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AIM: To investigate whether brain ischemia induces serine phosphorylation of neuronal nitric oxide synthase(nNOS) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the interaction between CaMKIIa and nNOSin rat hippocampus. METHODS: Brain ischemia was induced by bilateral carotid artery occlusion procedure.Phosphorylation and the interaction of proteins were studied by immunoprecipitation and immunoblotting. Weinvestigated during brain ischemia serine phosphorylation and amount of nNOS in crude membranes fraction (P)and cytosolic fraction (S), interaction between CaMKIIa and nNOS, and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62, a selective inhibitor of CaMKII) on phos-phorylation and the interaction of proteins in P. RESULTS: Serine phosphorylation of nNOS in P increased persis-tently during brain ischemia, and 15 min ischemia-induced serine phosphorylation of nNOS was attenuated signifi-cantly by KN-62. But there was no serine phosphorylation of nNOS in S. The distributions of nNOS were notaffected by ischemia and KN-62. However, the binding levels of both CaMKIIa with nNOS and Thr286autophosphorylated CaMKIIa with nNOS increased after ischemia, and were diminished by KN-62. CONCLUSION:CaMKII interacted with nNOS and regulated serine phosphorylation of nNOS during brain ischemia.
AIM: To investigate whether brain ischemia induces serine phosphorylation of neuronal nitric oxide synthase (nNOS) by Ca2 + / calmodulin-dependent protein kinase II (CaMKII) and the interaction between CaMKIIa and nNOSin rat hippocampus. METHODS: Brain ischemia was induced by bilateral carotid artery occlusion procedure. Phosphorylation and the interaction of proteins were studied by immunoprecipitation and immunoblotting. Weinvestigated during brain ischemia serine phosphorylation and amount of nNOS in crude membranes fraction (P) and cytosolic fraction (S), interaction between CaMKIIa and nNOS, and the effects of 1- [N, O-bis- (5-isoquinolinesulfonyl) -N-methyl-L-tyrosyl] -4-phenylpiperazine (KN-62, a selective inhibitor of CaMKII) on phos-phorylation and the interaction of proteins in P. RESULTS: Serine phosphorylation of nNOS in P increased persis-tently during brain ischemia, and 15 min ischemia-induced serine phosphorylation of nNOS was attenuated signifi-cantly by KN-62. But there was no serine phosphorylation of nNOS in S. The distributions of nNOS were not affected by ischemia and KN-62. However, the binding levels of both CaMKIIa with nNOS and Thr286autophosphorylated CaMKIIa with nNOS increased after ischemia, and were diminished by KN-62. CONCLUSION: CaMKII interacted with nNOS and regulated serine phosphorylation of nNOS during brain ischemia.