一氧化氮合酶抑制剂阻断阿片耐受和依赖的信号转导途径

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

目的探讨一氧化氮合酶（NOS）抑制剂NG-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、Ca2+系统和一氧化氮-环磷酸鸟苷（NO-cGMP）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+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-胍氨酸方法，放射免疫测定和激光共聚焦显微镜技术，测定cAMP含量，NOS活性，cGMP生成量和[Ca2+]i水平。免疫组织化学检测iNOS蛋白表达。结果稳定表达iNOS基因的NG-LNCXiNOS细胞在高选择性δ-受体激动剂δ-阿片受体高选择性激动剂（DPDPE）长时程作用及纳洛酮戒断时，胞内cAMP水平和[Ca2+]i增加，胞浆相iNOS活性和cGMP生成增多，与DPDPE预处理剂量成正比。10-4mol/LL-NNA能降低吗啡、DPDPE、DADLE诱发的cAMP、iNOS活性和蛋白及cGMP的升高，但不能降低[Ca2+]i。结论NOS抑制剂延缓阿片耐受和依赖的发生，可能是负调控AC-cAMP系统和NO-cGMP系统，为临床应用NOS抑制剂防治阿片耐受和成瘾提供了实验依据。

Objective To investigate the role of adenylate crclase (AC)-cAMP system and Ca2+ system and NG-cGMP sled 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:coned group; opioid agonist group; opioid agonist + nalonoxe group; LNNA + opioid agonist group and L-NNA + opioid agonist + nalonoxe group. The intracellular cALMP and cGMP levels were measured by 3 H-cAMP protein binding assay and s H-cGMP radioimmunoassay , respectively. NOS activity was determined by the conversion of 3 H-arginine to 3 H-citrulline. The change of [ Ca2+ ] i was studied by the laser scanning confocal microscopy thchnique . iNOS protein expression was detected using immunohistochemistry with monoclonal antibody of iNOS,and imaging analysis was performed. Results lung-term administration of high-selective δ-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 AG-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-spectific protein expession in DPDPE-induced tolerance and naloxone-prmipited withdrawal cells. Conclusion NOS inhibitor may attenuate the development of opioid tolerance and withthawal via the negative regulation of AG-cAMP system and NG-cGMP system. It can be clinically used to prevent opiate tolerance and addiction.