N-Methyl-D-Aspartate Receptor-Dependent Denitrosylation of Neuronal Nitric Oxide Synthase Increase the Enzyme Activity
Figure 3
Effects of different drugs on the S-nitrosylation of nNOS in primary cortical neurons induced by oxygen-glucose deprivation/reperfusion (OGD/reoxygenation).
A, Time course analysis of the S-nitrosylation and phosphorylation levels of nNOS in primary cortical neurons at various reperfusion time points after OGD. The data shown are the means ± S.D. from three independent experiments. aP<0.05 versus the control group; bP<0.05 versus the R6h group. B, The effects of GSNO (100 μΜ), or co-treatment with DTT (10 mM), and MK801 (20 μΜ) on the Ser847 phosphorylation and S-nitrosylation levels for nNOS induced by OGD followed by 6 hours of reoxygenation in primary cortical neurons. The blots shown are representative of three independent experiments (n = 3); aP<0.05 versus the control group; bP<0.05 versus the PBS group. C, Optimization of the auranofin concentration in the nNOS S-nitrosylation experiment in primary cortical neurons induced by OGD followed by 6 hours of reoxygenation. The bands shown are representative of three independent experiments. aP<0.05 versus the 0 μΜ group. D-F, The effects of auranofin (4 μΜ), DNCB (2 μΜ), BAPTA (4 μΜ), EGTA (25 mΜ) and W-7 (40 μΜ) on the Ser847 phosphorylation and S-nitrosylation of nNOS induced by OGD followed by 6 hours of reoxygenation in primary cortical neurons. The bands shown are representative of three independent experiments; aP<0.05 versus the control group; bP<0.05 versus the DMSO group. G, TrxR1 AS, TrxR1 MS oligonucleotide, or vehicle (TE) was administrated at a concentration of 1 µM to rat primary cortical neurons every 24 hours for five days prior to OGD. After 6 hours of reoxygenation, the cells were harvested and TrxR1 was analyzed by western blotting (n = 3); aP<0.05 versus the vehicle group. H, The effects of TrxR1 AS on the phosphorylation and S-nitrosylation of nNOS induced by OGD followed by 6 hours of reoxygenation in primary cortical neurons. The blots shown are representative of three independent experiments; aP<0.05 versus the control group; bP<0.05 versus the vehicle group.