一氧化氮合酶抑制剂阴断可片耐受和依赖的信号转导途径

目的 探讨一氧化氮合酶（ＮＯＳ）抑制剂Ｎ＾Ｇ－硝基－Ｌ－精氨酸（Ｌ－ＮＮＡ）对阿片耐受和依赖机制中腺苷酸环化酶－环磷酸腺苷（ＡＣ－ｃＡＭＰ）系统、Ｃａ＾２＋系统和一氧化氮－环磷酸鸟苷（ＮＯ－ｍＧＭＰ）系统变化的影响。方法 实验共分为对照组，阿片激动剂组，阿片激动剂组，阿片激动剂＋纳洛酮组；Ｌ－ＮＮＡ＋阿片激动剂组和Ｌ－ＮＮＡ＋阿片激动剂＋纳洛酮组。采用竞争性蛋白结合试验，＾３Ｈ－精氨酸转化成＾３Ｈ

Effect of nitric oxide synthase inhibitor on signal transduction pathway of opiate tolerance and dependence in NG108-15 cells expressing iNOS gene]

OBJECTIVE: To investigate the role of adenylate cyclase (AC)-cAMP system and Ca2+ system and NO-cGMP signal system and the effects of a NOS inhibitor, NG-nitro-L-arginine (L-NNA) in the neuronal mechanisms of opioid tolerance and dependence. METHODS: The experiments were performed in five groups: control group; opioid agonist group; opioid agonist + nalonoxe group; L-NNA + opioid agonist group and L-NNA + opioid agonist + nalonoxe group. The intracellular cAMP and cGMP levels were measured by 3H-cAMP protein binding assay and 3H-cGMP radioimmunoassay, respectively. NOS activity was determined by the conversion of 3H-arginine to 3H-citrulline. The change of Ca2+]i was studied by the laser scanning confocal microscopy technique. iNOS protein expression was detected using immunohistochemistry with monoclonal antibody of iNOS, and imaging analysis was performed. RESULTS: Long-term administration of high-selective delta-opioid receptor agonist DPDPE and precipitation of opioid withdrawal by naloxone significantly induced increase of cAMP level and Ca2+]i in NG-LNCXiNOS cells with stable expression of iNOS gene. The cytosolic iNOS activity and cGMP generation were enhanced by DPDPE dose-dependently. 10(-4) mol/L L-NNA could block opioid agonist-induced AC-cAMP desensitization and activity of NO-cGMP second messenger pathway, but it could not reduce opioid-induced elevation of Ca2+]i. Furthermore, L-NNA decreased iNOS-specific protein expression in DPDPE-induced tolerance and naloxone-precipited withdrawal cells. CONCLUSION: NOS inhibitor may attenuate the development of opioid tolerance and withdrawal via the negative regulation of AC-cAMP system and NO-cGMP system. It can be clinically used to prevent opiate tolerance and addiction.