PEROXYNITRITE-INDUCED CYTOTOXICITY IN PC12 CELLS - EVIDENCE FOR AN APOPTOTIC MECHANISM DIFFERENTIALLY MODULATED BY NEUROTROPHIC FACTORS

Peroxynitrite is a powerful oxidant formed by the near-diffusion-limit ed reaction of nitric oxide with superoxide, Large doses of peroxynitr ite (> 2 mM) resulted in rapid cell swelling and necrosis of undiffere ntiated PC12 cells, However, brief exposure to lower concentrations of peroxynitrite (EC(50) = 850 mu M) initially (3-4 h) caused minimal da mage to low-density cultures. By 8 h, cytoplasmic shrinkage with nucle ar condensation and fragmentation became increasingly evident, After 2 4 h, 36% of peroxynitrite-treated cells demonstrated these features as sociated with apoptosis, In addition, 46% of peroxynitrite-treated cel ls demonstrated DNA fragmentation (by terminal-deoxynucleotide transfe rase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintrica rboxylic acid, Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment, Although peroxynitrite is known to be toxic t o cells, the present study provides a first indication that peroxynitr ite induces apoptosis, Furthermore, pretreatment of cells with nerve g rowth factor or insulin, but not epidermal growth factor, was protecti ve against peroxynitrite-induced apoptosis, However, both acidic and b asic fibroblast growth factors greatly increased peroxynitrite-initiat ed apoptosis, to 63 and 70%, respectively, Thus, specific trophic fact ors demonstrate differential regulation of peroxynitrite-induced apopt osis in vitro.