Regulation of nitric oxide/reactive oxygen species redox signaling by nNOS splicing variants

We previously demonstrated different expression patterns of the neuronal nitric oxide synthase (nNOS) splicing variants, nNOS-μ and nNOS-α, in the rat brain; however, their exact functions have not been fully elucidated. In this study, we compared the enzymatic activities of nNOS-μ and nNOS-α and investigated intracellular redox signaling in nNOS-expressing PC12 cells, stimulated with a neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP⁺), to enhance the nNOS uncoupling reaction. Using in vitro studies, we show that nNOS-μ produced nitric oxide (NO), as did nNOS-α, in the presence of tetrahydrobiopterin (BH₄), an important cofactor for the enzymatic activity. However, nNOS-μ generated more NO and less superoxide than nNOS-α in the absence of BH₄. MPP ⁺ treatment induced more reactive oxygen species (ROS) production in nNOS-α-expressing PC12 cells than in those expressing nNOS-μ, which correlated with the intracellular production of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a downstream messenger of nNOS redox signaling, and apoptosis in these cells. Furthermore, post-treatment with 8-nitro-cGMP aggravated MPP⁺-induced cytotoxicity via activation of the H-Ras/extracellular signal-regulated kinase signaling pathway. In conclusion, our results provide strong evidence that nNOS-μ exhibits distinctive enzymatic properties of NO/ROS production, contributing to the regulation of intracellular redox signaling, including the downstream production of 8-nitro-cGMP.