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Function of Uptake Transporters for Taurocholate and Estradiol 17β-D-Glucuronide in Cryopreserved Human Hepatocytes

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Summary:

The uptake properties of taurocholate (TC) and estradiol 17β-D-glucuronide (E217βG) were examined in freshly isolated and cryopreserved human hepatocytes to discover if active transport is retained in cryopreserved human hepatocytes. Firstly, the uptake of TC and E217βG was measured before and after cryopreservation. The uptake of TC was found to be Na+-dependent both in fresh and cryopreserved hepatocytes. The uptake activity in cryopreserved hepatocytes was found to range from 10 to 200% of that observed in freshly isolated cells. A kinetic analysis was performed to evaluate the transport activity of TC and E217βG and revealed that the Michaelis constant (Km) for these compounds in cryopreserved human hepatocytes was 2–8 and 3–18 μM, respectively. This was within the range of Km values previously found in human Na+-taurocholate cotransporting polypeptides (NTCP) and organic anion transporting polypeptides (OATP) 2 and 8, respectively. The kinetic analyses also showed that the species difference between human and rat hepatocytes was more marked for the maximal uptake rate (Vmax) (> 22 and > 22 times higher for TC and E217βG in rats than in humans, respectively) than that for Km (2–12 and 0.7–4 times higher, respectively), compared with earlier data we obtained in primary cultured rat hepatocytes. Hence, we conclude that cryopreserved human hepatocytes, at least in part, retain their transporter functions and, therefore, can be a useful experimental system for examining the mechanism of the hepatic uptake of drugs.

References (45)

  • B. Hsiang et al.

    A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liver-specific human organic anion transporting polypeptide and identification of rat and human hydroxymethylglutaryl-CoA reductase inhibitor transporters

    J. Biol. Chem.

    (1999)
  • J. König et al.

    Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide

    J. Biol. Chem.

    (2000)
  • J.E. Race et al.

    Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3)

    Biochem. Biophys. Res. Commun.

    (1999)
  • S.H. Cha et al.

    Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta

    J. Biol. Chem.

    (2000)
  • G.W. Sandker et al.

    Characterization of transport in isolated human hepatocytes: A study with the bile acid taurocholic acid, the unchanged ouabain and the organic cations vecuronium and rocuronium

    Biochem. Pharmacol.

    (1994)
  • R.G. Tirona et al.

    Polymorphisms in OATP-C. Identification of multiple allelic variants associated with altered transport activity among European- and American-Americans

    J. Biol. Chem.

    (2001)
  • Μ. Müller et al.

    Molecular aspects of hepatobiliary transport

    Am. J. Physiol.

    (1997)
  • Μ. Yamazaki et al.

    Recent advances in carrier-mediated hepatic uptake and biliary excretion of xenobiotics

    Pharm. Res.

    (1996)
  • Μ. Yamazaki et al.

    Uptake is the rate-limiting step in the overall hepatic elimination of pravastatin at steady state in rats

    Pharm. Res.

    (1996)
  • S. Miyauchi et al.

    Conjuigative metabolism of 4-methylumbelliferone in the rat liver. Verification of the sequestration process in the multiple indicator dilution experiments

    Chem. Pharm. Bull.

    (1987)
  • S. Miyauchi et al.

    Dose-dependent hepatic handling of 1-propranolol determined by multiple indicator dilute method: Influence of tissue binding of 1-propranolol on its hepatic elimination

    Biol. Pharm. Bull.

    (1993)
  • M. Inoue et al.

    Taurocholate transport by rat liver sinusoidal membrane vesicles: Evidence of sodium co-transport

    Hepatology

    (1982)
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    a

    Current address: Faculty of Pharmaceutical Sciences, Showa University, Tokyo, Japan

    b

    Current address: Phase-1 Molecular Toxicology, Inc., Santa Fe, NM, USA

    c

    Current address: Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan

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