Human B2 Receptor Research Articles

A novel nonpeptide antagonist of the kinin B1 receptor: effects at the rabbit receptor.

The kinin B1 receptor (B1R) has attracted interest as a potential therapeutic target because this inducible G protein-coupled receptor is involved in sustained inflammation and inflammatory pain production. Compound 11 (2-[(2R)-1-[(3,4-dichlorophenyl) sulfonyl]-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]acetamide) is a high-affinity nonpeptide antagonist for the human B1R, but it is potent at the rabbit B1R as well: its Ki value for the inhibition of [3H]Lys-des-Arg9-BK (bradykinin) binding to a novel myc-labeled rabbit B1R expressed in COS-1 is 22 pM. In contractility tests (organ bath pharmacology), we found that compound 11 is an apparently surmountable antagonist of des-Arg9-BK- or Lys-des-Arg9-BK-induced contraction of the rabbit isolated aorta (pA2 values of 10.6+/-0.14 and 10.4+/-0.12, respectively). It did not influence contractions induced by angiotensin II in the rabbit aorta or by BK or histamine in the jugular vein, but it suppressed the prostaglandin-mediated relaxant effect of des-Arg9-BK on the rabbit isolated mesenteric artery. Compound 11 (1 nM) inhibited both the phosphorylation of the extracellular signal-regulated kinase1/2 mitogen-activated protein kinases induced by Lys-des-Arg9-BK in serum-starved rabbit aortic smooth muscle cells and the agonist-induced translocation of the fusion protein B1R-yellow fluorescent protein expressed in human embryonic kidney (HEK) 293 cells. Compound 11 does not importantly modify the expression of myc-B1R over 24 h in HEK 293 cells (no detectable action as "pharmacological chaperone"). The present results support that compound 11 is a potent and highly selective antagonist suitable for further investigations of the role of the kinin B1R in models of inflammation, pain, and sepsis based on the rabbit.

Discovery of a potent, non-peptide bradykinin B1 receptor antagonist.

Bradykinin (BK) plays an important role in the pathophysiological processes accompanying pain and inflammation. Selective bradykinin B1 receptor antagonists have been shown to be anti-nociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. We have explored chemical modifications in a series of dihydroquinoxalinone sulfonamides to evaluate the effects of various structural changes on biological activity. The optimization of a screening lead compound, facilitated by a homology model of the BK B1 receptor, culminated in the discovery of a potent human BK B1 receptor antagonist. Results from site-directed mutagenesis studies and experiments in an animal pain model are presented.

Cardiac hypertrophy and microvascular deficit in kinin B2 receptor knockout mice.

Experimental and clinical evidence suggests kinin involvement in adaptive myocardial growth. Kinins are growth-inhibitory to cardiomyocytes. Knockout of kinin B2 receptor (B2R) signaling causes dilated and failing cardiomyopathy in 129/J mice, and a 9-bp deletion polymorphism of human B2R is associated with reduced receptor expression and exaggerated left ventricular growth response to physical stress. We reasoned that genetic background and aging may significantly influence the impact of B2R mutation on cardiac phenotype. The theory was challenged in C57BL/6 mice, a strain that naturally differs from the 129/J strain, carrying 1 instead of 2 renin genes. C57BL/6 B2R knockouts (B2R-KO) showed higher blood pressure and heart rate levels (P<0.05) compared with wild-type controls (WT) at all ages examined. At 12 months, left ventricular contractility and diastolic function were mildly altered (P<0.05) and histological and morphological analyses revealed ventricular hypertrophy and cardiomyocyte enlargement in B2R-KO (P<0.01). Reparative fibrosis was enhanced by 208% and capillary density reduced by 38% (P<0.01). Functional and structural alterations induced by B2R deletion in C57BL/6 mice were less severe than those reported previously in the 129/J strain. We conclude that interaction of B2R signaling with other genetic determinants influences aging-related changes in myocardial structure and function. These findings may help us understand the role of kinins in the development of cardiac failure.

Do angiotensin-converting enzyme inhibitors directly stimulate the kinin B1receptor?

It has been recently claimed that the human B1 receptors for kinins bind angiotensin-converting enzyme (ACE) inhibitors via a potential zinc-binding domain and are pharmacologically stimulated by these drugs. We verified whether ACE inhibitors stimulate B1 receptors in vitro. The isolated rabbit aorta or mouse stomach responded by negligible contractions to the application of captopril, enalaprilat, or zofenoprilat. The human isolated umbilical vein also failed to respond to enalaprilat. All of these preparations were responsive to the B1 receptor agonists des-Arg9-bradykinin (BK) or Lys-des-Arg9-BK. Furthermore, enalaprilat applied continuously had no significant interaction with the effects of Lys-des-Arg9-BK on the rabbit aorta. Enalaprilat failed to stimulate [3H]arachidonate release, translocate the receptors (confocal microscopy), or stimulate ERK1/2 phosphorylation (immunoblot) in HEK-293 cells stably expressing the rabbit B1 receptor conjugated to yellow fluorescent protein. The phospho-ERK1/2 content of arterial smooth muscle cells of human or rabbit origin was increased by treatment with Lys-des-Arg9-BK but not with enalaprilat. ACE inhibitors do not act as bona fide agonists of the kinin B1 receptors.

Development of an intact cell reporter gene β-lactamase assay for G protein-coupled receptors for high-throughput screening

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a β-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells. The β-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous β-lactamase activity in mammalian cells. The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors. Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the β-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The β-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-β-lactamase assay is well suited to automation and miniaturization required for high-throughput screening.

Molecular and pharmacological diversity of the kinin B1 receptor

The pharmacological properties of the kinin B1 receptor in binding the endogenous kinin peptides are known to differ across species. Molecular cloning has revealed that these pharmacological differences arise from the diversity within the BDKRB1 gene. In this report, the molecular diversity of the human BDKRB1 gene is expanded by the identification of eight single nucleotide polymorphisms (SNPs) in the coding sequence of the receptor, three of which change the amino acid sequence of the receptor. The molecular cloning and pharmacological characterization of two primate B1 receptors, rhesus and African Green monkey, reveals that they exhibit the same high degree of selectivity for des-Arg 10kallidin(Lys-bradykinin) relative to des-Arg 9bradykinin that is observed with the human kinin B1 receptor. Previous mutagenesis studies of the human B1 receptor have implicated extracellular domain (EC) IV in conferring this selectivity for des-Arg 10kallidin, by interacting with the N-terminal Lys residue of the peptide. The pharmacological analysis of chimeric B1 receptors, in which EC-IV of the human B1 receptor is replaced with the corresponding domain of either rat or dog, supports the proposal that EC-IV is an important determinant in conferring ligand selectivity.

Correction to “Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G9/11-mediated phospholipase Cβ activity ”

Correction to “Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G_9/11-mediated phospholipase Cβ activity ”

Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G(q/11)-mediated [correction of G(9/11)-mediated] phospholipase Cbeta activity.

The human B1 bradykinin (BK) receptor (B1R) is more efficacious than the human B2 BK receptor (B2R) in both ligand-independent and agonist-dependent coupling to G(q/11)-mediated phospholipase Cbeta activity. In fact, B1R is constitutively active, whereas B2R exhibits little if any constitutive activity. To evaluate the role of the C-terminal domain in receptor G(q/11) coupling, we constructed chimeric and C-terminally truncated receptors. The slopes of the increase in basal and agonist-dependent cellular phosphoinositide hydrolysis as a function of receptor density in transiently transfected human embryonic kidney 293 cells provided parameters of receptor coupling. Exchanging the C-terminal domains between the two receptors revealed that these domains are largely responsible for the difference in coupling. B1R truncation showed that this receptor does not directly depend on the C-terminal domain for efficient coupling, although coupling is dramatically augmented by residues in the membrane-distal portion of the domain downstream from Tyr(327). On the other hand, coupling of B2R is absolutely dependent on a membrane-proximal epitope in the C-terminal domain upstream from Lys(315). This epitope is adjacent to a basic residue, Arg(311), which exerts an inhibitory effect on coupling. Arg(311) is not conserved in B1R, and complementary mutations in B2R and B1R showed that this residue, together with previously identified serines and threonines, acts to attenuate the coupling efficacy of B2R. Therefore, the C-terminal domain participates intimately in the efficacy of B1R and B2R G(q/11) coupling by contributing both positive and negative regulatory epitopes.

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.

Preliminary mutational analysis of the human kinin B2 receptor for nonpeptide antagonist ligands recognition.

FR173657, LF16,0335, and LF16,0687 are nonpeptide antagonists, endowed with high affinity and selectivity for the human kinin B2 receptor. The kinin B2 receptor belongs to the family of G-protein-coupled receptors with seven transmembrane (TM) helices. In the present study, we aimed, through computer-assisted modeling and mutagenesis, to identify residues in the human B2 receptor (hB2R) amino acid sequence that are involved in nonpeptide antagonist binding in order to build up experimental data as a first step towards a molecular model of nonpeptide ligands binding site. Fourteen amino acid residues within the TM segments were mutated to alanine. The wild type and mutant receptors were stably expressed in Chinese hamster ovary (dhfr-) cells and tested for their ability to bind agonist ([3H]bradykinin) and peptide antagonist ([3H]MENI 1270) radioligands. The affinity of nonpeptide ligands was determined by heterologous competition experiments using the above radioligands. We found that some mutations in TM2 (W86A) and TM7 (Y295A, N297A) impair the binding affinity of the three nonpeptide antagonists. On the other hand, some mutated residues in TM3 (S1 17A) and TM6 (W256A) reduce the affinity of LF16,0335 and LF16,0687 only. Results are discussed in order to build up a hypothesis for the likely different interactions of various nonpeptide ligands with the B2 receptor.

In vitro and in vivo activity of analogues of the kinin B2 receptor antagonist MEN1 1270.

In this study, we describe the in vitro and in vivo activities of a series of cyclic peptide analogues of the selective kinin B2 receptor antagonist MEN11270 on Chinese hamster ovary cells expressing the human B2 receptor (hB2R), the human isolated umbilical vein (hUV), the isolated guinea pig ileum (gpI), and bradykinin (BK) induced bronchoconstriction (BC) and hypotension in anaesthetized guinea pigs. Substitutions in the backbone of MEN1 1270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10alpha)) aimed to increase the potency in inhibiting bronchospasm versus hypotension following the topical (intratracheal (i.t.)) or systemic (intravenous (i.v.)) application of these antagonists. A series of analogues were left unprotected from N-terminal cleavage by aminopeptidases (MEN12739, MEN13052, MEN13346, and MEN13371): these compounds maintained sizeable affinities for the hB2R (pKi = 9.4, 9.6, 9.7, and 8.6, respectively) and antagonist activities toward BK in the hUV (pA2 = 7.9, 8.3, 8.2, and 7.5) and gpI assays (pK(B) = 7.4, 7.8, 7.9, and 7.9), but the inhibition of BK-induced BC and hypotension in vivo was negligible following either i.v. or i.t. administration. Two analogues (MEN12388 and MEN13405) could be potential substrates of angiotensin-converting enzyme: these have good activity in the hB2R (pKi = 9.5 and 8.9, respectively), hUV (pA2 = 8.2 for MEN12388), and gpI assays (pK(B) = 8.4 and 8.0) but an in vivo activity 10- to 30-fold lower than the parent compound MEN1 1270 (pKi = 9.4, pA2 = 8.1, pKB = 8.3) when given by either the i.v. or the i.t. route. Other analogues were functionalized with a quaternary ammonium Lys derivative (MEN13031, MEN12374, and the previously mentioned MEN13052) or with an ethyl group on Arg (MEN13655 and the previously mentioned MEN13346 and MEN13405) in order to hinder or facilitate local absorption. MEN13346 and MEN13031 (pKi = 9.7and 9.5, pA2 = 8.2 and 7.9, pKB = 7.9 and 8.5, respectively) were 10- to 30-fold less active in vivo than MEN1 1270, without improving the discrimination between BK-induced BC and hypotension after either systemic or topical administration. It is concluded that the decreased in vivo activities of cyclic analogues of MEN11270 on BK-induced BC and hypotension following either their intratracheal or their intravenous routes of administration might be due in large part to metabolic degradation.

Mediator caused induction of a human bradykinin B1 receptor minigene: participation of c-Jun in the process.

The bradykinin B1 receptor (BKB1R) gene is expressed in selected tissues such as lung and kidney. In these tissues it is expressed at a very low level until induced by inflammatory mediators. Our aim has been to understand the mechanism of this regulatory process. A human BKB1R minigene was constructed. It contained a 1.8 kb promoter, the entire exon I, 1.5 kb of intron I, the entire exon II and intron II, and the luciferase gene as a reporter. Transient transfection of the minigene into SV40-transformed IMR90 cells (IMRSV) resulted in a promoter activity which was activated by the mediators, lipopolysaccharide and (LPS) desArg(10)-kallidin. In contrast, these mediators did not induce the activity of the 1.8 kb promoter construct alone. Thus, motifs exclusive of the promoter such as 5'-UTR and/or intron regions are required for mediator-induced expression of this gene. Promoter activities of both the minigene and the 1.8 kb promoter construct were enhanced in a dose-dependent manner upon cotransfection with c-Jun. Furthermore, cotransfecting c-Jun with the minigene achieved the maximal promoter activity with no further increase in response to mediators. Conversely, the induction of the minigene promoter activity by mediators was abolished upon cotransfection with a dominant negative mutant of c-Jun. Other experiments suggest that multiple AP-1 sites are interactive with the c-Jun upregulation of this gene. Taken together, these results point to c-Jun as a key intermediary in the activation of the expression of this gene by mediators. However, participation of motifs outside of the promoter are necessary to obtain this inducible expression.

New conformationally homogeneous beta-turn antagonists of the human B2 kinin receptor.

We have designed and synthesized a conformationally homogeneous series of cyclic pentapeptides of the general structure c[Pro-aa(i)-D-Tic-Oic-aa(i + 3)] which adopt a type-II' beta-turn conformation believed important for high affinity antagonism of the bradykinin (BK) B2 receptor. We incorporated D-Tic and octahydroindole-2-carboxylic acid (Oic) residues (present in known active antagonists) in a cyclic pentapeptide that would place the D-aa in the i + 1 position of the beta-turn and a proline as a bridge between the C- and N-termini sides of the turn. In positions i and i + 3 alkyl, aromatic, polar or charged amino acids could be introduced without dramatically changing the overall structure. Ten analogues were studied using 1H nuclear magnetic resonance (NMR) and evaluated for their binding affinity for the human B2 receptor. The NMR data in dimethylsulfoxide (DMSO) confirmed the structural homogeneity within the class and, on the basis of this, one representative member of the series was chosen for a detailed structure determination using NMR data in sodium dodecylsulphate (SDS) micelles and molecular dynamics calculations. Despite the structural similarity, the binding affinity of the ten analogues was strongly influenced by the nature of the side-chains in positions i and i + 3, with the doubly charged analogue 49 (pKi = 6.2) proving best. This compound may serve as the starting point for the discovery of new non-peptide bradykinin B2 receptor antagonists.

Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells.

In this study, we analysed the agonist-promoted trafficking of human B(2) (B(2)R) and B(1) (B(1)R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B(2)R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B(2)R using radioligand binding and by imaging of B(2)R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B(1)R was sequestered to a considerably lesser extent upon binding of des-Arg(10)-kallidin. B(2)R sequestration was rapid (half-life approximately 5 min) and reached a steady-state level that was significantly lower than that of BK sequestration. B(2)R sequestration was minimally inhibited by K44A dynamin (22.4+/-3.7%), and was insensitive to arrestin-(319-418), which are dominant-negative mutants of dynamin I and beta-arrestin respectively. Furthermore, the B(2)R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the beta(2)-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2+/-16.3%) and by arrestin-(319-418) (36.9+/-4.4%). Our results show that B(2)R is sequestered to a significantly greater extent than is B(1)R upon agonist treatment in HEK293 cells. Furthermore, B(2)R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and beta-arrestin-independent manner and, at least in part, involves caveolae.

In vivo footprinting analysis of the human kinin B1 receptor gene promoter

In vivo footprinting analysis of the human kinin B1 receptor gene promoter

Adenovirus-mediated human tissue kallikrein gene delivery inhibits neointima formation induced by interruption of blood flow in mice.

Tissue kallikrein cleaves kininogen to produce vasoactive kinin peptides. Binding of kinins to bradykinin B(2) receptors on vascular endothelial cells stimulates the release of nitric oxide and prostacyclin, thus activating the cGMP and cAMP pathways. In this study, we evaluated the effects of adenovirus-mediated human tissue kallikrein gene (Ad.CMV-cHK) delivery in a mouse model of arterial remodeling induced by permanent alteration in shear stress conditions. Mice underwent ligature of the left common carotid artery and were injected intravenously with saline or 1.8 x 10(9) plaque-forming units of Ad.CMV-cHK or control virus (Ad.CMV-LacZ). Fourteen days after surgery, morphometric analysis revealed that Ad. CMV-cHK reduced neointima formation by 52% (P<0.05) compared with Ad. CMV-LacZ. Expression of human tissue kallikrein (HK) mRNA was detected in mouse carotid artery, aorta, kidney, heart, and liver, and recombinant HK was present in the urine and plasma of mice receiving HK gene. Kallikrein gene transfer resulted in increases in urinary kinin, cGMP, and cAMP levels. The protective action of Ad. CMV-cHK on neointima formation was significantly reduced (P<0.05) in mice with knockout of the kinin B(2) receptor gene compared with wild-type control mice (J129Sv mice). In contrast, the effect of Ad. CMV-cHK was amplified (P<0.05) in transgenic mice overexpressing human B(2) receptor compared with wild-type control mice (c57/Bl6 mice). Thus, the inhibitory effect of recombinant kallikrein on structural alterations caused by the interruption of blood flow appears to be mediated by the B(2) receptor. These results provide new insight into the role of the tissue kallikrein-kinin system in vascular remodeling and suggest the application of HK gene therapy to treat restenosis and atherosclerosis.

G(-699)/C polymorphism in the bradykinin-1 receptor gene in patients with renal failure.

Bradykinin is thought to have protective effects on the progression of renal failure. Of particular interest, it has been reported that one polymorphism in the promoter region of the human kinin B1-receptor gene which is associated with higher activity, is less frequently found in patients with end-stage renal failure. The present study was performed to independently confirm these results. Cross-sectional study on 376 healthy controls, 262 non-diabetic dialysis patients and 175 patients with type 1 diabetes >/=10 years and microalbuminuria (of whom 21 were dialysis-dependent) and 334 patients with type 2 diabetes >/=10 years and nephropathy (of whom 61 were dialysis-dependent). Genotyping was performed by polymerase chain reaction, followed by restriction enzyme analysis. All groups were in Hardy Weinberg equilibrium. The study showed no significant difference in the frequency of the C-allele between controls (0.093) and non-diabetic dialysis patients (0.095). No significant difference in C-allele frequency was observed between controls and patients with type 1 diabetes and microalbuminuria (0.092) or patients with type 2 diabetes and nephropathy (0.099). In large cohorts of patients with non-diabetic end-stage renal disease and diabetic renal disease with and without end-stage renal failure, no change in the frequency of the C-699 allele of the B-1-receptor gene was found.

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: A possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.

Nonpeptide mimic of bradykinin with long-acting properties

Kinins, members of a family of peptides released from kininogens by the action of kallikreins, have been implicated in a variety of biological activities including vasodilation, increased vascular permeability, contraction of smooth muscle cells and activation of sensory neurons. However, investigation of the physiological actions of kinins have been greatly hampered because its effects are curtailed by rapid proteolytic degradation. We examined the pharmacological characteristics of the first nonpeptide bradykinin receptor agonist 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline (FR190997). FR190997, whose structure is quite different from the natural peptide ligand, but is similar to the nonpeptide antagonists FR165649, FR167344 and FR173657, potently and selectively interacts with the human B2 receptor and markedly stimulates inositol phosphate formation in transfected Chinese hamster ovary (CHO) cells. FR190997 induces concentration-dependent contraction of isolated guinea pig ileum. In vivo, FR190997 mimics the biological action of bradykinin and induces hypotensive responses in rats with prolonged duration, presumably as a consequence of its resistance to proteolytic degradation. Therefore, FR190997 is a highly potent and subtype-selective nonpeptide agonist which displays high intrinsic activity at the bradykinin B2 receptor. This compound represents a powerful tool for further investigation of the physiology and pathophysiology of bradykinin receptors.

Homologous and heterologous induction of the human bradykinin B1-receptor and B1-receptor localisation along the rat nephron

The kinin B1-receptor which is absent or expressed at very low levels under physiological conditions is strongly induced under inflammatory conditions. It has been shown that B1-receptor induction during inflammation involves interleukin-1β (IL-1β) production and activation of nuclear factor-κB (NF-κB). Since bradykinin (BK), the B2-receptor agonist induces IL-1β expression and activates NF-κB, we have analysed the effect of B2-receptor activation in cultured human lung fibroblasts cells on B1-receptor expression by a semiquantitative RT-PCR analysis. Treatment with BK resulted in a significant increase in the expression of B1-receptor mRNA which was abolished by a specific B2-receptor antagonist. This result suggests that B2-receptor activation can prime the expression of B1-receptors. Although the renal localisation of the B2-receptor has been thoroughly studied, nothing is known about the distribution of the B1-receptor in the kidney. Using a combination of microdissection and a semiquantitative RT-PCR/Southern blot analysis we showed the absence of B1-receptors under physiological conditions in 10 microdissected rat nephron segments. However, 18 h LPS-treatment induced significant expression of the B1-receptor in all, but one segment. These studies provide the first molecular basis for the observed changes in renal haemodynamics after B1-agonist infusion in animal kidney models.