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

INTRODUCTION:We previously demonstrated that cyclic AMP-dependent protein kinase 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), and 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 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 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.

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