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 2

Activation of A2A AR downmodulates P-gp expression and function in human brain endothelial cells.

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Activation of A2A AR downmodulates P-gp expression and function in human...
(A) Western blot analysis of P-gp expression in HCMEC-D3 cells treated with Lexiscan (LEX) (1 μM) for up to 72 hours. GAPDH was used as loading control. (B) For densitometric analysis, the intensity of P-gp upon Lexiscan treatment was divided by that of DMSO control and graphed. Densitometric analysis of short-time point (dotted box) is depicted as an inset. (C) Rho123 uptake assay of HCMEC-D3 cells treated with Lexiscan (1 μM) was performed. Concentrations of Rho123 accumulation in brain endothelial cells were analyzed by fluorometry (Synergy, Biotek), with excitation at 488 nm and emission at 523 nm. *P < 0.05; **P < 0.01 (n = 4, 2-tailed Student’s t test, 1 representative result of 3 different experiments). (D) Western blot analysis depicting P-gp expression in human primary brain endothelial cells treated with Lexiscan (1 μM) for up to 72 hours. GAPDH was used as loading control. (E) Densitometric analysis was used to measure the intensity of P-gp expression with Lexiscan or NECA treatment and was divided by the DMSO control and graphed. Densitometric analysis of short-time point (dotted box) is depicted as an inset. (F) Rho123 uptake assay of human primary brain endothelial cells treated with Lexiscan (1 μM). The concentration of Rho123 accumulation in endothelial cells was quantified by fluorometry (Synergy, Biotek), with excitation at 488 nm and emission at 523 nm. *P < 0.05; **P < 0.01 (n = 4, 2-tailed Student’s t test, 1 representative result of 3 different experiments). (G) Schematic diagram of Rho123 accumulation assay from C and F.