Apoptosis in Ca2+ reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-kappa B activation

We previously reported that incubation of cultured astrocytes in Ca2+-conta ining 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+ reperfu sion injury of astrocytes appears to be mediated by apoptosis, as demonstra ted by DNA fragmentation and prevention of death by caspase-3 inhibitors. P aradoxical 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 hy drogen peroxide (H2O2) for 30 min and then incubated without H2O2 for 1-5 d ays, cell toxicity including apoptosis was observed. Ca2+ reperfusion injur y induced by Ca2+ depletion or H2O2 exposure was blocked by the iron chelat or 1,10-phenanthroline, the NF-kappa B inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H2O 2 exposure rapidly increased the level of nuclear NF-kappa B p65 subunit, a nd the effect was blocked by 1,10-phenanthroline, pyrrolidinedithiocarbamat e and FK506. These findings indicate that Ca2+ reperfusion-induced apoptosi s is mediated at least partly by ROS production and ROS cause NF-kappa B ac tivation in cultured astrocytes.