Apoptosis in Ca2 + reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-kappaB activation.
We previously reported that incubation of cultured astrocytes in Ca2 + -containing medium after exposure to Ca2 + -free medium caused Ca2 + influx followed by delayed cell death. Here, we studied the mechanisms underlying the Ca2 + -mediated injury of cultured astrocytes. Our results show that Ca2 + reperfusion injury of astrocytes appears to be mediated by apoptosis, as demonstrated by DNA fragmentation and prevention of death by caspase-3 inhibitors. Paradoxical Ca2 + challenge stimulated rapidly reactive oxygen species (ROS) production. Ca2 + reperfusion injury of astrocytes was influenced by several reagents which modified ROS production. When astrocytes were exposed to hydrogen peroxide (H2O2) for 30 min and then incubated without H2O2 for 1-5 days, cell toxicity including apoptosis was observed. Ca2 + reperfusion injury induced by Ca2 + depletion or H2O2 exposure was blocked by the iron chelator 1, 10-phenanthroline, the NF-kappaB inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H2O2 exposure rapidly increased the level of nuclear NF-kappaB p65 subunit, and the effect was blocked by 1,10-phenanthroline, pyrrolidinedithiocarbamate and FK506. These findings indicate that Ca2 + reperfusion-induced apoptosis is mediated at least partly by ROS production and ROS cause NF-kappaB activation in cultured astrocytes.
Takuma
K
K
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences,Kobe Gakuin University, Japan.
Lee
E
E
Kidawara
M
M
Mori
K
K
Kimura
Y
Y
Baba
A
A
Matsuda
T
T
D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D053938:DNA Fragmentation / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism*
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; C505410:Casp3 protein, rat; D053148:Caspase 3; D002118:Calcium