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Bradykinin Mediates Phosphorylation of eNOS in Odontoblasts

¹ Department of Operative and Preventive Dentistry and Endodontics, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany;
² Department of Molecular and Cellular Sport Medicine, German Sport University, Cologne, Germany;
³ Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany;
⁴ Department of Operative Dentistry and Periodontology, University of Cologne, Germany; and
⁵ Department I of Anatomy, University of Cologne, Germany

View larger version (146K): [in a new window]   Figure 1. Localization of t-eNOS, p-eNOS at Ser1177, Ser116, t-Akt/PKB, p-Akt/PKB, t-ERK1/2, and p-ERK1/2 in odontoblasts. In odontoblasts and in their processes, localization of total (t)-eNOS was detected in predentin and initial dentin tubules (A). Odontoblasts revealed reaction products for phosphorylated (p)-eNOS at Ser1177 (B) and at Ser116 (C) in predentin. Localization of p-eNOS at Thr495 was not identifiable in odontoblasts and in their processes in predentin (D). Localization of t-Akt/PKB was detected in odontoblasts and in their processes in predentin and initial dentin tubules (E). In the odontoblast layer, p-Akt/PKB was identifiable (F). Immunohistochemical reaction product for t-ERK1/2 was localized in predentin (G). A subpopulation of odontoblasts and their processes in predentin revealed reaction product for p-ERK1/2 (H). Confocal images of 20-µm-thick sections revealed an intense staining for t-eNOS in odontoblasts and in their processes within dentinal tubules (I). The reaction product for p-eNOS at Ser1177 was detected in odontoblasts and in their processes in predentin and in initial dentin tubules (J). Localization of p-eNOS at Ser116 was detected in odontoblast cell bodies and in odontoblast processes in predentin and dentin (K). Localization of p-eNOS at Thr495 was not detectable in odontoblasts (L). Existence of p-eNOS at Thr495 in pulpal blood vessels (D,L; arrows) served as positive controls. Bar = (A-H) 50 µm, (I-L) 10 µm. p = dental pulp, pd = predentin, d = dentin, o = odontoblasts.

View larger version (92K): [in a new window]   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.

View larger version (54K): [in a new window]   Figure 3. Time (5 and 10 min)-dependent effects of bradykinin on the t-Akt/PKB, p-Akt/PKB, t-ERK1/2, and p-ERK1/2 in odontoblasts. In odontoblasts treated with BK for 5 (I, control, 52.78 ± 13.42/BK, 83.55 ± 22.42) and 10 (J, control [A], 113.35 ± 5.30/BK [B], 110.06 ± 9.15) min, there was no significant difference in the staining intensities for total (t)-Akt/PKB between control and treated odontoblasts. Localization of phosphorylated (p)-Akt/PKB was not identifiable in the control odontoblasts, while the signal intensities were increased after 5 (I, control, 33.54 ± 10.18/BK, 156.75 ± 6.90; asterisks) and 10 (J, control [C], 35.95 ± 5.66/BK [D], 77.38 ± 4.63; asterisks) min of BK treatment. 5 (I, control, 159.80 ± 7.81/BK, 154.98 ± 3.53) and 10 (J, control [E], 150.57 ± 2.14/BK [F], 157.72 ± 2.99) min of BK treatment did not affect staining intensities of t-ERK1/2 in either control or treated odontoblasts. The staining intensities for p-ERK1/2 were not detectable in control (G) or in odontoblasts treated with BK after 5 (I, control, 28.47 ± 2.68/BK, 34.46 ± 11.19) or 10 (J, control [G], 17.72 ± 1.97/BK [H], 29.15 ± 7.33) min. Data are mean ± SD; n = 4 for each group. Significant differences were considered at a P value < 0.05. Bar = 50 µm.

View larger version (79K): [in a new window]   Figure 4. Effects of B2 antagonist HOE 140 on phosphorylation sites of eNOS. Experiments were performed for 5 min in the absence and presence of HOE 140 (10–6 M). In comparison with untreated odontoblasts, HOE 140 attenuated signal intensities for t-eNOS after 5 min in treated odontoblasts (control [A], 120.51 ± 8.41/HOE 140 [B], 104.21 ± 5.24). HOE 140 treatment induced a significant decrease in the signal intensities of p-eNOS at Ser1177 (control [C], 134.23 ± 2.28/HOE 140 [D], 94.29 ± 17.88). HOE 140 treatment decreased staining intensities of p-eNOS at Thr495 (control [E], 57.44 ± 10.77/HOE 140 [F], 28.31 ± 13.08). Incubations without primary antibodies resulted in an abolition of specific reaction products in odontoblasts of control (G) and HOE 140 treatment (H) conditions. The HOE 140 treatment did not significantly affect staining intensities of t-Akt/PKB in either control or treated odontoblasts (control [I], 143.14 ± 20.27/HOE 140 [J], 101.54 ± 28.80). The staining intensities of p-Akt/PKB in odontoblasts were significantly decreased after 5 min of HOE 140 treatment (control [K], 56.41 ± 9.30/HOE 140 [L], 28.74 ± 0.75). There were no significant differences in staining intensities for t-ERK1/2 (control [M], 131.64 ± 8.82/HOE 140 [N], 123.05 ± 11.37) and p-ERK1/2 (control [O], 27.65 ± 8.82/HOE 140 [P], 29.57 ± 9.38) between control and HOE 140-treated odontoblasts. Data are mean ± SD; n = 3 for each group. Significant differences were considered at a P value < 0.05. Bar = 70 µm.