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Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1)

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Abstract

An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 μM and 4.4 μM, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammonium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK-OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates.

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References

  • Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM (1999) Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 39:361–398

    Article  PubMed  CAS  Google Scholar 

  • Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM (2003) P-glycoprotein: from genomics to mechanism. Oncogene 22:7468–7485

    Article  PubMed  CAS  Google Scholar 

  • Bronger H, König J, Kopplow K, Steiner HH, Ahmadi R, Herold-Mende C, Keppler D, Nies AT (2005) ABCC drug efflux pumps and organic anion uptake transporters in human gliomas and the blood-tumor barrier. Cancer Res 65:11419–11428

    Article  PubMed  CAS  Google Scholar 

  • Chen CM, Chang HC (1995) Determination of berberine in plasma, urine and bile by high-performance liquid chromatography. J Chromatogr B Biomed Appl 10:117–123

    Article  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Dantzig AH, Shepard RL, Cao J, Law KL, Ehlhardt WJ, Baughman TM, Bumol TF, Starling JJ (1996) Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979. Cancer Res 56:4171–4179

    PubMed  CAS  Google Scholar 

  • Dantzig AH, Shepard RL, Law KL, Tabas L, Pratt S, Gillespie JS, Binkley SN, Kuhfeld MT, Starling JJ, Wrighton SA (1999) Selectivity of the multidrug resistance modulator, LY335979, for P-glycoprotein and effect on cytochrome P-450 activities. J Pharmacol Exp Ther 290:854–862

    PubMed  CAS  Google Scholar 

  • den Dunnen JT, Antonarakis SE (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15:7–12

    Article  Google Scholar 

  • Fujita T, Urban TJ, Leabman MK, Fujita K, Giacomini KM (2006) Transport of drugs in the kidney by the human organic cation transporter, OCT2 and its genetic variants. J Pharm Sci 95:25–36

    Article  PubMed  CAS  Google Scholar 

  • Gorboulev V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U, Quester S, Baumann C, Lang F, Busch AE, Koepsell H (1997) Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol 16:871–881

    Article  PubMed  CAS  Google Scholar 

  • Gründemann D, Gorboulev V, Gambaryan S, Veyhl M, Koepsell H (1994) Drug excretion mediated by a new prototype of polyspecific transporter. Nature 372:549–552

    Article  PubMed  Google Scholar 

  • Gründemann D, Babin-Ebell J, Martel F, Ording N, Schmidt A, Schömig E (1997) Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells. J Biol Chem 272:10408–10413

    Article  PubMed  Google Scholar 

  • Gründemann D, Liebich G, Kiefer N, Köster S, Schömig E (1999) Selective substrates for non-neuronal monoamine transporters. Mol Pharmacol 56:1–10

    PubMed  Google Scholar 

  • Grycova L, Dostal J, Marek R (2007) Quaternary protoberberine alkaloids. Phytochemistry 68:150–175

    Article  PubMed  CAS  Google Scholar 

  • Hayer-Zillgen M, Brüss M, Bönisch H (2002) Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3. Br J Pharmacol 136:829–836

    Article  PubMed  CAS  Google Scholar 

  • Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H, Bruford EA (2004) The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Introduction. Pflügers Arch 447:465–468

    Article  PubMed  CAS  Google Scholar 

  • Iwunze MO (2000) Media influence on the enhancement of the fluorescence of berberine hydrochloride. Chemical Monthly 131:429–435

    Article  CAS  Google Scholar 

  • Kimura N, Masuda S, Tanihara Y, Ueo H, Okuda M, Katsura T, Inui K (2005) Metformin is a superior substrate for renal organic cation transporter OCT2 rather than hepatic OCT1. Drug Metab Pharmacokinet 20:379–386

    Article  PubMed  CAS  Google Scholar 

  • Koepsell H, Schmitt BM, Gorboulev V (2003) Organic cation transporters. Rev Physiol Biochem Pharmacol 150:36–90

    Article  PubMed  CAS  Google Scholar 

  • Koepsell H, Lips K, Volk C (2007) Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm Res 24:1227–1251

    Article  PubMed  CAS  Google Scholar 

  • König J, Cui Y, Nies AT, Keppler D (2000) A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol 278:G156–G164

    PubMed  Google Scholar 

  • Letschert K, Komatsu M, Hummel-Eisenbeiss J, Keppler D (2005) Vectorial transport of the peptide CCK-8 by double-transfected MDCKII cells stably expressing the organic anion transporter OATP1B3 (OATP8) and the export pump ABCC2. J Pharmacol Exp Ther 313:549–556

    Article  PubMed  CAS  Google Scholar 

  • Maeng HJ, Yoo HJ, Kim IW, Song IS, Chung SJ, Shim CK (2002) P-glycoprotein-mediated transport of berberine across Caco-2 cell monolayers. J Pharm Sci 91:2614–2621

    Article  PubMed  CAS  Google Scholar 

  • Meyer-Wentrup F, Karbach U, Gorboulev V, Arndt P, Koepsell H (1998) Membrane localization of the electrogenic cation transporter rOCT1 in rat liver. Biochem Biophys Res Commun 248:673–678

    Article  PubMed  CAS  Google Scholar 

  • Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, Inui K (2002) Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J Am Soc Nephrol 13:866–874

    PubMed  CAS  Google Scholar 

  • Nies AT, Keppler D (2007) The apical conjugate efflux pump ABCC2 (MRP2). Pflügers Arch 453:643–659

    Article  PubMed  CAS  Google Scholar 

  • Nies AT, König J, Cui Y, Brom M, Spring H, Keppler D (2002) Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2). Eur J Biochem 269:1866–1876

    Article  PubMed  CAS  Google Scholar 

  • Okuda M, Saito H, Urakami Y, Takano M, Inui K (1996) cDNA cloning and functional expression of a novel rat kidney organic cation transporter, OCT2. Biochem Biophys Res Commun 224:500–507

    Article  PubMed  CAS  Google Scholar 

  • Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, Moriyama Y (2005) A human transporter protein that mediates the final excretion step for toxic organic cations. Proc Natl Acad Sci USA 102:17923–17928

    Article  PubMed  CAS  Google Scholar 

  • Pan GY, Wang GJ, Liu XD, Fawcett JP, Xie YY (2002a) The involvement of P-glycoprotein in berberine absorption. Pharmacol Toxicol 91:193–197

    Article  PubMed  CAS  Google Scholar 

  • Pan JF, Yu C, Zhu DY, Zhang H, Zeng JF, Jiang SH, Ren JY (2002b) Identification of three sulfate-conjugated metabolites of berberine chloride in healthy volunteers’ urine after oral administration. Acta Pharmacol Sin 23:77–82

    PubMed  CAS  Google Scholar 

  • Ren J, Chaires JB (1999) Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry 38:16067–16075

    Article  PubMed  CAS  Google Scholar 

  • Sasaki M, Suzuki H, Ito K, Abe T, Sugiyama Y (2002) Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2). J Biol Chem 277:6497–6503

    Article  PubMed  CAS  Google Scholar 

  • Schaub TP, Kartenbeck J, König J, Spring H, Dörsam H, Stähler G, Störkel S, Thon WF, Keppler D (1999) Expression of the MRP2 gene-encoded conjugate export pump in human kidney proximal tubules and in renal-cell carcinoma. J Am Soc Nephrol 10:1159–1169

    PubMed  CAS  Google Scholar 

  • Schein FT, Hanna C (1960) The absorption, distribution and excretion of berberine. Arch Int Pharmacodyn Ther 124:317–325

    PubMed  CAS  Google Scholar 

  • Schömig E, Lazar A, Gründemann D (2006) Extraneuronal monoamine transporter and organic cation transporters 1 and 2: a review of transport efficiency. Handb Exp Pharmacol 151–180

  • Shepard RL, Cao J, Starling JJ, Dantzig AH (2003) Modulation of P-glycoprotein but not MRP1- or BCRP-mediated drug resistance by LY335979. Int J Cancer 103:121–125

    Article  PubMed  CAS  Google Scholar 

  • Shitan N, Tanaka M, Terai K, Ueda K, Yazaki K (2007) Human MDR1 and MRP1 recognize berberine as their transport substrate. Biosci Biotechnol Biochem 71:242–245

    Article  PubMed  CAS  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  PubMed  CAS  Google Scholar 

  • Suzuki M, Suzuki H, Sugimoto Y, Sugiyama Y (2003) ABCG2 transports sulfated conjugates of steroids and xenobiotics. J Biol Chem 278:22644–22649

    Article  PubMed  CAS  Google Scholar 

  • Takeda M, Khamdang S, Narikawa S, Kimura H, Kobayashi Y, Yamamoto T, Cha SH, Sekine T, Endou H (2002) Human organic anion transporters and human organic cation transporters mediate renal antiviral transport. J Pharmacol Exp Ther 300:918–924

    Article  PubMed  CAS  Google Scholar 

  • Tsai PL, Tsai TH (2004) Hepatobiliary excretion of berberine. Drug Metab Dispos 32:405–412

    Article  PubMed  CAS  Google Scholar 

  • Yonezawa A, Masuda S, Yokoo S, Katsura T, Inui KI (2006) Cisplatin and oxaliplatin, but not carboplatin and nedaplatin, are substrates for human organic cation transporters (SLC22A1-3 and MATE family). J Pharmacol Exp Ther 319:879–886

    Article  PubMed  CAS  Google Scholar 

  • Zhang L, Dresser MJ, Gray AT, Yost SC, Terashita S, Giacomini KM (1997) Cloning and functional expression of a human liver organic cation transporter. Mol Pharmacol 51:913–921

    PubMed  CAS  Google Scholar 

  • Zhang L, Schaner ME, Giacomini KM (1998) Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa). J Pharmacol Exp Ther 286:354–361

    PubMed  CAS  Google Scholar 

  • Zhang S, Lovejoy KS, Shima JE, Lagpacan LL, Shu Y, Lapuk A, Chen Y, Komori T, Gray JW, Chen X, Lippard SJ, Giacomini KM (2006) Organic cation transporters are determinants of oxaliplatin cytotoxicity. Cancer Res 66:8847–8857

    Article  PubMed  CAS  Google Scholar 

  • Zuo F, Nakamura N, Akao T, Hattori M (2006) Pharmacokinetics of berberine and its main metabolites in conventional and pseudo germ-free rats determined by liquid chromatography/ion trap mass spectrometry. Drug Metab Dispos 34:2064–2072

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We gratefully acknowledge Dr. J. König for his contributions to cloning efforts during the early phase of this work and Johanna Hummel-Eisenbeiss for excellent technical assistance during HPLC analyses.

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Correspondence to Anne T. Nies.

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This study was supported in part by the Deutsche Krebshilfe (Bonn, Germany).

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Nies, A.T., Herrmann, E., Brom, M. et al. Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1). Naunyn-Schmied Arch Pharmacol 376, 449–461 (2008). https://doi.org/10.1007/s00210-007-0219-x

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