S-nitrosylation, as a post-translational protein modification, recently has been paid more and more attention in
stroke research. S-nitrosylation regulates
protein function by the mechanisms of covalent attachment that control the addition or the removal of
nitric oxide (NO) from a
cysteine thiol. The derivation of NO is established by the demonstration that, in cerebral neurons, NO mainly generates from
neuronal nitric oxide synthase (nNOS) during the early stages of reperfusion. In the past researches, we demonstrate that global
ischemia-reperfusion facilitates the activation of
glutamate receptor 6 (GluR6) -mediated
c-Jun N-terminal kinase (JNK) signaling pathway. The objective of this study is primarily to determine, during the early stages of reperfusion in rat four-vessel occlusion (4-VO) ischemic model, whether nNOS-derived NO affects the GluR6-mediated JNK signaling route via S-nitrosylation which is performed mainly by the
biotin switch assay. Here, we show that administration of
7-nitroindazole, an inhibitor of nNOS, or
ketamine, an antagonist of
N-methyl-d-aspartate receptor (NMDAR), diminishes the increased S-nitrosylation of GluR6 induced by
cerebral ischemia-reperfusion. In contrast, 2-amion-5,6-dihydro-6-methyl-4H-1,3-thiazine, an inhibitor of inducible
NO synthase does not affect S-nitrosylation of GluR6. Moreover, treatment with
sodium nitroprusside (SNP), an exogenous NO donor, increases the S-nitrosylation and phosphorylation of nNOS, leading to the attenuation of the increased S-nitrosylation of GluR6 and the assembling of GluR6*
postsynaptic density protein 95 (PSD95)*
mixed lineage kinase 3 (MLK3) signaling module induced by
cerebral ischemia-reperfusion. The results also show that GluR6 downstream MLK3*
mitogen activated protein kinase kinase 4/7* JNK signaling module and nuclear or non-nuclear apoptosis pathways are involved in the above signaling route. However,
dithiothreitol (DTT) antagonizes the neuroprotection of SNP. Treatment with DTT alone, as a negative control, prevents S-nitrosylation of
proteins, which indicates the existence of endogenously produced S-nitrosylation. These data suggest that GluR6 is S-nitrosylated by endogenous NO in
cerebral ischemia-reperfusion, which is possibly correlated with NMDAR* PSD95* nNOS signaling module, and further activates GluR6* PSD95* MLK3 signaling module and JNK signaling pathway. In contrast, exogenous NO donor antagonizes the above action of endogenous NO generated from nNOS. Thus, our results provide the coupling of nNOS with GluR6 by S-nitrosylation during the early stages of
ischemia-reperfusion, which can be a new approach for
stroke therapy.