The longitudinal muscle of rat ileum as a sensitive monoreceptor assay for bradykinin B-1 receptors

1 Various bradykinin derivatives, acting preferentially at B-1 or B-2 receptors, were tested in the isolated longitudinal smooth muscle of rat ileum. Experiments were carried out in the presence of chlorpheniramine and atropine (both 1 mu M), guanethidine and indomethacin (both 3 mu M) and of the peptidase inhibitors (captopril, bestatin and thiorphan, all 1 mu M). 2 The rank order of potency was (pD(2) values+/-s.e.mean, n = 5 in parentheses, at 5 h from set-up): [des-Arg(9)]-BK (8.27+/-0.11)greater than or equal to [des-Arg(10)]-kallidin (7.67+/-0.24) > bradykinin (6.69+/-0.25). The B-2 receptor selective agonist, [Hyp(3),Tyr(Me)(8)]-BK, was approximately 10 fold less active than bradykinin. Contractile responses to all agonists increased with time. The maximal response to the B-1 receptor agonist, [desArg(9)]-BK at 5 h (94+/-2%) was significantly (P<0.05) greater than that measured at 2 h (74+/-2%). 3 The B-2 receptor antagonist, D-Arg[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-BK (Hoe 140, 0.1 mu M) did not affect responses to the B-1 receptor agonist [des-Arg(9)]-BK (0.1 nM-1 mu M) nor those to the B-2 receptor agonist, [Hyp(3),Tyr(Me)(8)]-BK (1 nM-10 mu M). In control experiments performed in the longitudinal smooth muscle of guinea-pig ileum and rat isolated urinary bladder as bioassays for B-2 receptors, the B-2 receptor antagonist Hoe 140 (0.1 mu M) antagonized bradykinin-induced contractions. 4 In the rat isolated ileum the B-1 receptor antagonist, D-Arg[Hyp(3), Thi(5), D-Tic(7), Oic(8), des-Arg(9)]-BK ([des-Arg(10)]-Hoe 140, 0.3-10 mu M) competitively antagonized contractile responses to [des-Arg(9)]-BK with an estimated pK(B) of 6.74+/-0.08 (Schild plot slope with confidence limits 1.22, (0.70-1.73) n=13). In control experiments in the guinea-pig isolated ileum and rat isolated urinary bladder, [des-Arg(10)]-Hoe 140 (1-10 mu M) did not inhibit B-2 receptor-mediated contractile responses. 5 The putative B-1 receptor antagonist, [Leu(8),des-Arg(9)]-BK, behaved as a partial agonist when responses were determined 2 h from set-up (pD(2) 6.43+/-0.21, n = 5; E(max) 30% of that evoked by [des-Arg(9)]-BK); at 5 h from set-up it behaved as a full agonist (pD(2) 7.48+/-0.12, n= 5; E(max) 90% of that evoked by [des-Arg(9)]-BK). At this time the response to [Leu(8),des-Arg(9)]-BK was antagonized in a concentration-dependent manner by [des-Arg(10)]-Hoe 140, which at 1 mu M and 10 mu M, produced dose-ratios of 6.33+/-3.66 (n = 4) and 103+/-40 (n = 4). 6 In view of the rank order of potency of agonists, the antagonist activity by [des-Arg(10)]-Hoe 140 and the lack of antagonist activity of Hoe 140, we conclude that the longitudinal smooth muscle of rat ileum, after histamine, acetylcholine, noradrenaline, and prostanoid production blockade, is a sensitive monoreceptor assay for studying the pharmacology of bradykinin B-1 receptors. Further the preparation can also be used as a sensitive bioassay to identify partial agonist activity of B-1 receptor antagonists such as [Leu(8),desArg(9)]-BK.