidrug resistance protein (MRP1) activity in human lung cancer cells. Br J Pharmacol 2001;132: 778-784.

35.

Leier I. , Hummel-Eisenbeiss J. , Cui Y. , Keppler D. : ATP-dependent paraaminohippurate transport by apical multidrug resistance protein MRP2. Kidney Int 2000;57:1636-1642.

36.

Mayer R. , Kartenbeck J. , Buchler M. , Jeditschky G. , Leier I. , Keppler D. : Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes. J Cell Biol 1995;131:137-150.

37.

Ishikawa T. , Muller M. , Klunemann C. , Schaub T. , Keppler D. : ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. role of the ATP-dependent transport system for glutathione S-conjugates . J Biol Chem 1990;265:19279-19286.

38.

Mayatepek E. , Lehmann WD : Defective hepatobiliary leukotriene elimination in patients with the Dubin-Johnson syndrome. Clin Chim Acta 1996;249:37-46.

39.

Cui Y. , Konig J. , Buchholz U. , Spring H. , Leier I. , Keppler D. : Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol 1999:55:929-937.