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

TY - JOUR

T1 - In vitro evidence for the brain glutamate efflux hypothesis

T2 - brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate

AU - Helms, Hans Christian

AU - Madelung, Rasmus

AU - Waagepetersen, Helle Sønderby

AU - Nielsen, Carsten Uhd

AU - Brodin, Birger

N1 - Copyright © 2012 Wiley Periodicals, Inc.

PY - 2012/5

Y1 - 2012/5

N2 - 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 cm 2, 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 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.

AB - 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 cm 2, 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 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.

U2 - 10.1002/glia.22321

DO - 10.1002/glia.22321

M3 - Journal article

C2 - 22392649

SN - 0894-1491

VL - 60

SP - 882

EP - 893

JO - GLIA

JF - GLIA

IS - 6

ER -