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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.

journal of cerebral blood flow and metabolism

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

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