title: Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1.
abstract: Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.
authors.affiliation: Brain Injury and Repair Program, Howard Florey Institute, Vic. 3010, Australia.
authors.forename: Ross D
authors.initials: RD
authors.lastname: O'Shea
authors.forename: Chew L
authors.initials: CL
authors.lastname: Lau
authors.forename: Mark C
authors.initials: MC
authors.lastname: Farso
authors.forename: Shanti
authors.initials: S
authors.lastname: Diwakarla
authors.forename: Chrissandra J
authors.initials: CJ
authors.lastname: Zagami
authors.forename: Brian B
authors.initials: BB
authors.lastname: Svendsen
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Feeney
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Callaway
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Jones
authors.forename: David V
authors.initials: DV
authors.lastname: Pow
authors.forename: Niels C
authors.initials: NC
authors.lastname: Danbolt
authors.forename: Bevyn
authors.initials: B
authors.lastname: Jarrott
authors.forename: Philip M
authors.initials: PM
authors.lastname: Beart
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects / Q000648:ultrastructure; D019921:Biotinylation; D015153:Blotting, Western; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D027341:Excitatory Amino Acid Transporter 1 / Q000096:biosynthesis; D027342:Excitatory Amino Acid Transporter 2 / Q000096:biosynthesis / Q000378:metabolism*; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000378:metabolism; D018613:Microscopy, Confocal; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010641:Phenotype; D021381:Protein Transport; D015854:Up-Regulation
chemical_list: D000199:Actins; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D008070:Lipopolysaccharides; C493393:Slc1a3 protein, mouse; D001224:Aspartic Acid