Figure 2. Time (1, 3, 5, and 10 min)-dependent effects of bradykinin (10^–7) on the phosphorylation sites of eNOS in odontoblasts. Experiments were performed in the absence (control) and presence of bradykinin (BK) (10^–7 M) for 1 min (A-F, M), 3 min (N), 5 min (O), and 10 min (G-L, P). In comparison with untreated odontoblasts, the stimulation of odontoblasts with BK did not significantly affect staining intensities of total (t) eNOS for 1 (M), 3 (N), 5 (O), and 10 (P) min. In odontoblasts treated with BK, staining levels of phosphorylated (p) eNOS at Ser¹¹⁷⁷ were elevated after 1 (M, control [A], 128.39 ± 0769/BK [B], 137.33 ± 0.80), 3 (N, control, 128.14 ± 13.16/BK, 140.05 ± 11.15; asterisks), and 5 (O, control: 99.92 ± 13.68/BK, 117.52 ± 5.83; asterisks) min. But there was no significance in difference after 10 (P, control [G], 117.26 ± 4.29/BK [H], 120.58 ± 3.49) min in staining intensities for Ser¹¹⁷⁷ between control and BK-treated odontoblasts. In odontoblasts, Thr⁴⁹⁵ was faintly detected after 1 (M, control [C], 54.74 ± 03.44/BK [D], 68.90 ± 9.67) min of BK treatment, and the signal was time-dependent, significantly increased after 3 (N, control, 33.43 ± 10.54/BK, 58.10 ± 18.65; asterisks), 5 (O, control, 47.97 ± 8.10/BK, 89.96 ± 7.55; asterisks), and 10 (P, control [I], 54.53 ± 15.33/BK (J), 108.30 ± 5.59; asterisks) min. The absence of primary antibodies in incubations resulted in a lack of specific reaction products in odontoblasts (control, E; BK, F). Although a basal phosphorylation of Ser¹¹⁶ was identified in odontoblasts (K), the treatment of odontoblasts with BK for 5 and 10 min (control, K; BK, L) did not affect the phosphorylation of eNOS at Ser¹¹⁶. Data are mean ± SD; n = 3 for groups of 1 and 3 min, n = 4 for groups of 5 and 10 min. Significant differences were considered at a P value < 0.05. Bar = 50 µm.