Marie-Thérèse Bawolak

B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin) Is an Inactivation-Resistant Agonist of the Bradykinin B2 Receptor Derived from the Peptide Antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-bradykinin): Pharmacologic Profile and Effective Induction of Receptor Degradation

The bradykinin B2 receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 (d-Arg-[Hyp3,Igl5,d-Igl7,Oic8]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B2 receptor and insurmountable at the rabbit B2 receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (d-Arg-[Hyp3,Igl5,Oic7,d-Igl8]-bradykinin; inverted sequence Oic7, d-Igl8) and B-9972 (d-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin); they are low- and high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B2 receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B2 receptors: conjugate of the B2 receptor with green fluorescent protein (B2R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B2 receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B2 receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B2R-GFP desensitization ([3H]bradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B2 receptors by the agonists. The results illustrate the agonist-antagonist transition in B2 receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist.

Role of Nuclear Factor-κB and Protein Kinase C Signaling in the Expression of the Kinin B1 Receptor in Human Vascular Smooth Muscle Cells

Kinin B1 receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of three previously proposed pathways [nuclear factor-κB (NF-κB), protein kinase C, and agonist autoregulation] that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real-time reverser transcription/polymerase chain reaction with an optional actinomycin D treatment period, and NF-κB immunofluorescence were primarily employed in these primary cell cultures. Various stimulatory compounds that increase receptor mRNA stability only (human and bovine sera, cycloheximide) or that stimulate NF-κB nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1β, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B1 receptor agonist [3H]Lys-des-Arg9-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-κB p65 is necessary for the effective expression of the cell surface B1 receptor under basal or IL-1β, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1β-, FBS-, or PMA-induced stabilization of B1 receptor mRNA was inhibited by the addition of the protein kinase C inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF-109203x), which also diminished the Bmax under FBS or PMA treatment. Lys-des-Arg9-bradykinin had little effect on NF-κB activation, the Bmax, or receptor mRNA abundance or stability. Both NF-κB and protein kinase C signaling are required for the effective expression of the kinin B1 receptor in human umbilical artery smooth muscle cells.

Effects of inactivation-resistant agonists on the signalling, desensitization and down-regulation of bradykinin B2 receptors

Background and purpose:  A peptide bradykinin (BK) B2 receptor agonist partially resistant to degradation, B‐9972, down‐regulates this receptor subtype. We have used another recently described non‐peptide agonist, compound 47a, as a tool to study further the effects of metabolically more stable and thus persistent, agonists of the BK B2 receptor on signalling, desensitization and down‐regulation of this receptor.

Fluorescent Ligands of the Bradykinin B1 Receptors: Pharmacologic Characterization and Application to the Study of Agonist-Induced Receptor Translocation and Cell Surface Receptor Expression

Unlike the widely distributed and preformed B2 receptors, the bradykinin B1 receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B1 receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-ϵ-aminocaproyl-Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-bradykinin] and the agonist B-10378 [5(6)CF-ϵ-aminocaproyl-Lys-des-Arg9-bradykinin]. B-10376 has a Ki of 10 to 20 nM to displace [3H]Lys-des-Arg9-bradykinin from rabbit or human recombinant B1 receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA2, 7.49). B-10378 was a full agonist at the naturally expressed B1 receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition Ki of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B1 receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B1 receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B1 receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B1 receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).

Design of Fluorescent Bradykinin Analogs: Application to Imaging of B2 Receptor-Mediated Agonist Endocytosis and Trafficking and Angiotensin-Converting Enzyme

The known structure-activity relationship and docking models for peptide ligands of the bradykinin B2 receptor indicate a certain tolerance to N-terminal extension. We took advantage of this by generating two fluorescent bradykinin analogs containing 5(6)-carboxyfluorescein (CF) optionally used with the ε-aminocaproyl spacer condensed at the N terminus of the agonist. Pharmacological studies indicated that CF-bradykinin was virtually inactive as a B2 receptor ligand and agonist, whereas CF-ε-aminocaproyl-bradykinin (CF-εACA-BK) was 400- to 1000-fold less potent than bradykinin (competition of [3H]bradykinin binding to B2 receptors, contractility of the human isolated umbilical vein). Nevertheless, CF-εACA-BK (5 μM) was taken up by human embryonic kidney 293a cells expressing recombinant B2 receptors, but not by those cotreated with an antagonist or expressing a truncated receptor that is pharmacologically intact but not phosphorylable. A higher-affinity CF-conjugated peptide, the antagonist CF-εACA-d-Arg-[Hyp3,Igl5,d-Igl7,Oic8]-bradykinin (B-10380), labeled both intact and truncated receptor forms at the cell surface. The fluorescent agonist CF-εACA-BK was found in vesicles positive for β-arrestin1, Rab5, and Rab7, then apparently degraded as a function of time because the fluorescence was transferred from the vesicles to the cytosol in a vesicular-ATPase-dependent process (3 h). The ectopeptidase angiotensin-converting enzyme (ACE) is a major kininase. The binding affinity of CF-εACA-BK for this carboxydipeptidase is identical to that of bradykinin ([3H]enalaprilat displacement assay). Recombinant ACE is essentially a plasma membrane protein in CF-εACA-BK imaging of intact cells. Micromolar CF-εACA-BK is a probe for the two major physiological targets of bradykinin, the B2 receptor and ACE. As an agonist, it is subjected to β-arrestin-mediated endocytosis, trafficking, and subsequent ligand degradation.

A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells

Angiotensin converting enzyme (ACE) is a drug target and an effective bradykinin (BK)-inactivating ectopeptidase. We exploited a recently described [(3)H]enalaprilat binding assay to quantify the full dynamic range of ACE expression in intact human umbilical vein endothelial cells (HUVECs) stimulated with known or novel modulators of ACE expression. Further, the affinities for ACE of a set of physiological substrates were determined using the same assay. BK has the highest affinity (K(i) 525 nM) among known substrates to displace [(3)H]enalaprilat binding from ACE. Tumor necrosis factor (TNF)-alpha repressed the expression of ACE in HUVECs while phorbol 12-myristate 13-acetate (PMA) upregulated it in 24h (approximately 12-fold dynamic range by [(3)H]enalaprilat binding, corroborated by ACE immunoblotting). Intermediate levels of ACE expression were seen in cells stimulated with both PMA and a cytokine. In contrast, high glucose, insulin or EGF failed to affect ACE expression. The effect of TNF-alpha was abated by etanercept, the IKK2 inhibitor TPCA-1, or a p38 inhibitor while that of PMA was reduced by inhibitors of PKC isoforms sensitive to phorbol esters and calcium. The short-term PKC- and MEK1-dependent increase of c-Fos expression was best correlated to PMA-induced ACE upregulation. The [(3)H]enalaprilat binding assay applied to HUVECs supports that ACE is a particularly active kininase and that endothelial ACE expression is dynamically and specifically regulated. This has potential importance in inflammatory diseases and diabetes.

Prolonged signalling and trafficking of the bradykinin B2 receptor stimulated with the amphibian peptide maximakinin: Insight into the endosomal inactivation of kinins

Maximakinin, a 19-residue peptide from the amphibian Bombina maxima, incorporates the full sequence of bradykinin (BK) at its C-terminus with a hydrophilic 10-residue N-terminal extension. As a putative venom component, it may stimulate BK B(2) receptors (B(2)Rs) in a distinct manner relative to the fragile mammalian agonist BK. Maximakinin affinity for B(2)Rs and angiotensin converting enzyme (ACE) and its pharmacological profile have been compared to those of BK. Maximakinin is an agonist of the human and rabbit B(2)R with a 8-12 fold lesser potency, but a prolonged duration of action relative to BK (ERK MAP kinase activation, c-Fos induction in HEK 293 cells). Maximakinin had a moderately inferior affinity (∼6-fold vs. BK) for recombinant ACE based on [(3)H]enalaprilat binding displacement. Unlike BK, maximakinin induced the internalization of the fusion protein B(2)R-green fluorescent protein (GFP) and the downregulation of this construction over a 12-h stimulation period, reproducing the effect of inactivation-resistant B(2)R agonists. Alternate homologues of BK extended at the N-terminus showed intermediate behaviours between BK and maximakinin in the B(2)R-GFP downregulation assay. The recycling of B(2)R-GFP at the cell surface after a 3-h BK treatment was notably inhibited by cotreatment with E-64 or bafilomycin A1, supporting that an endosomal cysteine protease degrades kinins in a process that determines the cycling and fate of the B(2)R. Maximakinin is the first known natural kinin sequence that elicits a prolonged cellular signalling, thus suggesting a possible basis for a venomous action and a naturally selected one for the design of B(2)R-transported biotechnological cargoes.

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

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) 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 ε-aminocaproyl (ε-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 (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-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 B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-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.

Vascular smooth muscle contractility assays for inflammatory and immunological mediators.

The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B(2) receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B(1) receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other.

Met-Lys-bradykinin-Ser-Ser, a peptide produced by the neutrophil from kininogen, is metabolically activated by angiotensin converting enzyme in vascular tissue

Bradykinin (BK) is a vasoactive nonapeptide cleaved from circulating kininogens and that is degraded by angiotensin converting enzyme (ACE). It has been reported that the PR3 protease from human neutrophil releases an alternate peptide of 13 amino acids, Met-Lys-BK-Ser-Ser, from high molecular weight kininogen. We have studied vascular actions of this kinin. Its affinity for recombinant B₁ and B₂ receptors is very low, as assessed by the binding competition of [³H]Lys-des-Arg⁹-BK and [³H]BK, respectively, but Met-Lys-BK-Ser-Ser effectively displaced a fraction of [³H]enalaprilat binding to recombinant ACE. Mutant recombinant ACE constructions revealed that affinity gap between BK and Met-Lys-BK-Ser-Ser is larger for the N-terminal catalytic site than for the C-terminal one, based on competition for the substrate Abz-Phe-Arg-Lys(Dnp)-Pro-OH in an enzymatic assay. Met-Lys-BK-Ser-Ser is a low potency stimulant of the rabbit aorta (bioassay for B₁ receptors), but the human isolated umbilical vein, a contractile bioassay for the B₂ receptors, responded to Met-Lys-BK-Ser-Ser more than expected from the radioligand binding assay, this agonist being ∼30-fold less potent than BK in the vein. Venous tissue treatment with the ACE inhibitor enalaprilat reduced the apparent potency of Met-Lys-BK-Ser-Ser by 15-fold, while not affecting that of BK. In the rabbit isolated jugular vein, Met-Lys-BK-Ser-Ser is nearly as potent as BK as a contractile stimulant of endogenous B₂ receptors (EC₅₀ values of 16.3 and 10.5 nM, respectively), but enalaprilat reduced the potency of Met-Lys-BK-Ser-Ser 13-fold while increasing that of BK 5.3-fold. In vascular tissue, ACE assumes a paradoxical activating role for Met-Lys-BK-Ser-Ser.