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

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).我们通过使用该离体系统检查了脊髓中NNOS激活与NNOS激活的N/OFQ信号途径。 N/OFQ以时间和剂量依赖性方式通过NADPH-DIAPHORASE组织化学评估，从而增强了脊髓浅表层中的NNOS活性。在3-10 nm处观察到最大效应。 NMDA受体拮抗剂MK-801和D-AP5抑制了N/OFQ刺激的NNOS活性，但不受NR2B选择性拮抗剂的抑制。在NR2D（ - / - ）小鼠中观察到刺激活性，但在NR2A（ - / - ）或NR2A（ - / - ）/NR2D（ - / - ）小鼠中观察到。 N/OFQ受体拮抗剂减弱了NNOS活性，而NNOS的活性则增加了N/OFQ，但不是NMDA。 N/OFQ通过激活含有NR2A的NMDA受体引发的生化级联反应刺激了NNOS活性。为了澄清NO本身是否影响脊髓中的NNOS活性作为逆行使者，我们检查了NO/CGMP信号传导NNOS活性调节中NO/CGMP信号传导途径的参与。没有产生的剂增强了脊髓8-BR-CGMP中的NADPH-肾脏酶染色，也增强了与No-no-no-BR-BR-CAMP和Forskolin（腺苷酸环化酶的激活剂）的相似之处，从而增强了它。没有产生的药物明显增加了脊髓中的CGMP水平。此外，NNOS激活被NMDA拮抗剂MK-801和D-AP5完全抑制，部分由NR2B选择性拮抗剂，并在NR2A（ - / - ）和NR2A（ - / - - - - ）/2D（ - 2D（ - / - ）中减弱。这些结果表明，通过激活含有NR2A和NR2B亚基的NMDA受体，NO可以调节NNOS活性为脊髓中的逆行信使。较少的