Characterization of des-Arg9-bradykinin-induced contraction in guinea-pig gallbladder in vitro

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Abstract

We have reported that bradykinin induces graded contraction in guinea-pig gallbladder in vitro through activation of bradykinin B2 receptors and prostanoid release, while des-Arg9-bradykinin, a selective bradykinin B1 receptor agonist, causes only a weak contraction, suggesting the presence of badykinin B1 receptors in this tissue. In the present study, we attempted to characterise the receptor subtype and the possible mechanism by which des-Arg9-bradykinin induces contraction in this preparation. Contractions induced by des-Arg9-bradykinin in guinea-pig gallbladder (1 pM to 1 μM) increased significantly as a function of time elapsed after setting up of the preparation, reaching the maximum after 6 h of equilibration (EC50 16.4 pM and Emax 0.6±0.08 g). Des-Arg9-bradykinin-induced contraction in guinea-pig gallbladder was totally prevented by cycloheximide (70 μM, an inhibitor of protein synthesis), indomethacin (3 μM), ibuprofen (30 μM), phenidone (30 μM) or Ca2+-free medium plus EGTA, and was partially antagonised by MK 571 ((3-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3-oxo-propyl) thio) methyl) propanoic acid, 0.1 μM) or by nicardipine (1 μM), but was not affected by dazoxiben (30 μM), staurosporine (100 nM) or L 655,240 (240 (3-[1-(4-clorobenzil)-5-fluoro-3-metilhyindol-2il] 2,2-dimetilpropanoic acid, 1 μM). Unexpectedly, des-Arg9-bradykinin-induced contraction was unaffected by the selective bradykinin B1 receptor antagonists, des-Arg9-[Leu8]-bradykinin and des-Arg9-NPC 17761 (des-Arg0-d-Arg [Hip3, d-HipE (transtiofenil)7, Oic8]-des-Arg9-bradykinin). However, the selective bradykinin B2 receptor antagonists, HOE 140 (d-Arg0-[Hyp3, Thi5, d-Tic7, Oic8]-bradykinin) and NPC 17731 (d-Arg0 [Hyp3, DHypE (transpropyl)7, Oic8]-bradykinin), completely blocked des-Arg9-bradykinin-mediated contraction. Pre-treatment of the animals with Escherichia coli endotoxin (lipopolysaccharide, 30 μg/animal, i.v., 24 h) did not significantly change the response to des-Arg9-bradykinin induction. It is concluded that des-Arg9-bradykinin-induced contractions in guinea-pig gallbladder are mediated primarily by the release of proinflammatory eicosanoid(s) derived from the cyclo-oxygenase pathway. These effects are unrelated to thromboxane A2 and do not seem to be coupled to activation of a protein kinase C-dependent mechanism. Response to des-Arg9-bradykinin increases as a function of the equilibration period of the preparation by a mechanism dependent on protein synthesis and seems to be mediated by activation of bradykinin B2 (but not B1) receptors. Finally, in contrast to that observed for bradykinin, the contraction induced by des-Arg9-bradykinin in guinea-pig galbladder is fully dependent on the influx of extracellular Ca2+, partially through L-type Ca2+ channels.

Introduction

Kinin action is mediated by the activation of two membrane receptors, denoted B1 and B2. Bradykinin preferentially acts through stimulation of constitutive B2 receptors which are widely distributed in both peripheral and central nervous systems (Hall, 1992). On the other hand, the kinins without the C-terminal arginine des-Arg9-bradykinin or des-Arg10-lys-bradykinin, two kininase I active metabolites, exhibit higher affinities for the kinin B1 than B2 receptor (Marceau, 1995). Much less is known about the bradykinin B1 receptors than for the B2 receptors. Bradykinin B1 receptors are rarely expressed in non-traumatized tissues and are up-regulated following in vitro incubation for long periods, after tissue trauma or infection or in vivo following treatment of animals with bacterial lipopolyssacharide endotoxin (Regoli and Barabé, 1980; Bhoola et al., 1992; Burch et al., 1993; Marceau, 1995). Both kinin B1 and B2 receptors have been cloned in most animal species and they possesses high homology in the amino acid sequence (Mceachern et al., 1991; Hess et al., 1992; Mcintyre et al., 1993; Hess et al., 1994; Pesquero et al., 1996).

In a recent study, we demonstrated that bradykinin and related kinins, after 2 h of equilibration of guinea-pig gallbladder, caused in this preparation graded contractions characterized by two distinct phases: high-affinity (0.1 pM to 1 nM) and low-affinity (3 nM to 3 μM) through activation of bradykinin B2 receptors (Cabrini et al., 1995). In addition, des-Arg9-bradykinin, the selective B1 receptor agonist, caused a weak contraction compared to bradykinin, suggesting the possible presence of B1 receptors in this preparations. The present study was therefore aimed at investigating the receptor subtype and also the mechanisms by which des-Arg9-bradykinin induces contraction in the guinea-pig gallbladder in vitro.

Section snippets

Tissue preparation

Guinea pigs of either sex (300–400 g), maintained on a 12 h light–dark cycle at 23±2°C and fed with standard commercial diet, were killed by stunning and exsanguination. The abdomen was opened and the gallbladder was removed. After washout in Krebs solution (see composition below), usually 4 strips of each gallbladder (3–4 mm wide and 10–15 mm long) were set up in individual 5 ml organ baths containing Krebs solution of the following composition (mM): NaCl, 113; KCl, 4.7; CaCl2, 2.5; NaHCO3,

Contraction induced by des-Arg9-bradykinin

The results in Fig. 1A show that the addition of des-Arg9-bradykinin (1 pM to 1 μM) caused a concentration-dependent contractile response in guinea-pig gallbladder. The results in Fig. 1A also show the concentration–response curves for des-Arg9-bradykinin obtained 3, 4, 5, 6 and 7 h after the equilibration period. As may be observed, the concentration–response curves elicited by des-Arg9-bradykinin increased significantly as a function of time elapsed after setting up the preparation, reaching

Discussion

The aim of this study was to investigate the possible presence of B1 receptors in the guinea-pig galbladder in vitro as we suggested previously (Cabrini et al., 1995). Our results indicate that des-Arg9-bradykinin induced a time-dependent contraction with high affinity but low efficacy in the guinea-pig gallbladder. The time-dependent increase in the des-Arg9-bradykinin response was completely inhibited by cycloheximide (an inhibitor of protein synthesis) when it was maintained in contact with

Acknowledgements

We wish to thank the pharmaceutical companies for the generous gifts of some of the drugs used in this work. This study was supported by grants from CNPq and FINEP (Brazil). D.A.C. is an M.Sc. student receiving a grant from CNPq.

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