New selective bradykinin receptor antagonists and bradykinin B2 receptor characterization

Substantial progress has been made recently in the field of kinin pharmacology with the identification of sensitive bioassay organs and the discovery of bradykinin B2 receptor antagonists. Data obtained with such compounds in various laboratories support the hypothesis that kinins act on multiple (at least two) receptor types. Domenico Regoli and colleagues review here the basic criteria of receptor characterization as they apply to kinins and present a critical analysis of the bioassay organs and B2 receptor antagonists currently used in kinin pharmacology.     

From bradykinin B2 receptor antagonists to orally active and selective bradykinin B1 receptor antagonists

The bradykinin (BK) B1 receptor is an attractive target for the treatment of chronic pain and inflammation. Starting from a dual B1 and B2 antagonist, novel antagonists were designed that display low-nanomolar affinity for human B1 receptor and selectivity over B2. Initially, potent imidazoline derivatives were studied, but these compounds suffered from low bioavailability. This issue could be overcome by the use of less basic amino derivatives leading to orally active compounds.     

Nonpeptide bradykinin B2 receptor antagonists: conversion of rodent-selective bradyzide analogues into potent,orally-active human bradykinin B2 receptor antagonists

The 1-(2-nitrophenyl)thiosemicarbazide (TSC) derivative, (S)-1-[4-(4-benzhydrylthiosemicarbazido)-3-nitrobenzenesulfonyl]pyrrolidine-2-carboxylic acid [2-[(2-dimethylaminoethyl)methylamino]ethyl]amide (bradyzide; (S)-4), was recently disclosed as a novel, potent, orally active nonpeptide bradykinin (BK) B2 receptor antagonist. The compound inhibited the specific binding of [3H]BK to NG108-15 cell membrane preparations (rodent neuroblastoma-glioma) expressing B2 receptors with a K(i) of 0.5 +/- 0.2 nM. Compound (S)-4 also demonstrated oral efficacy against Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in rats with an ED50 value of 0.84 micromol/kg. After we optimized the terminal binding determinants projecting from the TSC framework, we found that it was possible to replace the potentially toxicophoric nitro and divalent sulfur moieties with only a 15-fold loss in binding affinity ((S)-14a). However, bradyzide and its congeners were found to have much lower affinities for cloned human B2 receptors, expressed in Cos-7 cells. The hitherto synthesized TSC series was screened against the human B2 receptor, and the dibenzosuberane (DBS) pharmacophore emerged as the key structural requirement for potency. Incorporation of this group resulted in a series of derivatives ((S)-14d,e and 19b-d) with K(i) ranges of 10.7-176 nM in NG108-15 cells (expressing the rodent B2 receptor) and 0.79-253 nM in Cos-7 cells (expressing the human B2 receptor). There was no evidence of agonist activity with any of the nonpeptides in any of the cell lines tested. In vivo, oral administration of compound 19c reversed FCA-induced and turpentine-induced mechanical hyperalgesia in rodents with ED50 values of 0.027 and 0.32 micromol/kg, respectively. The selectivity profiles of compounds (S)-14f and (S)-14g were also assessed to determine the conformational and/or steric preferences of the double-ring arrangement. The affinity of (S)-14 g for the human B2 receptor suggested that it may be a hydrophobic interaction with the ethane bridge of the DBS moiety that accounts for the increased potency of compounds (S)-14d,e and 19b,c at this receptor, by favoring a binding mode inaccessible to the unsubstituted diphenylmethyl derivative, (S)-4.     

Intramolecular signal transduction by the bradykinin B2 receptor

To study the intramolecular signal transduction, we performed single point and cassette mutations in transmembranal and intracellular regions of the bradykinin B2 receptor. We studied the influence of the two intramembranal Cys residues at positions 304 and 348, the role of Arg at position 177 in the highly conserved tripeptide sequence Asp-Arg-Tyr, the cytosolic G-protein binding area, and attempted to verify the general hypothesis of an ion tunnel-like interface in GPCRs. Wild type receptor, His-tagged receptor, and His-tagged mutant receptors were expressed in COS-7 cells and functionally compared by bradykinin-induced formation of inositolphosphate and arachidonic acid. To investigate the expression, all mutants were modified at the N-terminus by insertion of two successive His-tags and detected with an anti-poly-His antibody. Replacement of the second and third cytosolic loop by a loop from another membrane protein as well as single replacement of Arg at position 177 by Ala leads to a fully inactive receptor mutant without any ligand binding affinity and stimulatory activity. Mutants with replacement of Cys residues 304 and 348 by Ser showed only moderate effects. Regardless of the replacement of Asp 407 by Ala, the receptor is able to increase the agonist-induced levels of inositolphosphate and of arachidonic acid, indicating that our studies can not verify the postulated ion tunnel hypothesis.     

New highly potent bradykinin B2 receptor antagonists.

pA2 values of new B2 receptor antagonists ranging from 7.51 to 8.86 were measured on the rabbit jugular vein, while lower values were observed in the other preparations (for instance, the hamster urinary bladder). The most potent antagonists were those containing a hydroxyproline (Hyp) in position 3, a D-Arg at the N-terminal and a Leu instead of a Phe in position 8, with or without other chemical changes. D-Arg[Hyp3,D-Phe7,Leu8]-BK was found to be competitive, selective for B2 receptors and specific for kinins since it was without effect against substance P and angiotensin II in the rabbit jugular vein. The essential feature for obtaining B2 receptor antagonists appears to be the replacement or reorientation of Phe8 of bradykinin. The rabbit jugular vein provides a sensitive bioassay in which the potency and specificity of B2 receptor antagonists can be adequately evaluated.     

Discovery of orally active nonpeptide bradykinin B2 receptor antagonists

Orally active nonpeptide bradykinin (BK) B2 receptor antagonists have been discovered by using directed random screening and chemical modification. These compounds displaced [3H]BK binding to B2 receptors in guinea-pig ileum membranes, rat uterus membranes and human lung fibroblasts with nanomolar IC50s. They did not inhibit different specific radio-ligand bindings to other receptor sites including B2 receptors. In isolated guinea-pig ileum preparations, these compounds had no agonistic effect on smooth muscle contraction at 10(-6) M, and caused parallel rightward shifts of the concentration-response curves to BK on contraction with higher p A2 values. They also blocked human B2 receptor-mediated phosphatidylinositol hydrolysis without agonistic effect. In vivo, the oral administrations of these antagonists potently inhibited BK-induced bronchoconstriction in guinea-pigs. They also reduced carrageenin-induced paw edema and caerulein-induced pancreatitis in rats. Moreover, these compounds alleviated kaolin-induced pain in mice by oral administration. These results show that our compounds are potent, selective, and orally active BK B2 receptor antagonists and that they may have therapeutic potential against inflammatory diseases and pain.     

Aspirin inhibits human bradykinin B2 receptor ligand binding function

The bradykinin B2 receptor, a member of the G protein-coupled receptors superfamily, is involved in a variety of physiological functions, including vasodilation, electrolyte transfer in epithelia, mediation of pain, and inflammation. The effect of aspirin on bradykinin binding to cell-surface receptor and on signal transduction were studied in CHO-K1 cells, stably expressing the human B2 receptor. Cell-surface organization of the receptor was assessed by immunoprecipitation and Western blot analysis in CHO-K1 cells expressing N-terminally V5-tagged B2 receptor. We found that the widely used analgesic, anti-thrombotic, and anti-inflammatory drug aspirin alters the B2 receptor ligand binding properties. Aspirin reduces the apparent affinity of the receptor for [3H]-bradykinin by accelerating the dissociation rate of [3H]-bradykinin-receptor complexes. In addition, aspirin reduces the capacity of unlabeled bradykinin or the B2 receptor antagonist icatibant to destabilize pre-formed [3H]-bradykinin-receptor complexes. Kinetic and reversibility studies are consistent with an allosteric type of mechanism. Aspirin effect on B2 receptor binding properties is not accompanied by alteration of the cell-surface organization of the receptor in dimers and monomers. Aspirin does not influence the receptor ability to transduce bradykinin binding into activation of G-proteins and phospholipase C. These results suggest that aspirin is an allosteric inhibitor of the B2 receptor, a property that may be involved in its therapeutic actions.     

2,3-diaminopyridine bradykinin B1 receptor antagonists

Bradykinin B1 receptor antagonists embody a potentially novel approach for the treatment of chronic pain and inflammation. A series of 2,3-diaminopyridine B1 antagonists was optimized to have sub-nanomolar affinity and good pharmacokinetic properties. Lead compounds were shown to exhibit good efficacy in rabbit in vivo models of pain and inflammation.     

2-Aminobenzophenones as a novel class of bradykinin B1 receptor antagonists

Selective bradykinin (BK) B 1 receptor antagonists could be novel therapeutic agents for the treatment of pain and inflammation. Elucidation of the structure activity relationships of the structurally novel HTS lead compound 1 provided potent hBK B 1 receptor antagonists with excellent receptor occupancy in the CNS of hBK B 1 transgenic rats.     

Pharmacological and functional characterization of bradykinin B2 receptor in human prostate

The objective of this study was to pharmacologically characterize bradykinin receptors, a component of the kallikrein-kinin system, in normal human prostate cells. In primary cultured human prostate stromal cells, bradykinin, but not [des-Arg9]bradykinin or [des-Arg10]kallidin, produced calcium mobilization or inositol phosphates accumulation with potencies (pEC50) of 8.8+/-0.2 and 8.2+/-0.2, respectively. This was consistent with abundance of bradykinin B2 mRNA over bradykinin B1 mRNA in prostate stromal cells. Although the prostate epithelial cells (prostate epithelium, BPH-1, and PC-3) expressed mRNA for bradykinin B2 receptors (albeit in lesser amounts than stromal cells), bradykinin was not functionally efficacious in the epithelial cells. Increasing concentrations of D-arginyl-L-arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahhydro-3-isoquinolinecarbonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1H-indole-2-carbonyl-L-arginine (HOE-140), a bradykinin B2-selective peptide antagonist, attenuated bradykinin concentration-response curves in human prostate stromal cells with apparent estimate of affinity similar to that for the human bradykinin B2 receptor. Bradykinin (10 nM) caused proliferation of prostate stromal cells and phosphorylated extracellular signal-regulated kinases (ERK-1 and ERK-2) that were blocked by HOE-140 (1 microM). This study demonstrated that, in primary cultures of normal human prostate stromal cells, bradykinin activates bradykinin B2 receptors that may play a significant role in proliferation via activation of ERK-1/2 pathways.     

Non-peptide antagonists and agonists of the bradykinin B(2) receptor

Stimulation of the bradykinin (BK) B(2) receptor by kinins is associated with pathophysiological as well as pronounced beneficial effects. Consequently, interference with BK B(2) receptors by either antagonism or agonism offers promising therapeutic approaches for the development of drugs for the treatment of various human diseases. BK B(2) receptor antagonists may prove useful for the treatment of pathological situations caused by excessively increased local kinin concentrations, such as inflammation, tissue injury and pain. Beneficial effects of peptide BK B(2) receptor antagonists in perennial rhinitis, asthma and brain edema have already been demonstrated in clinical trials. On the other hand, kinins have also been identified as potent vasodilatory and organ-protective peptides. Therefore, BK B(2) receptor agonists may have the potential to become valuable therapeutics in the treatment of cardiovascular diseases such as hypertension, myocardial hypertrophy, myocardial infarction and arrhythmias as well as diabetic disorders. For both approaches, potent, selective and even orally active non-peptide compounds have been discovered recently. Prototypes of these novel third generation classes of compounds are the alkylphosphonium salt WIN-64338, the pseudopeptide NPC-18884, the thiosemicarbazide bradyzide and especially the imidazo[1,2-a]pyridine FR-167344 and the quinolines FR-173657 and LF-16.0687 as non-peptide BK B(2) receptor antagonists, whereas the 4-(2-pyridylmethoxy)-substituted quinoline FR-190997 and the 3-(2-pyridylmethyl)-substituted benzimidazole FR-191413 emerged as non-peptide BK B(2) receptor agonists. These antagonists and agonists of the BK B(2) receptor have already demonstrated efficacy a various animal models of human diseases, which offers promising therapeutic approaches for the development of drugs for the treatment or even prevention of a variety of severe human diseases either via stimulation or via blockade of BK B(2) receptors.     

Two B1 and B2 bradykinin receptor antagonists fail to inhibit the Ca2+ response elicited by bradykinin in human skin fibroblasts

The elevation of intracellular [Ca2+] induced by bradykinin (Bk) was monitored with fura-2 fluorescence in human skin fibroblasts. Neither [des-Arg10][Leu9]kallidin nor D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE140) inhibited the Ca2+ response stimulated by Bk. Moreover, each behaved as a partial agonist causing the elevation of intracellular [Ca2+].     

Evidence for a pulmonary B3 bradykinin receptor

We have examined pulmonary effects of bradykinin (Bk) in vivo and in vitro in guinea pigs and their potential inhibition by antagonists of Bk B1 and B2 receptors. Bk was a potent bronchoconstrictor in vivo and caused contractions of isolated, epithelium-denuded trachealis. D-Arg[Hyp3,D-Phe7]-Bk (NPC567) and D-arg[Hyp3,Thi5,8,D-Phe7]-Bk (NPC349), B2 receptor antagonists, were weak inhibitors of Bk-induced bronchoconstriction in vivo and were virtually inactive as antagonists of Bk-induced airway smooth muscle contraction. Several other B2 antagonists as well as B1 antagonist, des-Arg9-[Leu8]-Bk, did not inhibit Bk-induced tracheal contraction. The B1 receptor agonist des-Arg9-Bk was without effect on tracheal tone. Tracheal responses to Bk were unaffected by antagonists of muscarinic, histamine, serotonin, and catecholamine receptors. The inability of the antagonists to inhibit Bk is unlikely to be due to their degradation, because NPC567 was only weakly active in the presence of inhibitors of kininase I (EC 3.4.11.2), kininase II (EC 3.4.15.1), and neutral endopeptidase (EC 3.4.24.11). These studies were corroborated by ligand binding experiments in guinea pig and ovine airways. In [3H]Bk binding, the Bk antagonists had no effect in guinea pig trachea, slightly displaced [3H]Bk in ovine trachea, and inhibited approximately 60% of total specific binding in lung. des-Arg9-[Leu8]-Bk and several other agents, including atropine, neurokinin A, substance P, and vasoactive intestinal peptide, had no effect on lung Bk binding. Bk and its analogs were not degraded during the binding assay. These data suggest that pulmonary tissue, particularly in the large airways, contains a novel Bk binding site, a B3 receptor, which may be involved in Bk-induced bronchoconstriction.     

Functional studies of bradykinin receptors in Chinese hamster ovary cells stably expressing the human B2 bradykinin receptor

Bradykinin B1 and B2 receptors, members of the G-protein coupled receptor superfamily, are involved in inflammation and pain. Chinese hamster ovary (CHO) cells stably expressing the human B2 bradykinin receptor (CHO-B2) were used to characterize the signal transduction pathways associated with this receptor and its regulation. The selective B2 antagonist [3H]NPC17731 but not the selective B1 antagonist [3,4-prolyl-3,4-(3)H(N)]-[des-Arg10,Leu9]kallidin ([3H]DALKD) bound to CHO-B2 cell membranes with a Kd of 0.77 nM and a Bmax of 1087 fmol/mg protein. [3H]NPC17731 binding was inhibited by bradykinin ligands in the order: NPC17731 > bradykinin > kallidin > DALKD > [des-Arg10] kallidin (DAKD), consistent with the pharmacological profile of B2 bradykinin receptors. The B2 agonist bradykinin and the B1/B2 agonist kallidin, but not the B1 agonist DAKD, increased [35S]GTP gamma S binding to the CHO-B2 cell membranes. The B2 bradykinin receptors were co-immunoprecipitated with G alpha q/11. In response to bradykinin stimulation, coupling of the B2 receptors to G alpha q/11 was increased by 10-fold. Bradykinin and kallidin, but not DAKD, induced intracellular calcium release in CHO-B2 cells, which was blocked by NPC17731 but not by DALKD. These results demonstrate that B2 bradykinin receptors directly coupled to G alpha q/11 to regulate intracellular calcium release. CHO-B2 cell is a useful system that can be applied to study the effect of potential agents that may influence the B2 receptor function.     

Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts

Background and purpose:

Bradykinin (BK) and B₂ receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B₂ receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells.

Experimental approach:

Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes.

Key results:

[³H]-BK saturation studies indicated receptor density (B_max) and K_d values of 121 550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK_i 8.9) was threefold more potent than icatibant (pK_i 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC₅₀ 0.45 nM), with pK_B values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC₅₀ 216 nM) and IL-8 (EC₅₀ 53 nM) release. Both MEN16132 (IL-6: pIC₅₀ 8.1; IL-8: pIC₅₀ 8.4) and icatibant (IL-6: pIC₅₀ 6.6; IL-8: pIC₅₀ 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002), NF-κb (BAY-117085) and by the glucocorticoid dexamethasone.

Conclusions and implications:

Bradykinin via B₂ receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B₂ receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.     

Effects of an orally active non-peptide bradykinin B2 receptor antagonist,FR173657, on plasma exudation in rat carrageenin-induced pleurisy

1. Effects of an orally active non-peptide (BK) B₂ receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 ((E)-3-(6-acetamido-3-pyridyl)- N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl] acrylamide) on the plasma exudation in rat carrageenin-induced pleurisy were investigated.
2. Plasma exudation induced by intrapleural injection of bradykinin (BK, 3 nmol per rat) into male SD strain rats (SPF, 8 weeks old) were significantly inhibited by oral administration of novel B₂ receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 (3–30 mg kg⁻¹, 1 h before BK injection) in a dose-dependent manner, whereas that induced by histamine was not.
3. The inhibitory effect of 30 mg kg⁻¹ {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 persisted for more than 4 h.
4. Intrapleural injection of λ-carrageenin (2% (w/v), 0.1 ml per rat) caused marked plasma exudation and accumulation of exudates from 1 h after carrageenin injection. The maximum plasma exudation response was observed 5 h after carrageenin. The oral administration of {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 to rats (30 mg kg⁻¹, 1 h before carrageenin) significantly (by 50–77%) blunted the plasma exudation 1, 3, 5, and 7 h after carrageenin, causing a significant parallel reduction (by 42–57%) in the volume of exudates.
5. The anti-inflammatory effect of {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 on rat carrageenin-induced pleurisy was almost equipotent with that of the peptide B₂ antagonist Hoe140 (1 mg kg⁻¹, i.v.), a plasma kallikrein inhibitor, soy bean trypsin inhibitor (0.3 mg per rat, intrapleural injection) and bromelain (10 mg kg⁻¹, i.v.).
6. In pleurisy induced by intrapleural injection of a histamine releaser, compound 48/80, the plasma exudation was observed only within 20 min after the injection. This plasma exudation was not affected by {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657, although it was completely inhibited by a mixture of pyrilamine (5 mg kg⁻¹, i.v.) and methysergide (3 mg kg⁻¹, i.v.).
7. These results indicate that {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 is an orally active, promising anti-inflammatory agent for kinin-dependent inflammation.

     

Effects of the orally active non-peptide bradykinin B2 receptor antagonist, FR173657, on plasma extravasation in guinea pig airways

We investigated the effect of the orally active non-peptide bradykinin B2 receptor antagonist, FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2.4-di-chloro-3-[(2-methyl-8-quinoli nyl)oxymethyl]phenyl]-N-methy-laminocarbonylmethyl] acrylamide), on plasma extravasation mediated by activation of sensory nerves in guinea pig airways. Plasma extravasation was assessed by the photometric measurement of the extravasated Evans blue after formamide extraction. We found that the increase in Evans blue dye extravasation evoked by an aerosol of bradykinin (0.1 mM, 2 min) in the presence of phosphoramidon (2.5 mg/kg, i.v.) was abolished completely by FR173657 (20 mg/kg, p.o.) in the trachea and main bronchi. In sensitized guinea pigs pretreated with phosphoramidon, FR173657 (20 mg/kg, p.o.) inhibited plasma extravasation evoked by ovalbumin aerosol (5%, 2 min) by 77+/-14.2% in the trachea and 65+/-11.2% in the main bronchi. FR173657 (20 mg/kg, p.o.) did not affect the plasma extravasation caused by aerosolised capsaicin. These findings suggest that FR173657 is an orally active, promising anti-inflammatory agent for kinin-dependent inflammation following antigen challenge.     

Localization of B2 bradykinin receptor mRNA in the rat retina and sclerocornea

The expression of the B2 bradykinin receptor (BKR) mRNA in the adult rat eye was investigated by RNA blot and in situ hybridization analyses. Blot hybridization analysis of poly(A)+ RNA from the whole eye identified RNA species of 6000 and 4000 nucleotides, consistent with those observed in brain, lung, kidney and uterus. In situ hybridization analysis using digoxigenin-labeled riboprobes revealed intense labeling in the retinal ganglion cell layer and in a population of cells adjacent to the sclerocorneal junction. These data suggest that the B2 BKR is involved in biological processes in both retina and sclerocornea.     

Pharmacological evidence for a single bradykinin B2 receptor in the guinea-pig.

1. The present study addresses the possibility of the existence of different kinin B2 receptor subtypes in the guinea-pig by evaluating the affinity of peptide and nonpeptide receptor antagonists. For this purpose, jugular vein rings, ileum segments, lung parenchymal and trachea strips were set up in organ baths for isometric tension measurements. The experiments were conducted in the presence o indomethacin (3 microM), atropine (10 microM) and captopril (10 microM). 2. BK contracted jugular vein (JV), ileum (GPI), parenchyma (LP) and trachea (GPT) with an EC50 of 13.2 +/- 1.4 nM (n=27), 11.2 +/- 2.1 (n=26), 23.6 +/- 6.3 (n=26), and 33.0 +/- 6.5 (n=27), respectively. Thiorphan, a neutral endopeptidase (EC 3.4.34.11) inhibitor and MERGETPA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a carboxypeptidase inhibitor, had no effect on the BK-induced contractions of JV, GPI and LP. In the GPT, thiorpan potentiated the contractile response to BK and was thus added in the corresponding experiments. 3. The peptide B2 receptor antagonist, Hoe 140 and the nonpeptide compound, WIN 64338, behaved as noncompetitive antagonists against contractile responses to cumulative BK in the four tissues although Hoe 140 appeared as a competitive inhibitor in the GPT only. IN order to compare the inhibitory potency of these compounds between tissues, pKB values were determined. Mean values of pKB for Hoe 140 were 8.05 +/- 0.07, 8.43 +/- 0.11, 8.13 +/- 0.18, and 8.52 +/- 0. 25 in the JV, GPI, GPT and LP, respectively. WIN 64338 gave mean pKB values of 6.89 +/- 0.10, 7.57 +/- 0.12, 7.36 +/- 0.12 adn 7.51 +/- 0.28 in the JV, GPI, LP and GPT, respectively. 4. D-Arg [Hyp3, D-Phe7, Leu8]BK and D-Arg[Hyp3, D-Phe7]BK (NPC 567) inhibited in a competitive fashion the concentration-response curves to BK. Values of pA2for each compound were not significantly different in the four tissues and were between 5.81 and 6.31 for D-Arg [Hyp3, D-Phe7, Leu8]BK and between 5.55 and 5.65 for NPC 567.