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.