CA2-INDUCED NEURONAL APOPTOSIS( AND REACTIVE OXYGEN SPECIES IN STAUROSPORINE)

Staurosporine (0.03-0.5 mu M) induced a dose-dependent, apoptotic dege neration in cultured rat hippocampal neurons that was sensitive to 24- h pretreatments with the protein synthesis inhibitor cycloheximide (1 mu M) or the cell cycle inhibitor mimosine (100 mu M). To investigate the role of Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D-28K, a Ca2+ binding p rotein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in th e hippocampal neurons using adenovirus-mediated gene transfer. Infecti on of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective prote ins assessed 48 h later. Overexpression of both calbindin D-28K and Cu /Zn superoxide dismutase significantly reduced staurosporine neurotoxi city compared with control cultures infected with a beta-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis w as also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage-sensitiv e Ca2+ channels reduces this apoptotic cell death. In contrast, neithe r the glutamate receptor agonist NMDA (1-10 mu M) nor the NMDA recepto r antagonist dizocilpine (MK-801; 1 mu M) was able to reduce staurospo rine neurotoxicity. Cultures treated with the antioxidants U-74500A (1 -10 mu M) and N-acetylcysteine (100 mu M) also demonstrated reduced st aurosporine neurotoxicity. These results suggest a fundamental role fo r both Ca2+ and reactive oxygen species in staurosporine-induced neuro nal apoptosis.