吗啡耐受和依赖时第二信使分子和阿片受体变化及与谷氨酸受体的关系

The relationship between changes of some second messengers and opioid receptor as well as activities of glutamate receptor in morphine tolerance and dependence

目的 研究吗啡耐受和依赖时某些第二信使分子和阿片受体变化及与谷氨酸受体的关系。方法 竞争性蛋白结合法、共聚焦显微镜技术和受体结合实验检测 c AMP水平、胞内钙和阿片受体变化。结果 在表达诱导型一氧化氮合酶 c DNA神经细胞中 ,吗啡急性刺激剂量依赖性抑制 c AMP生成 ,慢性用药引起 c AMP代偿性增加。谷氨酸受体拮抗剂 MK80 1增强吗啡的急性抑制效应 ,抑制阿片受体介导 c AMP系统脱敏。对吗啡慢性给药细胞 ,用吗啡刺激和纳络酮戒断引起 [Ca2 + ]i增加 ,阿片受体 Kd值增加和 Bmax值减少 ,MK80 1对这些指标的改变没有影响。结论 MK80 1减轻阿片耐受和依赖的机制可能不是改变阿片受体特性 ,主要与受体后信号转导有关。

Objective To investigate the relationship between changes of some second messengers and opioid receptors,as well as activities of glutemate receptors in morphine tolerance and dependence.Methods Intracellular cAMP content was detected by 3H cAMP protein binding assay.Laser scanning confocal microscopy was applied to monitoring the cytosolic free calcium([Ca 2+ ] i)in Fluo3/AM loaded cells.The changes of opioid receptors were determined by 3H Etorphine binding assay with intact cell.Results Incubation of NG LNCXiNOS neuronal cells expressing iNOS cDNA with morphine (1nmol·L -1 10μmol·L -1 )for 15min dose dependently inhibited forskolin stimulated cAMP accumulation.Chronic treatment of cells with 10μmol·L -1 morphine for 48h resulted in the compensatory increase in cAMP production in a time dependent manner.1μmol·L -1 MK801,a glutamate receptor antagonist,co administrated with morphine,enhanced acute inhibitory effect of morphine and inhibited cAMP system desensitization mediated by opioid receptos,but could not affect the elevation of [Ca 2+ ] i and K d value in the cell pretreated with morphine.Conclusion The mechanisms underlying attenuation of opiate tolerance and dependence by MK801 may not be associated with the opioid receptors and binding affinity,mostly associated with the regulation of post receptor transdution system,which significantly blocks opioid receptor desensitization.