论文部分内容阅读
目的 观察阿片类依赖时Ca2 + /钙调蛋白依赖的蛋白激酶Ⅱ (CaMKⅡ )信息通路的变化 ,以及Ca2 + /CaMKⅡ信息通路与cAMP水平之间的关系。方法 以NG10 8 15细胞作为体外的细胞模型 ,分别采用竞争性蛋白结合法及放射免疫法、PDE法、γ 3 2 P参入法以及RT PCR测定cAMP水平、钙调蛋白 (CaM)活性、CaMKⅡ活性和mDOR的mRNA表达水平的变化。结果 DPDPE长时程作用NG10 8 15细胞 ,cAMP水平升高 ,形成阿片依赖 ;细胞CaM活性和CaMKⅡ活性也升高 ,该升高可被CaM拮抗剂W 7所抑制 ,而CaMKⅡ活性的升高可被CaMKⅡ特异性抑制剂KN 6 2所抑制。W 7或KN 6 2可抑制阿片依赖导致的细胞cAMP水平的升高。阿片依赖时 ,加入纳洛酮诱发戒断 ,CaM活性、CaMKⅡ活性进一步增高。而在阿片依赖时 ,δ阿片受体的mRNA表达水平无明显变化。结论 Ca2 + /钙调蛋白依赖的蛋白激酶Ⅱ信息通路可通过调节cAMP水平参与阿片依赖的机制。
Objective To investigate the changes of Ca2 + / calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) pathway and the relationship between Ca2 + / CaMKⅡ pathway and cAMP level during opioid dependence. Methods NG10 8 15 cells were used as a cell model in vitro. The levels of cAMP, CaM and CaMKⅡ were determined by competitive protein binding assay, radioimmunoassay, PDE, γ 3 2 P incorporation and RT PCR, respectively And mDOR mRNA expression level changes. Results DPDPE long-term effect on NG10 8 15 cells, cAMP levels increased opioid dependence; CaM activity and CaMK Ⅱ cell activity also increased, the increase can be CaM antagonist W 7 inhibition, and CaMK Ⅱ activity increased By CaMK Ⅱ specific inhibitor KN 6 2 inhibition. W 7 or KN 6 2 inhibited opioid-dependent elevation of cellular cAMP levels. Opioid dependence, add naloxone induced withdrawal, CaM activity, CaMK Ⅱ activity was further increased. In opioid dependence, δ opioid receptor mRNA expression levels did not change significantly. Conclusion Ca2 + / calmodulin-dependent protein kinase Ⅱ signaling pathway may be involved in opioid dependence by regulating cAMP levels.