BCL-2 PROTECTS AGAINST APOPTOSIS IN NEURONAL CELL-LINE CAUSED BY THAPSIGARGIN-INDUCED DEPLETION OF INTRACELLULAR CALCIUM STORES

The toxicity of thapsigargin, a selective inhibitor of endoplasmic ret icular Ca2+-ATPase, was investigated in GT1-7 cells, a murine hypothal amic cell line. Treatment of these cells with 50 or 100 nM thapsigargi n greatly reduced cell viability at 24 and 48 h. These doses of thapsi gargin induced a rapid rise in free cytosolic Ca2+([Ca2+](i)), followe d by a sustained increase. Addition of EGTA to chelate extracellular C a2+ diminished somewhat the size of the initial increase of [Ca2+](i) caused by thapsigargin, and abolished the sustained increase. The sust ained increase could also be abolished by addition of La3+ and by SKF 96365, a drug selective for receptor-mediated calcium entry, but not b y verapamil or flunarizine. Pretreatment with 50 mu M BAPTA/AM, a cyto solic Ca2+ chelator, inhibited the peak [Ca2+](i) caused by thapsigarg in but did not inhibit the sustained elevation of [Ca2+](i). Neither E GTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (''laddering''), nu clear condensation and fragmentation, and was inhibited by protein syn thesis inhibitor cycloheximide, all characteristic of apoptotic cell d eath. Overexpression of the protooncogene bcl-2 in GT1-7 cells inhibit ed significantly DNA fragmentation, nuclear condensation and fragmenta tion, and cell death induced by thapsigargin. However, Bcl-2 did not a lter either basal [Ca2+](i) or the elevation of [Ca2+](i) induced by t hapsigargin. Our results suggest that abnormal Ca2+ release from endop lasmic reticulum caused by thapsigargin induces GT1-7 death by apoptos is and that this effect does not depend on Ca2+ influx from the extrac ellular space. Bcl-2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca 2+ release in GT1-7 neuronal cells.