Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710, South Korea.
Biochem Biophys Res Commun. 2013 Jul 12;436(4):601-6. doi: 10.1016/j.bbrc.2013.06.003. Epub 2013 Jun 10.
Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function.
远红外（FIR）辐射的反复热疗可改善冠心病患者和小鼠模型的血管功能，但其潜在机制尚不完全清楚。本研究采用 FIR 作为热疗剂，研究其对内皮型一氧化氮合酶（eNOS）活性和一氧化氮（NO）产生的影响的分子机制。FIR 辐射可在时间依赖性方式（高达 40min 的 FIR 辐射）下增加牛主动脉内皮细胞（BAEC）中 eNOS 丝氨酸 1179 位的磷酸化（eNOS-Ser(1179)），而不改变 eNOS 的表达。这一增加伴随着 NO 产生和细胞内 Ca(2+)水平的增加。Ca(2+)/钙调蛋白依赖性蛋白激酶 II（CaMKII）的选择性抑制剂 KN-93 和蛋白激酶 A 抑制剂 H-89 处理可抑制 FIR 辐射刺激的 eNOS-Ser(1179)磷酸化。FIR 辐射本身也会增加培养基的温度。由于瞬时受体电位香草酸（TRPV）离子通道已知是温度敏感的钙通道，因此我们探讨 TRPV 通道是否介导这些观察到的效应。逆转录-PCR 分析显示 BAEC 中有两种 TRPV 同工型，TRPV2 和 TRPV4。尽管钌红（pan-TRPV 抑制剂）完全逆转了 FIR 辐射的观察到的作用，但在用 TRPV2 抑制剂 Tranilast 处理的细胞中发现了部分衰减（约 20%）。然而，TRPV2 的 siRNA 转染没有明显改变 FIR 辐射刺激的 eNOS-Ser(1179)磷酸化。本研究表明，FIR 辐射通过增加 CaMKII 介导的 eNOS-Ser(1179)磷酸化来增加 NO 产生，但 TRPV 通道可能不参与这一途径。我们的结果可能为 FIR 辐射改善内皮功能的分子机制提供了依据。
