Published Online
on September 16, 2004

A more recent version of this article appeared on November 1, 2004

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Submitted on May 14, 2004
Revised on July 6, 2004
Accepted on August 3, 2004

Modification of Endothelial NO Synthase Through Protein Phosphorylation After Forebrain Cerebral Ischemia/Reperfusion

Koji Osuka MD; Yasuo Watanabe MD, PhD^*; Nobuteru Usuda MD, PhD; Ayami Nakazawa PhD; Masaaki Tokuda MD, PhD; and Jun Yoshida MD, PhD

From the Department of Neurosurgery (K.O., J.Y.), Nagoya University Graduate School of Medicine, Japan; the Department of Cell Physiology (Y.W., M.T.), Kagawa University Faculty of Medicine, Japan; and the Department of Anatomy II (N.U., A.N.), Fujita Health University School of Medicine, Aichi, Japan.

^* To whom correspondence should be addressed. E-mail: yasuwata{at}kms.ac.jp.

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 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.

Key words: cerebral blood flow • nitric oxide • stroke

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