Journal of Cerebral Blood Flow and Metabolism 2010-09-01

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

Brian T Hawkins, Robert R Rigor, David S Miller

Index: J. Cereb. Blood Flow Metab. 30(9) , 1593-7, (2010)

Full Text: HTML

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.