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.
Highlights
The transmembrane drug efflux transporter P-glycoprotein (Pgp; MDR1; ABCB1) contributes to the disposition of a wide variety of drugs of different therapeutic categories due to its extensive tissue distribution and broad substrate specificity [1,2]
The successful infection was detected by EGFP fluorescence and Western blot analysis of the Pgp-EGFP fusion protein
Functional analysis of Pgp trafficking by the calcein AM extrusion assay In the calcein AM extrusion assay, we studied the effect of mitomycin C (MMC) (1 mM) following 20 h of 4 h exposure with MMC (Figure 2D)
Summary
The transmembrane drug efflux transporter P-glycoprotein (Pgp; MDR1; ABCB1) contributes to the disposition of a wide variety of drugs of different therapeutic categories due to its extensive tissue distribution and broad substrate specificity [1,2]. Multiple extracellular and intracellular signals regulate the expression and functionality of Pgp, including transcriptional modulation via nuclear receptors, like the pregnane-X receptor, which are involved in drug-induced changes in Pgp expression [4,5]. Modulation of trafficking of Pgp from intracellular reservoirs to the cell surface alters post-transcriptional Pgp expression, and may be an effective and rapid way of the cell to respond to potentially toxic compounds by functional membrane insertion of the efflux transporter [7]. Intracellular trafficking of Pgp was investigated in a human brain capillary endothelial cell line (hCMEC/D3)[13], using a Pgp and enhanced green fluorescent fusion protein (Pgp-EGFP) inducible by doxycycline. To study drug-induced trafficking of Pgp, we used the chemotherapeutic agent mitomycin C (MMC), which has previously been shown to increase membrane-associated Pgp by inducing Pgp trafficking in Madin-Darby canine kidney (MDCK) and rat hepatoma cells [7]
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