A fundamental study for application of nitric oxide to a diagnosis and treatments of the chronic pain

一氧化氮在慢性疼痛诊断和治疗中应用的基础研究

基本信息

批准号：
18613023
负责人：
ABE Tetsuya
金额：
$ 2.5万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18613023/
关键词：
nitric oxide neuropathic pain primary afferent NMDA receptor spinal cord enzymohistochemistry Imaging mice 神経因性痺痛ノシセプチン

项目摘要

We established an ex vivo system to elucidate biochemical and molecular mechanisms for nNOS activation by the use of a combination of isolated intact spinal cord preparations and NADPH-diaphorase histochemistry.Nociceptin/orphanin FQ (N/OFQ) was earlier shown to be involved in the maintenance of neuropathic pain by activating neuronal nitric oxide synthase (nNOS). We examined the N/OFQ signal pathways coupled to nNOS activation in the spinal cord by using this ex vivo system. N/OFQ enhanced nNOS activity in the superficial layer of the spinal cord, as assessed by NADPH-diaphorase histochemistry, in a time- and dose-dependent manner. The maximum effect was observed at 3-10 nM. The N/OFQ-stimulated nNOS activity was inhibited by NMDA receptor antagonists MK-801 and D-AP5, but not by the NR2B-selective antagonist; and the stimulated activity was observed in NR2D(-/-) mice, but not in NR2A(-/-) or NR2A(-/-)/NR2D(-/-) mice. N/OFQ receptor antagonists attenuated the nNOS activity stimulated … More by N/OFQ, but not that by NMDA. N/OFQ stimulated nNOS activity by a biochemical cascade initiated by activation of NMDA receptors containing NR2A.To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity. NO-generating agents enhanced NADPH-diaphorase staining in the spinal cord 8-Br-cGMP also enhanced it similar to that by NO-generating agents, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NO-generating agents markedly increased the cGMP level in the spinal cord. Additionally, the nNOS activation was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the NR2B-selective antagonist, and was attenuated in NR2A(-/-) and NR2A(-/-)/2D(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing NR2A and NR2B subunits. Less

我们建立了一个离体系统，通过使用分离的完整脊髓制剂和 NADPH-心肌黄酶组织化学的组合来阐明 nNOS 激活的生化和分子机制。痛敏肽/孤啡肽 FQ (N/OFQ) 较早被证明参与通过激活神经元一氧化氮合酶 (nNOS) 维持神经性疼痛我们检查了与脊柱中 nNOS 激活相关的 N/OFQ 信号通路。通过使用这种离体系统，N/OFQ 增强了脊髓表层的 nNOS 活性，通过 NADPH-心肌黄酶组织化学评估，以时间和剂量依赖性方式观察到最大效果。 N/OFQ 刺激的 nNOS 活性被 NMDA 受体拮抗剂 MK-801 和 D-AP5 抑制，但不被 NR2B 选择性拮抗剂和在 NR2D(-/-) 小鼠中观察到刺激的活性，但在 NR2A(-/-) 或 NR2A(-/-)/NR2D(-/-) 小鼠中观察到 N/OFQ 受体拮抗剂减弱了刺激的 nNOS 活性。 N/OFQ 刺激 nNOS 活性，N/OFQ 则不通过 NMDA 受体激活引发的生化级联刺激 nNOS 活性。 NR2A.为了阐明NO是否作为逆行信使影响脊髓中的nNOS活性，我们检查了NO/cGMP信号通路在调节nNOS活性中的参与，NO生成剂增强了脊髓中的NADPH-心肌黄酶染色。 8-Br-cGMP 也增强了它，类似于 NO 生成剂，8-Br-cAMP 和毛喉素（腺苷酸环化酶的激活剂）增强了它NO生成剂显着增加了脊髓中的cGMP水平，此外，nNOS激活被NMDA拮抗剂MK-801和d-AP5完全抑制，部分被NR2B选择性拮抗剂抑制，并且在NR2A(-)中被减弱。 /-) 和 NR2A(-/-)/2D(-/-) 小鼠这些结果表明 NO 可能作为逆行信使调节 nNOS 活性。脊髓通过激活含有 NR2A 和 NR2B 亚基的 NMDA 受体。