Bradykinin B 1 receptors in the rabbit urinary bladder: induction of responses, smooth muscle contraction, and phosphatidylinositol hydrolysis

1 The aim of this study was to analyse the pharmacological characteristics, and second‐messenger coupling‐mechanisms, of bradykinin B 1 receptors in an intact tissue, the rabbit urinary bladder; and to investigate the influence of inhibition of endogenous peptidases on kinin activities. 2 In preparations of rabbit mucosa‐free urinary bladder, at 90 min after mounting of the preparations, bradykinin (1 n m − 10 μ m ) evoked contractile responses. In contrast, the B 1 receptor‐selective agonist [des‐Arg 9 ]‐BK (10 n m − 10 μ m ) was only weakly active at this time. Contractile responses to [des‐Arg 9 ]‐BK increased with time of tissue incubation in the organ bath, reaching a maximum after 3 h, when the pD 2 estimates were 6.4 ±0.3 for bradykinin, and 6.9 ± 0.2 for [des‐Arg 9 ]‐BK. 3 Once stabilized, responses to [des‐Arg 9 ]‐BK in the bladder were competitively antagonized by the B 1 receptor‐selective antagonists [Leu 8 , des‐Arg 9 ]‐BK and d ‐Arg‐[Hyp 3 , Thi 5 , d ‐Tic 7 , Oic 8 , des‐Arg 9 ]‐BK ([des‐Arg 10 ]‐Hoe140) (p K B estimates were 6.1 ± 0.1 and 7.1 ± 0.1, respectively; n = 17–21), but responses were unaffected by the B 2 receptor‐selective antagonist d ‐Arg‐[Hyp 3 , Thi 5 , d ‐Tic 7 , Oic 8 ]‐BK (Hoe140) (100 n m ; n = 4). Contractile responses to bradykinin itself were partially, but significantly, inhibited by the B 1 receptor‐selective antagonist, [Leu 8 , des‐Arg 9 ]‐BK (10 μ m ) ( P < 0.05), or by the B 2 receptor‐selective antagonist Hoe140 (100 n m ) ( P < 0.005) alone, and were largely blocked by a combination of the two antagonists ( P < 0.0001). 4 The combined presence of the carboxypeptidase inhibitor dl ‐2‐mercaptomethyl‐3‐guanidino‐ethylthiopropanoic acid (mergetpa; 10 μ m ), the neutral endopeptidase inhibitor, phosphoramidon (1 μ m ), and the angiotensin‐converting enzyme inhibitor, enalaprilat (1 μ m ) increased the potency of bradykinin 17 fold ( P < 0.001), but that of [des‐Arg 9 ]‐BK was unchanged ( P >0.05): pD 2 estimates were 7.6 ± 0.1 and 6.8 ± 0.1 for bradykinin and [des‐Arg 9 ]‐BK, respectively, in treated preparations. In the presence of peptidase inhibitors, the affinities of the antagonists [Leu 8 , des‐Arg 9 ]‐BK and [des‐Arg 10 ]‐Hoe140 were unchanged as compared with those determined in the absence of peptidase inhibitors ( P >0.05). [Leu 8 , des‐Arg 9 ]‐BK inhibited responses to bradykinin under these conditions ( n = 4). 5 In endothelium‐denuded preparations of the rabbit isolated aorta, an archetypal B 1 receptor preparation, contractile responses to the B 1 receptor‐selective agonist [des‐Arg 9 ]‐BK (10 n m − 10 μ m ) (and to bradykinin) increased progressively with time of tissue incubation; and [des‐Arg 9 ]‐BK responses were completely antagonized by the B 1 receptor antagonist [Leu 8 , des‐Arg 9 ]‐BK (p K B 6.3 ± 0.2; n = 13). 6 In experiments measuring stimulation of hydrolysis of phosphatidylinositol in rabbit urinary bladder, [des‐Arg 9 ]‐BK (10 μ m − 1 m m ), and bradykinin (100 μ m ) significantly increased accumulation of inositol phosphates ( P < 0.0001). The increase in accumulation of inositol phosphates evoked by [des‐Arg 9 ]‐BK (10 μ m − 1 m m ) was significantly inhibited by [des‐Arg 10 ]‐Hoe140 (10 μ m ) ( P < 0.01). 7 We conclude that in the mucosa‐free rabbit urinary bladder, [des‐Arg 9 ]‐BK evokes contraction largely via activation of B 1 receptors which have similar properties, including time‐dependent induction, to B 1 receptors in the rabbit isolated aorta. Bradykinin evokes contraction via stimulation of both B 1 and B 2 receptors, but does not require conversion by peptidases in order to activate B 1 receptors. We demonstrate, for the first time, B 1 receptor‐coupling to phosphatidylinositol hydrolysis in an intact tissue preparation.