N-Methyl-D-Aspartate Receptor-Dependent Denitrosylation of Neuronal Nitric Oxide Synthase Increase the Enzyme Activity
Figure 2
Identification of the nNOS S-nitrosylated site and examination of the regulation of nNOS S-nitrosylation.
A, Wild type and mutant pME18S-nNOS plasmids were transfected into HEK293 cells for 48 hours, then the cells were then treated with GSNO (100 μΜ) or not for 1 hour. The blot shown is representative of three independent experiments that yielded equivalent results. B, Transfected HEK293 cells were incubated with or without GSNO and protein extracts were used to determine the nNOS activity levels. The enzyme activity levels were determined using the Nitric Oxide Synthase Kit (Nanjing Jiancheng Bioengineering Institute) in accordance with the manufacturer’s protocol. The data shown are the mean ± SD of five independent experiments (n = 5). *P<0.02 versus WT/C326G nNOS GSNO+ groups or WT nNOS GSNO- groups. C, D and E, Characterization of the S-nitrosylation and phosphorylation status of wild type nNOS (WT-nNOS), C331G nNOS and S847A nNOS exogenously expressed in HEK293 cells. The cells were incubated for 30 minutes in the presence or absence of GSNO (100μΜ) before treatment with or without A23187 (10μΜ) for an additional 30 minutes. The phosphorylation of nNOS was tested by immunoblotting with an anti-NP847 antibody. The blots shown are representative of three independent experiments.