Characterization of des-Arg 9-bradykinin-induced contraction in guinea-pig gallbladder in vitro

Abstract

We have reported that bradykinin induces graded contraction in guinea-pig gallbladder in vitro through activation of bradykinin B 2 receptors and prostanoid release, while des-Arg 9-bradykinin, a selective bradykinin B 1 receptor agonist, causes only a weak contraction, suggesting the presence of badykinin B 1 receptors in this tissue. In the present study, we attempted to characterise the receptor subtype and the possible mechanism by which des-Arg 9-bradykinin induces contraction in this preparation. Contractions induced by des-Arg 9-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 (EC 50 16.4 pM and E max 0.6±0.08 g). Des-Arg 9-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 Ca 2+-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-Arg 9-bradykinin-induced contraction was unaffected by the selective bradykinin B 1 receptor antagonists, des-Arg 9-[Leu 8]-bradykinin and des-Arg 9-NPC 17761 (des-Arg 0- d-Arg [Hip 3, d-HipE (transtiofenil) 7, Oic 8]-des-Arg 9-bradykinin). However, the selective bradykinin B 2 receptor antagonists, HOE 140 ( d-Arg 0-[Hyp 3, Thi 5, d-Tic 7, Oic 8]-bradykinin) and NPC 17731 ( d-Arg 0 [Hyp 3, DHypE (transpropyl) 7, Oic 8]-bradykinin), completely blocked des-Arg 9-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-Arg 9-bradykinin induction. It is concluded that des-Arg 9-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 A 2 and do not seem to be coupled to activation of a protein kinase C-dependent mechanism. Response to des-Arg 9-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 B 2 (but not B 1) receptors. Finally, in contrast to that observed for bradykinin, the contraction induced by des-Arg 9-bradykinin in guinea-pig galbladder is fully dependent on the influx of extracellular Ca 2+, partially through L-type Ca 2+ channels.