Rapid Loss of Blood–Brain Barrier P-Glycoprotein Activity through Transporter Internalization Demonstrated Using a Novel in Situ Proteolysis Protection Assay

Blood–brain barrier (BBB) P-glycoprotein activity is rapidly reduced by vascular endothelial growth factor (VEGF) acting via Src and by tumor necrosis factor-α acting via protein kinase C (PKC)β1. To probe underlying mechanism(s), we developed an in vivo, immunoblot-based proteinase K (PK) protection assay to assess the changes in the P-glycoprotein content of the BBB's luminal membrane. Infusion of PK into the brain vasculature selectively cleaved luminal membrane P-glycoprotein, leaving intracellular proteins intact. Intracerebroventricular injection of VEGF partially protected P-glycoprotein from proteolytic cleavage, consistent with transporter internalization. Activation of PKCβ1 did not protect P-glycoprotein. Thus, VEGF and PKCβ1 reduce P-glycoprotein activity by distinct mechanisms.

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2,4,6-Tribromophenol Exposure Decreases P-Glycoprotein Transport at the Blood-Brain Barrier

Toxicological Sciences ◽

10.1093/toxsci/kfz155 ◽

2019 ◽

Vol 171 (2) ◽

pp. 463-472 ◽

Cited By ~ 4

Author(s):

Andrew W Trexler ◽

Gabriel A Knudsen ◽

Sascha C T Nicklisch ◽

Linda S Birnbaum ◽

Ronald E Cannon

Keyword(s):

Blood Brain Barrier ◽

Ex Vivo ◽

Female Rats ◽

Brain Barrier ◽

Transport Activity ◽

Fish Food ◽

P Glycoprotein ◽

Blood Brain ◽

Male And Female Rats

Abstract 2,4,6-Tribromophenol (TBP, CAS No. 118-79-6) is a brominated chemical used in the production of flame-retardant epoxy resins and as a wood preservative. In marine environments, TBP is incorporated into shellfish and consumed by predatory fish. Food processing and water treatment facilities produce TBP as a byproduct. 2,4,6-Tribromophenol has been detected in human blood and breast milk. Biologically, TBP interferes with estrogen and thyroid hormone signaling, which regulate important transporters of the blood-brain barrier (BBB). The BBB is a selectively permeable barrier characterized by brain microvessels which are composed of endothelial cells mortared by tight-junction proteins. ATP-binding cassette (ABC) efflux transporters on the luminal membrane facilitate the removal of unwanted endobiotics and xenobiotics from the brain. In this study, we examined the in vivo and ex vivo effects of TBP on two important transporters of the BBB: P-glycoprotein (P-gp, ABCB1) and Multidrug Resistance-associated Protein 2 (MRP2, ABCC2), using male and female rats and mice. 2,4,6-Tribromophenol exposure ex vivo resulted in a time- (1–3 h) and dose- (1–100 nM) dependent decrease in P-gp transport activity. MRP2 transport activity was unchanged under identical conditions. Immunofluorescence and western blotting measured decreases in P-gp expression after TBP treatment. ATPase assays indicate that TBP is not a substrate and does not directly interact with P-gp. In vivo dosing with TBP (0.4 µmol/kg) produced decreases in P-gp transport. Co-treatment with selective protein kinase C (PKC) inhibitors prevented the TBP-mediated decreases in P-gp transport activity.

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In vitro and in vivo study of dolichyl phosphate on the efflux activity of P‐glycoprotein at the blood–brain barrier

International Journal of Developmental Neuroscience ◽

10.1016/j.ijdevneu.2013.10.005 ◽

2013 ◽

Vol 31 (8) ◽

pp. 828-835 ◽

Cited By ~ 3

Author(s):

Bian‐Sheng Ji ◽

Juan Cen ◽

Lu Liu ◽

Ling He

Keyword(s):

Blood Brain Barrier ◽

Brain Barrier ◽

In Vivo Study ◽

P Glycoprotein ◽

Dolichyl Phosphate ◽

Blood Brain

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Comparative vulnerability of PET radioligands to partial inhibition of P-glycoprotein at the blood-brain barrier: A criterion of choice?

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x211045444 ◽

2021 ◽

pp. 0271678X2110454

Author(s):

Louise Breuil ◽

Solène Marie ◽

Sébastien Goutal ◽

Sylvain Auvity ◽

Charles Truillet ◽

...

Keyword(s):

Blood Brain Barrier ◽

Pet Imaging ◽

Brain Barrier ◽

Maximal Response ◽

Half Maximum ◽

Partial Inhibition ◽

P Glycoprotein ◽

Blood Brain

Only partial deficiency/inhibition of P-glycoprotein (P-gp, ABCB1) function at the blood-brain barrier (BBB) is likely to occur in pathophysiological situations or drug-drug interactions. This raises questions regarding the sensitivity of available PET imaging probes to detect moderate changes in P-gp function at the living BBB. In vitro, the half-maximum inhibitory concentration (IC50) of the potent P-gp inhibitor tariquidar in P-gp-overexpressing cells was significantly different using either [11C]verapamil (44 nM), [11C] N-desmethyl-loperamide (19 nM) or [11C]metoclopramide (4 nM) as substrate probes. In vivo PET imaging in rats showed that the half-maximum inhibition of P-gp-mediated efflux of [11C]metoclopramide, achieved using 1 mg/kg tariquidar ( in vivo IC50 = 82 nM in plasma), increased brain exposure by 2.1-fold for [11C]metoclopramide (p < 0.05, n = 4) and 2.4-fold for [11C]verapamil (p < 0.05, n = 4), whereby cerebral uptake of the “avid” substrate [11C] N-desmethyl-loperamide was unaffected (p > 0.05, n = 4). This comparative study points to differences in the “vulnerability” to P-gp inhibition among radiolabeled substrates, which were apparently unrelated to their “avidity” (maximal response to P-gp inhibition). Herein, we advocate that partial inhibition of transporter function, in addition to complete inhibition, should be a primary criterion of evaluation regarding the sensitivity of radiolabeled substrates to detect moderate but physiologically-relevant changes in transporter function in vivo.

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Tariquidar, a Selective P-glycoprotein Inhibitor, Does Not Potentiate Loperamide’s Opioid Brain Effects in Humans despite Full Inhibition of Lymphocyte P-glycoprotein

Anesthesiology ◽

10.1097/aln.0b013e31818d8f28 ◽

2008 ◽

Vol 109 (6) ◽

pp. 1092-1099 ◽

Cited By ~ 21

Author(s):

Daniel Kurnik ◽

Gbenga G. Sofowora ◽

John P. Donahue ◽

Usha B. Nair ◽

Grant R. Wilkinson ◽

...

Keyword(s):

Blood Brain Barrier ◽

Pupil Diameter ◽

Ex Vivo ◽

Area Under The Curve ◽

Brain Barrier ◽

Double Blind ◽

P Glycoprotein ◽

Blood Brain ◽

Double Blind Crossover Study

Background Loperamide, a potent opioid, has been used as an in vivo probe to assess P-glycoprotein activity at the blood-brain barrier, because P-glycoprotein inhibition allows loperamide to cross the blood-brain barrier and exert its central opioid effects. In humans, studies with nonselective and moderately potent inhibitors resulted in mild opioid effects but were confounded by the concurrent inhibition of loperamide's metabolism. The authors studied the effect of the highly selective, potent P-glycoprotein inhibitor tariquidar on loperamide's central opioid effects. Methods In a randomized, double-blind, crossover study, nine healthy subjects received on 2 study days oral loperamide (32 mg) followed by an intravenous infusion of either tariquidar (150 mg) or placebo. Central opioid effects (pupil diameter, sedation) were measured for 12 h, and blood samples were drawn up to 48 h after drug administration to determine plasma loperamide concentrations and ex vivo P-glycoprotein activity in T lymphocytes. Values for pupil diameter and loperamide concentrations were plotted over time, and the areas under the curves on the tariquidar and placebo study day were compared within each subject. Results Tariquidar did not significantly affect loperamide's central effects (median reduction in pupil diameter area under the curve, 6.9% [interquartile range, -1.4 to 12.1%]; P = 0.11) or plasma loperamide concentrations (P = 0.12) but profoundly inhibited P-glycoprotein in lymphocytes by 93.7% (95% confidence interval, 92.0-95.3%). Conclusion These results suggest that despite full inhibition of lymphocyte P-glycoprotein, the selective P-glycoprotein inhibitor tariquidar does not potentiate loperamide's opioid brain effects in humans.

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