Subarachnoid hemorrhage induces neuronal nitric oxide synthase phosphorylation at Ser1412 in the dentate gyrus of the rat brain.

ABSTRACT

INTRODUCTION: We previously demonstrated that cyclic AMP-dependent protein kinase (PKA) phosphorylates neuronal nitric oxide synthase (nNOS) at Ser1412 in the hippocampal dentate gyrus after forebrain ischemia; this phosphorylation event activates NOS activity and might contribute to depression after cerebral ischemia. In this study, we revealed chronological and topographical changes in the phosphorylation of nNOS at Ser1412 immediately after subarachnoid hemorrhage (SAH). METHODS: 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 autologous blood were used as controls. NOS was partially purified from crude samples via an ADP-agarose gel. Levels of nNOS, nNOS phosphorylated at Ser1412 (p-nNOS), PKA, and p-PKA at Thr197 were studied in the rat hippocampus and cortex using Western blot analyses and immunohistochemistry. RESULTS: According to the Western blot analysis, levels of p-nNOS at Ser1412 were significantly increased in the hippocampus, but not in the cortex, between 1 and 3 h after SAH. Immunohistochemistry revealed the phosphorylation of nNOS at Ser1412 and PKA at Thr197 in the dentate gyrus, but not in the CA1 area, 1 h after SAH. An injection of saline instead of blood also significantly increased levels of p-nNOS at Ser1412 in the hippocampus 1 h after the injection. CONCLUSIONS: An immediate increase in intracranial pressure (ICP) might induce transient cerebral ischemia and promote the PKA-mediated phosphorylation of nNOS at Ser1412 in the dentate gyrus. This signal transduction pathway induces the excessive production of nitric oxide (NO) and might be involved in cognitive dysfunction after SAH.