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‐10μ 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 μm). 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 KC1) 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 μ 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 μ m ) or ibuprofen (30 μ m ), and was partially reduced by phenidone (30 μ m ), but was not affected by atropine (1 μ m ), nicardipine (1 μ m ), Ca 2+ ‐free medium plus EGTA, dazoxiben (30 μ m ), L‐655,240 (10 nM, a selective receptor antagonist of thromboxane A 2 ), MK‐571 (0.1 μ m , a selective leukotriene D 4 receptor antagonist), tetrodotoxin (0.3 μ m ), CP 96,345 (0.3 μ m , a NK 1 receptor antagonist), mepyramine (1 μ m ), glibenclamide (1 μ m ), H‐7 (3 μ m ), staurosporine (100 nM), or phorbol 12‐myristate 13‐acetate (1 μ m ). However, BK‐induced contractions in GPG maintained in Ca 2+ ‐free medium were markedly attenuated by ryanodine (10 μ m ). 4 Prostaglandin E 2 , prostaglandin F 2α or U46619 (0.1 nM to 100 μ m ), caused concentration‐dependent contractions in GPG with mean EC 50 S of 3.1 μ m ; 1.7 μ 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 μ 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 μ 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 Ca 2+ 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.