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Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions

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Abstract

Confluent cell monolayers of brain capillary endothelial cells (BCEC) are used widely as an in vitro cell culture model of the blood–brain barrier. The present study describes the influence of cell-culture conditions on tight junctions, filamentous-actin cytoskeleton, and expression of ATP-binding cassette (ABC) transporters in primary cell cultures of porcine BCEC. Astrocyte as well as C6 glioma-conditioned cell culture medium was used in combination with retinoic acid, dexamethasone, cyclic adenosine monophosphate (cAMP) analogs, or 1,25-dihydroxyvitamin D3. It was shown that C6-conditioned medium led to a reorganization of filamentous actin and to an improved staining of zonula occludens-associated protein-1 (ZO-1). Further optimization of these culture conditions was achieved with cAMP analogs and dexamethasone. Retinoic acid, as well as 1,25-dihydroxyvitamin D3, did not improve cellular tight junctions as judged by filamentous actin, ZO-1 rearrangement, and transcellular electrical resistance (TER) measurements. However, these morphological changes did not influence the paracellular permeability of the extracellular marker sucrose. Expression of ABC transporters such as P-glycoprotein, multidrug resistance-associated protein-1( MRP1), and MRP2 were compared by measuring messenger RNA (mRNA) levels in whole-brain tissue, isolated brain capillaries, and cultured cells. In freshly isolated BCEC, mRNA levels of MRP2 and P-glycoprotein dropped by two- to sevenfold, respectively, whereas MRP1 mRNA levels were slightly increased. During cell culture, mRNA levels of MRP1 and MRP2 decreased by up to fivefold, while P-glycoprotein levels remained constant. These results were unaltered by different cell-culture conditions. In conclusion, the present study suggests that paracellular permeability, as well as mRNA expression of the studied ABC transporters in primary cultures, of porcine BCEC are insensitive toward changes in cell-culture conditions.

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Fig 1A, B.
Fig 2A-E.
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Abbreviations

ACEM:

Astrocyte-conditioned endothelial medium

BCEC:

Brain capillary endothelial cells

C6CEM:

C6-conditioned endothelial medium

TER:

Transcellular electrical resistance

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Acknowledgements

This work was supported by the Swiss National Science Foundation (grant 32-052918.97). We thank U. Behrens for excellent technical assistance. We would also like to thank S. Brill and Dr. P. Roberts for careful revision of this manuscript

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Authors and Affiliations

  1. Division of Clinical Pharmacology, University Hospital, Petersgraben 4, 4031 Basel, Switzerland

    Michael Török, Heike Gutmann & Jürgen Drewe

  2. Pharmaceuticals Division, F. Hoffmann-La Roche Ltd, Basel, Switzerland

    Jörg Huwyler

  3. Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany

    Gert Fricker

Authors
  1. Michael Török
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  2. Jörg Huwyler
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  3. Heike Gutmann
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  4. Gert Fricker
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  5. Jürgen Drewe
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Correspondence to Jürgen Drewe.

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Török, M., Huwyler, J., Gutmann, H. et al. Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions. Exp Brain Res 153, 356–365 (2003). https://doi.org/10.1007/s00221-003-1620-4

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