Glutamate-induced increases in transglutaminase activity in primary cultures of astroglial cells.
Glutamate exposure of astroglial cells caused ligand-gated channel receptor activation, associated with excitotoxic cell response. We investigated the effects of 24 h glutamate exposure on transglutaminase in astrocytes primary cultures at 7, 14, and 21 days in vitro (DIV). Increases in enzyme activity were observed as a function of cell differentiation stage in glutamate-treated cultures. These effects were significantly reduced when GYKI 52466, an AMPA/KA receptors inhibitor, was added to the culture medium prior to incubation with glutamate. Microscopy observation on transglutaminase-mediated, fluorescent dansylcadaverine incorporation in living cells was consistent with these results. Western blotting analysis with monoclonal antibody showed that glutamate also up-regulated tissue transglutaminase expression, which reached the highest values in 14 DIV cultures. Confocal laser scanning microscopy analysis of immunostained astroglial cells showed a mainly cytoplasmic localisation of the enzyme both in control and treated cultures; nevertheless, counterstaining with the nuclear dye acridine orange demonstrated the presence of tissue transglutaminase also into the nucleus of glutamate-exposed and 21 DIV cells. The increases in enzyme expression and localisation in the nucleus of glutamate-treated astroglial cells may be part of biochemical alterations induced by excitotoxic stimulus.
Campisi
A
A
Department of Biological Chemistry, Medical Chemistry and Molecular Biology, University of Catania, Catania, Italy.
Caccamo
D
D
Raciti
G
G
Cannavò
G
G
Macaione
V
V
Currò
M
M
Macaione
S
S
Vanella
A
A
Ientile
R
R
D000818:Animals; D000831:Animals, Newborn; D014151:Anti-Anxiety Agents / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D001569:Benzodiazepines; D015153:Blotting, Western / Q000379:methods; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D018683:Excitatory Amino Acid Agents / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D018698:Glutamic Acid / Q000494:pharmacology*; D007150:Immunohistochemistry / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D011503:Transglutaminases / Q000032:analysis / Q000378:metabolism*
D014151:Anti-Anxiety Agents; D018683:Excitatory Amino Acid Agents; D018691:Excitatory Amino Acid Antagonists; C061950:GYKI 52466; D001569:Benzodiazepines; D018698:Glutamic Acid; D011503:Transglutaminases; D005974:Glutamate-Ammonia Ligase; C008542:monodansylcadaverine; D002103:Cadaverine