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 μ m ), guanethidine and indomethacin (both 3 μ m ) and of the peptidase inhibitors (captopril, bestatin and thiorphan, all 1 μ 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)≥ [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 μ m ) did not affect responses to the B 1 receptor agonist [des‐Arg 9 ‐BK (0.1 nM‐1 μ m ) nor those to the B 2 receptor agonist, [Hyp 3 , Tyr(Me) 8 ]‐BK (1 nM‐10 μ 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 μ 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 μ m ) competitively antagonized contractile responses to [des‐Arg 9 ]‐BK with an estimated p K 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 μ 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 μ m and 10 μ 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.