Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions.
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.
Török
Michael
M
Division of Clinical Pharmacology, University Hospital, Petersgraben 4, 4031 Basel, Switzerland.
Huwyler
Jörg
J
Gutmann
Heike
H
Fricker
Gert
G
Drewe
Jürgen
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000098:blood supply*; D018929:Cell Culture Techniques / Q000379:methods*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D026901:Membrane Transport Proteins; D008954:Models, Biological; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D034421:Sus scrofa; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D017077:Culture Media, Conditioned; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D062826:Zonula Occludens-1 Protein; C421109:multidrug resistance-associated protein 1