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

我们建立了一个离体系统来阐明nNOS激活的生物化学和分子机制，通过使用分离的完整脊髓标本和NADPH-黄递酶组织化学的组合，Nociceptin/nociceptin FQ（N/OFQ）早期被证明通过激活神经元型一氧化氮合酶（nNOS）参与维持神经病理性疼痛。我们研究了N/OFQ信号通路耦合到nNOS激活在脊髓中使用这个离体系统。N/OFQ增强nNOS活性在脊髓浅层，由NADPH-黄递酶组织化学，在时间和剂量依赖性的方式进行评估。在3-10 nM下观察到最大效应。NMDA受体拮抗剂MK-801和D-AP 5可抑制N/OFQ刺激的nNOS活性，但NR 2B选择性拮抗剂则无此作用; NR 2D（-/-）小鼠可观察到N/OFQ刺激的nNOS活性，而NR 2A（-/-）或NR 2A（-/-）/NR 2D（-/-）小鼠则无此作用。N/OFQ受体拮抗剂可减弱nNOS活性 关于我们 N/OFQ对NMDA无明显影响。N/OFQ通过激活含有NR 2A的NMDA受体启动的生化级联反应刺激nNOS活性。为了阐明NO本身是否作为逆行信使影响脊髓nNOS活性，我们研究了NO/cGMP信号通路在nNOS活性调节中的作用。NO产生剂增强脊髓NADPH-黄递酶染色，8-Br-cGMP也增强它类似于NO产生剂，和8-Br-cAMP和forskolin，腺苷酸环化酶的激活剂，适度增强它。NO产生剂显著增加脊髓cGMP水平。NMDA拮抗剂MK-801和d-AP 5可完全抑制nNOS的激活，NR 2B选择性拮抗剂可部分抑制nNOS的激活，NR 2A（-/-）和NR 2A（-/-）/2D（-/-）小鼠的nNOS激活减弱。提示NO可能通过激活含有NR 2A和NR 2B亚基的NMDA受体，作为脊髓内的逆行信使调节nNOS活性。少

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