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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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 1

P-gp is expressed mostly in the cytoplasm of human brain endothelial cells and is colocalized with caveolae.

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P-gp is expressed mostly in the cytoplasm of human brain endothelial cel...
(A) P-gp (green) is expressed in the cytoplasm of a human brain endothelial cell line, HCMEC-D3 (top panels), and in human primary brain endothelial cell, HBMVEC (middle and bottom panels). HCMEC-D3 (top panels) were counterstained with WGA (red) to depict cell membrane. HBMVEC were stained with an antibody to caveolin-1 (middle panels) (red) or antibody to CD31 (bottom panels) (red) as markers for caveolae and cell membrane, respectively. Nucleus was counterstained with DAPI (blue). Scale bars: 25 μm. (B) Representative enlarged images from inset in A of HBMVEC cells. Scale bar: 10 μm. (C) P-gp was pulled down (immunoprecipitated) using anti–P-gp antibody and immunoblotted (IB) with an anti–caveolin-1 antibody. (D) HBMVEC was treated with Rho123 (green, top panel) for 1 hour, then fixed and stained with anti–P-gp (red in top panel or green in bottom panel).Caveolae were counterstained with anti–caveolin-1 (green, bottom panel). Scale bar: 25 μm. (E) Enlarged images from inset in D depicting P-gp or caveolae colocalization. Scale bar: 10 μm. (F and G) Inhibition of P-gp with PSC833, a functional inhibitor of P-gp in Rho123 uptake in human brain endothelial cell line HCMEC-D3 (F) and primary human brain endothelial cells (G). **P < 0.01 (n = 3, 2-tailed Student’s t test, 1 representative result from 3 different experiments).