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  1. Xiaochun Hu,
  2. Haoyu Liu,
  3. Dongying Zhang,
  4. Jian Liu,
  5. Dongying Zhang
  1. Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University


    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.

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