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，通过使用分离的完整脊髓制剂和NADPH-diaphorase组织化学相结合来阐明nNOS激活的生化和分子机制。疼痛肽/孤啡肽FQ （N/OFQ）通过激活神经元一氧化氮合酶（nNOS）参与神经性疼痛的维持。我们利用这种离体系统研究了脊髓中与nNOS激活相关的N/OFQ信号通路。通过NADPH-diaphorase组织化学评估，N/OFQ以时间和剂量依赖性的方式增强脊髓浅层nNOS活性。在3 ~ 10 nM处效果最大。NMDA受体拮抗剂MK-801和D-AP5可抑制N/ ofq刺激的nNOS活性，但nr2b选择性拮抗剂不能抑制nNOS活性；在NR2D（-/-）小鼠中观察到刺激的活性，而在NR2A（-/-）或NR2A(-/-)/NR2D（-/-）小鼠中没有。N/OFQ受体拮抗剂对nNOS活性的抑制作用大于N/OFQ对NMDA的抑制作用。N/OFQ通过激活含有NR2A的NMDA受体引发的生化级联刺激nNOS活性。为了阐明NO本身是否作为逆行信使影响nNOS在脊髓中的活性，我们研究了NO/cGMP信号通路在nNOS活性调控中的作用。no生成剂对脊髓NADPH-diaphorase染色的增强作用与no生成剂类似，8-Br-cAMP和腺苷酸环化酶激活剂forskolin对NADPH-diaphorase染色有中度增强作用。no生成剂显著提高脊髓cGMP水平。此外，NMDA拮抗剂MK-801和d-AP5完全抑制nNOS的激活，nr2b选择性拮抗剂部分抑制nNOS的激活，NR2A（-/-）和NR2A(-/-)/2D（-/-）小鼠的nNOS活性减弱。这些结果表明，NO可能通过激活含有NR2A和NR2B亚基的NMDA受体来调节nNOS在脊髓中的逆行信使活性。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