Phosphorylation of neuronal nitric oxide synthase at Ser1412 in the dentate gyrus of rat brain after transient forebrain ischemia

Abstract

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser847 in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser1412in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser1412 after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24h, after 15min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser1412, PKA, and p-PKA at Thr197 were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser1412 significantly increased between 1 and 6h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser1412 and PKA at Thr197 occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr197, which then phosphorylates nNOS at Ser1412 in the subgranular layer of the dentate gyrus.