Modification of endothelial NO synthase through protein phosphorylation after forebrain cerebral ischemia/reperfusion.
OBJECTIVE
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 Ser1177 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 Ser437, eNOS, phospho-eNOS at Ser1177, and VEGF expression, respectively. Immunohistochemical studies were performed to examine the spatial expression patterns of phospho-Akt at Ser437 and phospho-eNOS at Ser1177.


RESULTS
Increase in phospho-Akt at Ser437 was observed transiently 0.5 to 2 hours after reperfusion, whereas elevation of phospho-eNOS at Ser1177 and VEGF expression was observed from 6 hours after reperfusion. Endothelial cells in the microvessels were the major source of eNOS phosphorylated at Ser1177 at the 12-hour time point.


CONCLUSIONS
Increase in Ser1177 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.
Osuka
Koji
K
Department of Neurosurgery, Nagoya University Graduate School of Medicine, Japan.
Watanabe
Yasuo
Y
Usuda
Nobuteru
N
Nakazawa
Ayami
A
Tokuda
Masaaki
M
Yoshida
Jun
J
D000818:Animals; D001921:Brain / Q000201:enzymology; D002545:Brain Ischemia / Q000201:enzymology*; D006706:Homeostasis; D008297:Male; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D042461:Vascular Endothelial Growth Factor A; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D011505:Protein-Tyrosine Kinases