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Characterization of the L-glutamate clearance pathways across the blood-brain barrier and the effect of astrocytes in an in vitro blood-brain barrier model.

ABSTRACT: The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood-brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co-application of transporter inhibitors and competing amino acids. Endothelial abluminal L-glutamate uptake was almost abolished by co-application of an EAAT-1 specific inhibitor, whereas luminal uptake was inhibited by L-glutamate and L-aspartate (1?mM). L-glutamate uptake followed Michaelis-Menten-like kinetics with high and low affinity at the abluminal and luminal membrane, respectively. This indicated that L-glutamate is taken up via EAAT-1 at the abluminal membrane and exits at the luminal membrane via a low affinity glutamate/aspartate transporter. Metabolism of L-glutamate and transport of metabolites was examined using [U-¹³C] L-glutamate. Intact L-glutamate and metabolites derived from oxidative metabolism were transported through the endothelial cells. High amounts of L-glutamate-derived lactate in the luminal medium indicated cataplerosis via malic enzyme. Thus, L-glutamate can be transported intact from brain to blood via the concerted action of abluminal and luminal transport proteins, but the total brain clearance is highly dependent on metabolism in astrocytes and endothelial cells followed by transport of metabolites.

SUBMITTER: Helms HC

PROVIDER: S-EPMC5718321 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Characterization of the L-glutamate clearance pathways across the blood-brain barrier and the effect of astrocytes in an in vitro blood-brain barrier model.

Helms Hans Cc HC Aldana Blanca I BI Groth Simon S Jensen Morten M MM Waagepetersen Helle S HS Nielsen Carsten U CU Brodin Birger B

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 20170201 12

The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood-brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co-application of transporter inhibitors and competing amino acids. Endothelial abluminal L-glutamate uptake was almost abolished by co-application of an EAAT-1 specific inhibitor, whereas luminal uptake was ...[more]

PMID: 28145808

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Project description:Background:The brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer's disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-? (A?). Methods:We used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to A?, both under resting or inflammatory conditions (TNF? and IFN?), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs). Results:In the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by A? treatment. On the contrary, in endothelial monocultures, A? induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by A?. This modulation by A? was not observed in endothelial monocultures. In addition, endothelial expression of ?-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by A? only in the co-culture model. miR-200b, increased in astrocytes following A? treatment and may represent one of the factors involved in the control of Gnt-III expression. Conclusion:These data point out that, at least in the early phases of A? exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer.

Dataset Information

Characterization of the L-glutamate clearance pathways across the blood-brain barrier and the effect of astrocytes in an in vitro blood-brain barrier model.

Publications

Characterization of the L-glutamate clearance pathways across the blood-brain barrier and the effect of astrocytes in an in vitro blood-brain barrier model.

Similar Datasets

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