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

1 The mechanisms underlying bradykinin (BK)-mediated contractions in s trips of guinea-pig gallbladder (GPG) were examined by use of selectiv e 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 p hases: high affinity (0.1 pM-1 nM) and low affinity (3 nM-10 mu M). Th e 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 (6 0.92)>BK (77.35). The contractile responses (g of tension) to 3 mu M o f 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-L ys-BK, 1.36+/-0.15. The selective B-1 agonist, des-Arg(9)-BK, caused o nly a weak contraction with maximal response (0.21+/-0.05 g), which co rresponded to approximately 10% of that induced by BK. 3 BK-induced co ntraction 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 no t affected by atropine (1 mu M), nicardipine (1 mu M), Ca2+-free mediu m 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 leukot riene 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-myrista te 13-acetate (1 mu M). However, BK-induced contractions in GPG mainta ined 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 me an 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 select ive B-2 receptor antagonists, Hoe 140, NPC 17731 and NPC 17761 (0.01-1 mu M), caused concentration-dependent displacements to the right of t he contractile concentration-response curve for BK. The selective B-1 receptor antagonist, des-Arg(9)-[Leu(8)]-BK (1 mu M), did not affect B K-induced GPG contraction. 6 These data suggest that both high and low affinity BK responses in GPG are mediated by activation of B-2 recept ors, and that BK-mediated contraction in GPG depends on the release of intracellular Ca2+ sources sensitive to ryanodine. In addition, BK-in duced contraction in GPG is mediated by release of proinflammatory eic osanoid(s) derived from the cyclo-oxygenase pathway from arachidonic a cid metabolism unrelated to thromboxane A(2), and seems not to be coup led to activation of a protein kinase C-dependent mechanism.