【摘要】 目的：利用稳定表达诱导型ＮＯ合酶的ＮＧＬＮＣＸｉＮＯＳ细胞，观察阿片类药物对受体介导ＡＣｃＡＭＰ系统的影响。方法：竞争性蛋白结合法和完整细胞受体结合实验测定ｃＡＭＰ含量和受体结合力。结果：阿片类药物短时程作用抑制ＡＣ活性，受阿片受体介导的、百日咳毒素敏感的Ｇ蛋白信号通路调节。阿片类药物长时程作用和纳洛酮急性戒断引起受体脱敏，呈时间和剂量依赖性，伴有受体下调。结论：成功地建立阿片耐受和依赖细胞模型，可用于研究ＡＣｃＡＭＰ系统和ＮＯｃＧＭＰ系统在阿片耐受和成瘾中的调节。更多还原

【Abstract】 AIM: To study the effect of opiate on receptor mediated adenylate cyclase (AC) cAMP signal system in NG LNCXiNOS cell with the stable expression of inducible nitric oxide synthase gene. METHODS: The intracellular cAMP content was measured with 3H cAMP protein binding assay. The effect of opioid agonists on binding properties of opioid receptor was assessed using 3H Etor binding assay with intact cells. RESULTS: The forskolin stimulated cAMP accumulation was inhibited by opioid agonists in a concentration dependent manner. The order of inhibitory potencies was DPDPE( D Pen 2, D Pen 5 enkephalin)>DADLE( D Ala 2, D Leu 5 enkephalin)>etorphine>morphine with IC 50 values of 2 0×10 -8 , 2 1×10 -8 , 3 7×10 -8 and 1 7×10 -7 mol·L -1 , respectively. Such acute inhibitory effect of opioid agonist on adenylate cyclase (AC) activity could be remarkably blocked by the addition of naloxone and completely abolished by pretreatment of the cells with pertussis toxin(PTX), suggesting that acute inhibition of opioid agonists on AC cAMP system may be regulated via a receptor mediated and PTX sensitive G protein pathway. After NG LNCXiNOS cells were pretreated with DPDPE and DADLE for 48 hours, opioid agonists elevated intracellular cAMP concentration in the presence and absence of naloxone. Chronic exposure of the cells to DPDPE and DADLE resulted in reduction of Bmax values and increase of K D values. Long term treatment of neuronal cells with opioid agonists and naloxone precipitated cell “withdrawal” can lead to receptor desensitization in a time dependent and a dose dependent manner, and the down regulation of opiate receptor. CONCLUSION: A cellular model in opioid tolerance and dependence was successfully established. It may be useful for studying the cross talk regulation of AC cAMP and NO cGMP signal pathway on the development of opiate tolerance and addiction.更多还原