Modification of endothelial NO synthase through protein phosphorylation after forebrain cerebral ischemia/reperfusion

Background and Purpose-Production of NO by endothelial NO synthase (eNOS) is thought to play a neuroprotective role after cerebral ischemia. The vascular endothelial growth factor (VEGF) contributes to activation of eNOS by Ca2+/calmodulin and also stimulates the protein kinase Akt, which directly phosphorylates eNOS on Ser(1177) and increases enzyme activity. Although the expression of VEGF has been studied in ischemic stroke models, the activation of eNOS after cerebral ischemia has not been investigated. The purpose of the present study was to clarify molecular mechanisms underlying the regulation of eNOS activity through protein phosphorylation in postischemic processes. Methods-Sprague-Dawley rats were subjected to forebrain cerebral ischemia for 15 minutes with hypotension and reperfusion for up to 24 hours. Western blot analysis and ELISAs were used to study the temporal profiles of Akt, phospho-Akt at Ser(437), eNOS, phospho-eNOS at Ser(1177), and VEGF expression, respectively. Immunohistochemical studies were performed to examine the spatial expression patterns of phospho-Akt at Ser(437) and phospho-eNOS at Ser(1177). Results-Increase in phospho-Akt at Ser(437) was observed transiently 0.5 to 2 hours after reperfusion, whereas elevation of phospho-eNOS at Ser(1177) and VEGF expression was observed from 6 hours after reperfusion. Endothelial cells in the microvessels were the major source of eNOS phosphorylated at Ser(1177) at the 12-hour time point. Conclusions-Increase in Ser(1177) phospho-eNOS occurs in endothelial cells of microvessels after ischemic episodes with temporal expression of VEGF, pointing to a contribution to the autoregulation of postischemic brain damage.