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

•Subarachnoid hemorrhage (SAH) induces cerebral ischemia immediately after onset.•Transient cerebral ischemia activates CaMKII in the hippocampus.•Activated CaMKII phosphorylates neuronal NOS at Ser847.•Neuronal NOS phosphorylation at Ser847 decreases its enzymatic activity.•This signal transduction attenuates early brain injury in the hippocampus after SAH.

Early brain injury has recently been identified as an indicator of poor prognosis after subarachnoid hemorrhage (SAH). Calmodulin-dependent protein kinase IIα (CaMKIIα) has been shown to phosphorylate neuronal NOS (nNOS) at Ser847, resulting in a reduction in nNOS activity. In this study, we revealed chronological changes in the phosphorylation of nNOS at Ser847 in the hippocampus and cortex immediately after SAH. In a rat single-hemorrhage model of SAH, the hippocampus and adjacent cortex were collected up to 24 h after SAH. 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 Ser847 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 Ser847 was observed in interneurons of the hippocampus at 1 h after SAH. Injection of saline instead of blood also significantly induced p-nNOS at Ser847 levels in the hippocampus at 1 h after injection. The colocalization of CaMKIIα and nNOS was transiently increased in the hippocampus at 0.5 h after SAH. Our data suggest that immediately after SAH, an increase in intracranial pressure might induce transient cerebral ischemia, potentially promoting the phosphorylation of nNOS at Ser847 by CaMKIIα in the hippocampus. The activation of p-nNOS at Ser847 in the hippocampus may alleviate ischemic insults immediately after SAH to exert a neuroprotective effect against early brain injury.