应用共聚焦激光扫描技术研究阿片受体介导胞内Ca^(2+)增高的调节机制

Mechanisms of Opioid Receptor-induced Elevation in Intracellular Calcium by Confocal Laser Scanning Microscopy

目的 观察阿片激动剂急性和长时程作用于稳定表达 i NOS基因的 NG- L NCXi NOS细胞 ,对细胞内游离钙 ([Ca2 + ]i)的影响及 G-蛋白的调节作用。方法 应用共聚焦显微镜和荧光探针 Fluo- 3,监测单细胞 [Ca2 + ]i 含量变化。结果 δ-阿片激动剂 DPDPE和吗啡急性刺激细胞诱发 [Ca2 + ]i 增加 ,纳洛酮对其起翻转作用 ;用百日咳毒素预处理细胞后 ,吗啡急性刺激不能引起 [Ca2 + ]i 增加 ;DPDPE和吗啡长时程处理细胞 ,再用相应的阿片激动剂急性刺激也造成 [Ca2 + ]i 增加 ,但 [Ca2 + ]i 增加水平较低 ,用纳洛酮戒断造成 [Ca2 + ]i 明显增加。结论 阿片激动剂诱发[Ca2 + ]i 增加是由阿片受体介导的 ,受对 PTX敏感的 G-蛋白调节 ,当细胞发生阿片耐受和依赖时 ,表现为受体介导 Ca2 + 系统的脱敏现象。

To determine the acute and chronic effects of opioid receptor agonists on the intracellular free calcium concentration ([Ca 2+] i) in NG-LNCXiNOS cells,stably expressing iNOS gene,and regulation of G-protein on opioid-induced response in [Ca 2+] i.Methods A single cell [Ca 2+] i is measured by confocal laser scanning microscopy using Ca 2+-sensitive dye Fluo-3 as an new calcium fluorescent probe.Results DPDPE(D-Pen 2 ,D-Pen 5-enkephalin),a δ-opioid receptor agonist, and morphine acutely induced the increase in [Ca 2+] i of NG-LNCXiNOS cells .The elevation in [Ca 2+] i by DPDPE could be abolished with naloxone.Pretreatment of the cells with pertussis toxin (PTX) at 100 ng/ml for 24 hours almost completely blocked morphine-evoked response.In contrast to acute effect of opioid agonists on [Ca 2+] i,the cells exposed to l μmol/L DPDPE or 10 μmol/L morphine for 48 hours also appeared to raise [Ca 2+] i.However,the elevation in [Ca 2+] i was not greater than that caused by acute effect of DPDPE or morphine. After cell "withdrawal" was precipitated by the addition of 10 μmol/L naloxone,the increasement of [Ca 2+] i could further be intensified. Conclusions The opioid agonist-induced increase in [Ca 2+] i is mediated by opioid receptor and regulated though PTX-sensitive G-protein.The attenuation of this response in chronically treated cells with opioid agonist is associated with receptor desensitization.