Rapid S-nitrosylation of actin by NO-generating donors and in inflammatory pain model mice.
Rapid S-nitrosylation of actin by NO-generating donors and in inflammatory pain model mice.
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DOI：
10.1186/1744-8069-7-101
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发表时间：
2011-12-22
期刊：
影响因子：
3.3
通讯作者：
Ito S
中科院分区：
文献类型：
--
作者：
Lu J;Katano T;Uta D;Furue H;Ito S
S-亚硝基化是蛋白质中半胱氨酸残基的可逆翻译后修饰，已成为NO作为信号分子的重要机制。我们最近证明，肌动蛋白是一个主要的S-亚硝基化蛋白在脊髓和建议，NO直接衰减多巴胺从PC 12细胞引起的F-肌动蛋白的分解释放。然而，在动物疼痛模型中，肌动蛋白的S-亚硝基化的发生仍不清楚。在本研究中，肌动蛋白的S-亚硝基化的动力学分析是通过使用NO生成供体。用生物素开关法和链霉亲和素-琼脂糖纯化法鉴定S-亚硝基化肌动蛋白。NOR 1（t1/2 = 1.8 min）可明显抑制PC 1/2细胞多巴胺的释放，NOR 3（t1/2 = 30 min）则抑制作用较小，而内源性NO供体S-亚硝基谷胱甘肽（S-nitroso-glutathione）对多巴胺的释放无明显影响。膜渗透性cGMP类似物不能取代NOR 1作为抑制剂，也不能可溶性鸟苷酸环化酶和cGMP依赖性蛋白激酶的抑制剂减弱抑制。在加入NOR 1后5分钟，通过生物素开关测定检测S-亚硝基化肌动蛋白。与动力学分析一致，在后爪注射2%福尔马林后5分钟，在炎性疼痛模型中，脊髓中的肌动蛋白被快速且最大程度地S-亚硝基化。在体内膜片钳记录的脊髓背角，NOR 3表现出抑制作用的抑制性突触传递中间神经元的胶状质。本研究表明，快速S-亚硝基化的肌动蛋白发生在体外的存在下，在外源性NO产生的捐助者和在体内的炎症疼痛模型小鼠。我们的数据表明，除了众所周知的cGMP依赖性蛋白激酶途径，S-亚硝基化参与疼痛的传递，通过抑制性神经元的去抑制。
S-Nitrosylation, the reversible post-translational modification of reactive cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. We recently demonstrated that actin is a major S-nitrosylated protein in the spinal cord and suggested that NO directly attenuates dopamine release from PC12 cells by causing the breakdown of F-actin. However, the occurrence of S-nitrosylation of actin remained unclarified in animal pain model. Kinetic analysis of S-nitrosylation of actin in the present study was made by using NO-generating donors. The biotin-switch assay and purification on streptavidin-agarose were employed for identification of S-nitrosylated actin. Dopamine release from PC12 cells was markedly attenuated by NOR1 (t1/2 = 1.8 min) and much less by NOR3 (t1/2 = 30 min), but not by S-nitroso-glutathione, an endogenous NO donor. A membrane-permeable cGMP analogue could not substitute for NOR1 as a suppressor nor could inhibitors of soluble guanylate cyclase and cGMP-dependent protein kinase attenuate the suppression. S-Nitrosylated actin was detected by the biotin-switch assay at 5 min after the addition of NOR1. Consistent with the kinetic analysis, actin in the spinal cord was rapidly and maximally S-nitrosylated in an inflammatory pain model at 5 min after the injection of 2% formalin into the hind paws. In vivo patch-clamp recordings of the spinal dorsal horn, NOR3 showed an inhibitory action on inhibitory synaptic transmission in interneurons of the substantia gelatinosa. The present study demonstrates that rapid S-nitrosylation of actin occurred in vitro in the presence of exogenous NO-generating donors and in vivo in inflammatory pain model mice. Our data suggest that, in addition to the well-known cGMP-dependent protein kinase pathway, S-nitrosylation is involved in pain transmission via disinhibition of inhibitory neurons.
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