Ihara Hideshi, Kitamura Atsushi, Kasamatsu Shingo, Ida Tomoaki, Kakihana Yuki, Tsutsuki Hiroyasu, Sawa Tomohiro, Watanabe Yasuo, Akaike Takaaki
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Biochem J. 2017 Mar 15;474(7):1149-1162. doi: 10.1042/BCJ20160999.
We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3',5'-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.
我们先前已证明神经元型一氧化氮（NO）合酶（nNOS）剪接变体nNOS-µ和nNOS-α在大脑中具有不同的空间表达谱；然而，它们的确切功能尚未完全明确。在此，我们使用电子顺磁共振来比较重组nNOS-µ和nNOS-α产生活性氧（ROS，在此情况下为超氧化物）的电子解偶联反应。nNOS-µ产生的超氧化物量为nNOS-α产生量的44%。我们还通过研究钙离子载体A23187诱导的这些电子解偶联反应，评估了稳定表达nNOS-α和nNOS-µ的HEK293细胞中的ROS生成情况。A23187处理在表达nNOS-α的HEK293细胞中诱导产生的ROS比表达nNOS-µ的细胞更多。此外，免疫细胞化学分析显示，经A23187处理的表达nNOS-α的细胞比表达nNOS-µ的细胞产生更多的8-硝基鸟苷3',5'-环一磷酸，其为NO/ROS氧化还原信号传导中的第二信使。分子进化分析显示，nNOS-µ特异性区域的非同义位点与同义位点的比率高于完整基因，这表明该区域的功能限制比完整基因少。这些观察结果揭示了nNOS-µ变体的生理相关性，并可能增进对nNOS依赖性NO/ROS氧化还原信号传导及其在神经系统中的病理生理后果的理解。
