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, 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 6 70 X cm 2 , and 14 C-D-mannitol permeability values of 0.88 6 0.13 3 10 26 cm s 21 . 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 6 15 lM and a J max of 44 6 3.1 pmol min 21 cm 22 for L-aspartate and a K m of 138 6 49 lM and J max of 28 6 3.1 pmol min 21 cm 22 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), 22 (Slc1a2), and 23 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and 23 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. V

The Efficiency of Glutamate Uptake Differs between Neonatal and Adult Cortical Microvascular Endothelial Cells

“…Taken together, these data suggest that the microvasculature of the immature cortex presents different functional characteristics from those in the mature brain, in particular regarding glutamate uptake that may impact the brainto-blood efflux of glutamate. [5][6][7][8] These results are in line with the higher sensitivity of nCMECs to glutamate. 4 Indeed, nCMECs express more NMDAR and are more prone to release vascular protease under glutamate stimulation than aCMECs.…”

“…5,6 In particular, brain endothelial cells appear to control extracellular glutamate concentrations by inducing the uptake and, subsequently, the efflux of glutamate across the blood-brain barrier. 7,8 To our knowledge, EAAT1-3 expression in neonatal endothelial cells has not been studied.…”

Inflammation, Glutamate, and Glia: A Trio of Trouble in Mood Disorders

“…Extracellular glutamate effluxes through astrocytes into the perivascular space and then through the endothelium into the peripheral blood. It is now known that much of extracellular glutamate is removed by EAATs expressed on the abluminal (brain side) of the endothelium of the cerebral blood vessels, crosses the blood vessel wall, and is released into the blood by glutamate transporters present on the luminal or blood side of the endothelium (Helms et al, 2012). Thus, rapid reduction in the concentrations of plasma glutamate leads to an aggressive mobilization of glutamate from the brain into the plasma and can result in rapid reduction of brain glutamate.…”

An Electrically Tight In Vitro Blood–Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

“…The co-cultured endothelial cells displayed high TEER values, low mannitol fluxes, and polarized transport of the small molecules, L-glutamate and L-aspartate favouring the abluminal-to-luminal direction [34].…”