Reactive nitrogen species in shear-dependent signaling

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Laminar shear stress protects arterial wall from the development of atherosclerotic plaques by controlling the structure and function of endothelial cells. The anti-atherogenic effects of laminar shear involve production of vasoactive factors such as NO, reactive oxygen species (ROS) and reactive nitrogen species (RNS) in endothelial cells. However, the mechanisms 1) by which laminar shear stimulates production of NO from eNOS, ROS from NADPH oxidases, and their reaction products, RNS, and 2) by which these free radicals mediate the anti-atherogenic effects of laminar shear are not known. As detailed in the Preliminary results, our recent results strongly suggest that shear stress regulates eNOS activity by an unexpected mechanisms. Unlike the previous assumption that protein kinase B(Akt) would be the protein kinase responsible for phosphorylation of eNOS at Ser1179 resi8due, we show that other kinases such as protein kinase A is responsible for phosphorylation of eNOS-Ser1179. Considering the presence of multiple potential phosphorylation sites found in eNOS, it is important to res examine the detailed mechanisms by which phosphorylation/dephosphorylation regulate eNOS in response to shear. To address these questions, we have developed mouse aortic endothelial cells from normal and several knockout (KO) mice deficient in eNOS and NADPH oxidase components (gp 91 and p47). Using these endothelial cells as well as the eNOS-KO cells transfected with mutated eNOS constructs, we will examines the hypotheses 1) that laminar shear stimulates NO production by controlling by phosphorylation of key regulatory sites of eNOS by the phosphatidyl-3-kinase and protein kinase A-dependent mechanisms, and 2) that shear prevents endothelial cell death induced by pro-atherogenic factors by the NO, ROS, and RNS- dependent mechanisms. These hypotheses will be examined by following the four aims: Aim 1: Determine the signaling pathways regulating shear- dependent phosphorylation and activation of eNOS. Aim 2: Determine the effect of laminar shear on RNS productions. Aim 4: Define the role of NO and RNS in laminar shear-dependent, anti-atherogenic responses of endothelial cells.