title: Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells.
abstract: 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.
authors: 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.
authors: Kimimasa
authors: K
authors: Sakata
authors: Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
authors: Takashi
authors: T
authors: Kondo
authors: Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
authors: Natsumi
authors: N
authors: Mizuno
authors: Life Science Research Center, University of Toyama, Toyama 930-0194, Japan.
authors: Miki
authors: M
authors: Shoji
authors: Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors: Hironobu
authors: H
authors: Yasui
authors: Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors: Tohru
authors: T
authors: Yamamori
authors: Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors: Osamu
authors: O
authors: Inanami
authors: Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
authors: Hiroki
authors: H
authors: Yokoo
authors: Department of Thoracic and Cardiovascular Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan.
authors: Naoki
authors: N
authors: Yoshimura
authors: Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan. Electronic address: yhattori@med.u-toyama.ac.jp.
authors: Yuichi
authors: Y
authors: Hattori
mesh_terms: D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000528:radiation effects*; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D064546:Protein Kinase C beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D000975:Antioxidants; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; C579054:PRKCB protein, human; D064546:Protein Kinase C beta; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Human endothelial cells; Protein kinase C; Reactive oxygen species; X-ray radiation; eNOS