A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier
Do-Geun Kim, Margaret S. Bynoe
Do-Geun Kim, Margaret S. Bynoe
Published April 4, 2016
Citation Information: J Clin Invest. 2016;126(5):1717-1733. https://doi.org/10.1172/JCI76207.
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Research Article Vascular biology

A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

Abstract

The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology.

Authors

Do-Geun Kim, Margaret S. Bynoe

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Figure 7

A2A receptor activation by Lexiscan induces rapid and reversible downmodulation of P-gp expression and function in brain vascular endothelial cells in WT mice.

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A2A receptor activation by Lexiscan induces rapid and reversible downmod...
(A) Immunofluorescence images of brains from Lexiscan-treated mice of WT brain at 15 and 30 minutes after Lexiscan treatment. Frozen brain sections were stained with GLUT1 (red), or P-gp (green) and counterstained with DAPI (blue). (B) Enlarged image of A. (C) Brain epirubicin accumulation assay in the Lexiscan- and vehicle-treated (control) mice. 10 mg/kg of epirubicin was injected intravenously with or without 0.05 mg/kg of Lexiscan. Mice were perfused with ice-cold PBS and sacrificed at different time points. The accumulation of epirubicin in the brain was measured using fluorometric excitation at 488 nm and emission at 590 nm. *P < 0.05 (n = 4, 2-tailed Student’s t test). (D) Fluorescent microscopic analysis of epirubicin accumulation in the brains of mice treated with Lexiscan or PSC833 (a functional P-gp inhibitor) compared with vehicle (control). 10 mg/kg of epirubicin was injected intravenously with or without 0.05 mg/kg of Lexiscan or 50 mg/kg of PSC833 for 15 minutes. Mice were perfused with ice-cold PBS and sacrificed. Brain was sectioned for microscopic analysis for full-brain image, and focal zoomed image from cortex was laid as an inset. Epirubicin is in red, and nucleus was counterstained with DAPI (blue). (E) Intensity of epirubicin from different regions of cortex (CTX), cerebellum (CRBL), and hippocampus (HPC) from brains of control, Lexiscan-, or PSC833-injected animals was quantified and depicted as graphs. Scale bars: 100 μm (A); 50 μm (B) 5 mm (D). ***P < 0.001 (n = 50, 2-tailed Student’s t test).