Increased ICP promotes CaMKII-mediated phosphorylation of neuronal NOS at Ser847 in the hippocampus immediately after subarachnoid hemorrhage

Early brain injury has recently been identified as an indicator of poor prognosis after subarachnoid hemorrhage (SAH). Calmodulin-dependent protein kinase II alpha (CaMKII alpha) has been shown to phosphorylate neuronal NOS (nNOS) at Ser(847), resulting in a reduction in nNOS activity. In this study, we revealed chronological changes in the phosphorylation of nNOS at Ser(847) in the hippocampus and cortex immediately after SAH. In a rat single-hemorrhage model of SAM, the hippocampus and adjacent cortex were collected up to 24 h after SAM. Samples from rats that were not injected with blood were used as controls. NOS was partially purified from the crude samples using ADP-agarose affinity chromatography. Western blot analysis revealed that nNOS phosphorylated (p-nNOS) at Ser(847) was significantly increased in the hippocampus, but not in the cortex, at 1 h after SAH compared with that resulting from the control treatment. Immunoreactivity of p-nNOS at Ser(847) was observed in intemeurons of the hippocampus at 1 h after SAH. Injection of saline instead of blood also significantly induced p-nNOS at Ser(847) levels in the hippocampus at 1 h after injection. The colocalization of CaMKII alpha and nNOS was transiently increased in the hippocampus at 0.5 h after SAM. Our data suggest that immediately after SAH, an increase in intracranial pressure might induce transient cerebral ischemia, potentially promoting the phosphorylation of nNOS at Ser(847) by CaMKIIa in the hippocampus. The activation of p-nNOS at Ser(847) in the hippocampus may alleviate ischemic insults immediately after SAM to exert a neuroprotective effect against early brain injury. (C) 2015 Elsevier B.V. All rights reserved.