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

doi:10.2133/dmpk.18.33

Drug Metabolism and Pharmacokinetics

Volume 18, Issue 1, 2003, Pages 33-41

https://doi.org/10.2133/dmpk.18.33 Get rights and content

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 (K_m) for these compounds in cryopreserved human hepatocytes was 2–8 and 3–18 μM, respectively. This was within the range of K_m 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 (V_max) (> 22 and > 22 times higher for TC and E₂17βG in rats than in humans, respectively) than that for K_m (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.

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Because of the loss of cellular polarity, ABC transporters expressed in the bile canaliculi (e.g., P-glycoprotein [P-gp] and multidrug resistance-associated protein 2 [MRP2]) are internalized in suspended rat hepatocytes [32], and the function is believed to be lost after isolation. To evaluate drug uptake in SHHs, human hepatocytes need to be separated from the buffer to quantify the compounds taken up by the human hepatocytes, for which the so-called oil-spin method was used [25]. The media-loss approach, in which drug disappearance from the buffer rather than uptake into hepatocytes is monitored, is also used for SHHs [33].
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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

^d: Current address: Millennium Pharmaceuticals Inc., Cambridge, MA, USA

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

Summary:

Chem.- Biol. Interact.

Chem. Biol. Interact

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Drug Discovery Today

Gastoroenterology

Biochem. Biopys. Res. Com.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Molecular aspects of hepatobiliary transport

Am. J. Physiol.

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

Pharm. Res.

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

Pharm. Res.

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

Chem. Pharm. Bull.

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.

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

Hepatology

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