*Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
†Department of Pharmacology, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
Biochem J. 2014 Apr 15;459(2):251-63. doi: 10.1042/BJ20131262.
Phosphorylation is considered a main mechanism modulating nNOS (neuronal nitric oxide synthase) function to reduce NO production. In the present study, the effects of nNOS phosphorylation on redox signalling, including that of NO, ROS (reactive oxygen species), and 8-nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate), a downstream messenger of redox signalling, were investigated. In vitro experiments revealed that a phosphorylation-mimic mutant of nNOS (Ser847 replaced with aspartic acid, 847D) increased uncoupling to produce a superoxide. In addition, nicotine, which triggers an influx of Ca2+, induced more ROS and 8-nitro-cGMP production in 847D-expressing PC12 cells than WT (wild-type)-expressing cells. Additionally, nicotine-induced phosphorylation of nNOS at Ser847 and increased ROS and 8-nitro-cGMP production in rat CGNs (cerebellar granule neurons). In CGNs, the NOS (nitric oxide synthase) inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and superoxide dismutase completely inhibited ROS and 8-nitro-cGMP production, whereas the CaMK (Ca2+/calmodulin-dependent protein kinase) inhibitor KN93 mildly reduced this effect. Nicotine induced HO-1 (haem oxygenase 1) expression in CGNs and showed cytoprotective effects against apoptosis. Moreover, 8-nitro-cGMP treatment showed identical effects that were attenuated by KN93 pre-treatment. The present paper provides the first substantial corroboration for the biological effects of nNOS phosphorylation at Ser847 on redox signalling, including ROS and intracellular 8-nitro-cGMP generation in neurons, which possibly play roles in neuroprotection.
磷酸化被认为是调节 nNOS（神经元型一氧化氮合酶）功能以减少 NO 产生的主要机制。在本研究中，研究了 nNOS 磷酸化对氧化还原信号转导的影响，包括 NO、ROS（活性氧）和 8-硝基-cGMP（氧化还原信号转导的下游信使）。体外实验表明，nNOS 的磷酸化模拟突变体（Ser847 被天冬氨酸取代，847D）增加解偶联以产生超氧阴离子。此外，尼古丁触发 Ca2+内流，在 847D 表达的 PC12 细胞中比 WT（野生型）表达的细胞诱导更多的 ROS 和 8-硝基-cGMP 产生。此外，尼古丁诱导 nNOS 在 Ser847 处磷酸化，并增加大鼠 CGNs（小脑颗粒神经元）中的 ROS 和 8-硝基-cGMP 产生。在 CGNs 中，NOS（一氧化氮合酶）抑制剂 L-NAME（NG-硝基-L-精氨酸甲酯）和超氧化物歧化酶完全抑制 ROS 和 8-硝基-cGMP 的产生，而 CaMK（钙/钙调蛋白依赖性蛋白激酶）抑制剂 KN93 轻度降低了这种作用。尼古丁诱导 CGNs 中的 HO-1（血红素加氧酶 1）表达，并表现出对细胞凋亡的保护作用。此外，8-硝基-cGMP 处理显示出与 KN93 预处理减弱的相同效果。本文首次提供了实质性证据，证明 nNOS 在 Ser847 处的磷酸化对氧化还原信号转导（包括神经元中 ROS 和细胞内 8-硝基-cGMP 的产生）具有生物学效应，这可能在神经保护中发挥作用。
