title: 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.
authors.affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Hawkins
authors.forename: Robert R
authors.initials: RR
authors.lastname: Rigor
authors.forename: David S
authors.initials: DS
authors.lastname: Miller
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects* / Q000378:metabolism*; D015153:Blotting, Western; D002196:Capillaries / Q000187:drug effects; D002560:Cerebrovascular Circulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D019286:Endopeptidase K / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007276:Injections, Intraventricular; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D018696:Neuroprotective Agents; D010703:Phorbol Esters / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008565:Membrane Proteins; D018696:Neuroprotective Agents; D010703:Phorbol Esters; D042461:Vascular Endothelial Growth Factor A; C029705:12-deoxyphorbolphenylacetate-20-acetate; D019286:Endopeptidase K