title: Blood-brain barrier cell line PBMEC/C1-2 possesses functionally active P-glycoprotein.
abstract: The blood-brain barrier (BBB) maintains the homeostasis between the central nervous system and the blood circulation. One of the main efflux transporter proteins at the BBB is P-glycoprotein (P-gP) also known as ABCB1 or MDR1. Due to the important role of P-gP for the transport barrier function of the BBB, the presence and functionality of P-gP was investigated in porcine cell line PBMEC/C1-2. Presence of P-gP was confirmed on the protein level by western blotting and immunofluorescence microscopy as well as on the mRNA level by qPCR. Functional assessment was accomplished by an established 96-well uptake assay using Rhodamine 123 and Doxorubicin as P-gP substrates and Verapamil as moderate P-gP inhibitor. In this regard, fluorescence microscopy confirmed a significant higher uptake of Rhodamine 123 into PBMEC/C1-2 cells when preincubated with Verapamil. Finally, knock-down of P-gP by antisense oligonucleotides revealed an increase of Rhodamine 123 uptake indicating decreased P-gP functionality. In summary, the presence and functionality of P-gP in the immortalised cell line PBMEC/C1-2 was proven with several techniques and assays. Thus, this cell line could be used for P-gP studies in the context of BBB relevant issues.
authors: PharmaCon GmbH, Riglergasse 4/5, 1180 Vienna, Austria.
authors: Winfried
authors: W
authors: Neuhaus
authors: Martina
authors: M
authors: Stessl
authors: Elisabeth
authors: E
authors: Strizsik
authors: Barbara
authors: B
authors: Bennani-Baiti
authors: Michael
authors: M
authors: Wirth
authors: Stefan
authors: S
authors: Toegel
authors: Maaya
authors: M
authors: Modha
authors: Johannes
authors: J
authors: Winkler
authors: Franz
authors: F
authors: Gabor
authors: Helmut
authors: H
authors: Viernstein
authors: Christian R
authors: CR
authors: Noe
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects; D002460:Cell Line; D002491:Central Nervous System Agents / Q000494:pharmacology; D004317:Doxorubicin / Q000493:pharmacokinetics; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D055785:Gene Knockdown Techniques; D008856:Microscopy, Fluorescence; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D020112:Rhodamine 123 / Q000493:pharmacokinetics; D013552:Swine; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002491:Central Nervous System Agents; D005456:Fluorescent Dyes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D020112:Rhodamine 123; D004317:Doxorubicin; D014700:Verapamil