Objectives To investigate the effects of Exendin-4 (Ex-4) on nitric oxide (NO) produced bypig iliac endothelium cells (PIEC) and the relationship with intracellular calcium ions change.
Methods GLP-1receptor (GLP-1R) in the PIEC membrane was detected by immunofluorescence. PIEC were cultured in RPMI 1640 (with calcium)/DMEM (non calcium) nutrient mediumseparately and were treated with various concentrations of Ex-4. NO production and the intracellular calcium concentration were measured using Griess reaction in cell culture supernatants and Flow-Cytometer with the fluorescence probeFluo-2 AM, respectively. The relationship between EX-4 and NO release wasevaluated by measuring NO production before and after pretreated by eNOSblocker L-NAME.
Results GLP-1R was expressed in the PIEC. PIEC cultured in DMEM or RPMI 1640 were incubated with six different concentration Ex-4 (0.5, 1, 2, 4, 8 µg/ml). The NO productions of PIEC in DMEM were 1.39±0.06, 1.38±0.01, 1.33±0.00, 1.61±0.01 and 2.42±0.08 µmol/l, and the productions of cells in RPMI 1640 were 1.41±0.08, 1.57±0.13, 1.46±0.09, 1.54±0.09 and 4.07±0.17. In different culture medium, the NO production of cells processing with 8 µg/ml Ex-4 were significantly higher than all the other concentrations (p<0.05). PICE were then treat with 8 µg/mlEx-4 at a series of time points from 2 h, 4 h, 8 h, 12 h to 24 h. The NO productions increased gradually (0.53±0.02, 0.61±0.04, 1.44±0.07, 1.02±0.06, 0.13±0.06) and reached the peak 8 h later (p<0.05 vs 0.53±0.02), and then slowly fell to the normal. Together with a higher intracellular calcium concentration when compared with the control group (57.46±1.56 vs 62.38±1.84, p<0.05).
No production in two L-NAME processing groups (L-NAME 10 µmol/land 100 µmol/l) were 2.21±0.02 and 2.08±0.31, which were significantly decreased when compared to the non L-NAME pretreated group (2.32±0.16, p<0.05). The no production of both L-NAME groups was not significant (p>0.05).
Conclusions GLP-1R was expressed in the PIEC. Ex-4 increased NO production in the endothelium cell through activation of eNOS in a dose and time dependent manner, and calcium maybe play an important role in the process.