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

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.

Figure 1

Proteinase K (PK) cleaves luminal membrane proteins in situ. (A) Representative blots of capillary proteins isolated from rats perfused with the concentration of PK indicated. (B) Densitometry of blots represented in (A). Immunoreactivity for P-glycoprotein and multidrug resistance-related protein 2 (Mrp2) significantly decreased with increasing concentration of PK. Immunoreactivity for the Na,K-ATPase α1 subunit (expressed primarily on the abluminal membrane) was unchanged. Data are mean relative band densities for the proteins indicated normalized to relative band densities for β-actin±s.e.m. Significance is determined by one-way analysis of variance with a Neuman–Keuls post hoc test. ^*P<0.05, n=3 for all experiments. (C) Proteinase K cleaves actin in permeabilized brain capillaries. Inclusion of the cell permeabilizing detergent Triton X-100 (TX, 0.1%) in the perfusate together with PK (60 U/mL) facilitates cleavage of actin, as seen by the low molecular weight band recognized by the actin antibody. Note that the lower molecular weight band is not present in any other condition.

Figure 2

Intracerebroventricular injection of vascular endothelial growth factor (VEGF) internalizes P-glycoprotein at the blood–brain barrier (BBB). (A) Animals were given an ICV injection of either VEGF or artificial cerebrospinal fluid (CON) before undergoing perfusion with proteinase K (PK). Capillaries were isolated from the hemispheres ipsilateral to the injection site. The VEGF treatment was associated with a significant (P<0.05) increase in immunoreactivity for P-glycoprotein after PK perfusion, indicating that P-glycoprotein was less available for cleavage by PK and implying that P-glycoprotein is internalized in response to VEGF. (B) The VEGF does not induce protection of multidrug resistance-related protein 2 (Mrp2) from proteolysis by PK. (C) Infusion of the protein kinase C (PKC)βI activating phorbol ester 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA) to the cerebral microvasculature before PK infusion has no effect on P-glycoprotein immunoreactivity. Representative blots are shown, each histogram shows mean normalized band densities±s.e.m. from three separate experiments. Significance is determined by Student's t-test; ^*P<0.05.