Department of Medical Chemistry, Kansai Medical University, Moriguchi, Japan.
Mol Pain. 2009 Sep 29;5:58. doi: 10.1186/1744-8069-5-58.
The role of the diffusible messenger nitric oxide (NO) in the regulation of pain transmission is still a debate of matter, pro-nociceptive and/or anti-nociceptive. S-Nitrosylation, the reversible post-translational modification of selective cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. The occurrence of S-nitrosylation in the spinal cord and its targets that may modulate pain transmission remain unclarified. The "biotin-switch" method and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were employed for identifying S-nitrosylated proteins.
Here we show that actin was a major protein S-nitrosylated in the spinal cord by the NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP). Interestingly, actin was S-nitrosylated, more in the S2 fraction than in the P2 fraction of the spinal homogenate. Treatment of PC12 cells with SNAP caused rapid S-nitrosylation of actin and inhibited dopamine release from the cells. Just like cytochalasin B, which depolymerizes actin, SNAP decreased the amount of filamentous actin cytoskeleton just beneath the membrane. The inhibition of dopamine release was not attenuated by inhibitors of soluble guanylyl cyclase and cGMP-dependent protein kinase.
The present study demonstrates that actin is a major S-nitrosylated protein in the spinal cord and suggests that NO directly regulates neurotransmitter release by S-nitrosylation in addition to the well-known phosphorylation by cGMP-dependent protein kinase.
扩散信使一氧化氮（NO）在疼痛传递调节中的作用仍存在争议，具有致痛和/或抗痛作用。S-亚硝基化，即蛋白质中选择性半胱氨酸残基的可逆翻译后修饰，已成为 NO 作为信号分子发挥作用的重要机制。脊髓中 S-亚硝基化的发生及其可能调节疼痛传递的靶标仍不清楚。“生物素开关”法和基质辅助激光解吸/电离飞行时间质谱法被用于鉴定 S-亚硝基化蛋白。
在这里，我们展示了一氧化氮供体 S-亚硝基-N-乙酰-DL-青霉胺（SNAP）可使脊髓中的肌动蛋白发生主要的蛋白质 S-亚硝基化。有趣的是，肌动蛋白在脊髓匀浆的 S2 部分比 P2 部分更容易发生 S-亚硝基化。PC12 细胞用 SNAP 处理会导致肌动蛋白快速 S-亚硝基化，并抑制多巴胺从细胞中释放。与细胞松弛素 B 一样，细胞松弛素 B 可使肌动蛋白解聚，SNAP 也会减少细胞膜下丝状肌动蛋白细胞骨架的数量。多巴胺释放的抑制作用不受可溶性鸟苷酸环化酶和 cGMP 依赖性蛋白激酶抑制剂的减弱。
本研究表明肌动蛋白是脊髓中主要的 S-亚硝基化蛋白，并表明 NO 通过 S-亚硝基化直接调节神经递质释放，除了众所周知的 cGMP 依赖性蛋白激酶磷酸化作用。
