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-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 microM) intially (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.
Estévez
A G
AG
Division Neurobiología Cellular y Molecular, Instituto Clemente Estable, Montevideo, Uruguay.
Radi
R
R
Barbeito
L
L
Shin
J T
JT
Thompson
J A
JA
Beckman
J S
JS
D000818:Animals; D017209:Apoptosis; D002470:Cell Survival; D004396:Coloring Agents; D004249:DNA Damage; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D009414:Nerve Growth Factors / Q000502:physiology*; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D009566:Nitrates / Q000494:pharmacology*; D016716:PC12 Cells; D051381:Rats
D004396:Coloring Agents; D009414:Nerve Growth Factors; D009566:Nitrates; D016222:Fibroblast Growth Factor 2; C039642:peroxynitric acid