Fig 2: NOS3 activation and NO production by endothelial cells treated with B2R and D2R agonists.Confluent cells were stimulated with 100 nM BK, 100 nM SUM, or both agonists simultaneously for the indicated times. (A) The protein expression levels of pNOS3 Ser1177 and NOS3 were analyzed by western blotting and normalized to the amount of β-actin. (B) The pNOS3/NOS3 ratio was quantified by densitometric analysis. The bars represent the mean values ± SD from three independent experiments as compared to the untreated cells, assumed to have a ratio equal to 1 (dashed line). (C) The percentage of changes in NO production is presented in comparison to the untreated cells, with an assumed reference control value of 100% (dashed line). The mean values ± SD from at least three experiments performed in duplicate are shown. *P < 0.001 versus BK-treated cells, #P < 0.001 versus SUM-treated cells.

Fig 3: ROS production by HUV-EC-Cs treated with B2R and D2R agonists.DCFH-DA-loaded endothelial cells (1.0 × 105) were stimulated with 100 nM BK, 100 nM SUM, or both agonists simultaneously. Some samples were additionally preincubated with 10 μM HOE 140 or 10 μM eticlopride for 30 minutes or 1 μM apocynin for 1 hour prior to agonist stimulation. The bars represent the mean percentage of the changes in ROS production relative to the untreated cells (with an assumed reference control value of 100%; dashed line). The more significant differences were indicated inside figure. *P < 0.001 versus BK-treated cells, #P < 0.001 versus SUM-treated cells.

Fig 4: Effect of B2R and D2R agonists on antioxidant enzyme activity.Endothelial cells (1 × 106) were treated with 100 nM BK, 100 nM SUM, or both agonists simultaneously for 2 hours. In some samples, the cells were additionally preincubated with 10 μM HOE 140 or 10 μM eticlopride for 30 minutes prior to receptor agonist stimulation. (A) The enzymatic activity of MnSOD and Cu/ZnSOD in cell lysates normalized to the amount of sample protein was measured after protein separation by native PAGE. (B) The changes in MnSOD and Cu/ZnSOD activity calculated after densitometric gel analysis are presented relative to the untreated cells (with an assumed reference control value of 100%; dashed line). (C) The enzymatic activity of CAT was spectrophotometrically measured and normalized to the amount of sample protein. The figures indicate changes in the percentage of CAT activity relative to the untreated cells (with an assumed reference control value of 100%; dashed line). The bars represent the mean values ± SD from at least three experiments performed in duplicate. *P < 0.001 versus BK-treated cells, #P < 0.001 versus SUM-treated cells.

Fig 5: Involvement of B2R and D2R in oxidative and inflammatory responses as well as in apoptotic processes in endothelial cells.BK–bradykinin, B2R –bradykinin B2 receptor, CAT–catalase, D2R- dopamine D2 receptor, ET-1 –endothelin-1, IL-6 –interleukin-6, MnSOD–mitochondrial superoxide dismutase, pNOS3 –phosphorylated endothelial nitric oxide synthase, ROS–reactive oxygen species, SUM–sumanirole. Thick arrows represent mechanisms that may not necessarily occur directly and in which additional factors may be involved.