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

我们建立了一个体外系统，通过结合分离的完整脊髓标本和NADPH-黄递酶组织化学来阐明nNOS激活的生化和分子机制。Nociceptin/Oranin FQ(N/OFQ)早期被证明通过激活神经元型一氧化氮合酶(NNOS)参与神经病理性疼痛的维持。我们利用这个体外实验系统研究了脊髓中与nNOS激活相关的N/OFQ信号通路。NADPH-黄递酶组织化学显示，N/OFQ以时间和剂量依赖的方式增强脊髓浅层nNOS活性。在3-10 nm处效果最好。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其活性，NR2D(-/-)小鼠有此激活作用，而NR2A(-/-)或NR2A(-/-)/NR2D(-/-)小鼠则无此激活作用。N/OFQ受体拮抗剂减弱…刺激的一氧化氮合酶活性更多的是N/OFQ，而不是NMDA。N/OFQ通过激活含有NR2a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生成剂增强脊髓NADPH-黄递酶的染色，8-BR-cGMP与NO生成剂相似，8-BR-cAMP和腺苷环化酶激活剂Forsklin适度增强脊髓NADPH-黄递酶的表达。NO生成剂可显著增加脊髓组织中cGMP水平。NMDA拮抗剂MK-801和d-AP5可部分抑制NR2B选择性拮抗剂对nNOS的激活，而NR2A(-/-)和NR2A(-/-)/2D(-/-)小鼠的nNOS活性则明显减弱。这些结果提示，NO可能通过激活含有NR2A和NR2B亚单位的NMDA受体，作为逆行信使调节脊髓内nNOS的活性。较少

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