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Keywords:

  • central nervous system;
  • L-glutamate pharmacology;
  • endothelial glutamate;
  • efflux;
  • blood-brain barrier model

Abstract

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.