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

Abstract

Peroxynitrite is a powerful oxidant formed by the near‐diffusion‐limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 µM) 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.

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells

“…5,7 Our current study confirms this interpretation by noting that peroxynitrite activates caspase 3 in a bell-shaped fashion with upstream translocation of Bax, release of cytochrome c and cleavage of caspase 9 occurring at concentrations which induce maximal activation of caspase 3.…”

“…Peroxynitrite has been shown to induce minimal necrosis within a few hours of treatment at concentrations producing biochemical and morphological markers of apoptosis 24 h later. 5 These results may explain the small but significant increase in TUNEL staining observed at 7 h following treatment with peroxynitrite, an increase that does not correlate temporally with caspase activation. It is well accepted that the TUNEL stain does not discriminate between apoptotic and necrotic nuclei.…”

“…5,[7][8][9] When peroxynitrite is directly added to preparations of isolated mitochondria, the permeability transition pore is activated and cytochrome c is released, suggesting that peroxynitrite-induced apoptosis may be the result of direct oxidative damage to the mitochondria. 17 However, other studies have shown that peroxynitrite-induced apoptosis could be either inhibited or enhanced by treatment with trophic factors, 5,6 suggesting a regulated process. In the current study, we report that peroxynitrite activation of the intrinsic or mitochondrial apoptotic pathway in PC12 cells is preceded by phosphorylation of p38 and JNK MAPK and by translocation of phosphorylated JNK to the mitochondria.…”

“…Peroxynitrite induces apoptosis in a number of cell types in culture, including pheochromocytoma-derived PC12 cells, 5,6 cortical neurons, 7 HL-60 cells 8 and rat thymocytes. 9 Peroxynitrite-induced apoptosis of PC12 cells can be prevented by pretreatment with nerve growth factor (NGF), insulin and caspase inhibitors.…”

“…9 Peroxynitrite-induced apoptosis of PC12 cells can be prevented by pretreatment with nerve growth factor (NGF), insulin and caspase inhibitors. 5 Treatment with peroxynitrite also decreases the activity of the phosphatidyl-inositol 3-kinase (PI3-K) pathway, 6 while stimulating the activity of p38 10 and c-Jun-N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. 11 Both the inactivation of PI3-K 12 and the activation of p38 and JNK/MAPK 13 have been shown to promote apoptosis.…”