title: The Rho kinase inhibitor Fasudil up-regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes.
abstract: OBJECTIVE
  Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology.
  
  
  METHODS
  The functional activity of glutamate transport was determined in murine cultured astrocytes after exposure to the ROCK inhibitors Fasudil (HA-1077) and Y27632 using biochemical, molecular and morphological approaches. Cytochemical analyses assessed changes in astrocytic morphology, F-/G-actin, and localizations of EAAT1/2.
  
  
  RESULTS
  Fasudil and Y27632 increased [(3)H]-D-aspartate (D-Asp) uptake into astrocytes, and the action of Fasudil was time-dependent and concentration-related. The rapid stellation of astrocytes (glial fibrillary acidic protein immunocytochemistry) induced by Fasudil was accompanied by reduced phalloidin staining of F-actin and increased V(max) for [(3)H]-D-Asp uptake. Immunoblotting after biotinylation demonstrated that Fasudil increased the expression of EAAT1 and EAAT2 on the cell surface. Immunocytochemistry indicated that Fasudil induced prominent labelling of astrocytic processes by EAAT1/2.
  
  
  CONCLUSIONS
  These data show for the first time that ROCK plays a major role in determining the cell surface expression of EAAT1/2, providing new evidence for an association between transporter function and astrocytic phenotype. ROCK inhibitors, via the actin cytoskeleton, effect a consequent elevation of glutamate transporter function - this activity profile may contribute to their beneficial actions in neuropathologies.
authors.affiliation: Molecular Neuropharmacology, Florey Neuroscience Institutes, Parkville, Australia.
authors.forename: C L
authors.initials: CL
authors.lastname: Lau
authors.forename: R D
authors.initials: RD
authors.lastname: O'Shea
authors.forename: B V
authors.initials: BV
authors.lastname: Broberg
authors.forename: L
authors.initials: L
authors.lastname: Bischof
authors.forename: P M
authors.initials: PM
authors.lastname: Beart
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000199:Actins / Q000648:ultrastructure*; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D001692:Biological Transport; D002478:Cells, Cultured; D027341:Excitatory Amino Acid Transporter 1 / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D005904:Glial Fibrillary Acidic Protein; D018698:Glutamic Acid / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016548:Prosencephalon / Q000166:cytology; D015854:Up-Regulation; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000199:Actins; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; C493396:Slc1a2 protein, mouse; C493393:Slc1a3 protein, mouse; C528900:glial fibrillary astrocytic protein, mouse; D001224:Aspartic Acid; D018698:Glutamic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054460:rho-Associated Kinases; C049347:fasudil