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. 2005 Jul;145(6):740-50.
doi: 10.1038/sj.bjp.0706222.

Mechanisms Underlying the Relaxation Response Induced by Bradykinin in the Epithelium-Intact Guinea-Pig Trachea in Vitro

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Mechanisms Underlying the Relaxation Response Induced by Bradykinin in the Epithelium-Intact Guinea-Pig Trachea in Vitro

Valfredo Schlemper et al. Br J Pharmacol. .
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Abstract

In this study, we investigated some of the signalling pathways involved in bradykinin (BK)-induced relaxation in epithelium-intact strips of the guinea-pig trachea (GPT + E). BK induced time- and concentration-dependent relaxation of GPT + E. Similar responses were observed for prostaglandin E2 (PGE2) or the combination of subthreshold concentrations of BK plus PGE2. The nonselective cyclooxygenase (COX) inhibitors indomethacin or pyroxicam, or the selective COX-2 inhibitors DFU, NS 398 or rofecoxib, but not the selective COX-1 inhibitor SC 560, all abolished BK-induced relaxation. The tyrosine kinase inhibitors herbimycin A and AG 490 also abolished BK-induced relaxation in GPT + E. The nonselective nitric oxide synthase (NOS) inhibitor 7-NINA concentration-dependently inhibited BK effects. BK-induced relaxation was prevented by the selective antagonists for EP3 (L 826266), but not by EP1 (SC 19221), EP1/EP2 (AH 6809) or EP4 (L161982) receptor antagonists. Otherwise, the selective inhibitors of protein kinases A, G and C, mitogen-activated protein kinases, phospholipases C and A2, nuclear factor-kappaB or potassium channels all failed to significantly interfere with BK-mediated relaxation.BK caused a marked increase in PGE2 levels, an effect that was prevented by NS 398, HOE 140 or AG 490. COX-2 expression did not differ in preparations with or without epithelium, and it was not changed by BK stimulation. However, incubation with BK significantly increased the endothelial NOS (eNOS) and neuronal NOS (nNOS) expression, independent of the epithelium integrity. Our results indicate that BK-induced relaxation in GPT + E depends on prostanoids (probably PGE2 acting via EP3 receptors) and NO release and seems to involve complex interactions between kinin B2 receptors, COX-2, nNOS, eNOS and tyrosine kinases.

Figures

Figure 1
Figure 1
Typical records showing the time-dependent increase in BK (100 nM)- (a) and PGE2 (300 nM)- (b) induced relaxation responses in the GPT+E. Relaxation concentration–response curves for BK and for PGE2 in the GPT+E under spontaneous tonus following 2 h of time equilibration (c). Relaxant effect observed with the combination of subthreshold concentrations of BK (0.01 nM) and PGE2 (0.01 nM) in the GPT+E after 2 h of time equilibration (d). Each point represents the mean of 6–7 experiments and the vertical lines indicate the s.e.m.
Figure 2
Figure 2
Inhibitory effect of the nonselective COX inhibitors indomethacin (a) and pyroxicam (b) on BK (100 nM)-induced relaxation in the GPT+E. Negative and positive values represent relaxation and contraction responses, respectively. The bars represent the mean of 5–7 experiments and the vertical lines indicate the s.e.m. Data differ significantly from control value, *P<0.05, **P<0.01.
Figure 3
Figure 3
Inhibitory effect of selective COX-2 inhibitors DFU (a), NS 398 (b) and rofecoxib (c) on BK (100 nM)-induced relaxation in the GPT+E. Negative and positive values represent relaxation and contraction responses, respectively. The bars represent the mean of 7–8 experiments and the vertical lines indicate the s.e.m. Data differ significantly from control values, **P<0.01, ***P<0.001.
Figure 4
Figure 4
Inhibitory effect of the nonselective NO synthase inhibitor 7-NINA on BK (100 nM)-induced relaxation in the GPT+E. The bars represent the mean of 7–8 experiments and the vertical lines indicate the s.e.m. Data differ significantly from control values, **P<0.01.
Figure 5
Figure 5
Effect of selective EP1/EP2 (SC 19220, 10 μM), EP2 (AH 6809, 10 μM), EP3 (L 826266, 30 μM) and EP4 (L 161982, 30 μM) receptor antagonists on (a) BK (100 nM)- or (b) PGE2 (300 nM)-induced relaxation in the GPT+E. Negative and positive values represent relaxation and contraction responses, respectively. The bars represent the mean of 7–8 experiments and the vertical lines indicate the s.e.m. Data differ significantly from control value, **P<0.01, ***P<0.001.
Figure 6
Figure 6
Relaxing effect of BK (100 nM) in the absence and presence of herbimycin A (a) and AG 490 (b) in the GPT+E. Negative and positive values represent relaxation and contraction responses, respectively. The bars represent the mean of 8–9 experiments and vertical lines are the s.e.m. Data differ significantly from control value, *P<0.05, ***P<0.01.
Figure 7
Figure 7
Effect of incubation with COX-1 (SC 560), COX-2 (NS 398), NO synthesis (aminoguanidine, AMG) and EGFR-Trk (AG 490) inhibitors or the selective B2 receptor antagonist (HOE 140) on BK (100 nM)-induced PGE2 generation in the GPT+E. PGE2 concentrations were measured by ELISA. Each point represents the mean±s.e.m. of three experiments. #P<0.05 vs nontreated (NT) preparations, **P<0.01, ***P<0.001 vs BK-stimulated GPT+E.
Figure 8
Figure 8
Representative Western blot showing the constitutive protein expression of COX-2 (a), eNOS (b) and nNOS (c) in the absence or presence of BK (100 nM; 10 min of incubation) in the GPT+E (+Ep) and GPT−E (−Ep). Results were normalized by arbitrarily setting the densitometry of the basal group and are expressed as the mean±s.e.m. (N=3). Data differ significantly from basal levels (−Ep and +Ep), **P<0.01.

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