Inhibition of nerve growth factor signaling by peroxynitrite

The reactive oxygen species peroxynitrite has been implicated in mediating oxidative damage within the brain, and in particular in those regions assoc iated with the pathology of Alzheimer disease. Evidence for peroxynitrite d amage includes the abundance of nitrated tyrosine residues within proteins of neural cells. Potential sites for peroxynitrite-induced cytotoxicity are the tyrosine residues of tyrosine kinase receptors that are crucial for th e maintenance of cholinergic neurons. The peroxynitrite generator 3-morphol inosydnonmine (SIN-1) was used to examine the effects of peroxynitrite gene ration on nerve growth factor (NGF)/TrkA signaling in PC12 pheochromocytoma cells that express a cholinergic phenotype. NGF produced a concentration-d ependent increase in PC12 cellular metabolism (EC50 = 15.2 ng/ml) measured in a microphysiometer. This action of NGF was inhibited in a concentration- dependent manner up to 67% of control by a brief (20 min) exposure of the c ells to SIN-1. This inhibition of the NGF cellular response by SIN-1 was no t related to generalized cellular toxicity. In fact, the per oxynitrite sca venger uric acid significantly attenuated the inhibitory actions of SIN-1. Pretreatment with SIN-1 also resulted in a decrease in the NGF-induced phos phorylation of TrkA protein. Furthermore, SIN-1 treatment reduced the activ ity of mitogen activated protein kinase (MAPK), a downstream kinase activat ed by TrkA receptor stimulation. These data suggest that SIN-1 treatment in hibits NGF signaling by inactivating TrkA receptors through the formation o f nitrotyrosine residues on the receptor. The inactivation of TrkA receptor s may contribute to the initial insult that eventually leads to neuronal ce ll death. (C) 2001 Wiley-Liss, Inc.