Rapid loss of blood-brain barrier P-glycoprotein activity through transporter internalization demonstrated using a novel in situ proteolysis protection assay.

Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism
Brian T HawkinsDavid S Miller

Abstract

Blood-brain barrier (BBB) P-glycoprotein activity is rapidly reduced by vascular endothelial growth factor (VEGF) acting via Src and by tumor necrosis factor-alpha acting via protein kinase C (PKC)beta1. 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 PKCbeta1 did not protect P-glycoprotein. Thus, VEGF and PKCbeta1 reduce P-glycoprotein activity by distinct mechanisms.

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Citations

Sep 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Ronald E CannonDavid S Miller
Jan 5, 2014·The Journal of Pharmacology and Experimental Therapeutics·David S Miller
Nov 22, 2012·Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism·Tara A CartwrightDavid S Miller
Jun 22, 2012·Journal of Neurochemistry·Gwen McCaffreyThomas P Davis
Feb 12, 2015·Clinical Pharmacology and Therapeutics·D S Miller
May 20, 2016·Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism·Vijay R MoreRonald E Cannon
Oct 23, 2018·Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism·Margaret E TomeThomas P Davis
Oct 1, 2010·American Journal of Physiology. Renal Physiology·Aihua DengRoland C Blantz
Aug 15, 2019·Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism·Yutaro HoshiTetsuya Terasaki
Sep 16, 2020·Fluids and Barriers of the CNS·Björn BauerAnika M S Hartz
Jan 12, 2021·Tissue Barriers·Guinever Eustaquio Do ImperioStephen G Matthews

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Methods Mentioned

BETA
electrophoresis

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