The nitrone spin trap PBN alters the cellular response to H2O2: activation of the EGF receptor/ERK pathway

The nitrone spin trap PBN has been shown to protect neuronal cells from reactive oxygen species both in culture and in vivo. As an approach to understanding the molecular mechanisms by which PBN may function to protect cells, we examined whether PBN alters the cellular response to reactive oxygen species. H2O2 stimulation of PC-12 cells results in weak activation of both the ERK and JNK signal transduction pathways. PBN pretreatment of PC-12 cells, followed by H2O2 stimulation, results in strong and selective activation of the pro-survival ERK pathway. H2O2 induction of ERK activity in PBN-pretreated cells was shown to be dependent on extracellular Ca+2 influx. Further analysis of the ERK pathway showed that in PBN-pretreated cells, EGF receptor and the adapter protein SHC were phosphorylated in a Ca+2-dependent, ligand-independent manner following H2O2 stimulation. Interestingly, H2O2 stimulation of PBN-pretreated cells results in only 30% of the increase in intracellular Ca+2 as compared to untreated cells following H2O2 stimulation. These data suggest a model in which PBN attenuates H2O2-induced Ca+2 entry, yet magnifies or alters Ca+2 action, resulting in the activation of the EGF receptor/ERK pathway.