NERVE GROWTH-FACTOR PROTECTS PC12 CELLS AGAINST PEROXYNITRITE-INDUCEDAPOPTOSIS VIA A MECHANISM DEPENDENT ON PHOSPHATIDYLINOSITOL 3-KINASE

Nerve growth factor (NGF) prevents apoptosis induced by the oxidant pe roxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previ ous studies have shown that activation of phosphatidylinositol 3-kinas e (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the prote ction NGF provided against peroxynitrite-induced apoptosis at concentr ations consistent with their effectiveness as Pi 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immuno precipitates after treatment of PC12 cells with peroxynitrite, PI 3-ki nase activity was reduced by 50% of that detected in control cells, wh ereas Pi 3-kinase activity in NGF-treated cells was unaffected by pero xynitrite. If an antibody against PI 3-kinase was used to immunoprecip itate the enzyme, treatment with peroxynitrite had no effect on activi ty. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibit ing the enzyme. NGF also activates p21(Ras)-dependent pathways, but th is did not appear to be required for NGF to exert its protective effec t against peroxynitrite. PC12 cells expressing a dominant inhibitory m utant of p21(Ras) were equally susceptible to peroxynitrite-induced ap optosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21(Ras) mutant cell line. Althou gh many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting pero xynitrite-induced apoptosis.