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
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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 4

P-gp downmodulation by Lexiscan is mediated by MMP9 cleavage and translocation to insoluble cytoskeletal fractions.

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P-gp downmodulation by Lexiscan is mediated by MMP9 cleavage and translo...
(A) Schematic diagram of experimental procedure for analysis of Lexiscan’s effect on P-gp solubility and excretion in primary brain endothelial cells (HBMVEC). (B and C) Western blot analysis of P-gp expression of HBMVEC upon activation with Lexiscan at different time points after lysis with RIPA buffer (B) or CSK buffer to measure P-gp levels in the cytoskeletal fraction (C). Band intensity was normalized to GAPDH and graphed. (D) Western blot analysis of MMP9 in HBMVEC upon activation with Lexiscan at different time points. Band intensity was normalized using GAPDH and plotted. (E) Western blot analysis of secreted P-gp in HBMVEC upon activation with Lexiscan in absence or presence of an MMP9 inhibitor: both short and long exposures of Western blot from MMP9 inhibitor–treated samples are shown. Band intensity was plotted as graph. (F–H) Immunoprecipitation assay (F) of MMP9 with lysate from HBMVEC treated with vehicle (control) or 1 μM of Lexiscan for 5 minutes. P-gp was pulled down using anti–P-gp antibody and immunoblotted with an anti-MMP9 antibody. IFA (G) of MMP9 on HBMVEC treated with control (top panel) or 1 μM of Lexiscan (middle panel) for 5 minutes. Cells were stained with P-gp (green) and MMP9 (red). Nucleus was counterstained with DAPI (blue). Boxed region (inset) of Lexiscan-treated sample was magnified and displayed separately (bottom panel). Cell-surface colocalization of P-gp and MMP9 is indicated by arrows. Scale bars: 25 μm (upper and middle panels); 5 μm (bottom panels). (H) Analysis of intensity of fluorescence of MMP9 from G. Fluorescent intensity of MMP9 was quantified and plotted as graph (n = 20). **P < 0.01 (2-tailed Student’s t test, 1 representative result of 3 different experiments).