title: Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.
abstract: P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.
authors: Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany.
authors: Andreas
authors: A
authors: Noack
authors: Department of Trauma Surgery, Hannover Medical School, Hannover, Germany.
authors: Sandra
authors: S
authors: Noack
authors: Department of Trauma Surgery, Hannover Medical School, Hannover, Germany.
authors: Andrea
authors: A
authors: Hoffmann
authors: Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
authors: Katia
authors: K
authors: Maalouf
authors: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany.
authors: Manuela
authors: M
authors: Buettner
authors: INSERM, U1016, Institut Cochin, Paris, France ; Centre National de la Recherche Scientifique (CNRS), UMR8104, Paris, France ; Université René Descartes, Paris, France.
authors: Pierre-Olivier
authors: PO
authors: Couraud
authors: Department of Biological Sciences, The Open University, Milton Keynes, United Kingdom.
authors: Ignacio A
authors: IA
authors: Romero
authors: Weill Medical College of Cornell University, New York, New York, United States of America.
authors: Babette
authors: B
authors: Weksler
authors: Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany.
authors: Dana
authors: D
authors: Alms
authors: Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany.
authors: Kerstin
authors: K
authors: Römermann
authors: Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
authors: Hassan Y
authors: HY
authors: Naim
authors: Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany ; Center for Systems Neuroscience, Hannover, Germany.
authors: Wolfgang
authors: W
authors: Löscher
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism; D001921:Brain / Q000187:drug effects* / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects*; D020112:Rhodamine 123
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020112:Rhodamine 123; D016685:Mitomycin; D002784:Cholesterol