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In vitro evidence for the brain glutamate efflux hypothesis: Brain endothelial cells cocultured with astrocytes display a polarized brain‐to‐blood transport of glutamate

The concentration of the excitotoxic amino acid, L‐glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood‐brain barrier endothelium in brain L‐glutamate homeostasis. Transendothelial transport‐ and accumulation studies of 3H‐L‐glutamate, 3H‐L‐aspartate, and 3H‐D‐aspartate in an electrically tight bovine endothelial/rat astrocyte blood‐brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm2, and 14C‐D‐mannitol permeability values of 0.88 ± 0.13 × 10−6 cm s−1. Unidirectional flux studies showed that L‐aspartate and L‐glutamate, but not D‐aspartate, displayed polarized transport in the brain‐to‐blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a Km of 69 ± 15 μM and a Jmax of 44 ± 3.1 pmol min−1 cm−2 for L‐aspartate and a Km of 138 ± 49 μM and Jmax of 28 ± 3.1 pmol min−1 cm−2 for L‐glutamate. The EAAT inhibitor, DL‐threo‐ß‐Benzyloxyaspartate, inhibited transendothelial brain‐to‐blood fluxes of L‐glutamate and L‐aspartate. Expression of EAAT‐1 (Slc1a3), −2 (Slc1a2), and −3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT‐1 and −3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood‐brain barrier itself may participate in regulating brain L‐glutamate concentrations. © 2012 Wiley Periodicals, Inc.