Coupling between neuronal nitric oxide synthase and glutamate receptor 6–mediated c-Jun N-terminal kinase signaling pathway via S-nitrosylation contributes to ischemia neuronal death

• Published in: Neuroscience Vol. 155; no. 4; pp. 1120 - 1132
• Main Authors: Yu, H.-M, Xu, J, Li, C, Zhou, C, Zhang, F, Han, D, Zhang, G.-Y
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 09-09-2008; Elsevier
• Subjects: Animals; Biological and medical sciences; Cell Death - drug effects; Cell Death - physiology; cerebral ischemia; Disease Models, Animal; Disks Large Homolog 4 Protein; Dithiothreitol - pharmacology; Enzyme Inhibitors - pharmacology; Excitatory Amino Acid Antagonists - pharmacology; Fundamental and applied biological sciences. Psychology; GluK2 Kainate Receptor; GluR6; Immunoprecipitation; Intracellular Signaling Peptides and Proteins - metabolism; Ischemia - pathology; JNK Mitogen-Activated Protein Kinases - metabolism; Ketamine - pharmacology; Male; Medical sciences; Membrane Proteins - metabolism; Models, Biological; Neurology; Neurons - drug effects; Neurons - physiology; neuroprotection; Nitric Oxide - metabolism; Nitric Oxide Synthase Type I - metabolism; nNOS; phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid - physiology; S-nitrosylation; Signal Transduction - drug effects; Signal Transduction - physiology; Vascular diseases and vascular malformations of the nervous system; Vertebrates: nervous system and sense organs; Fas ligand; eNOS; CaMK II; MLK3; DMSO; JNK; 7-NI; ketamine hydrochloride; sodium nitroprusside; S-nitrosylation; Tris-buffered saline with 0.1% Tween 20; 7-nitroindazole; endothelial nitric oxide synthase; iNOS; phosphorylation; c-Jun N-terminal kinase; SDS; MKK4/7; AMT; glutamate receptor 6; GluR6; EGTA; Ca 2+/calmodulin-dependent kinase II; HPDP; nNOS; biotin-hexyl pyridyldithiopropionamide; NMDAR; DTT; cytochrome c oxidase subunit IV; postsynaptic density protein 95; COXIV; ethylenediamine tetraacetic acid disodium; ethylene glycol-bis(β-aminoethyl)-N,N,N,N tetraacetic acid; inducible nitric oxide synthase; dimethyl sulfoxide; SNP; cerebral ischemia; mixed lineage kinase 3; sodium dodecyl sulfate; 2-amion-5,6-dihydro-6-methyl-4H-1,3-thiazine; dithiothreitol; TBST; KT; neuronal nitric oxide synthase; EDTA; N-methyl- d-aspartate receptor; PSD95; FasL; neuroprotection; mitogen activated protein kinase kinase 4/7; Neuroprotection; Phosphorylation; Enzyme; Transferases; Central nervous system; Mitogen-activated protein kinase; Cardiovascular disease; Glutamate receptor; Nitric-oxide synthase; Encephalon; Signal transduction; Neuron; Ischemia; Cell death; Oxidoreductases
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2008.03.061

Abstract 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.