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Research ArticleArticle

Contribution of Organic Anion Transporting Polypeptide to Uptake of Its Possible Substrates into Rat Hepatocytes

Hirokazu Kouzuki, Hiroshi Suzuki, Kousei Ito, Rui Ohashi and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics February 1999, 288 (2) 627-634;
Hirokazu Kouzuki
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Hiroshi Suzuki
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Kousei Ito
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Rui Ohashi
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Yuichi Sugiyama
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Abstract

Organic anion transporting polypeptide (oatp1) has been cloned from rat liver as one of the transporters responsible for the hepatic uptake of ligands, and its substrate specificity has been determined. However, the contribution of oatp1 to the Na+-independent uptake of ligands into rat hepatocytes remains to be investigated. In the present study, we determined the contribution of oatp1 and examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells transiently expressing oatp1 and normalized using estradiol-17β-d-glucuronide as a reference compound. Western blot analysis indicated that oatp1 was less extensively glycosylated in transfected COS-7 cells, and the expression level in transfectant was one-seventh that in rat liver. TheKm values for the uptake of estradiol-17β-d-glucuronide were similar for cultured hepatocytes and oatp1-transfected COS-7 cells (Km = 12.3 versus 20.4 μM), although theVmax value for oatp1-transfected COS-7 cells was one-seventh that for cultured hepatocytes (Vmax = 1.30 versus 0.175 nmol/min/mg protein). The contribution of oatp1 to the Na+-independent uptake of taurocholic acid and cholic acid into rat hepatocytes was more than 50 to 60%, whereas the corresponding values for the sulfate-conjugates of estrone and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole were 20 to 30%. In addition, the analysis indicated that the contribution of oatp1 to the Na+-independent uptake of several ligands [glucuronide-conjugate of 6-hydroxy-5,7-dimethyl2-methylamino-4-(3-pyridylmethyl)benzothiazole, ibuprofen, pravastatin, ouabain, and 2,4-dinitrophenyl-S-glutathione] was minimal. Collectively, the transfected COS-7 cells may be used to quantitatively predict oatp1 activity in hepatocytes after correction of its expressed amount. It is also suggested that multiple transport mechanisms are responsible for the Na+-independent uptake of organic anions into hepatocytes.

Footnotes

  • Send reprint requests to: Yuichi Sugiyama, Ph.D., Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

  • ↵1 This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, and the Core Research for Evolutional Sciences and Technology of Japan Sciences and Technology Corporation.

  • Abbreviations:
    Ntcp
    sodium taurocholate cotransporting polypeptide
    oatp
    organic anion transporting polypeptide
    E217βG
    estradiol-17βd-glucuronide
    TC
    taurocholate, taurocholic acid
    CA
    cholate, cholic acid
    DNP-SG
    2,4-dinitrophenyl-S-glutathione
    E3040
    6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole
    BSP
    bromosulfophthalein
    DBSP
    dibromosulfophthalein
    DMEM
    Dulbecco’s modified Eagle’s medium
    SSC
    saline sodium citrate
    SDS
    sodium dodecyl sulfate
    BSA
    bovine serum albumin
    TBS-T
    Tris-buffered saline containing 0.05% Tween 20
    SD
    Sprague-Dawley
    Km
    Michaelis constant
    Vmax
    maximum transport velocity
    CLuptake
    uptake clearance
    • Received March 11, 1998.
    • Accepted August 31, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 288 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 288, Issue 2
1 Feb 1999
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Research ArticleArticle

Contribution of Organic Anion Transporting Polypeptide to Uptake of Its Possible Substrates into Rat Hepatocytes

Hirokazu Kouzuki, Hiroshi Suzuki, Kousei Ito, Rui Ohashi and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics February 1, 1999, 288 (2) 627-634;

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Research ArticleArticle

Contribution of Organic Anion Transporting Polypeptide to Uptake of Its Possible Substrates into Rat Hepatocytes

Hirokazu Kouzuki, Hiroshi Suzuki, Kousei Ito, Rui Ohashi and Yuichi Sugiyama
Journal of Pharmacology and Experimental Therapeutics February 1, 1999, 288 (2) 627-634;
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