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.