Bcl-2 and Bcl-X-L block thapsigargin-induced nitric oxide generation, c-Jun NH2-terminal kinase activity, and apoptosis

The proteins Bcl-2 and Bcl-X-L prevent apoptosis, but their mechanism of ac tion is unclear. We examined the role of Bcl-2 and Bcl-X-L in the regulatio n of cytosolic Ca2+, nitric oxide production (NO), c-Jun NH2-terminal kinas e (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca2+ ATPase, was used to disrupt Ca2+ homeostasis. TG acutely elevated intracellular free Ca2+ and m itochondrial Ca2+ levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca2+ response wit h 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxym ethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N-G-nitro-L -arginine methyl ester hydrochloride (L-NAR IE) blocked TG-induced NO produ ction and apoptosis in JT/Neo cells. By contrast, while TG produced compara ble early changes in the Ca2+ level (i.e., within 3 h) in Jurkat cells over expressing Bcl-2 and Bcl-X-L (JT/Bcl-2 or JT/Bcl-X-L), NO production, late (36-h) Ca2+ accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X-L, cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis com parable to that seen in JT/Neo cells. TG also activated the JNK pathway, wh ich was blocked by L-NAME. Transient expression of a dominant negative muta nt SEK1 (Lys --> Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduce d TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X-L inhibited TG-ind uced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocke d TG-induced JNK activation, suggesting that JNK activation occurred downst ream of caspase-3. Thus, TG-induced Ca2+ release leads to NO generation fol lowed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway an d apoptosis. In summary, Ca2+-dependent activation of NO production mediate s apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X-L, cel ls are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X-L protect the cells against TG-induced apoptosis by negatively regulating Ca2+-sensit ive NO synthase activity or expression.