Brain ischemia induces serine phosphorylation of neuronal nitric oxide synthase by Ca(2+)/calmodulin-dependent protein kinase II in rat hippocampus.
OBJECTIVE
To investigate whether brain ischemia induces serine phosphorylation of neuronal nitric oxide synthase (nNOS) by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the interaction between CaMKII? and nNOS in 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. We investigated during brain ischemia serine phosphorylation and amount of nNOS in crude membranes fraction (P) and cytosolic fraction (S), interaction between CaMKIIalpha 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 phosphorylation and the interaction of proteins in P.


RESULTS
Serine phosphorylation of nNOS in P increased persistently during brain ischemia, and 15 min ischemia-induced serine phosphorylation of nNOS was attenuated significantly 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 CaMKIIalpha with nNOS and Thr(286) autophosphorylated CaMKIIalpha with nNOS increased after ischemia, and were diminished by KN-62.


CONCLUSIONS
CaMKII interacted with nNOS and regulated serine phosphorylation of nNOS during brain ischemia.
Yan
Xue-bo
XB
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, China.
Meng
Fan-jie
FJ
Song
Bo
B
Zhang
Guang-yi
GY
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002545:Brain Ischemia / Q000201:enzymology* / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006624:Hippocampus / Q000201:enzymology*; D008297:Male; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism*
D012694:Serine; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases