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Partial Maintenance of Taurocholate Uptake by Adult Rat Hepatocytes Cultured in a Collagen Sandwich Configuration

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Abstract

Purpose. This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions.

Methods. Hepatocytes isolated from male Wistar rats (230−280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein, Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5.

Results. By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was ∼13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 μM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (32−41 μM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were ∼5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film.

Conclusions. A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

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Authors and Affiliations

  1. Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599

    Xingrong Liu, Kim L. R. Brouwer & Edward L. LeCluyse

  2. Division of Bioanalysis and Drug Metabolism, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina, 27709

    Liang-Sheng L. Gan & Kenneth R. Brouwer

  3. Division of Clinical Pharmacology, Department of Medicine, University Hospital, CH-8091, Zurich, Switzerland

    Bruno Stieger & Peter J. Meier

  4. Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, 66047

    Kenneth L. Audus

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  1. Xingrong Liu
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  2. Kim L. R. Brouwer
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  3. Liang-Sheng L. Gan
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  4. Kenneth R. Brouwer
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  5. Bruno Stieger
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  6. Peter J. Meier
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  7. Kenneth L. Audus
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  8. Edward L. LeCluyse
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Liu, X., Brouwer, K.L.R., Gan, LS.L. et al. Partial Maintenance of Taurocholate Uptake by Adult Rat Hepatocytes Cultured in a Collagen Sandwich Configuration. Pharm Res 15, 1533–1539 (1998). https://doi.org/10.1023/A:1011994831139

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  • DOI: https://doi.org/10.1023/A:1011994831139

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