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.