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 8

Broad-spectrum AR agonist NECA induces gradual and delayed downmodulation of P-gp expression and function in brain vascular endothelial cells in WT mice.

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Broad-spectrum AR agonist NECA induces gradual and delayed downmodulatio...
(A) Western blot analysis of P-gp and BCRP1 from mouse brain at 2 and 18 hours after NECA treatment. (B) Enumeration of expression intensity of P-gp (top graph) or BCRP1 (bottom graph) bands from NECA treatment from Western blot analysis. Intensity of bands from NECA treatment group was divided by that of DMSO control. Acquired values were normalized by GAPDH and graphed. *P < 0.05 (n = 3, 2-tailed Student’s t test). (C) IFA of P-gp in NECA-treated mouse brain at 2 and 18 hours after treatment. For IFA, brain frozen section were stained with GLUT1 (red) or P-gp (green) and counterstained with DAPI (blue). Scale bar: 100 μm. (D) Epirubicin brain accumulation assays in NECA-treated mice were performed after 0.08 mg/kg of NECA was injected intravenously for indicated time and, subsequently, 10 mg/kg of epirubicin was intravenously injected. At 15 minutes after epirubicin treatment, mice were perfused with ice-cold PBS and sacrificed at different time points. The accumulation of epirubicin in the brain was measured using fluorometry, with excitation at 488 nm and emission at 590 nm. *P < 0.05 (n = 4, 2-tailed Student’s t test).