title: In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate.
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 (3) H-L-glutamate, (3) H-L-aspartate, and (3) H-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 cm(2) , and (14) C-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 K(m) of 69 ± 15 μM and a J(max) of 44 ± 3.1 pmol min(-1) cm(-2) for L-aspartate and a K(m) of 138 ± 49 μM and J(max) 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.
authors.affiliation: Department of Pharmacy, The Faculty of Medicines and Health, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Hans Christian
authors.initials: HC
authors.lastname: Helms
authors.forename: Rasmus
authors.initials: R
authors.lastname: Madelung
authors.forename: Helle Sønderby
authors.initials: HS
authors.lastname: Waagepetersen
authors.forename: Carsten Uhd
authors.initials: CU
authors.lastname: Nielsen
authors.forename: Birger
authors.initials: B
authors.lastname: Brodin
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001253:Astrocytes / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D001812:Blood-Brain Barrier / Q000378:metabolism*; D001921:Brain / Q000166:cytology*; D002417:Cattle; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D017097:Electric Impedance; D042783:Endothelial Cells / Q000502:physiology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D050491:Glutamate Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D008353:Mannitol / Q000378:metabolism; D012333:RNA, Messenger / Q000592:standards; D051381:Rats; D012964:Sodium / Q000378:metabolism; D014316:Tritium / Q000378:metabolism
chemical_list: D000596:Amino Acids; D005904:Glial Fibrillary Acidic Protein; D050491:Glutamate Plasma Membrane Transport Proteins; D012333:RNA, Messenger; D014316:Tritium; D001224:Aspartic Acid; D018698:Glutamic Acid; D008353:Mannitol; D012964:Sodium