在高浓度过氧化氢作用下，苏氨酸495位点的内皮型一氧化氮合酶磷酸化及线粒体活性氧生成。

Endothelial nitric oxide synthase phosphorylation at Threonine 495 and mitochondrial reactive oxygen species formation in response to a high H₂O₂ concentration.

作者信息

Guterbaum T J, Braunstein T H, Fossum A, Holstein-Rathlou N-H, Torp-Pedersen C T, Domínguez H

机构信息

The Danish National Research Foundation Center for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark.

出版信息

J Vasc Res. 2013;50(5):410-20. doi: 10.1159/000354225. Epub 2013 Sep 3.

DOI:10.1159/000354225

PMID:24008236

Abstract

BACKGROUND

Hydrogen peroxide (H₂O₂) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂.

METHODS

HUVECs were exposed to 400 μM H₂O₂ for 30 min. Phosphorylation at Thr(495) was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-β inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632.

RESULTS

H₂O₂ increased eNOS phosphorylation at Thr(495) (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr(495).

CONCLUSION

H₂O₂-mediated phosphorylation of eNOS Thr(495) is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.

摘要

背景

过氧化氢（H₂O₂）在缺血/再灌注期间以及炎症过程中于血管内产生，二者均会导致血管功能障碍。我们研究了人脐静脉内皮细胞（HUVECs）暴露于H₂O₂时，参与内皮型一氧化氮合酶（eNOS）第495位苏氨酸（Thr(495)）磷酸化的细胞途径。

方法

将HUVECs暴露于400 μM H₂O₂中30分钟。通过蛋白质印迹法评估Thr(495)的磷酸化情况，并通过流式细胞术监测活性氧（ROS）。通过用蛋白激酶C（PKC）-β抑制剂鲁布前列素或泛PKC抑制剂GF109203X预处理来研究PKC途径。此外，我们通过MEKK1/2抑制剂U0126和ROCK抑制剂Y27632研究了ROCK和ERK途径。

结果

H₂O₂使Thr(495)处的eNOS磷酸化增加（与对照（100%）相比增至176%，p < 0.001），同时线粒体ROS生成增加（从19.7%增至45.3%，p < 0.01）。U0126和Y27632可以剂量依赖性方式阻止这种磷酸化增加，但不会导致线粒体ROS生成降低。相反，添加抗氧化剂N-乙酰-L-半胱氨酸仅能阻止线粒体ROS生成，但不能阻止eNOS Thr(495)的磷酸化。