title: Regulation of endocytosis into human brain-microvascular endothelial cells by inhibition of efflux proteins.
abstract: The expressions of P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) in the blood-brain barrier (BBB) were regulated by verapamil and probenecid. The two protein interveners inhibited the efflux-pumping capability for permeating calcein-AM in the monolayer of human brain-microvascular endothelial cells (HBMECs). The immunochemical staining revealed that the order in the integrity of tight junction was human astrocyte (HA)-regulated HBMECs>HBMECs cultured with 100% astrocyte-conditioned medium (ACM)>HBMECs cultured with 50% ACM>HBMECs. The viability of HBMECs was higher than 94% when the concentrations of verapamil and probenecid were lower than 50 μM and 1000 μM, respectively. The culture using ACM negligibly affected the activity of P-gp and MRPs on HBMECs after the suppression with verapamil and/or probenecid. However, the double culture of HA-regulated HBMECs promoted the quantity of P-gp and MRPs and reduced the endocytosis of calcein-AM. The inhibitive and endocytotic analysis can unveil the role of HAs in the protein expressions on HBMECs for establishing a reliable BBB model in vitro.
authors.affiliation: Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan, ROC. chmyck@ccu.edu.tw
authors.forename: Yung-Chih
authors.initials: YC
authors.lastname: Kuo
authors.forename: Chin-Hsun
authors.initials: CH
authors.lastname: Lu
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism; D001921:Brain / Q000166:cytology*; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D055806:Microvessels / Q000166:cytology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011339:Probenecid / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011339:Probenecid; D014700:Verapamil; C085925:calcein AM