title: Thrombin decreases expression of the glutamate transporter GLAST and inhibits glutamate uptake in primary cortical astrocytes via the Rho kinase pathway.
abstract: Astrocyte glutamate transporters GLAST and GLT1 play a key role in regulating neuronal excitation and their levels are altered in patients with epilepsy, and after traumatic brain injury. The mechanisms which regulate their expression are not well understood. We tested the hypothesis that exposure of astrocytes to high levels of thrombin, as may occur after a compromise of the blood-brain barrier, would reduce astrocyte glutamate transporter levels. In isolated rat cortical astrocytes we examined the effects of thrombin on the expression and function of glutamate transporters, and the signaling pathways involved in these responses by using Western blotting and selective inhibitors. Thrombin induced a selective decrease in the expression of GLAST but not GLT1, with a corresponding decrease in the capacity of astrocytes to take up glutamate. Activation of the thrombin receptor PAR-1 with an activating peptide induced a similar decrease in the expression of GLAST and compromise of glutamate uptake. The downregulation of GLAST induced by thrombin was mediated by the mitogen activated protein kinases p38 MAPK, ERK and JNK, but inhibition of these kinases did not prevent the decrease in glutamate uptake induced by thrombin. In contrast, inhibition of the Rho kinase pathway using the specific inhibitor, Y27632, suppressed both the decrease in the expression of GLAST and the decrease in glutamate uptake induced by thrombin. In hippocampal astrocyte cultures, thrombin caused a decrease in both GLAST and GLT1. In tissue resected from brains of children with intractable epilepsy, we found a decrease in the integrity of the blood-brain barrier along with a reduction in immunoreactivity for both transporters which was associated with an increase in cleaved thrombin and reactive astrogliosis. The in vitro results suggest a specific mechanism by which thrombin may lead to a compromise of astrocyte function and enhanced synaptic excitability after the blood-brain barrier is compromised. The human in vivo results provide indirect support evidence linking the compromise of the blood-brain barrier to thrombin-induced reduction in glutamate transporter expression and an increase in neuronal excitation.
authors.affiliation: Department of Pediatrics, Division of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Department of Pediatrics, Ruth D. & Ken M. Davee Pediatric Neurocritical Care Program, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Chunshu
authors.initials: C
authors.lastname: Piao
authors.affiliation: Department of Pediatrics, Division of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Department of Pediatrics, Ruth D. & Ken M. Davee Pediatric Neurocritical Care Program, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Hantamalala
authors.initials: H
authors.lastname: Ralay Ranaivo
authors.affiliation: Department of Pediatrics, Division of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Department of Pediatrics, Ruth D. & Ken M. Davee Pediatric Neurocritical Care Program, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Rusie
authors.affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Nitin
authors.initials: N
authors.lastname: Wadhwani
authors.affiliation: Department of Pediatrics, Division of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Sookyong
authors.initials: S
authors.lastname: Koh
authors.affiliation: Department of Pediatrics, Division of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Department of Pediatrics, Division of Critical Care, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Department of Pediatrics, Ruth D. & Ken M. Davee Pediatric Neurocritical Care Program, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. Electronic address: m-wainwright@northwestern.edu.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Wainwright
mesh_terms: D000293:Adolescent; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000503:physiopathology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D004827:Epilepsy / Q000473:pathology; D027341:Excitatory Amino Acid Transporter 1 / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D050491:Glutamate Plasma Membrane Transport Proteins / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors; D055815:Young Adult; D062826:Zonula Occludens-1 Protein / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D005904:Glial Fibrillary Acidic Protein; D050491:Glutamate Plasma Membrane Transport Proteins; C493397:SLC1A2 protein, human; C493392:Slc1a3 protein, rat; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D018698:Glutamic Acid; D054460:rho-Associated Kinases; D013917:Thrombin
keywords: Blood–brain barrier; Glutamate transporter; Kinases; Protease