Development of a binding assay for the B1 receptors for kinins

doi:10.1016/0162-3109(94)00053-I

Immunopharmacology

Volume 29, Issue 2, March 1995, Pages 141-147

Immunopharmacology

https://doi.org/10.1016/0162-3109(94)00053-I Get rights and content

Abstract

A novel binding assay to kinin B₁ receptors was developed, based on the design of a high-affinity agonist ligand, [¹²⁵I]Tyr-Gly-Lys-Aca-Lys-des-Arg⁹-BK. Binding to rabbit aortic smooth muscle cells is highly temperature-dependent (optimal at 37 °C); apparent binding equilibrium is reached within 30 min, and competition by kinin analogs reveals the expected correlation with the B₁ receptor pharmacology. The dissociation constant (K_d) of the labeled ligand is approx. 0.2 nM and this value does not change significantly as a function of cytokine pretreatment. However, the receptor abundance (B_max) is significantly increased (1.5-fold) by pretreating the cells with interleukin-1 (IL-1), while oncostatin M (OSM) produces a marginal increase of the B_max. This assay may be useful in documenting the regulation of B₁ receptors in pathology.

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Cited by (39)

Synthesis and evaluation of a68Ga-labeled bradykinin B1 receptor agonist for imaging with positron emission tomography
2017, Bioorganic and Medicinal Chemistry
Citation Excerpt :
Since Sar-[Hyp4,Cha6,d-Phe9,des-Arg10]kallidin was previously reported as a potent B1R agonist,31 our data showed that replacing Sar (sarcosine) with Ga-DOTA-Ahx did not change their agonist characteristics. In addition, our data also confirmed the reports by others that modification at the N-terminus of B1R-targeting peptides was tolerable without significantly affecting their binding affinities.32–34 To assess the stability of 68G-Z01115, both in vitro and in vivo studies were conducted and monitored by HPLC.
A novel ⁶⁸Ga-labeled bradykinin B1 receptor (B1R) agonist, ⁶⁸Ga-Z01115, was synthesized and evaluated for imaging with positron emission tomography (PET). Z01115 exhibited good binding affinity (K_i = 25.4 ± 5.1 nM) to hB1R. ⁶⁸Ga-Z01115 was prepared in 74 ± 5 decay-corrected radiochemical yield with >99% radiochemical purity and 155 ± 89 GBq/µmol (4.2 ± 2.4 Ci/μmol) specific activity. ⁶⁸Ga-Z01115 was stable in vitro in mouse plasma (93% remaining intact after 60 min incubation), and relatively stable in vivo (51 ± 5% remaining intact at 5 min post-injection). PET imaging and biodistribution studies in mice showed that ⁶⁸Ga-Z01115 cleared rapidly from nontarget tissues/organs, and generated high target-to-nontarget contrast images. The uptake of ⁶⁸Ga-Z01115 in B1R-positive (B1R+) tumor was 5.65 ± 0.59%ID/g at 1 h post-injection. Average contrast ratios of B1R+ tumor-to-B1R− tumor, -to-blood and -to-muscle were 24.3, 24.4 and 82.9, respectively. Uptake of ⁶⁸Ga-Z01115 in B1R+ tumors was reduced by ∼90% with co-injection of cold standard, confirming it was mediated by B1R. Our data suggest that ⁶⁸Ga-Z01115 is a promising tracer for imaging the expression of B1R that is overexpressed in a variety of cancers.
N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: Structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes
2012, Peptides
Citation Excerpt :
Maximakinin, a natural sequence isolated from amphibian skin, is composed of the full BK sequence at its C-terminal region with a 10-residue N-terminal extension (DLPKINRKGPRPPGFSPFR); this peptide has pharmacological activity in mammalian tissues [27,7] (see also Section 3.1.1). N-terminal extension of both agonist and antagonist B1R ligands is illustrated by development of the high affinity radioligand [125I]-Tyr-Gly-Lys-ɛ-aminocaproyl-Lys-des-Arg9-BK [22], successfully used in tumor autoradiography [25], or of the highly potent antagonist B-10324 (2,3,4,5,6-pentafluorocinnamoyl-B-9958) [16]. In this paper we review the properties of a series of recently produced and characterized kinin receptor ligands.
Peptide agonists and antagonists of both bradykinin (BK) B₁ and B₂ receptors (B₁R, B₂R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ɛ-aminocaproyl (ɛ-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B₁R: B-10376: CF-ɛ-ACA-Lys-Lys-[Hyp³, CpG⁵, d-Tic⁷, CpG⁸]des-Arg⁹-BK; B₂R: B-10380: CF-ɛ-ACA-d-Arg-[Hyp³, Igl⁵, d-Igl⁷, Oic⁸]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B₂R agonists CF-ɛ-ACA-BK, AF350-ɛ-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B₁R agonist B-10378 (CF-ɛ-ACA-Lys-des-Arg⁹-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ɛ-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.
Novel kinin B1 receptor agonists with improved pharmacological profiles
2009, Peptides
There is some evidence to suggest that inducible kinin B1 receptors (B1R) may play beneficial and protecting roles in cardiovascular-related pathologies such as hypertension, diabetes, and ischemic organ diseases. Peptide B1R agonists bearing optimized pharmacological features (high potency, selectivity and stability toward proteolysis) hold promise as valuable therapeutic agents in the treatment of these diseases. In the present study, we used solid-phase methodology to synthesize a series of novel peptide analogues based on the sequence of Sar[dPhe⁸]desArg⁹-bradykinin, a relatively stable peptide agonist with moderate affinity for the human B1R. We evaluated the pharmacological properties of these peptides using (1) in vitro competitive binding experiments on recombinant human B1R and B2R (for index of selectivity determination) in transiently transfected human embryonic kidney 293 cells (HEK-293T cells), (2) ex vivo vasomotor assays on isolated human umbilical veins expressing endogenous human B1R, and (3) in vivo blood pressure tests using anesthetized lipopolysaccharide-immunostimulated rabbits. Key chemical modifications at the N-terminus, the positions 3 and 5 on Sar[dPhe⁸]desArg⁹-bradykinin led to potent analogues. For example, peptides 18 (SarLys[Hyp³,Cha⁵, dPhe⁸]desArg⁹-bradykinin) and 20 (SarLys[Hyp³,Igl⁵, dPhe⁸]desArg⁹-bradykinin) outperformed the parental molecule in terms of affinity, functional potency and duration of action in vitro and in vivo. These selective agonists should be valuable in future animal and human studies to investigate the potential benefits of B1R activation.
B-1 bradykinin receptor
2007, xPharm: The Comprehensive Pharmacology Reference
The B₁ receptors for kinins are expressed by vascular, epithelial, nervous, and other cell types and are responsive to a class of kinin metabolites generated by carboxypeptidases N and M, which remove the C-terminal Arg residue (hence the des-Arg nomenclature of ligands). A special feature of this receptor type is its strongly regulated expression in inflammatory tissues. Chronic pain and inflammatory responses could be mediated by the B₁ receptors.
Des-Arg9-bradykinin increases intracellular Ca2+ in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice
2003, European Journal of Pharmacology
The effects of the selective bradykinin B₁ receptor agonist, des-Arg⁹-bradykinin and the bradykinin B₂ receptor agonist, bradykinin were studied on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in murine bronchoalveolar lavage cells from control and ovalbumin-sensitized mice using fura-2 microfluorimetry. The bronchoalveolar lavage cells of control mice, which were predominantly alveolar macrophages, showed an increase in [Ca²⁺]_i in response to bradykinin (1 μM) but not to des-Arg⁹-bradykinin (1 μM), indicating the presence of functional bradykinin B₂ receptors and the absence of B₁ receptors. Such elevation in [Ca²⁺]_i induced by bradykinin was totally inhibited by the selective bradykinin B₂ receptor antagonist, d-Arg⁰-Hyp³-Thi⁵-d-Tic⁷-Oic⁸-bradykinin (HOE-140; 10 μM). In contrast, bronchoalveolar lavage cells from ovalbumin-sensitized and -challenged mice significantly responded to both bradykinin and des-Arg⁹-bradykinin, indicating the presence of both functional bradykinin B₁ and B₂ receptors. Eosinophils exhibited higher response to des-Arg⁹-bradykinin (1 μM; 485% increase in [Ca²⁺]_i) compared to bradykinin (1 μM; 163% increase in [Ca²⁺]_i). This des-Arg⁹-bradykinin-induced [Ca²⁺]_i increase was markedly inhibited by the selective bradykinin B₁ receptor antagonist, Ac-Lys-[d-βNal⁷, Ile⁸]des-Arg⁹-bradykinin (R-715; 10 μM). Des-Arg⁹-bradykinin neither modified the basal [Ca²⁺]_i in lymphocytes nor in mononuclear cells from ovalbumin-sensitized and challenged mice, while bradykinin produced a [Ca²⁺]_i increase in both cell types. Our results further support the implication of the inducible bradykinin B₁ receptors in airway inflammatory response in ovalbumin-sensitized and challenged mice.
Kinin receptors: Functional aspects
2002, International Immunopharmacology
Two types of receptors (B₁R, B₂R) for kinins are defined in mammalian species. Comparative experiments involving recombinant fusion proteins consisting of rabbit B₁R or B₂R fused to GFP-related proteins are exploited to study the regulation of the response to kinins at the receptor level. The following points will be briefly reviewed and supported by some novel data. (1) The constitutive B₂Rs are internalized upon agonist stimulation, but completely recycled to the cell surface; however, B₂R destruction can be achieved following limited proteolysis (extracellular trypsin, neutrophil proteases), a plausible down-regulation mechanism in pathology. (2) The inducible B₁Rs, stimulated by des-Arg⁹-kinins, are not phosphorylated nor internalized upon agonist stimulation, but rather undergo a reversible redistribution to caveolae-related rafts. B₂Rs are also subjected to this translocation, but only transiently (before endocytosis). (3) Based on the analysis of rabbit aortic smooth muscle cells, B₁R induction by cytokines is dependent on nuclear factor κB in rabbit vascular tissue, but exogenous kinins acting on either receptor type do not induce B₁R expression.

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Immunopharmacology

Abstract

References (21)

Recent developments in the understanding of bradykinin receptors

Life Sci

Synthesis of bradykinin analogs

Methods Enzymol

Protein and cell membrane iodinations with a sparingly soluble chloroamide 1,3,4,6-letrachloro-3a,6a-diphrenylglycoluril

Biochem Biophys Res Commun

Receptor-mediated internalization of bradykinin

J Biol Chem

Bradykinin B₁ receptors in rabbit aorta smooth muscle cells in culture

Eur J Pharmacol

Solid phase peptide synthesis

Evidence for existence of two distinct bradykinin receptors on rat mesangial cells

Am J Physiol

Methods in binding studies

The involvement of bradykinin B₁ and B₂ receptor mechanisms in cytokine-induced mechanical hyperlagesia in the rat

Br J Pharmacol

Pulse exposure to protein synthesis inhibitors enhances vascular responses to des-Arg⁹-bradykinin: possible role of interleukin-1

Br J Pharmacol

Cited by (39)

Synthesis and evaluation of a<sup>68</sup>Ga-labeled bradykinin B1 receptor agonist for imaging with positron emission tomography

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: Structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Novel kinin B1 receptor agonists with improved pharmacological profiles

B-1 bradykinin receptor

Des-Arg<sup>9</sup>-bradykinin increases intracellular Ca<sup>2+</sup> in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice

Kinin receptors: Functional aspects

Development of a binding assay for the B1 receptors for kinins

Abstract

Life Sci

Methods Enzymol

Biochem Biophys Res Commun

J Biol Chem

Eur J Pharmacol

Solid phase peptide synthesis

Evidence for existence of two distinct bradykinin receptors on rat mesangial cells

Am J Physiol

Methods in binding studies

The involvement of bradykinin B1 and B2 receptor mechanisms in cytokine-induced mechanical hyperlagesia in the rat

Br J Pharmacol

Pulse exposure to protein synthesis inhibitors enhances vascular responses to des-Arg9-bradykinin: possible role of interleukin-1

Br J Pharmacol

Development of a binding assay for the B₁ receptors for kinins

The involvement of bradykinin B₁ and B₂ receptor mechanisms in cytokine-induced mechanical hyperlagesia in the rat

Pulse exposure to protein synthesis inhibitors enhances vascular responses to des-Arg⁹-bradykinin: possible role of interleukin-1