Sakata Kimimasa, Kondo Takashi, Mizuno Natsumi, Shoji Miki, Yasui Hironobu, Yamamori Tohru, Inanami Osamu, Yokoo Hiroki, Yoshimura Naoki, Hattori Yuichi
Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan; Department of Thoracic and Cardiovascular Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
Vascul Pharmacol. 2015 Jul;70:55-65. doi: 10.1016/j.vph.2015.03.016. Epub 2015 Apr 11.
Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤ 20 Gy) and time-dependent (6-72 h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-L-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other.
血管内皮细胞比体内其他任何组织都能吸收更高剂量的辐射，辐射后内皮型一氧化氮合酶（eNOS）功能受损可能是血管损伤发病机制的一个潜在因素。在本研究中，我们调查了暴露于X射线辐射的人脐静脉内皮细胞（HUVECs）中eNOS信号的早期变化。我们发现，电离辐射以剂量依赖性（≤20 Gy）和时间依赖性（6 - 72小时）的方式增加了HUVECs中eNOS在Ser-1177位点的磷酸化以及在Thr-495位点的去磷酸化。辐射后eNOS的总表达水平没有变化。尽管照射后观察到细胞外信号调节蛋白激酶1/2（ERK1/2）磷酸化短暂但显著增加，以及Akt磷酸化呈双相下降，但这些抑制剂对辐射诱导的eNOS磷酸化变化没有影响。蛋白激酶C-βII（PKC-βII）表达增加，通过小干扰RNA（siRNA）消除PKC-βII可消除辐射对这两个eNOS磷酸化事件的影响。此外，当抗氧化剂N-乙酰-L-半胱氨酸阻止辐射诱导的活性氧（ROS）生成增加时，照射的HUVECs中eNOS的Ser-1177磷酸化和Thr-495去磷酸化显著降低。然而，转染PKC-β siRNA并没有改变照射后的ROS产生，NAC也未能阻断辐射诱导的PKC-βII表达增加。综上所述，我们的结果表明，电离辐射诱导人血管内皮细胞中的eNOS激活归因于PKC-βII的上调和ROS生成的增加，二者相互独立。
