Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on August 15, 2007; DOI: 10.1124/jpet.107.123422
0022-3565/07/3232-534-546$20.00
JPET 323:534-546, 2007
CARDIOVASCULAR
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
Marie-Thérèse Bawolak,
Lajos Gera,
Guillaume Morissette,
John M. Stewart, and
François Marceau
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec and Department of Medicine, Université Laval, Quebec City, Quebec, Canada (M.-T.B., G.M., F.M.); and Department of Biochemistry, University of Colorado Health Sciences Center, Denver, Colorado (L.G., J.M.S.)
Received for publication
March 26, 2007
Accepted
August 14, 2007.
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Abstract
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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.
The blood-derived peptide bradykinin and its widely expressed B2 receptor subtype constitute together an excellent example of a cycling system of ligand-G protein-coupled receptor (GPCR) complex with agonist-induced receptor phosphorylation by GPCR kinases (Blaukat et al., 2001
), receptor-mediated ligand endocytosis and degradation (Munoz and Leeb-Lundberg, 1992), and, subsequently, very extensive receptor recycling at the cell surface upon agonist washout (Blaukat et al., 1996). In previous studies, a green fluorescent protein conjugate of the rabbit B2 receptor, B2R-GFP, has been shown to be internalized and extensively recycled when the agonist was degraded in the extracellular space (evidence based on immunoblotting using anti-GFP tag antibodies, imaging, and bradykinin concentration measurements; Bachvarov et al., 2001; Houle et al., 2003). Thus, bradykinin clearance, mainly by angiotensin I-converting enzyme (ACE) present in the serum-containing culture medium of HEK 293 cells expressing B2R-GFP, was sufficient for the recycling of the receptor construction at the cell surface in 
60 min (Bachvarov et al., 2001). Pharmacological ACE inhibition with captopril or the use of a carboxypeptidase- and ACE-resistant bradykinin analog, [Phe8
(CH2 NH)Arg9 ]bradykinin (Drapeau et al., 1988), led to a prolonged period (3 h) of B2R-GFP endocytosis in HEK 293 cells, but without degradation (down-regulation) (Bachvarov et al., 2001; Marceau et al., 2002; Houle and Marceau, 2003). Multiple cycles of receptor endocytosis and recycling may be suspected in these experiments. Bradykinin in the presence of captopril or the bradykinin C-protected analog may not be resistant to the multiple degradation pathways operating in endosomes (Munoz and Leeb-Lundberg, 1992). In recent experiments from another laboratory, the essentially complete recycling of human B2R-GFP was attributed to both a high level of surface expression and to a structural effect of GFP that favored receptor recycling over degradation, relative to wild-type human B2 receptors for which bradykinin-induced degradation was significant in less than 2 h (Kalatskaya et al., 2006).
B-9430 (structure in Fig. 1) is a structurally constrained and sequence-related bradykinin antagonist with high affinity at the B2 receptor (Stewart et al., 1996). The drug has been widely exploited in studies attempting to define a role for the kallikrein-kinin system in physiology and pathology (Pan et al., 2001; Uknis et al., 2001; Leeb-Lundberg et al., 2005). Interestingly, an exact isomer of B-9430, B-9972 (Fig. 1; Oic7,Igl8 sequence instead of D-Igl7,Oic8), is reportedly an agonist or a partial agonist possessing a large intrinsic activity (Bironaite et al., 2004; Taraseviciene-Stewart et al., 2005). The transition from an agonist to an antagonist peptide at the B2 receptor may be dependent on the spatial orientation of the C-terminal sequence (Vavrek and Stewart, 1985) and structural constraint in the backbone is not incompatible with the agonist status, as shown with B-9972. This peptide integrates several substitutions that should make it resistant to multiple peptidases/proteases, including aminopeptidases (Fig. 1). In a rare attempt to evaluate therapeutic actions of bradykinin receptor agonists, B-9972 was found to alleviate experimental pulmonary hypertension and its cardiac complications via the classical vasodilator effect mediated by endothelial B2 receptors (Taraseviciene-Stewart et al., 2005).
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Fig. 1. Structure and stereochemistry in the C-terminal region of three kinin receptor ligands that are isomers, B-9430, B-9972, and B-10344. The reference structure is that of bradykinin (BK) (H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH).
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The present work had three general aims: firstly, to extend the pharmacological characterization of B-9972 at the human and rabbit receptors, including some recombinant receptor systems. To further define the effect of the peptide C-terminal stereochemistry on receptor function, an intermediate isomer, B-10344, has been synthesized and evaluated. The second aim was to verify the hypothetical effect of the C-terminal receptor fusion with GFP on the recycling of the rabbit B2 receptor. A construction that included an N-terminal antigenic tag, myc-B2R, has been produced and evaluated for that purpose. Finally, the cycling of traceable forms of the rabbit B2 receptor in HEK 293 cells, B2R-GFP and the myc-B2R construction, have been examined as a function of the agonist structure. As a peptide ligand with a predicted extended resistance to intracellular degradation, notably if compared with [Phe8(CH2 NH)Arg9 ]bradykinin, B-9972 might exert distinctive effects on receptor cycling. Long-term in vivo stimulation with B-9972 reportedly reduced the receptor protein in rats (Taraseviciene-Stewart et al., 2005), raising the possibility to pharmacologically influence the degree of GPCR degradation over recycling.
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Materials and Methods
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Drugs and Reagents. Indanylglycine-containing B2 receptor antagonists were first reported in a series that included B-9430 (Gera and Stewart, 1996; Stewart et al., 1996). B-10344 and B-9972 were designed by swapping the amino acids at the positions 7 and 8 in B-9430 and modifying the stereochemistry of indanylglycine (Fig. 1). B-9972 was previously reported (Bironaite et al., 2004; Taraseviciene-Stewart et al., 2005). B-10344 was synthesized and purified using the same general methods. Several compounds were gifts: [Phe8(CH2 NH)Arg9 ]bradykinin (Drapeau et al., 1988) was from Prof. D. Regoli (University of Sherbrooke, Canada), the B2 receptor antagonists icatibant (Hoe 140) and LF16-0687 were from Laboratoires Fournier (Daix, France), the B2 receptor antagonist bradyzide was from Novartis (London, UK), and the B1 receptor antagonist compound 11 was from Merck Research Laboratories (West Point, PA) (Leeb-Lundberg et al., 2005). The other drugs were purchased from Sigma-Aldrich (St. Louis, MO).
Binding Competition Assay. The binding of 3 nM [3H]bradykinin (PerkinElmer Life and Analytical Sciences, Boston, MA; 90 Ci/mmol) to adherent intact HEK 293 cells stably expressing B2R-GFP was evaluated as described previously (Houle et al., 2000). The assay was applied to construct binding competition curves for a series of unlabeled peptides. A variation of this protocol (radioreceptor assay) was used to quantify the inactivation of bradykinin and of B-9972 (5 µM of either) after a 12-h incubation at 37°C with HEK 293 cells (72-cm2 flasks) maintained in their regular serum-containing culture medium (10 ml). Samples of the final cell supernatants, further diluted 1:25 in the buffer specific for the binding assay, were applied as unknown in a further [3H]bradykinin (3 nM) binding competition assay involving HEK 293 cells expressing B2R-GFP (0°C, in the presence of peptidase and protease inhibitors; Houle et al., 2000). The sample concentrations were expressed as bradykinin or B-9972 concentrations using standard competition curves obtained with each unlabeled authentic peptide. This assay is based on the knowledge of the structure-activity relationship at the rabbit B2 receptor; any bradykinin fragment has negligible affinity.
Contractility Assay: Rabbit Jugular Vein. A local ethics committee approved the animal experimentation. The external jugular vein, an established bioassay for the rabbit bradykinin B2 receptor (Houle et al., 2000), was isolated from male New Zealand White rabbits (1.5–2 kg). The vessels were spirally cut into strips (3 cm long) and were suspended between a metal hook and a thread loop under a tension of 0.5 g in thermostated (37°C) 5-ml tissue baths containing oxygenated (95% O2/5% CO2) Krebs' solution. The composition of Krebs was 117.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.18 mM MgSO4, 2.5 mM CaCl2, 25.0 mM NaHCO3, and 5.5 mM D-glucose. Captopril (1 µM) was continuously present in the bathing fluid. Isometric changes in vascular tone were measured by force transducers (model 52-9545; Harvard Apparatus, South Natick, MA) coupled to chart recorders. These studies aimed to investigate the nature and specificity of bradykinin B2 receptor ligands in the vascular smooth muscle preparation. Each tissue was subjected to the construction of two full cumulative concentration-response curves at times 1 and 4 h postmounting for an agonist (bradykinin, B-9972, or B-10344) in random sequence (the maximal effect of bradykinin helping to detect the full or partial agonist status of the other peptides in a preparation that essentially exhibits stable responses). In other tissues assigned to characterize the antagonist effect of B-9430, the control concentration-effect curve for bradykinin was obtained at time 1 h, the antagonist was applied at time 3.5 h, and the construction of the bradykinin concentration-effect curve was repeated at time 4 h. Contractility results are expressed as a percentage of the maximal response recorded when constructing the control curve (1 h). Results were expressed as mean ± S.E.M., and statistics were calculated with the use of the InStat 3.0 computer program (GraphPad Software Inc., San Diego, CA).
Contractility Assay: Rabbit Aorta. B-9430 was evaluated as an antagonist of the des-Arg9-bradykinin-induced contraction mediated by B1 receptors in this preparation precisely as described previously (Fortin et al., 2005).
Contractility Assay: The Human Umbilical Vein. The institutional research ethics board approved the anonymous use of human umbilical cord segments obtained after elective cesarean section deliveries. Human umbilical cords stored at 4°C were obtained within 24 h from cesareans. Segments of umbilical veins were dissected carefully from the cords, and a metal rod was inserted into the lumen. Excess connective tissue was excised, and rings (2–3 mm wide) were cut. Rings of umbilical veins were suspended under 2 g of baseline tension in 5-ml organ chambers containing oxygenated (95% O2-5% CO2) and warmed (37°C) Krebs' solution, as described above. Tissues randomly assigned to agonist peptides and equilibrated for 3 h before the construction of cumulative concentration curves. In other tissues used to characterize the antagonist effect of B-9430, this drug or saline vehicle was applied at time 2.5 h post-tissue mounting, and the bradykinin concentration-effect curve was constructed at time 3 h.
Construction and Expression of the Rabbit myc-B2R Conjugate. To assess the abundance of kinin receptors in cells in a radioligand-independent manner, we produced and characterized a rabbit B2 receptor labeled with the N-terminal (extracellular) myc epitope. Using the pCDNA3-based vector for the wild-type rabbit B2 receptor (Bachvarov et al., 1995) as a template, the coding region of the B2 receptor gene was amplified by polymerase chain reaction (PCR). 5'-GAACGAATTCGATGCTCAACATCACCTCTCAA-3' and 5'-TGATTCTAGATCACTGTTTGTTCCTCGTCCA-3' were used as sense and antisense PCR primers, respectively (primers derived from the rabbit receptor sequence, Bachvarov et al., 1995). The pair of primers contained additional EcoRI and XbaI sites, respectively (underlined), needed for the directional cloning of the rabbit B2 receptor coding regions in the eukaryotic expression vector pCI-neo (Promega, Madison, WI) to which an N-terminal myc epitope has been added (pCI-neo-myc vector, gift from Dr. Josée Lavoie, Le Centre Hospitalier Universitaire de Québec, Quebec City, QC, Canada). The PCR product and the pCI-neo-myc vector were digested with EcoRI and XbaI (Invitrogen, Carlsbad, CA) and ligated at room temperature for 2 h. The resultant vector (myc-B2R) contain the myc epitope peptide (MEQKLISEEDLNS) fused in frame with the corresponding rabbit receptor coding sequence at its carboxyl terminus. The sequence of the construction was verified (RSVS Core Laboratory, Pavillon Marchand, Laval University, Quebec City, QC, Canada).
Cells, Transfection, and Microscopy. COS-1 cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen). HEK 293 cells, originally obtained from American Type Culture Collection (Manassas, VA), were grown in 
-minimal essential medium supplemented with FBS (5%), horse serum (5%), and penicillin-streptomycin. The derivation of a HEK 293 cell line stably expressing B2R-GFP and its properties are described elsewhere (Houle et al., 2000; Bachvarov et al., 2001). Nontransfected HEK 293 cells were used in control experiments. A novel subclone of HEK 293 cells, dubbed HEK 293a, was obtained from Sigma-Aldrich and used in some experiments. The latter cells were grown and maintained in a different culture medium (Dulbecco's modified Eagle's medium supplemented with 10% FBS, L-glutamine, and antibiotics). COS-1 and HEK 293 cells grown until 70% confluent were transiently (48 h) transfected with the myc-B2R coding vector or the empty vector (pCI-neo-myc) with the use of the EX-Gen 500 transfection reagent (MBI Fermentas Inc., Flamborough, ON, Canada) as directed. COS-1 cells were primarily used for the characterization of the construction with radiolabeled bradykinin, allowing comparison with the wild-type rabbit B2 receptor expressed in the same cell type (Bachvarov et al., 1995). HEK 293a cells stably expressing myc-B2R or untransfected cells were fixed and submitted to indirect immunofluorescence using an anti-myc monoclonal antibody (clone 4A6; Millipore Corporation, Billerica, MA; dilution 1:1000) revealed using an Alexa-fluor 488-labeled anti-mouse goat antibody (Molecular Probes-Invitrogen, Eugene, OR; dilution 1:1000). The cells were further observed in epifluorescence microscopy.
Immunoblots. Immunoblots for B2R-GFP were performed as previously reported (Bachvarov et al., 2001), and the monoclonal anti-GFP antibody JL8 was used (Clontech, Palo Alto, CA). In brief, HEK 293 cells or HEK 293 cells stably expressing B2R-GFP grown in 25-cm2 flasks (80% confluence) were optionally treated with the ACE inhibitor captopril, combined or not with bradykinin, or with another B2 receptor ligand for 3 or 12 h in the regular, serum-containing culture medium. Total cell extracts were recovered and analyzed after SDS-polyacrylamide gel electrophoresis (7.5% gel) and protein transfer using the GFP-specific monoclonal antibody (dilution, 1:1000). Similar treatments were applied to HEK 293a cells transiently expressing myc-B2R; 7% gels were transferred and analyzed using the anti-myc monoclonal antibody 4A6 (Upstate Biotechnology, Lake Placid, NY; dilution, 1:1000). Equal track loading was verified in both types of immunoblots by migrating and transferring the same samples separately and immunoblotting for 
-actin (monoclonal from Sigma-Aldrich; dilution, 1:1000).
[3H]Bradykinin Endocytosis. One of the functions mediated by B2R-GFP in HEK 293 cells is the endocytosis of [3H]bradykinin into a cell organelle fraction that contains endosomes (Bachvarov et al., 2001). This assay was performed as described previously (10 nM radiolabeled agonist, 15-min incubation in regular culture medium in cells previously washed twice with culture medium, followed by extraction) to study whether long (12-h) pretreaments with agonists (bradykinin or B-9972, 100 nM in the regular culture medium) reduce cell surface receptors.
ERK Phosphorylation. An ERK1/2 MAP kinase phosphorylation assay was performed as described previously (Morissette et al., 2007; the same antibodies are currently sold by Cell Signaling Technology Inc., Beverly, MA) to gain insight into receptor desensitization following prolonged treatments with agonists (bradykinin or B-9972, 12 h before acute stimulation). It involved maintaining HEK 293 cells in medium with decreased serum (0.5% for 24 h), an optional treatment with bradykinin, combined or not with captopril, or B-9972 for the last 12 h followed by a 30-min incubation period with fresh low serum (0.5%) culture medium containing 71 µM cycloheximide, rinsing with the same medium, 10-min stimulation with 10 nM of bradykinin or 100 ng/ml epidermal growth factor (EGF) in the same medium, and, finally, cell extraction.
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Results
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Radioligand Binding Competition Assay. The specific binding of [3H]bradykinin (3 nM) was displaced from HEK 293 cells stably expressing B2R-GFP by unlabeled bradykinin, B-9430, and its two isomers B-9972 and B-10344; the three latter peptides were 2.9-, 7-, and 36-fold, respectively, less potent than bradykinin (Fig. 2).
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Fig. 2. Competition of [3H]bradykinin (3 nM) binding to HEK 293 cells stably expressing B2R-GFP by a panel of unlabeled BK-related peptides. Values are the means ± S.E.M. of six to eight experiments composed of duplicate determinations. Average specific binding without competitor (100%) was 91 fmol/well.
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Organ Bath Pharmacology. In the rabbit jugular vein contractility assay, B-9972 is apparently a full agonist with slightly reduced potency relative to the reference B2 receptor agonist, bradykinin (2-fold less potent; Fig. 3A). The isomer B-10344 has a low potency as an agonist because less than 50% of the contraction obtained with bradykinin was reached for a B-10344 cumulative concentration of 6.2 µM. A partial agonist status for this drug may be compatible with the observation of a low slope of its concentration-effect curve and its 2000-fold inferior potency relative to bradykinin (Fig. 3A), whereas B-10344 was only 36-fold less potent than bradykinin in the binding assay (Fig. 2). The other member of the isomer triad, B-9430, had no direct contractile effect on the vein but behaved as a potent and insurmountable antagonist of bradykinin (Fig. 3B), reminiscent of other structurally constrained peptide antagonists tested at the rabbit B2 receptors (Hoe 140 and NPC 17731) (Houle et al., 2000). The tissues treated with B-9430 exhibited only a small residual maximal effect for bradykinin but fully responded to histamine (data not shown), supporting their functional preservation.
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Fig. 3. Effect of the isomer triad on the rabbit jugular vein contractility assay, a bioassay for B2 receptors. A, agonist effect of BK, B-9972, and B-10344 on the preparation. B, antagonist effect of B-9430 on bradykinin-induced contraction. Values are the means ± S.E.M. of the number of determinations indicated by n.
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The isolated rabbit aorta is the contractile bioassay that initially served to define the pharmacological profile of the B1 receptors (Leeb-Lundberg et al., 2005). As reported previously (Stewart et al., 1996), B-9430 retains some affinity for this receptor with concentration-related rightward shift of the agonist concentration-effect curve at antagonist concentration levels of 100 nM and 1 µM (Fig. 4), a surmountable behavior and a calculated pA2 value of 6.49 (Schild plot, not shown). This value is practically the same (6.5) reported for this antagonist in the same bioassay by Stewart et al. (1996); the present experimental protocol, based on the full concentration-effect curves for the agonist, established the full surmountability of B-9430 at the rabbit B1 receptor. In the rabbit aorta, B-9972 had no significant agonist or antagonist action up to 6 µM (data not shown).
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Fig. 4. Antagonist effect of B-9430 on the rabbit aorta contractility assay stimulated with des-Arg9-bradykinin, a bioassay for the B1 receptors. Values are the means ± S.E.M. of the number of determinations indicated by n. For analysis, see Results.
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The same set of peptides was tested in the isolated human umbilical vein, with the addition of an agonist, [Phe8(CH2 NH)Arg9 ]bradykinin (Fig. 5; the effect of the latter peptide on the rabbit isolated jugular vein was reported elsewhere, Drapeau et al., 1988). The same order of agonist potency as in the rabbit vein was observed for bradykinin, B-9972 (4-fold less potent), and B-10344 (200-fold less potent than bradykinin; Fig. 5A). The full agonist status of B-10344 was supported in the human preparation by the recording of a maximal contractile effect that did not differ significantly from those obtained with the other agonists, when expressed in force units (gram weight, data not shown) or in percentage of an internal contractile standard (maximal response to 5-hydroxytryptamine recorded at the end of the experiments, Fig. 6A). B-9430 had no direct contractile effect on the isolated human umbilical veins but competitively antagonized bradykinin-induced contraction (Fig. 5B) with a calculated pA2 value of 7.70 (slope of Schild plot, –1.10).
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Fig. 5. Effect of the isomer triad on the human umbilical vein contractility assay. A, agonist effect of BK, [Phe8(CH2NH)Arg9]bradykinin, B-9972, and B-10344 on the preparation. The concentration-effect curves recorded at time 3 h are shown and were constructed in different tissues the presence of an antagonist (applied 30 min before) or its vehicle (saline). Values are means ± S.E.M. of the number of determinations indicated by n. For analysis, see Results.
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Fig. 6. Additional parameters derived from the cumulative concentration-effect curves constructed for four B2 receptor agonists (Fig. 5A). A, maximal kinin-induced effect expressed as a percentage of the maximal response to 5-hydroxytryptamine (5-HT, 10 µg/ml) recorded at the end of the experiment. B, time for half-relaxation from the maximal response for each kinin after washout of the maximal concentration. ANOVA indicated that the four groups were heterogeneous (P < 0.01). Further comparison with the values for BK using Dunnett's test indicated statistically different values for B-9972 and B-10344 (P values reported in figure).
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[Phe8(CH2NH)Arg9]bradykinin, with hypothetical inferior resistance to peptidases compared with B-9972, has a potency 3.5-fold inferior to the latter peptide (Fig. 5A), making B-9972 a more valuable term of comparison with bradykinin. In this set of experiments, the agonists were compared for the time for half-relaxation from maximal effect upon agonist washout. Bradykinin contracted with the maximal cumulative concentration (10.5 µM) was half-relaxed in 17.2 ± 2.9 min (Fig. 6B); the t1/2 for [Phe8(CH2NH)Arg9]bradykinin (10.5 µM) was not significantly different, but those for B-9972 (6.2 µM) or B-10344 (20.1 µM) were much higher and similar between them (Fig. 6B). Thus, the measurement of relaxation velocity from the maximal response of the agonists may be related to the predicted resistance to peptidases independently of potency, B-9972 and B-10344 being the most resistant peptides.
To identify pathways that could inactivate bradykinin in the human umbilical vein, concentration-effect curves were constructed in the presence of the ACE inhibitor enalaprilat, the neutral endopeptidase inhibitor phosphoramidon, or the aminopeptidase P inhibitor apstatin (Prechel et al., 1995). The peptidase inhibitors failed to potentiate bradykinin (Fig. 7A) or to influence the relaxation t1/2 measured after the washout of the maximal bradykinin concentration (Fig. 7B), suggesting that the corresponding peptidases are not of ut-most importance for bradykinin metabolism in this system.
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Fig. 7. Effect of enalaprilat, phosphoramidon, and apstatin on the concentration-effect relationship of BK as a contractile agent in the human umbilical vein (A) and the time for half-relaxation from the maximal response following bradykinin washout (B). Peptidase inhibitor concentrations are indicated. Presentation as in Figs. 5A and 6B.
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Effect of Agonists at B2R-GFP Expressed in HEK 293 Cells. As previously reported, the fluorescence associated with the B2R-GFP construction is mainly associated with the plasma membrane of HEK 293 cells (epifluorescence microscopy, Fig. 8). Although the antagonist B-9430 (5 µM) or the peptidase inhibitor captopril (1 µM) exerted no important effect on the fluorescence distribution, B2 receptor agonists translocated the fluorescence in a typical manner (Fig. 8A). After 30 min of incubation with bradykinin (100 nM), combined or not with captopril, or with B-9972 (100 nM), the fluorescence was redistributed from the plasma membrane, which became in general discontinuously labeled or unlabeled, to ill-defined intracellular structures of various sizes. These changes were largely prevented in cells cotreated with the antagonist B-9430 (5 µM). Longer treatments (12 h) revealed the reversibility of bradykinin-induced internalization of B2R-GFP, the loss of membrane localization of the fluorescence in B-9972-treated cells, and a partial recovery in most cells exposed to the bradykinin-captopril combination (Fig. 8B). Half-lives of 7.7 and 102 min have been found previously for bradykinin (100 nM) in the employed serum-containing culture medium at 37°C in the absence or presence of captopril, respectively (Bachvarov et al., 2001). Cells stimulated with B-9972 (100 nM) for 30 min and further washed with the complete culture medium and incubated for 11.5 h at 37°C were indistinguishable from control cells, suggesting that B2R-GFP molecules bound to this peptide are not committed to permanent sequestration and may recycle back to the plasma membrane. Experimental evidence of B-9972 degradation was obtained, but it was slower than that of bradykinin; after a 12-h incubation period of agonist peptides (5 µM) in the complete culture medium in the presence of HEK 293 cells that expressed B2R-GFP, the residual concentration of B-9972 was 100 nM; that of bradykinin was below the limit of detection (<10 nM; radioreceptor assay).
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Fig. 8. Epifluorescence microscopy studies of live HEK 293 cells stably expressing B2R-GFP and stimulated for 30 min (A) or 12 h (B) with B2R ligands and captopril at the indicated concentrations (double photographic samples shown for agonists). In B, some cells were stimulated with an agonist for 30 min, followed by an 11.5-h washout (w/o) period in complete culture medium. Control cells generally exhibit sharply defined plasma membrane-associated fluorescence. Typical results of 2 to 3 independent days of observation in multiple microscopic fields. Original magnification, 1000x.
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The fluorescent fusion protein B2R-GFP is efficiently internalized by agonist treatment but not significantly reduced in abundance by 3-h treatments with bradykinin or [Phe8(CH2NH)Arg9]bradykinin (see Introduction). Some of these results have been replicated in HEK 293 cells stably expressing B2R-GFP and treated with receptor ligands and/or captopril in the presence of cycloheximide (71 µM) to prevent the synthesis of novel receptors (immunoblot based on anti-GFP antibodies, Fig. 9, left; statistical analysis of four replicates applied to the agonists in Fig. 10A). The 101-kDa band corresponding to the fusion protein B2R-GFP was not degraded by 3-h treatments with bradykinin (100 nM) and captopril (1 µM; alone or combined) or the isomer peptides B-9972 (100 nM) or B-9430 (5 µM). Untransfected cell extracts reacted very little with the anti-GFP antibody. A longer (12-h) treatment of intact cells maintained at 37°C in the regular culture medium with bradykinin did not induce receptor degradation (tests run without cycloheximide due to long term toxicity of the latter). However, bradykinin combined with captopril or B-9972 alone induced the partial degradation of B2R-GFP, based on the parallel emergence of GFP-sized metabolites in the total cell extracts (Fig. 9, center and right). B-9972 was the most active agent in this respect, also significantly capable of decreasing the quantity of the fusion protein B2R-GFP in the corresponding cell extract. The effect of the agonists on immunoblot band intensity was quantified in four experiments (two of which are shown in Fig. 9) and presented in Fig. 10B. Either a nonpeptide B2 receptor antagonist (LF16-0687, 5 µM) or the peptide B-9430 (5 µM) abated the degradation of B2R-GFP induced by bradykinin + captopril or B-9972 (Fig. 9, middle and right, respectively). The antagonists alone may have promoted this degradation to a small extent, especially B-9430.
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Fig. 9. Effect of agonists on B2R-GFP expressed in HEK 293 cells: immunoblot of total cell extracts based on anti-GFP antibodies. The cells were submitted to the indicated treatments for 3 h in the presence of 71 µM cycloheximide (left) or 12 h (without cycloheximide, middle and right) in the regular culture medium with heat-inactivated FBS before extraction.
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Validation of a myc-B2R Construction. A novel N-terminally tagged myc-B2R construction, based on the rabbit B2R, was pharmacologically characterized. COS-1 cells transiently transfected with the pCI-neo-myc vector (sham transfection) bound little [3H]bradykinin, whereas cells that expressed myc-B2R exhibited specific and saturable binding (Fig. 11A). The affinity estimate derived from Scatchard plot analysis (Fig. 11B) was Kd = 2.38 nM; this value is close to the previously reported estimate (2.1 nM) in COS-1 cells based on similar techniques for the wild-type receptor (Bachvarov et al., 1995). The Bmax estimate was 34.3 fmol/well (Fig. 11, A and B). The pharmacological profile of the myc-B2R construction transiently expressed in COS-1 cells was investigated by the competition of [3H]bradykinin binding to cells by a panel of unlabeled bradykinin-related drugs used at 1 µM (Fig. 11C). The binding of 1 nM [3H]bradykinin was virtually abolished by 1 µM unlabeled bradykinin, Lys-bradykinin, or by any of the three B2 receptor antagonists, Hoe 140, bradyzide, and LF16-0687 (Fig. 11C). The des-Arg9 fragments of the native kinins bradykinin or Lys-bradykinin (selective B1 receptor agonists) were only marginally active in this respect. Compound 11, a high-affinity B1R antagonist, was inactive to displace [3H]bradykinin from myc-B2Rs.
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Fig. 11. Pharmacological characterization of recombinant myc-B2R transiently expressed in COS-1 cells. A, saturation curves of [3H]BK in cells transfected with the indicated vector. Values are the mean of duplicate determinations. The experiment shown is representative of two experiments. B, Scatchard plot derived from binding data in A for myc-B2R. C, competition of the binding of [3H]bradykinin (1 nM) to COS-1 cells transiently expressing myc-B2R by a panel of unlabeled drugs (1 µM each; average ± S.E.M. of two experiments composed of duplicate determinations). The total residual binding is presented.
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Anti-myc antibody may be used to assess the abundance of the surface myc-B2R receptor construction in a manner independent from ligands. The feasibility of this approach is shown in fixed and optionally permeabilized transfected HEK 293a cells (Fig. 12). Indirect immunofluorescence based on the monoclonal antibody 4A6 showed the abundant expression of myc-B2R in stably transfected HEK 293a cells, but much of the signal was intracellular (Fig. 12, left), consistent with the prominent amount of receptor protein present in the secretory pathway, as previously observed with other constructions (Fortin et al., 2006). A treatment with the agonist bradykinin translocated some of the surface fluorescence into granular material distinct from the more diffuse baseline intracellular fluorescence; in cells that have not been permeabilized, a decrease of the average fluorescence intensity at the cell surface was observed, consistent with receptor internalization.
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Fig. 12. Immunofluorescence of the myc epitope in HEK 293a cells expressing myc-B2R (epifluorescence based on the 4A6 antibody with phase contrast microscopy). All cells were fixed and optionally permeabilized, as indicated. Original magnification, 400x. Some cells were exposed to the agonist BK, added to the culture medium, and further incubated at 37°C for 10 min before observation, as indicated.
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HEK 293a cells transiently expressing myc-B2R expressed enough recombinant protein for immunoblotting detection based on the myc tag (Fig. 13). The 4A6 antibody was used to detect myc-B2R in total HEK 293a cell extracts. Twin bands (63 and 69 kDa) were considered to be the mature receptors, consistent with findings from several laboratories and confirmed with the highly purified form of the human B2 receptor (60–70 kDa; Camponova et al., 2007, and literature cited herein). Lower molecular weight bands were not informative. Digitized results from four experiments with 3- or 12-h agonist treatments are presented in Fig. 10, C and D, with statistical analysis. The 63- to 69-kDa myc-B2R bands were significantly reduced only by the 12-h treatments with B-9972 (100 nM). B-9430 prevented agonist-induced degradation (Fig. 13). Three-hour agonist treatments were not active to degrade myc-B2R (Figs. 10C and 13).
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Fig. 13. Effect of agonists on myc-B2R transiently expressed in HEK 293a cells: immunoblot of total cell extracts based on anti-myc antibodies. The cells were submitted to the indicated treatments for 3 (left, with 71 µM cycloheximide) or 12 h (right, without cycloheximide) in the regular culture medium with heat-inactivated FBS before extraction. In the left panel, some conditions are duplicated.
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Functional Desensitization of B2R-GFP by B-9972. A documented function of cell surface B2R-GFP is the rapid internalization of [3H]bradykinin into a fraction of HEK 293 cell organelles that contains endosomes (Bachvarov et al., 2001). This assay was adapted to probe the residual function of receptors in cells pretreated for 12 h with bradykinin or B-9972 (100 nM; Fig. 14A). Untransfected HEK 293 cells did not appreciably take up [3H]bradykinin, but the presence of B2R-GFP in stable transfectant cells determined a large up-take; the latter was not modified by bradykinin pretreatment, but long exposure to B-9972 reduced the uptake significantly by 80% (Fig. 14A).
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Fig. 14. Functional desensitization of B2R-GFP in HEK 293 cells submitted to 12-h treatments with agonists. A, [3H]bradykinin incorporation (10 nM, 15 min) in the 15,000-g pellet of the cell extract as a function of receptor presence and agonist pretreatment (12-h exposure to 100 nM BK or 100 nM B-9972). ANOVA indicated that the amount of captured radioactivity varied significantly as a function of treatments (P < 0.001). *, P < 0.01 versus control-recombinant B2 receptor value, Dunnett's test. B, immunoblots for phospho-ERK1/2 and total ERK1/2 in HEK 293 cells stably expressing B2R-GFP in response to bradykinin (10 nM, 10 min) or EGF (100 ng/ml) as a function of receptor presence and pretreatments (12-h exposure to bradykinin 100 nM, combined or not with 1 µM captopril, or B-9972 100 nM). One typical experiment is shown, and average densitometry values of four experiments are reported as histograms. ANOVA indicated that the quantity of p-ERK varied significantly as a function of treatments (P < 0.001). *, P < 0.05; **, P < 0.01 versus the maximum signal (bradykinin-stimulated B2R-GFP-expressing cells, fourth lane, Dunnett's test).
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B2R-GFP activation in HEK 293 cells is coupled to the phosphorylation of the ERK1/2 MAP kinases (Morissette et al., 2007). A standard 10-min stimulation of cells with bradykinin (10 nM) was applied to cells as a function of B2R-GFP expression or pretreatments (Fig. 14B). Untransfected cells failed to respond to bradykinin by ERK1/2 phosphorylation; those expressing B2R-GFP were responsive, as previously reported. However, a 12-h pretreatment with agonists variably decreased the acute effect of bradykinin: B-9972 (100 nM) or the combination bradykinin + captopril abolished it, whereas bradykinin alone reduced it partially (Fig. 14). When the phosphorylation was obtained with acute treatment with EGF, the 12-h B-9972 pretreatment did not significantly inhibit the response, showing that the desensitization was homologous.
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Discussion
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Pharmacological Profile of a Triad of Isomer Peptides. The structurally constrained B2 receptor antagonist B-9430 is a known antagonist of the B2 receptors (Leeb-Lundberg et al., 2005) but has not been characterized in useful bioassays of this receptor type, the rabbit jugular and human umbilical veins (Marceau et al., 1994, 2003; Rizzi et al., 1997; Pruneau et al., 1999; Houle et al., 2000). The insurmountable effect of B-9430 at the rabbit B2 receptor combined with the competitive and reversible effect at the human B2 receptor is a peculiar profile previously observed for another constrained peptide, icatibant (Marceau et al., 1994; Houle et al., 2000). The species-specific insurmountable behavior of icatibant has been verified at the recombinant rabbit B2 receptor (Bachvarov et al., 1995). B-9430 may be, like icatibant, an antagonist of low reversibility at the rabbit B2 receptor because an i.v. bolus of this drug inhibited the hypotensive effect of bradykinin for 24 h in rabbits (Stewart et al., 1996). The noncompetitive antagonism exerted by B-9430 and icatibant at the rabbit B2 receptor must be structurally explained by some of the 16% of amino acids that differ between this receptor and the human ortholog (Bachvarov et al., 1995), at which both peptides are competitive. Other antagonists, such as the nonpeptide LF16-0687, are competitive at both the rabbit and human B2 receptors (Pruneau et al., 1999; Houle et al., 2000; Bawolak and Marceau, 2007).
B-9870 (CU201) consists of a dimer of B-9430 molecules linked at their N terminus by the suberimidyl moiety (Gera et al., 1996). This large peptide exerts at micromolar concentrations antiproliferative effects not shared by B-9430 on tumor-derived cell lines (Chan et al., 2002; Thomas et al., 2006) and dissociable from kinin receptor binding (Morissette et al., 2007). However, B-9870 has much in common with B-9430 as a kinin receptor antagonist with nanomolar potency. Thus, B-9870 is an insurmountable antagonist of bradykinin in the rabbit jugular vein but a relatively low affinity (pA2, 7.4) competitive antagonist at the rabbit aorta B1 receptors (Morissette et al., 2007). B-9430 is also a surmountable B1 receptor antagonist (pA2, 6.5; Fig. 4). Recent evidence indicates that the insurmountable behavior of the constrained peptides at the rabbit B2 receptors is akin to a partial agonist activity detectable in cells that overexpress recombinant receptors of this type (MAP kinase activation, calcium transients, slow and partial endocytosis of surface receptors) (Morissette et al., 2007). This has been confirmed with B-9430 in present experiments (minor long-term degradation of B2R-GFP, Fig. 9; partial agonist behavior in calcium transient experiments, data not shown).
The isomer obtained by exchanging the residues in the seventh and eighth positions of B-9430, B-10344, was a low-affinity agonist in both venous contractility assays. B-10344 may be a partial agonist in the jugular vein, with a low slope of the concentration-effect curve relative to that of bradykinin and residual agonist potency (Fig. 3A) much lower than its binding potency relative to bradykinin (Fig. 2). However, this agent was a full agonist in the human umbilical vein based on similarity of Emax with bradykinin. A further modification, L isomerization of D-Igl8 in B-9972, yielded a full agonist at both the human and rabbit receptors. The step-wise modifications of pharmacological properties in the isomer peptide triad illustrate that the spatial orientation of the C terminus determines the transition from a B2 receptor agonist to an antagonist (Vavrek and Stewart, 1985; Fortin and Marceau, 2006); accordingly, the docking model for icatibant based on mutagenesis of the rat B2 receptor showed preferential interaction of the C terminus with residues from receptor transmembrane domain 7, whereas that of bradykinin interacted more with transmembrane domain 6 (Jarnagin et al., 1996). B-9972 was an agonist distinctively more potent at the B2 receptors than the older analog [Phe8(CH2NH)Arg9]bradykinin and, theoretically, integrates a more complete protection relative to hypothetical peptidases (aminopeptidases, endopeptidases). The radioreceptor assay approach showed that B-9972 is inactivated to a large extent in the presence of cells over a 12-h period, but less completely than bradykinin. Resistance to inactivation may explain the large potency of B-9972 relative to bradykinin in the rabbit jugular vein assay (1:2), whereas the analog retains only a smaller fraction of the affinity of bradykinin in the binding assay to rabbit B2R-GFP (1:7; Fig. 2). The jugular vein contractility assay is routinely run in the presence of the ACE inhibitor captopril, an agent that increases the apparent potency of bradykinin in this preparation (Gaudreau et al., 1981), but not in the human umbilical vein (Marceau et al., 1994). The slow relaxation of the washed human umbilical vein prestimulated with either B-9972 or its equally protected isomer B-10344, in comparison with preparations precontracted with either bradykinin or [Phe8(CH2NH)Arg9]bradykinin, further supports that B-9972 is resistant to multiple pathways of intracellular or extracellular degradation. These pathways may be different from ACE, neutral endopeptidase, and aminopeptidase P, based on the lack of potentiation of bradykinin by inhibiting these peptidases and on their absence of effect on the relaxation kinetics upon bradykinin washout (Fig. 7).
Degradation of B2 Receptors by B-9972. The fusion protein B2R-GFP (101 kDa) is a functional, high-affinity receptor with an intact pharmacological profile. The GFP label has the triple advantage of supporting imaging in intact cells, being an excellent antigenic tag in immunoblot experiments, and remaining as a stable reaction product after receptor degradation (Houle et al., 2003; Houle and Marceau, 2003). Generation of free GFP or its yellow variant has also been observed with the fusion proteins made from a receptor prone to degradation, the endothelin ETB receptor (Oksche et al., 2000) or the short-lived kinin B1 receptor (Fortin et al., 2003). Both bradykinin and B-9972 acutely promoted B2R-GFP endocytosis (Fig. 8; arrows 1 and 2 in the schematic representation, Fig. 15), but the amount of fusion protein in the total cell extracts remained approximately constant even after 3 h of stimulation (Figs. 9 and 10). B-9972 degraded this construction, but only for long treatments (12 h), and with cytosolic GFP-sized reaction products (imaging, Fig. 8; immunoblots, Figs. 9 and 10; arrow 4 in Fig. 15). This specific effect was abated by the peptide antagonist B-9430 or the nonpeptide antagonist LF16-0687. Bradykinin alone was inactive but, when combined with the ACE inhibitor captopril, could promote a fraction of the degradation induced by B-9972, based on the generation of free GFP. The same relationships (time, effect of agonists) were corroborated with the alternate construction myc-B2R (Figs. 10 and 13), but the N-terminal position of the antigenic tag was not favorable to detect reaction products. Thus, in the absence of cycloheximide during the 12-h treatments, the receptors are presumably resynthesized at a constant rate driven by the viral promoter of the expression vectors, but a superior degradation rate accounts for the slow receptor decline if cells are treated with B-9972. The decreased function of cell surface B2R-GFP following long treatments with B-9972 was confirmed using a [3H]bradykinin endocytosis assay and an ERK1/2 phosphorylation assay (Fig. 14), observations compatible with receptor degradation but that do not exclude agonist-induced internalization. In low-serum medium used for the phosphorylation assay, the bradykinin/captopril combination was relatively effective to functionally desensitize B2R-GFP. ACE is the major degradation pathway in the serum-containing medium of HEK 293 cells (Bachvarov et al., 2001; Fig. 15), but is not likely to be important for the degradation of the ligand internalized with the receptors. B-9972's structurally determined resistance to multiple hypothetical peptidases (including amino and endopeptidases) may be crucial for a stronger capacity of this peptide to promote agonist-induced degradation in high-serum medium, compared with the bradykinin-captopril combination. Only long cell treatments with a synthetic agonist degraded the largely recycled 2-adrenoceptors (Moore et al., 1999); the present results suggest that the bradykinin B2 receptors behave in a similar manner if the inherently fragile agonist is stabilized. However, the HEK 293 cells expressing B2R-GFP have been used to show more rapid and efficient B2 receptor degradation mechanisms in response to extracellular proteases, including those secreted by neutrophil leukocytes (Marceau et al., 2002; Houle et al., 2003).
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Fig. 15. Schematic representation of B2R-GFP cycling as a function of agonist treatment. For description, see Discussion.
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That C-terminal GFP tagging of the B2 receptors redirects them toward recycling over degradation (Kalatskaya et al., 2006) is not supported by the present data because immunoblots suggest an inferior proportion of degraded myc-B2Rs relative to B2R-GFPs (Fig. 10). However, given the back-ground expression of immunoreactive myc-B2R in cytosolic granules (Fig. 12), the proportion of mature receptors susceptible to degradation is uncertain in this transient expression system. Moreover, the species, technique for receptor detection, and expression levels were different in the previous report (Kalatskaya et al., 2006), making a detailed comparison risky. Nevertheless, the nature of the stimulation that leads to degradation is similar for both B2R-GFP and myc-B2R constructions, the most effective treatment being 12-h cell incubation with B-9972. In summary, the results illustrate the agonist-antagonist transition in B2 receptor peptide ligands with a constrained C-terminal structure, the importance of species in the receptor pharmacological profile, and the possibility of degrading receptors in a selective manner using a peptidase-resistant agonist.
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Acknowledgements
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We thank Johanne Bouthillier for excellent technical help.
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Footnotes
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This study was supported by the Canadian Institutes of Health Research (Operating Grant MOP-14077 to F.M. and Canada Graduate Scholarships Doctoral Award to G.M.).
M.-T.B. and L.G. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.123422.
ABBREVIATIONS: GPCR, G protein-coupled receptor; B2R-GFP, conjugate of the B2 receptor with green fluorescent protein; ACE, angiotensin I-converting enzyme; HEK, human embryonic kidney; B-9430, D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-bradykinin; B-9972, D-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin; B-10344, D-Arg-[Hyp3,Igl5,Oic7,D-Igl8]-bradykinin; myc-B2R, conjugate of the myc epitope with the B2 receptor; Hoe 140, D-Arg[Hyp3,Thi5, D-Tic7,Oic8]-bradykinin; LF16-0687, 1-[[2,4-dichloro-3-[(2,4-dimethylquinolin-8-yl)oxy]methyl]phenyl]sulfonyl]-N-[3-[[4-(aminoiminomethyl]-phenyl]carbonylamino]propyl]-2(S)-pyrrolidinecarboxamide, mesylate salt; bradyzide, (2S)-1-[4-(4-benzhydrylthiosemicarbazido)-3-nitrobenzenesulfonyl]-pyrrolidine-2-carboxylic acid {2-(2-dimethylaminoethyl)methylamino]ethyl}amide; 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; PCR, polymerase chain reaction; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; EGF, epidermal growth factor; BK, bradykinin; ANOVA, analysis of variance; NPC 17731, D-Arg[Hyp3,D-HypE(trans-propyl)7,Oic8]-bradykinin; B-9870, (CU201) dimer of B-9430 linked at their N terminus by suberidimyl.
Address correspondence to: Dr. François Marceau, Centre de Recherche en Rhumatologie et Immunologie, Room T1–49, Centre Hospitalier Universitaire de Québec, 2705 Laurier Boulevard, Quebec, Canada G1V 4G2. E-mail: francois.marceau{at}crchul.ulaval.ca
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Abstract
Materials and Methods
, captopril control tested only once (excluded from statistics). 
, n = 4 for this value.