Evaluation of blood-brain barrier transporters by co-culture of brain capillary endothelial cells with astrocytes

摘要

To investigate the transport function of the blood-brain barrier (BBB),we employed an in vitro model of the BBB,consisting of a co-culture of porcine brain capillary endothelial cells (BCECs) with rat astrocytes.Porcine BCECs were cultured on a filter insert with rat astrocytes on the underlying plastic well.Rat astrocytes induced characteristic BBB properties of porcine BCECs,such as #gamma#-glutamyl-transpeptidase activity and intercellular adhesion of porcine BCECs.Next,the transport properteis of P-glycoprotein (P-gp) substrate and several anionic compounds across the co-cultured porcine BCECs were characterized.Expression of P-gp was detected by immunocytochemistry,and efflux-directed transport of the P-gp substrate [~3H]daunomycin was observed.Luminal-to-abluminal transport of the monocarboxylic acid transporter 1 (MCT1) substrate [~(14)C]benzoic acid was saturable,and the K_m value (3.05mM) was also saturable,indicating that the monocarboxylic acid transporter of the BBB contributes to the efflux from the brain as well as to blood-to-brain influx.Abluminal-to-luminal transport of organci anions,[~3H]dehydroepiandrosterone sulfate,[~3H]estrone sulfate and [~3H]estradiol 17#beta3-D-glucuronide was significnatly higher than the corresponding luminal-to-abluminal transport.These results demonstrate the presence of multiple efflux transport pathways in this in vitro model.