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-mu and nNOS-alpha, in the rat brain; however, their exact functions have not been fully elucidated. In this study, we compared the enzymatic activities of nNOS-mu and nNOS-alpha 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-mu produced nitric oxide (NO), as did nNOS-alpha, in the presence of tetrahydrobiopterin (BH4), an important cofactor for the enzymatic activity. However, nNOS-mu generated more NO and less superoxide than nNOS-alpha in the absence of BH4. MPP (+) treatment induced more reactive oxygen species (ROS) production in nNOS-alpha-expressing PC12 cells than in those expressing nNOS-mu, which correlated with the intracellular production of 8-nitroguanosine 3 & PRIME;,5 & PRIME;-cyclic mono phosphate (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-mu exhibits distinctive enzymatic properties of NO/ROS production, contributing to the regulation of intracellular redox signaling, including the downstream production of 8-nitro-cGMP.