Peroxynitrite‐Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors

Abstract

Abstract: Peroxynitrite is a powerful oxidant formed by the near‐diffusion‐limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 m M) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC 50 = 850 µ M) initially (3–4 h) caused minimal damage to low‐density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite‐treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite‐treated cells demonstrated DNA fragmentation (by terminal‐deoxynucleotide transferase‐mediated dUTP‐digoxigenin nick end‐labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic 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 to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite‐induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite‐initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite‐induced apoptosis in vitro.