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 timepoints 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.