title: Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123.
abstract: P-glycoprotein (P-gp) expression at the blood-brain barrier prevents unwanted blood-borne toxins and signalling molecules from entering the brain. Primary and immortalised human brain endothelial cells (BECs) represent two suitable options for studying P-gp function in vitro. The limited supply of primary human BECs and their instability over passage number make this choice unattractive for medium/high throughput studies. The aim of this study was to further characterise the expression of P-gp by an immortalised human BEC line, hCMEC/D3, in order to evaluate their use as an in vitro human blood-brain barrier model. P-gp expression was stable over a high passage number (up to passage 38) and was polarised on the apical plasma membrane, consistent with human BECs in vivo. In addition, hCMEC/D3 cell P-gp expression was comparable, albeit slightly lower to that observed in primary isolated human BECs although P-gp function was similar in both cell lines. The P-gp inhibitors tariquidar and vinblastine prevented the efflux of rhodamine 123 (rh123) from hCMEC/D3 cells, indicative of functional P-gp expression. hCMEC/D3 cells also displayed polarised P-gp transport, since both tariquidar and vinblasine selectively increased the apical-to-basolateral permeability of hCMEC/D3 cells to rh123. The results presented here demonstrate that hCMEC/D3 cells are a suitable model to investigate substrate specificity of P-gp in BECs of human origin.
authors.affiliation: Department of Life Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.
authors.forename: Leon M
authors.initials: LM
authors.lastname: Tai
authors.forename: P Sreekanth
authors.initials: PS
authors.lastname: Reddy
authors.forename: M Alejandro
authors.initials: MA
authors.lastname: Lopez-Ramirez
authors.forename: Heather A
authors.initials: HA
authors.lastname: Davies
authors.forename: David K
authors.initials: DK
authors.lastname: Male
authors.forename: A David K
authors.initials: AD
authors.lastname: Male
authors.forename: A Jane
authors.initials: AJ
authors.lastname: Loughlin
authors.forename: Ignacio A
authors.initials: IA
authors.lastname: Romero
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D001812:Blood-Brain Barrier / Q000502:physiology; D001921:Brain / Q000187:drug effects / Q000502:physiology*; D002199:Capillary Permeability; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology*; D002478:Cells, Cultured; D002491:Central Nervous System Agents / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D006801:Humans; D008954:Models, Biological; D011804:Quinolines / Q000494:pharmacology; D020112:Rhodamine 123 / Q000493:pharmacokinetics*; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002491:Central Nervous System Agents; D005456:Fluorescent Dyes; D011804:Quinolines; D020112:Rhodamine 123; D014747:Vinblastine; C402343:tariquidar