MECHANISMS OF BRADYKININ-INDUCED CONTRACTION OF THE GUINEA-PIG GALLBLADDER IN-VITRO

1 The mechanisms underlying bradykinin (BK)-mediated contractions in strips of guinea-pig gallbladder (GPG) were examined by use of selective bradykinin (BK) receptor agonists and antagonists. 2 Addition of BK and related kinins (0.1 pM-10 mu M) after 2 h of equilibration of the preparation caused graded contractions characterized by two distinct phases: high affinity (0.1 pM-1 nM) and low affinity (3 nM-10 mu M). The rank order of potency for the first phase (mean EC(50), pM) was: BK (1.36) = Hyp(3)-BK (1.44)=Lys-BK (1.54)>Tyr(8)-BK (2.72)>Met-Lys-BK (4.30). The rank order of potency for the second phase (mean EC(50), nM, at concentration producing 50% of the contraction caused by 80 mM KCl) was: Hyp(3)-BK (8.95)>Met-Lys-BK (12.78)>Tyr(8)-BK (33.75)>Lys-BK (60.92)>BK (77.35). The contractile responses (g of tension) to 3 mu M of BK (the highest concentration tested) were: Hyp(3)-BK, 1.76+/-0.09; BK, 1.65+/-0.12; Lys-BK, 1.45+/-0.13; Tyr(8)-BK, 1.36+/-0.15 and Met-Lys-BK, 1.36+/-0.15. The selective B-1 agonist, des-Arg(9)-BK, caused only a weak contraction with maximal response (0.21+/-0.05 g), which corresponded to approximately 10% of that induced by BK. 3 BK-induced contraction in GPG was inhibited by indomethacin (3 mu M) or ibuprofen (30 mu M), and was partially reduced by phenidone (30 mu M), but was not affected by atropine (1 mu M), nicardipine (1 mu M), Ca2+-free medium plus EGTA, dazoxiben (30 nM), L-655,240 (10 nM, a selective receptor antagonist of thromboxane A(2)), MK-571 (0.1 mu M, a selective leukotriene D-4 receptor antagonist), tetrodotoxin (0.3 mu M), CP 96,345 (0.3 mu M, a NK1 receptor antagonist), mepyramine (1 mu M), glibenclamide (1 mu M), H-7 (3 mu M), staurosporine (100 nM), or phorbol 12-myristate 13-acetate (1 mu M). However, BK-induced contractions in GPG maintained in Ca2+-free medium were markedly attenuated by ryanodine (10 mu M). 4 Prostaglandin E(2), prostaglandin F-2 alpha or U46619 (0.1 nM to 100 mu M), caused concentration-dependent contractions in GPG with mean EC(50)s of 3.1 mu M; 1.7 mu M and 0.47 nM and maximal responses of 1.36+/-0.15; 1.32+/-0.20 and 0.96+/-0.09 g, respectively. 5 The selective B-2 receptor antagonists, Hoe 140, NPC 17731 and NPC 17761 (0.01-1 mu M), caused concentration-dependent displacements to the right of the contractile concentration-response curve for BK. The selective B-1 receptor antagonist, des-Arg(9)-[Leu(8)]-BK (1 mu M), did not affect BK-induced GPG contraction. 6 These data suggest that both high and low affinity BK responses in GPG are mediated by activation of B-2 receptors, and that BK-mediated contraction in GPG depends on the release of intracellular Ca2+ sources sensitive to ryanodine. In addition, BK-induced contraction in GPG is mediated by release of proinflammatory eicosanoid(s) derived from the cyclo-oxygenase pathway from arachidonic acid metabolism unrelated to thromboxane A(2), and seems not to be coupled to activation of a protein kinase C-dependent mechanism.