Bradykinin B1 receptors in the rabbit urinary bladder: induction of responses, smooth muscle contraction, and phosphatidylinositol hydrolysis

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Abstract
1 The aim of this study was to analyse the pharmacological characteristics, and second-messenger coupling-mechanisms, of bradykinin B1 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 nm − 10 μm) evoked contractile responses. In contrast, the B1 receptor-selective agonist [des-Arg9]-BK (10 nm − 10 μm) was only weakly active at this time. Contractile responses to [des-Arg9]-BK increased with time of tissue incubation in the organ bath, reaching a maximum after 3 h, when the pD2 estimates were 6.4 ±0.3 for bradykinin, and 6.9 ± 0.2 for [des-Arg9]-BK. 3 Once stabilized, responses to [des-Arg9]-BK in the bladder were competitively antagonized by the B1 receptor-selective antagonists [Leu8, des-Arg9]-BK and d-Arg-[Hyp3, Thi5,d-Tic7, Oic8, des-Arg9]-BK ([des-Arg10]-Hoe140) (pKB estimates were 6.1 ± 0.1 and 7.1 ± 0.1, respectively; n = 17–21), but responses were unaffected by the B2 receptor-selective antagonist d-Arg-[Hyp3, Thi5,d-Tic7, Oic8]-BK (Hoe140) (100 nm; n = 4). Contractile responses to bradykinin itself were partially, but significantly, inhibited by the B1 receptor-selective antagonist, [Leu8, des-Arg9]-BK (10 μm) (P < 0.05), or by the B2 receptor-selective antagonist Hoe140 (100 nm) (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 μm), the neutral endopeptidase inhibitor, phosphoramidon (1 μm), and the angiotensin-converting enzyme inhibitor, enalaprilat (1 μm) increased the potency of bradykinin 17 fold (P < 0.001), but that of [des-Arg9]-BK was unchanged (P>0.05): pD2 estimates were 7.6 ± 0.1 and 6.8 ± 0.1 for bradykinin and [des-Arg9]-BK, respectively, in treated preparations. In the presence of peptidase inhibitors, the affinities of the antagonists [Leu8, des-Arg9]-BK and [des-Arg10]-Hoe140 were unchanged as compared with those determined in the absence of peptidase inhibitors (P>0.05). [Leu8, des-Arg9]-BK inhibited responses to bradykinin under these conditions (n = 4). 5 In endothelium-denuded preparations of the rabbit isolated aorta, an archetypal B1 receptor preparation, contractile responses to the B1 receptor-selective agonist [des-Arg9]-BK (10 nm − 10 μm) (and to bradykinin) increased progressively with time of tissue incubation; and [des-Arg9]-BK responses were completely antagonized by the B1 receptor antagonist [Leu8, des-Arg9]-BK (pKB 6.3 ± 0.2; n = 13). 6 In experiments measuring stimulation of hydrolysis of phosphatidylinositol in rabbit urinary bladder, [des-Arg9]-BK (10 μm − 1 mm), and bradykinin (100 μm) significantly increased accumulation of inositol phosphates (P < 0.0001). The increase in accumulation of inositol phosphates evoked by [des-Arg9]-BK (10 μm − 1 mm) was significantly inhibited by [des-Arg10]-Hoe140 (10 μm) (P < 0.01). 7 We conclude that in the mucosa-free rabbit urinary bladder, [des-Arg9]-BK evokes contraction largely via activation of B1 receptors which have similar properties, including time-dependent induction, to B1 receptors in the rabbit isolated aorta. Bradykinin evokes contraction via stimulation of both B1 and B2 receptors, but does not require conversion by peptidases in order to activate B1 receptors. We demonstrate, for the first time, B1 receptor-coupling to phosphatidylinositol hydrolysis in an intact tissue preparation.

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