Exendin-4介导的猪髋动脉内皮细胞NO释放与细胞内钙离子浓度变化的关系

Relationship between nitric oxide release from pig iliac endothelial cells caused by Exendin-4 and change in intracellular calcium

目的观察Exendin-4（Ex-4）介导的猪髋动脉内皮细胞（PIEC）内一氧化氮（NO）释放与钙离子变化的关系,探讨Ex-4影响内皮细胞功能的机制。方法免疫荧光法检测确认PIEC上存在胰高血糖素样肽-1受体（GLP-1R）。NO荧光探针（DAF-FM DA）标记细胞内NO,荧光酶标仪检测Ex-4作用内皮细胞后不同时间点[加药前（0h）,加药后1、2、3、4、5、6、7、8、9、10、11、12h,共13个时点]细胞内NO释放情况;钙离子荧光探针（Fura-2 AM）标记细胞内钙,荧光酶标仪检测细胞内3个不同时段（0~30min,4~4.5h,8~8.5h）钙浓度变化情况;评价Ex-4诱导PIEC释放NO与细胞内钙的关系。结果 PIEC上有GLP-1R表达。Ex-4引起PIEC内NO增加,且有时间依赖关系,与0h比较,4h开始NO明显增高（P〈0.05）,8h时细胞内NO含量较其他各时点均明显增高（P〈0.05）。Ex-4干预后0~30min、4~4.5h和8~8.5h 3个时段细胞内钙均较对照组高（2.042±2.115 vs 0.634±0.352,P〈0.01;0.413±0.154 vs 0.113±0.111,P〈0.01;0.309±0.133 vs 0.063±0.120,P〈0.01）。Ex-4干预PIEC后NO释放前期前段（0~30min）,细胞内钙逐渐上升（拟合曲线：y=0.106x＋1.326）;NO释放中期前段（4~4.5h,y=0.003x＋0.374）和NO释放后期前段（8~8.5h,y=-0.001x＋0.324）细胞内钙浓度虽同NO一样也高于对照组,但没有明显的变化趋势。结论 Ex-4可增加内皮细胞内NO释放,该过程与细胞内钙变化密切相关。

Objective To study the effects of Exendin-4（Ex-4） on the function of endothelial cells by investigating the relationship of Ex-4 induced nitric oxide（NO） release from pig iliac endothelial cells（PIEC） and changes in intracellular calcium.Methods Glucagon-like peptide-1 receptor（GLP-1R） was confirmed to be present in PIEC by immunofluorescence assay.Intracellular NO production was probed using DAF-FM DA,and then measured with fluorescence microplate reader at the time-points of 0,1,2,3,4,5,6,7,8,9,10,11 and 12h.The intracellular Ca2＋ concentration was probed with Fura-2 AM at 0-30min,4-4.5h and 8-8.5h and measured with fluorescence microplate reader.The relationship between Ex-4 induced NO release in PIEC and intracellular Ca2＋ concentration was evaluated.Results GLP-1R was expressed in PIEC.Ex-4 increased the intracellular NO level in PIEC in a time-dependent manner.NO level was significantly higher 4 hours after Ex-4 treatment compared with that of 0-hour group（P〈0.05）.The intracellular NO level reached the top value 8 hours after Ex-4 treatment（P〈0.05）.The intracellular Ca2＋ concentrations were significantly higher during the periods of 0-30min,4-4.5h and 8-8.5h after Ex-4 treatment than those of their control groups（2.042±2.115 vs 0.634±0.352,P〈0.01;0.413±0.154 vs 0.113±0.111,P〈0.01;0.309±0.133 vs 0.063±0.120,P〈0.01）.During the period of 0-30min after Ex-4 treatment,the intracellular Ca2＋ concentration increased gradually（fitted curve： y=0.106x＋1.326）;while during the periods of 4-4.5h（y=0.003x＋0.374） and 8-8.5h（y=-0.001x＋0.324） after Ex-4 treatment,the intracellular Ca2＋ concentrations,and the NO levels were higher than those of their control groups,but without significant variation.Conclusions Ex-4 may increase the NO release in endothelial cells,and intracellular calcium ions may participate in the process.