THE REGULATION OF REACTIVE OXYGEN SPECIES PRODUCTION DURING PROGRAMMED CELL-DEATH

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxi dative glutamate toxicity was studied in an immortalized mouse hippoca mpal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and c ytosolic Ca2+ concentration was examined. An initial 5-10-fold increas e in ROS after glutamate addition is temporally correlated with GSH de pletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this bu rst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin Ip-conver ting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in th e death program. Inhibition of intracellular Ca2+ cy cling and the inf lux of extracellular Ca2+ also blocks maximum ROS production and prote cts the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is a n early requirement for protease activation, changes in gene expressio n, and a late requirement for Ca2+ mobilization.