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

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-IR).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l、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+l.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.

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

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.