Nitric oxide inhibition of ERK1/2 activity in cells expressing neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is a constitutively expressed enzyme responsible for the production of nitric oxide (NO.) from L-arginine and O-2. Nitric oxide is an intra- and intercellular messenger that mediates a diversity of signaling pathways in target cells. In the absence of L-arginine, nNOS has been shown to generate superoxide (O-2(radical anion)). Superoxide, either directly or through its self-dismutation to H2O2, is likewise believed to be a cell-signaling agent. Because nNOS can generate NO. and O-2(radical anion), we examined the activation of cellular signal transduction pathways in nNOS-transfected cells grown in the presence or absence of L-arginine. Spin trapping/ EPR spectroscopy confirmed that stimulated nNOS-transfected cells grown in an L-arginine environment secreted NO. into the surrounding milieu. Production of NO. blocked Ca2+ ionophore-induced activation of the ERK1/2 through a mechanism involving inhibition of the Ras G-protein and Raf-1 kinase. In contrast, ERK activation was largely unaffected in nNOS-transfected cells grown in L-arginine-free media. Inhibition of nNOS-generated NO. with the competitive NOS inhibitor, N-G-nitro-L-arginine methyl ester, in cells grown in L-arginine restored ERK1/2 activation to levels similar to that found when nNOS was activated in L-arginine-free media. These findings indicate that nNOS can differentially regulate the ERK signal transduction pathway in a manner dependent on the presence of L-arginine and the production of NO..