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

摘要

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.Sh and measured with fluorescence microplate reader. The relationship between Ex-4 induced NO release in PIEC and intracellular Ca + 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.Sh 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=0.106χ+1.326); while during the periods of 4-4.Sh (y=0.003x+0.374) and 8-8.5h [y=- 0.001lχ0.324] 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.%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.