Synergism between Angiotensin receptors ligands: Role of Angiotensin‐(1‐7) in modulating AT2R agonist response on nitric oxide in kidney cells

Figure 1. Synergistic effect of ang‐(1‐7) preincubation on NO• response to AT1R/AT2R agonist ang‐II in HK‐2 cells. (A) Cells were preincubated with ang‐(1‐7) (10‐12‐10‐5 M) for 10 min followed by incubation with ang‐II (10‐12‐10‐5 M) for another 1 h (i‐viii). The data points of ligand combinations and single ligands are shown with open (□) and closed (●, ang‐(1‐7); ■, ang‐II) symbols, respectively. Results are mean ± SEM; *P < .05 vs. single‐agonist concentration‐response based on two‐way ANOVA followed by Tukey's posthoc test (n = 3‐11). (B) Surface plots show the concentration‐response matrix and overall Bliss energy score (δ = 162). (C) Effect of the antagonist of AT2R (PD123319), MasR (A‐779), and AT1R (candesartan) on NO• response in HK‐2 cells. Cells were incubated with PD123319, A779, or candesartan (all 10‐5 M) for 10 minutes followed by incubation with ang‐(1‐7) (10‐10 M) for 10 min, then incubation with the AT2R agonist C21 (10‐10 M) for another 1 h. Results are mean ± SEM; *P < .05 vs. single agonist based on one‐way ANOVA followed by Fisher's Least Significance Difference test (n = 6‐7)

Figure 2. Synergistic effect of ang‐(1‐7) preincubation on NO• response to AT2R agonist C21 in HK‐2 cells. (A) Cells were preincubated with ang‐(1‐7) (10‐12‐10‐5 M) for 10 minutes followed by incubation with C21 (10‐12‐10‐5 M) for another 1 h (i‐viii). The data points of ligand combinations and single ligands are shown with open (□) and closed (●, ang‐(1‐7); ■, C21) symbols, respectively. Results are mean ± SEM; *P < .05 vs. single‐agonist concentration‐response based on two‐way ANOVA followed by Tukey's posthoc test (n = 3‐11). (B) Surface plots show the concentration‐response matrix and overall Bliss energy score (δ = 304). (C) Effect of the antagonist of AT2R (PD123319), MasR (A‐779), and AT1R (candesartan) on NO• response in HK‐2 cells. Cells were incubated with PD123319, A779, or candesartan (all 10‐5 M) for 10 min followed by incubation with ang‐(1‐7) (10‐10 M) for 10 min, then incubation with the AT2R agonist C21 (10‐10 M) for another 1 h. Results are mean ± SEM; *P < .05 based on one‐way ANOVA followed by Fisher's Least Significance Difference test (n = 6‐16)

Figure 3. Synergistic effect of C21 preincubation on NO• response to ang‐(1‐7) in HK‐2 cells. (A) Cells were preincubated with C21 (10‐12‐10‐5 M) for 10 min followed by incubation with ang‐(1‐7) (10‐12‐10‐5 M) for another 1 hour (i‐viii). The data points of ligands combinations and single ligands are shown with open (□) and closed (●, ang‐(1‐7); ■, C21) symbols, respectively. Results are mean ± SEM; *P < .05 vs. single‐agonist concentration‐response based on two‐way ANOVA followed by Tukey's posthoc test (n = 3‐12). (B) Surface plots show the concentration‐response matrix and overall Bliss energy score (δ = 484)

Figure 4. Reduction in synergy upon incubation of HK‐2 cells with a mixture of ang‐(1‐7) and C21. (A) Cells were incubated with a mixture of various concentrations of ang‐(1‐7) and C21 (10‐10‐10‐5 M) for 1 hour (i‐vi). The data points of ligands combinations and single ligands are shown with open (□) and closed (●, ang‐(1‐7); ■, C21) symbols, respectively. Results are mean ± SEM (n = 3‐12). (B) Surface plots show the concentration‐response matrix and overall Bliss energy score (δ = 76)

Figure 5. Reduction in synergy upon preincubation with nonpeptidic MasR agonist AVE0991 (AVE) followed by incubation with AT2R agonist C21 in HK‐2 cells. (A) Cells were pre‐incubated with AVE (10‐10‐10‐5 M) for 10 minutes followed by incubation with C21 (10‐10‐10‐5 M) for another 1 hour (i‐vi). The data points of ligand combinations and single ligands are shown with open (□) and closed (●, AVE; ■, C21) symbols, respectively. Results are mean ± SEM (n = 3‐12). (B) Surface plots show the concentration‐response matrix and overall Bliss energy score (δ = 45)

Figure 6. Competition binding experiment of 5(6)‐carboxyfluorescein (FAM)‐labeled ang‐(1‐7) in HK‐2 cells. Cells were seeded in glass‐bottom, black, 24‐well Sensoplate. Competition binding was carried out with FAM‐ang‐(1‐7) (10‐9 M) without or with unlabeled ang‐(1‐7), A779, candesartan, PD123319, ang‐II or C21 (all 10‐6 M). Cells were washed with PBS and analyzed through Expression Analysis application and bright field segmentation algorithm of Nexcelom Celigo Cytometer. The resulting images were subjected to sequential gating (integrated and mean intensity and size) on the GFP channel for the identification of FAM‐ang‐(1‐7) labeled cells. The percentages of FAM‐ang‐(1‐7) labeled cells in the presence of unlabeled ang‐(1‐7) are considered nonspecific, which was subtracted from the total for the specific binding which was considered as 100% for comparison with other ligands. Results are mean ± SEM (n = 4‐7)