Regulation of endocytosis into human brain-microvascular endothelial cells by inhibition of efflux proteins

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.

.Figure optionsDownload as PowerPoint slideHighlights
► Human astrocyte-regulated HBMECs produce an integral tight junction in the blood–brain barrier.
► HBMECs express P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs).
► Astrocyte-conditioned medium yields little variation in the expression of P-gp and MRPs.
► Verapamil and probenecid inhibit P-gp and MRPs and increase endocytosis of calcein-AM.