Nitric oxide activation of TrkB through peroxynitrite

NIH-3T3 cells stably transfected with TrkB, the receptor for brain-derived neurotrophic factor (BDNF), were used to study the effects of NO and peroxynitrite on TrkB. 3-Morpholinosydnonimine (SIN-1), a donor of NO and O2 – which immediately react to form peroxynitrite, induced TrkB tyrosine phosphorylation in a dose-dependent relationship from 2 to 40 mM. TrkB phosphorylation by SIN-1 was blocked by superoxide dismutase, which converts O2 – to H2O2 and prevents its reaction with NO to form peroxynitrite, and by K252a, an inhibitor of TrkB phosphorylation by BDNF. Treatment with NO or O2 – alone did not activate TrkB. Treatment directly with 1–4 mM peroxynitrite resulted in a dose-dependent increase in tyrosine phosphorylation of TrkB. SIN-1 treatment induced tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) and induced its binding with activated TrkB, similar to that seen with BDNF downstream signaling pathways. These studies demonstrate activation of TrkB through peroxynitrite.