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Does zaltoprofen antagonize the bradykinin receptors?
Regulatory Peptides 140 (2007) 125 – 130 www.elsevier.com/locate/regpep
Does zaltoprofen antagonize the bradykinin receptors? Marie-Thérèse Bawolak, François Marceau ⁎ Centre de recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, 2705 Laurier Blvd., Québec (Québec), Canada G1V 4G2 Department of Medicine, Université Laval, Canada Received 20 October 2006; accepted 19 November 2006 Available online 26 January 2007
Abstract Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B2 receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B2 receptor antagonists (contractility, human isolated umbilical vein) or B1 receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 μM) failed to antagonize BK or des-Arg9-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 μM) abated des-Arg9-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 μM) did not inhibit kininstimulated phospholipase A2 activity in HEK 293 cells expressing recombinant forms of the rabbit B1 or B2 receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems. © 2006 Elsevier B.V. All rights reserved. Keywords: B2 receptors; B1 receptors; Phospholipase A2; Cyclooxygenase
1. Introduction Zaltoprofen is a nonsteroidal antiinflammatory drug (NSAID) related to ibuprofen and other “propionic acid” derivatives, with a minor selectivity for COX-2 [1]. Indeed, IC50 values of 1.3 and 0.34 μM have been obtained for zaltoprofen in intact cellular systems against COX-1 and -2, respectively [1]. Its original sulfur-containing aromatic heterocycle may confer to this molecule novel and unforeseen properties (Fig. 1). As an analgesic drug, zaltoprofen has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B2 receptor, a G protein coupled receptor [2–5], and thus to exceed the efficacy of more conventional NSAIDs. Zaltoprofen did not displace cognate radioligands from the kinin receptors of either the B1 or B2 types, although the drug was able to inhibit BK-induced calcium signaling in sensory neurons [5]. Thus, it appears reasonable to hypothesize that zaltoprofen inhibits with selectivity the signaling of the B2 receptors, either by binding to the receptor molecule itself at a site that does not influence the ⁎ Corresponding author. Tel.: +1 418 525 4444x46155; fax: +1 418 654 2765. E-mail address: [email protected] (F. Marceau). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.025
docking of [3H]BK or by interfering with a biochemical pathway selectively recruited by these receptors. If the claim that zaltoprofen antagonizes BK has any predictive power, the drug should block the corresponding receptors in nonneuronal systems. In order to clarify the status of zaltoprofen as a direct or indirect BK antagonist, we applied the drug to an assay previously exploited for the characterization of B2 receptor antagonists, the contractility of the human isolated umbilical vein [6–9]. The effects of BK on this bioassay are not influenced by COX blockade or endothelium removal [6] and the stimulation of smooth muscle cells by B2 receptors is presumably direct; this is one of the few large blood vessels completely devoid of sympathetic innervation [10]. Other vascular preparations isolated from rabbits have also been exploited: the isolated aorta (direct contractile effect of kinins) and mesenteric artery (indirect prostaglandin-mediated relaxation) [11] are well adapted to evaluate drugs that bind to the alternate kinin receptor type, the B1 receptor selectively sensitive to kinin fragments devoid of the C-terminal arginine residue, such as des-Arg9-BK and Lys-des-Arg9-BK. The effect of zaltoprofen on phospholipase A2 activity induced by the stimulation of recombinant forms of the rabbit B1 and B2
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Fig. 1. Effect of drugs on BK- or 5-HT-induced contraction in the human isolated umbilical vein. Each tissue was subjected to the construction of two full cumulative concentration–effect curves (for each agonist) in the presence of zaltoprofen or LF 16-0687 at the indicated concentration or of their vehicle (DMSO or saline, respectively) applied 30 min before each construction. Values are the means ± s.e.m. of the number determinations indicated by n.
receptors has been tested in order to identify the level at which the drug inhibits the arachidonate cascade.
was a gift from Berlex (Lachine, QC, Canada). Other drugs were purchased from Sigma-Aldrich (St. Louis, MO).
2. Methods
2.2. Organ bath pharmacology
2.1. Drugs
A local ethic committee approved the procedures based on human tissues. Small segments of human umbilical cords stored at 4 °C were obtained within 24 h from normal deliveries. Rings of human umbilical vein were prepared and mounted in organ baths for contractility studies as described previously [6], except that 2 g of resting tension was applied to the rings. Changes in tension were recorded using isometric force transducers [6]. For the evaluation of drugs as B2 receptor antagonists and to establish their selectivity, the tissues were equilibrated for 3 h before
Zaltoprofen was a generous gift from Nippon Chemiphar Research Laboratories (Saitama, Japan). LF 16-0687 (anatibant), a known nonpeptide B2 receptor antagonist [8], was a gift from Laboratoires Fournier (Daix, France). Compound 11, a high affinity nonpeptide antagonist of the human and rabbit B1 receptor [11], has been obtained from Merck Research Laboratories (West Point, PA). The prostacyclin analog iloprost
M.-T. Bawolak, F. Marceau / Regulatory Peptides 140 (2007) 125–130
Fig. 2. Effect of zaltoprofen (30 μM) on the contractile responses mediated by the kinin B1 receptors (agonist des-Arg9-BK) in the rabbit aorta. Values are the means ± s.e.m. of the number of determinations indicated by n.
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starting the experiments, which consisted of the construction of a full cumulative concentration–effect curve for BK at time 3 h and of a second one for 5-hydroxytryptamine (5-HT), a powerful contractile agent in this tissue [12], at time 5 h. The tested antagonist drugs or their vehicle were applied to the bathing fluid 30 min before the construction of either concentration–effect curve. The effect of each drug at each tested concentration was calculated as the rightward shift (dose ratio, DR) of the averaged concentration–effect curve relative to the EC50 established in matched control tissues exposed only to the vehicle of the drugs. When applicable, Schild plot parameters were estimated using a computerized procedure [13]. A local ethics committee approved procedures based on animals. Rabbit aortic rings (New Zealand white, 1.5–2 kg, Charles River, St. Constant, Canada) were suspended under a tension of 2 g in 5 ml tissue baths containing oxygenated (95% O2:5% CO2) and warmed (37 °C) Krebs solution as described [11]. Some experiments were also performed in rings of rabbit
Fig. 3. Top: representative tracings of the tension developed by rings of rabbit mesenteric artery sequentially treated with an inhibitory drug or their DMSO vehicle, phenylephrine (PE, 4 μM) and the B1 receptor agonist des-Arg9-BK (100 nM, administered on a stable PE-induced contraction plateau). Abscissa scale, time; ordinate, isometric contraction (g). The closed symbols refer to the application of agents and the open symbols, to washout of stimulants. Bottom: effect of zaltoprofen or ibuprofen on des-Arg9-BK-induced changes of tone in the mesenteric artery preparation. Results are expressed as a positive (early contractile phase) and negative (slow, late relaxation) percent of PE-induced plateau. Values are the means ± s.e.m. of 7 determinations. Mann–Whitney U test showed that zaltoprofen exerted a significant effect on the aptitude of the relaxation relative to control tissues.
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inhibition of the effect of the B1 receptor antagonist des-Arg9-BK (100 nM) applied when the contraction plateaued. 2.3. Phospholipase A2 assay HEK 293 cell lines stably expressing fluorescent and fully functional rabbit B1 or B2 receptors, conjugated with yellow fluorescent protein or green fluorescent protein, respectively (B1RYFP, B2R-GFP), have been described elsewhere [17,18]. An arachidonic acid release assay was performed to evaluate the effect of zaltoprofen on kinin-induced phospholipase A2 activity in HEK 293 cells. 2.5 × 105 cells were seeded in 2 cm2 wells (24-well plates) containing 1 ml of the complete culture medium. 24 h later, as the cells were 50–60% confluent, 0.1 μCi of [3H]arachidonic acid (PerkinElmer Life Sciences, Boston, MA; specific activity 60–62.5 Ci/mmol) was added to each well. The cells were further incubated for 18 h, then washed 3 times with Earle's balanced salt solution containing 2 mg/ml of BSA. 1 ml of this medium was left in each well and inhibitory drugs were optionally introduced at this moment in the wells. The B1 agonist Lys-des-Arg9-BK (10 nM) of B2 receptor agonist BK (10 nM) or vehicle was added 30 min later. The plates were further incubated at 37 °C for 30 min, at which point 500 μl of the medium from each well was recovered in 1.5 ml conical tubes and centrifuged for 5 min at 15,000 g. 400 μl of the supernatants was transferred in vials for scintillation counting of the released arachidonate. 3. Results Fig. 4. [3H]Arachidonate released by HEK 293 cells stably expressing B1R-YFP (A) or B2R-GFP (B) and exposed to the cognate agonist peptides (10 nM; Lysdes-Arg9-BK or BK, respectively) for the last 30 min of incubation. Antagonist drugs (zaltoprofen 10 μM, compound 11 100 nM, LF 16-0687 1 μM) or their DMSO vehicle were optionally introduced 30 min before the agonist. Results are expressed as means ± s.e.m. of the number of determinations indicated by n. Mann–Whitney tests were performed to determine the effect of the agonist relative to the baseline release with the same receptor type and inhibitory drug (contiguous bar pairs in graphs). P values are indicated for significant effects of the agonists.
mesenteric arteries (suspended under a tension of 1 g [14]). Contractility studies in the aortic preparation were based upon the construction of cumulative concentration–responses curves for des-Arg9-BK (a B1 receptor agonist on this tissue) at time 5.5 h from the beginning of the incubation of the preparation. Zaltoprofen or its DMSO vehicle in control tissues were introduced at time 5 h and maintained in the bathing fluid during the construction of the curve to detect any antagonist effect. Contractility results were expressed as a percent of the maximal response recorded in each tissue. Precontracted rabbit mesenteric artery rings respond to desArg9-BK mostly by a prostaglandin (PG)-mediated relaxation and this response is acquired in vitro as a function of time and mediated by both COX-1 and COX-2 [11,15,16]. In tissues incubated for 5 h in organs baths and periodically washed with fresh Krebs solution, zaltoprofen, the alternate NSAID ibuprofen or their DMSO vehicle were introduced 30 min before the phenylephrine stimulation (4 μM) used to induce tissue precontraction in order to detect
Zaltoprofen (0.3–30 μM) failed to influence BK- or 5-HTinduced contraction of the human isolated umbilical vein (Fig. 1, top). By contrast, LF 16-0687 exerted a concentration-dependent surmountable antagonism of BK-induced contractility in this preparation (Fig. 1, bottom left). This effect has been previously reported by another laboratory [8] and was selective, as the contractile action of 5-HT was not consistently influenced by LF 16-0687 (Fig. 1, bottom right). The calculated pA2 value for LF 16-0687 against BK was 8.27 ± 0.19 (slope of the Schild regression −1.04 ± 0.13). Zaltoprofen (30 μM) also failed to antagonize the typical B1 receptor agonist in the rabbit aorta contractility assay (Fig. 2), a
Fig. 5. Schematic model of the action of drugs in the present experimental systems.
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prostaglandin-independent classical response and a system well adapted to characterize both peptide and nonpeptide B1 receptor antagonists [11,19]. The isolated rabbit mesenteric artery preparation is highly responsive to vasorelaxant prostaglandins, as shown here by the complete relaxation of the precontracted artery by the prostacyclin mimetic iloprost (5–250 nM; Fig. 3, top, left tracing). The B1 receptor agonist des-Arg9-BK (100 nM) exerted biphasic effects on the precontracted rabbit mesenteric arteries (Fig. 3, top, middle tracing), the relaxing phase amounting to 61% of the contraction plateau (statistics, Fig. 3, bottom). The major mechanical response, the relaxation, was significantly reduced by treatment with a low concentration of zaltoprofen (1 μM; sample record, Fig. 3, top right; statistical analysis, bottom). The alternate propionic acid NSAID, ibuprofen, was inactive in this respect at equimolar concentration, consistent with reported lower potency at both COX-1 and -2 [1]. A phospholipase A2 assay was applied to determine the level at which zaltoprofen inhibits the kinin-stimulated arachidonate cascade. HEK 293 cell lines that stably express either the recombinant receptors B1R-YFP or B2R-GFP responded to their respective agonists Lys-des-Arg9-BK (10 nM) or BK (10 nM) (Fig. 4); non-transfected HEK 293 cells are known to be refractory to these peptides in this assay [17,20]. A relatively high concentration of zaltoprofen (10 μM) failed to inhibit Lys-desArg9-BK-induced [3H]arachidonate release from HEK 293 cells expressing B1R-YFP, or BK-induced release from B2R-GFP expressing cells (Fig. 4). Nonpeptide antagonists of the B1 and B2 receptors, compound 11 and LF 16-0687, respectively, inhibited the kinin-stimulated phospholipase A2 with the expected selectivity.
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aorta is refractory to them. True B1 receptor antagonists, such as compound 11, prevent all components of des-Arg9-BK-induced response in precontracted rabbit mesenteric arteries [11], but inhibitors of COX-1 or -2, only the slow relaxant phase. Zaltoprofen, as expected from a nonselective COX inhibitor, potently suppressed the relaxant response recruited by the kinin. Zaltoprofen was more potent than ibuprofen in this respect. The rabbit recombinant B1 and B2 receptors have been shown to be coupled to phospholipase A2 [17,18] and zaltoprofen failed to inhibit the kinin-stimulated arachidonate cascade at this level, unlike true receptor antagonists (Fig. 4). Combined with the observation that zaltoprofen does not compete for radioligand binding at the B1 or B2 receptors [5], the findings altogether support that zaltoprofen is a COX inhibitor without proximal or specific inhibitory effect on the kinin receptors that can stimulate prostaglandin release (Fig. 5). If it is true that effects of kinins are inhibited with selectivity by zaltoprofen in primary sensory neurons, it may rather relate to a relatively selective effector role of COX in the response or affect pathways that are cell type-specific and not represented in vascular smooth muscle. Such hypothetical sites of actions have been previously proposed (12-lipoxygenase, specialized ion channels) [2]. Acknowledgements We thank Ms. Johanne Bouthillier for expert technical assistance. Supported by the Canadian Institutes of Health Research (grant MOP-14077). References
4. Discussion Zaltoprofen is an NSAID already in clinical use in Asia. BK receptor antagonism has not been applied to large populations of patients so far [21], unless the claims of selective BK receptor antagonism by zaltoprofen can be verified (see Introduction). It is intriguing that zaltoprofen contains a sulfur atom, unlike most nonselective NSAIDs, but like many recently developed nonpeptide kinin receptor antagonists (Fig. 1) [21]. In a bioassay that has allowed characterizing in a robust manner several peptide and nonpeptide B2 receptor antagonists, such as Hoe 140 (icatibant), LF 16-0687 and others [6–9], zaltoprofen (0.3–30 μM) failed to inhibit BK-induced human umbilical vein contractility. This vascular smooth muscle system expresses the classical B2 receptor and the necessary components (G protein, phospholipase C etc.) to couple it to sustained contraction. Zaltoprofen (30 μM) also failed to antagonize the prostaglandin-independent contractile effect of des-Arg9-BK mediated by the B1 receptors in the rabbit aortic preparation. On the other hand, the NSAID was highly effective to inhibit des-Arg9-BK-induced relaxation in the isolated rabbit mesenteric artery, a fact that may be interpreted as the interruption of autocrine signaling by relaxant prostanoids released by the kinin. One of the major pharmacological differences between the rabbit aorta and mesenteric artery preparations is the high sensitivity of the latter to relaxant prostaglandins such as prostacyclin [11] or iloprost (Fig. 3); the
[1] Kawai S. Cyclooxygenase selectivity and the risk of gastro-intestinal complications of various non-steroidal anti-inflammatory drugs: a clinical consideration. Inflamm Res 1998;47(suppl 2):S102–6. [2] Tang HB, Inoue A, Oshita K, Hirate K, Nakata Y. Zaltoprofen inhibits bradykinin-induced responses by blocking the activation of second messenger signaling cascades in rat dorsal root ganglion cells. Neuropharmacology 2005;48:1035–42. [3] Muratani T, Doi Y, Nishimura W, Nishizawa M, Minami T, Ito S. Preemptive analgesia by zaltoprofen that inhibits bradykinin action and cyclooxygenase in a post-operative pain model. Neurosci Res 2005;51:427–33. [4] Matsumo M, Inoue M, Ueda H. NSAID zaltoprofen possesses novel antinociceptive mechanism through blockage of B2-type bradykinin receptor in nerve endings. Neurosci Lett 2006;397:249–53. [5] Hirate K, Uchida A, Ogawa Y, Arai T, Yoda K. Zaltoprofen, a non-steroidal anti-inflammatory drug, inhibits bradykinin-induced pain response without blocking bradykinin receptors. Neurosci Res 2006;54:288–94. [6] Marceau F, Levesque L, Drapeau G, Rioux F, Salvino JM, Wolfe HR, Seoane PR, Sawutz DG. Effects of peptide and nonpeptide antagonists of bradykinin B2 receptors on the venoconstrictor action of bradykinin. J Pharmacol Exp Ther 1994;269:1136–43. [7] Rizzi A, Gobeil F, Calo G, Inamura N, Regoli D. FR 173657: a new, potent, nonpeptide kinin B2 receptor antagonist. An in vitro study. Hypertension 1997;29:951–6. [8] Pruneau D, Paquet JL, Luccarini JM, Defrêne E, Fouchet C, Franck RM, Loillier B, Robert C, Bélichard P, Duclos H, Cremers B, Dodey P. Pharmacological profile of LF 16-0687, a new potent non peptide bradykinin B2 receptor antagonist. Immunopharmacology 1999;43:187–94. [9] Marceau F, Fortin JP, Morissette G, Dziadulewicz EK. A non-peptide antagonist unusually selective for the human form of the bradykinin B2 receptor. Int Immunopharmacol 2003;3:1529–36.
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[10] Tuvemo T. Role of prostaglandins, prostacyclin, and thromboxanes in the control of the umbilical–placental circulation. Semin Perinatol 1980;4:91–5. [11] Morissette G, Fortin JP, Otis S, Bouthillier J, Marceau F. A novel nonpeptide antagonist of the kinin B1 receptor: effects at the rabbit receptor. J Pharmacol Exp Ther 2004;311:1121–30. [12] Altura BM, Malaviya D, Reich CF, Orkin LR. Effects of vasoactive agents on isolated human umbilical arteries and veins. Am J Physiol 1972;222:345–55. [13] Tallarida RJ, Murray RB. Manual of pharmacologic calculations with computer programs. New York: Springer-Verlag; 1987. [14] Petitclerc E, Poubelle PE, Marceau F. Rapid protein synthesis and turnover is involved in interleukin-1-induced relaxation of the rabbit isolated mesenteric artery. Analysis of the arachidonate cascade. J Pharmacol Exp Ther 1994;268:1419–25. [15] Churchill L, Ward PE. Relaxation of isolated mesenteric arteries by des-Arg9bradykinin stimulation of B1 receptors. Eur J Pharmacol 1986;130:11–8.
[16] Deblois D, Marceau F. The ability of des-Arg9-bradykinin to relax rabbit isolated mesenteric arteries is acquired during in vitro incubation. Eur J Pharmacol 1987;142:141–4. [17] Fortin JP, Dziadulewicz EK, Gera L, Marceau F. A nonpeptide antagonist reveals a highly glycosylated state of the rabbit kinin B1 receptor. Mol Pharmacol 2006;69:1146–57. [18] Houle S, Larrivée JF, Bachvarova M, Bouthillier J, Bachvarov DR, Marceau F. Antagonist-induced intracellular sequestration of the rabbit bradykinin B2 receptor. Hypertension 2000;35:1319–25. [19] Gera L, Fortin JP, Adam A, Stewart JM, Marceau F. Discovery of a dualfunction peptide that combines aminopeptidase N inhibition and kinin B1 receptor antagonism. J Pharmacol Exp Ther 2006;317:300–8. [20] Houle S, Molinaro G, Adam A, Marceau F. Tissue kallikrein actions at the rabbit natural or recombinant kinin B 2 receptors. Hypertension 2003;41:611–7. [21] Marceau F, Regoli D. Bradykinin receptor ligands: therapeutic perspectives. Nat Rev Drug Discov 2004;3:845–52.
Does zaltoprofen antagonize the bradykinin receptors? Marie-Thérèse Bawolak, François Marceau ⁎ Centre de recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, 2705 Laurier Blvd., Québec (Québec), Canada G1V 4G2 Department of Medicine, Université Laval, Canada Received 20 October 2006; accepted 19 November 2006 Available online 26 January 2007
Abstract Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B2 receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B2 receptor antagonists (contractility, human isolated umbilical vein) or B1 receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 μM) failed to antagonize BK or des-Arg9-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 μM) abated des-Arg9-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 μM) did not inhibit kininstimulated phospholipase A2 activity in HEK 293 cells expressing recombinant forms of the rabbit B1 or B2 receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems. © 2006 Elsevier B.V. All rights reserved. Keywords: B2 receptors; B1 receptors; Phospholipase A2; Cyclooxygenase
1. Introduction Zaltoprofen is a nonsteroidal antiinflammatory drug (NSAID) related to ibuprofen and other “propionic acid” derivatives, with a minor selectivity for COX-2 [1]. Indeed, IC50 values of 1.3 and 0.34 μM have been obtained for zaltoprofen in intact cellular systems against COX-1 and -2, respectively [1]. Its original sulfur-containing aromatic heterocycle may confer to this molecule novel and unforeseen properties (Fig. 1). As an analgesic drug, zaltoprofen has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B2 receptor, a G protein coupled receptor [2–5], and thus to exceed the efficacy of more conventional NSAIDs. Zaltoprofen did not displace cognate radioligands from the kinin receptors of either the B1 or B2 types, although the drug was able to inhibit BK-induced calcium signaling in sensory neurons [5]. Thus, it appears reasonable to hypothesize that zaltoprofen inhibits with selectivity the signaling of the B2 receptors, either by binding to the receptor molecule itself at a site that does not influence the ⁎ Corresponding author. Tel.: +1 418 525 4444x46155; fax: +1 418 654 2765. E-mail address: [email protected] (F. Marceau). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.025
docking of [3H]BK or by interfering with a biochemical pathway selectively recruited by these receptors. If the claim that zaltoprofen antagonizes BK has any predictive power, the drug should block the corresponding receptors in nonneuronal systems. In order to clarify the status of zaltoprofen as a direct or indirect BK antagonist, we applied the drug to an assay previously exploited for the characterization of B2 receptor antagonists, the contractility of the human isolated umbilical vein [6–9]. The effects of BK on this bioassay are not influenced by COX blockade or endothelium removal [6] and the stimulation of smooth muscle cells by B2 receptors is presumably direct; this is one of the few large blood vessels completely devoid of sympathetic innervation [10]. Other vascular preparations isolated from rabbits have also been exploited: the isolated aorta (direct contractile effect of kinins) and mesenteric artery (indirect prostaglandin-mediated relaxation) [11] are well adapted to evaluate drugs that bind to the alternate kinin receptor type, the B1 receptor selectively sensitive to kinin fragments devoid of the C-terminal arginine residue, such as des-Arg9-BK and Lys-des-Arg9-BK. The effect of zaltoprofen on phospholipase A2 activity induced by the stimulation of recombinant forms of the rabbit B1 and B2
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Fig. 1. Effect of drugs on BK- or 5-HT-induced contraction in the human isolated umbilical vein. Each tissue was subjected to the construction of two full cumulative concentration–effect curves (for each agonist) in the presence of zaltoprofen or LF 16-0687 at the indicated concentration or of their vehicle (DMSO or saline, respectively) applied 30 min before each construction. Values are the means ± s.e.m. of the number determinations indicated by n.
receptors has been tested in order to identify the level at which the drug inhibits the arachidonate cascade.
was a gift from Berlex (Lachine, QC, Canada). Other drugs were purchased from Sigma-Aldrich (St. Louis, MO).
2. Methods
2.2. Organ bath pharmacology
2.1. Drugs
A local ethic committee approved the procedures based on human tissues. Small segments of human umbilical cords stored at 4 °C were obtained within 24 h from normal deliveries. Rings of human umbilical vein were prepared and mounted in organ baths for contractility studies as described previously [6], except that 2 g of resting tension was applied to the rings. Changes in tension were recorded using isometric force transducers [6]. For the evaluation of drugs as B2 receptor antagonists and to establish their selectivity, the tissues were equilibrated for 3 h before
Zaltoprofen was a generous gift from Nippon Chemiphar Research Laboratories (Saitama, Japan). LF 16-0687 (anatibant), a known nonpeptide B2 receptor antagonist [8], was a gift from Laboratoires Fournier (Daix, France). Compound 11, a high affinity nonpeptide antagonist of the human and rabbit B1 receptor [11], has been obtained from Merck Research Laboratories (West Point, PA). The prostacyclin analog iloprost
M.-T. Bawolak, F. Marceau / Regulatory Peptides 140 (2007) 125–130
Fig. 2. Effect of zaltoprofen (30 μM) on the contractile responses mediated by the kinin B1 receptors (agonist des-Arg9-BK) in the rabbit aorta. Values are the means ± s.e.m. of the number of determinations indicated by n.
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starting the experiments, which consisted of the construction of a full cumulative concentration–effect curve for BK at time 3 h and of a second one for 5-hydroxytryptamine (5-HT), a powerful contractile agent in this tissue [12], at time 5 h. The tested antagonist drugs or their vehicle were applied to the bathing fluid 30 min before the construction of either concentration–effect curve. The effect of each drug at each tested concentration was calculated as the rightward shift (dose ratio, DR) of the averaged concentration–effect curve relative to the EC50 established in matched control tissues exposed only to the vehicle of the drugs. When applicable, Schild plot parameters were estimated using a computerized procedure [13]. A local ethics committee approved procedures based on animals. Rabbit aortic rings (New Zealand white, 1.5–2 kg, Charles River, St. Constant, Canada) were suspended under a tension of 2 g in 5 ml tissue baths containing oxygenated (95% O2:5% CO2) and warmed (37 °C) Krebs solution as described [11]. Some experiments were also performed in rings of rabbit
Fig. 3. Top: representative tracings of the tension developed by rings of rabbit mesenteric artery sequentially treated with an inhibitory drug or their DMSO vehicle, phenylephrine (PE, 4 μM) and the B1 receptor agonist des-Arg9-BK (100 nM, administered on a stable PE-induced contraction plateau). Abscissa scale, time; ordinate, isometric contraction (g). The closed symbols refer to the application of agents and the open symbols, to washout of stimulants. Bottom: effect of zaltoprofen or ibuprofen on des-Arg9-BK-induced changes of tone in the mesenteric artery preparation. Results are expressed as a positive (early contractile phase) and negative (slow, late relaxation) percent of PE-induced plateau. Values are the means ± s.e.m. of 7 determinations. Mann–Whitney U test showed that zaltoprofen exerted a significant effect on the aptitude of the relaxation relative to control tissues.
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inhibition of the effect of the B1 receptor antagonist des-Arg9-BK (100 nM) applied when the contraction plateaued. 2.3. Phospholipase A2 assay HEK 293 cell lines stably expressing fluorescent and fully functional rabbit B1 or B2 receptors, conjugated with yellow fluorescent protein or green fluorescent protein, respectively (B1RYFP, B2R-GFP), have been described elsewhere [17,18]. An arachidonic acid release assay was performed to evaluate the effect of zaltoprofen on kinin-induced phospholipase A2 activity in HEK 293 cells. 2.5 × 105 cells were seeded in 2 cm2 wells (24-well plates) containing 1 ml of the complete culture medium. 24 h later, as the cells were 50–60% confluent, 0.1 μCi of [3H]arachidonic acid (PerkinElmer Life Sciences, Boston, MA; specific activity 60–62.5 Ci/mmol) was added to each well. The cells were further incubated for 18 h, then washed 3 times with Earle's balanced salt solution containing 2 mg/ml of BSA. 1 ml of this medium was left in each well and inhibitory drugs were optionally introduced at this moment in the wells. The B1 agonist Lys-des-Arg9-BK (10 nM) of B2 receptor agonist BK (10 nM) or vehicle was added 30 min later. The plates were further incubated at 37 °C for 30 min, at which point 500 μl of the medium from each well was recovered in 1.5 ml conical tubes and centrifuged for 5 min at 15,000 g. 400 μl of the supernatants was transferred in vials for scintillation counting of the released arachidonate. 3. Results Fig. 4. [3H]Arachidonate released by HEK 293 cells stably expressing B1R-YFP (A) or B2R-GFP (B) and exposed to the cognate agonist peptides (10 nM; Lysdes-Arg9-BK or BK, respectively) for the last 30 min of incubation. Antagonist drugs (zaltoprofen 10 μM, compound 11 100 nM, LF 16-0687 1 μM) or their DMSO vehicle were optionally introduced 30 min before the agonist. Results are expressed as means ± s.e.m. of the number of determinations indicated by n. Mann–Whitney tests were performed to determine the effect of the agonist relative to the baseline release with the same receptor type and inhibitory drug (contiguous bar pairs in graphs). P values are indicated for significant effects of the agonists.
mesenteric arteries (suspended under a tension of 1 g [14]). Contractility studies in the aortic preparation were based upon the construction of cumulative concentration–responses curves for des-Arg9-BK (a B1 receptor agonist on this tissue) at time 5.5 h from the beginning of the incubation of the preparation. Zaltoprofen or its DMSO vehicle in control tissues were introduced at time 5 h and maintained in the bathing fluid during the construction of the curve to detect any antagonist effect. Contractility results were expressed as a percent of the maximal response recorded in each tissue. Precontracted rabbit mesenteric artery rings respond to desArg9-BK mostly by a prostaglandin (PG)-mediated relaxation and this response is acquired in vitro as a function of time and mediated by both COX-1 and COX-2 [11,15,16]. In tissues incubated for 5 h in organs baths and periodically washed with fresh Krebs solution, zaltoprofen, the alternate NSAID ibuprofen or their DMSO vehicle were introduced 30 min before the phenylephrine stimulation (4 μM) used to induce tissue precontraction in order to detect
Zaltoprofen (0.3–30 μM) failed to influence BK- or 5-HTinduced contraction of the human isolated umbilical vein (Fig. 1, top). By contrast, LF 16-0687 exerted a concentration-dependent surmountable antagonism of BK-induced contractility in this preparation (Fig. 1, bottom left). This effect has been previously reported by another laboratory [8] and was selective, as the contractile action of 5-HT was not consistently influenced by LF 16-0687 (Fig. 1, bottom right). The calculated pA2 value for LF 16-0687 against BK was 8.27 ± 0.19 (slope of the Schild regression −1.04 ± 0.13). Zaltoprofen (30 μM) also failed to antagonize the typical B1 receptor agonist in the rabbit aorta contractility assay (Fig. 2), a
Fig. 5. Schematic model of the action of drugs in the present experimental systems.
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prostaglandin-independent classical response and a system well adapted to characterize both peptide and nonpeptide B1 receptor antagonists [11,19]. The isolated rabbit mesenteric artery preparation is highly responsive to vasorelaxant prostaglandins, as shown here by the complete relaxation of the precontracted artery by the prostacyclin mimetic iloprost (5–250 nM; Fig. 3, top, left tracing). The B1 receptor agonist des-Arg9-BK (100 nM) exerted biphasic effects on the precontracted rabbit mesenteric arteries (Fig. 3, top, middle tracing), the relaxing phase amounting to 61% of the contraction plateau (statistics, Fig. 3, bottom). The major mechanical response, the relaxation, was significantly reduced by treatment with a low concentration of zaltoprofen (1 μM; sample record, Fig. 3, top right; statistical analysis, bottom). The alternate propionic acid NSAID, ibuprofen, was inactive in this respect at equimolar concentration, consistent with reported lower potency at both COX-1 and -2 [1]. A phospholipase A2 assay was applied to determine the level at which zaltoprofen inhibits the kinin-stimulated arachidonate cascade. HEK 293 cell lines that stably express either the recombinant receptors B1R-YFP or B2R-GFP responded to their respective agonists Lys-des-Arg9-BK (10 nM) or BK (10 nM) (Fig. 4); non-transfected HEK 293 cells are known to be refractory to these peptides in this assay [17,20]. A relatively high concentration of zaltoprofen (10 μM) failed to inhibit Lys-desArg9-BK-induced [3H]arachidonate release from HEK 293 cells expressing B1R-YFP, or BK-induced release from B2R-GFP expressing cells (Fig. 4). Nonpeptide antagonists of the B1 and B2 receptors, compound 11 and LF 16-0687, respectively, inhibited the kinin-stimulated phospholipase A2 with the expected selectivity.
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aorta is refractory to them. True B1 receptor antagonists, such as compound 11, prevent all components of des-Arg9-BK-induced response in precontracted rabbit mesenteric arteries [11], but inhibitors of COX-1 or -2, only the slow relaxant phase. Zaltoprofen, as expected from a nonselective COX inhibitor, potently suppressed the relaxant response recruited by the kinin. Zaltoprofen was more potent than ibuprofen in this respect. The rabbit recombinant B1 and B2 receptors have been shown to be coupled to phospholipase A2 [17,18] and zaltoprofen failed to inhibit the kinin-stimulated arachidonate cascade at this level, unlike true receptor antagonists (Fig. 4). Combined with the observation that zaltoprofen does not compete for radioligand binding at the B1 or B2 receptors [5], the findings altogether support that zaltoprofen is a COX inhibitor without proximal or specific inhibitory effect on the kinin receptors that can stimulate prostaglandin release (Fig. 5). If it is true that effects of kinins are inhibited with selectivity by zaltoprofen in primary sensory neurons, it may rather relate to a relatively selective effector role of COX in the response or affect pathways that are cell type-specific and not represented in vascular smooth muscle. Such hypothetical sites of actions have been previously proposed (12-lipoxygenase, specialized ion channels) [2]. Acknowledgements We thank Ms. Johanne Bouthillier for expert technical assistance. Supported by the Canadian Institutes of Health Research (grant MOP-14077). References
4. Discussion Zaltoprofen is an NSAID already in clinical use in Asia. BK receptor antagonism has not been applied to large populations of patients so far [21], unless the claims of selective BK receptor antagonism by zaltoprofen can be verified (see Introduction). It is intriguing that zaltoprofen contains a sulfur atom, unlike most nonselective NSAIDs, but like many recently developed nonpeptide kinin receptor antagonists (Fig. 1) [21]. In a bioassay that has allowed characterizing in a robust manner several peptide and nonpeptide B2 receptor antagonists, such as Hoe 140 (icatibant), LF 16-0687 and others [6–9], zaltoprofen (0.3–30 μM) failed to inhibit BK-induced human umbilical vein contractility. This vascular smooth muscle system expresses the classical B2 receptor and the necessary components (G protein, phospholipase C etc.) to couple it to sustained contraction. Zaltoprofen (30 μM) also failed to antagonize the prostaglandin-independent contractile effect of des-Arg9-BK mediated by the B1 receptors in the rabbit aortic preparation. On the other hand, the NSAID was highly effective to inhibit des-Arg9-BK-induced relaxation in the isolated rabbit mesenteric artery, a fact that may be interpreted as the interruption of autocrine signaling by relaxant prostanoids released by the kinin. One of the major pharmacological differences between the rabbit aorta and mesenteric artery preparations is the high sensitivity of the latter to relaxant prostaglandins such as prostacyclin [11] or iloprost (Fig. 3); the
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