Xu Li, Mabuchi Tamaki, Katano Tayo, Matsumura Shinji, Okuda-Ashitaka Emiko, Sakimura Kenji, Mishina Masayoshi, Ito Seiji
Department of Medical Chemistry, Kansai Medical University, 10-15 Fumizono, Moriguchi 570-8506, Japan.
Nitric Oxide. 2007 Aug;17(1):18-24. doi: 10.1016/j.niox.2007.04.004. Epub 2007 May 5.
We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. 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 by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.
我们最近证实，脊髓中神经元型一氧化氮合酶（nNOS）产生的一氧化氮（NO）参与了神经性疼痛的维持。为了阐明NO本身是否作为逆行信使影响脊髓中的nNOS活性，我们通过NADPH-黄递酶组织化学研究了NO/cGMP信号通路在nNOS活性调节中的作用。产生NO的药物NOR3（半衰期=30分钟）和SNAP（半衰期=5小时），而不是NOR1（半衰期=1.8分钟），显著增强了脊髓中的NADPH-黄递酶染色。8-溴-cGMP也能像NOR3一样增强染色，8-溴-cAMP和腺苷酸环化酶激活剂福斯可林则适度增强染色。NOR1和NOR3显著提高了脊髓中的cGMP水平。NO清除剂CPTIO、可溶性鸟苷酸环化酶抑制剂ODQ和cGMP依赖性蛋白激酶抑制剂KT5823显著抑制了NOR3对NADPH-黄递酶染色的增强作用。此外，NMDA拮抗剂MK-801和d-AP5完全抑制了NOR3增强的nNOS活性，GluRepsilon2选择性拮抗剂CP-101,606部分抑制，在GluRepsilon1（-/-）和GluRepsilon1（-/-）/epsilon4（-/-）小鼠中减弱。这些结果表明，NO可能通过激活含有GluRepsilon1和GluRepsilon2亚基的NMDA受体，作为脊髓中的逆行信使调节nNOS活性。
