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

Nitric Oxide. 2022 Mar 1:120:44-52. doi: 10.1016/j.niox.2022.01.004. Epub 2022 Jan 14.


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.

Keywords: 8-Nitro-cGMP; MPP(+); NO/ROS redox signaling; Neurotoxicity; Tetrahydrobiopterin; nNOS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Methyl-4-phenylpyridinium / pharmacology
  • Animals
  • Apoptosis / drug effects
  • Cyclic GMP / analogs & derivatives
  • Cyclic GMP / metabolism
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type I / metabolism*
  • Oxidation-Reduction
  • PC12 Cells
  • Phosphorylation / drug effects
  • Protein Isoforms / metabolism
  • Rats
  • Superoxides / metabolism*


  • 8-nitroguanosine 3',5'-cyclic monophosphate
  • Protein Isoforms
  • Superoxides
  • Nitric Oxide
  • Nitric Oxide Synthase Type I
  • Nos1 protein, rat
  • Mapk1 protein, rat
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Cyclic GMP
  • 1-Methyl-4-phenylpyridinium