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Pharmacological profile of hemokinin 1: a novel member of the tachykinin family
Life Sciences 71 (2002) 363 – 370 www.elsevier.com/locate/lifescie
Pharmacological profile of hemokinin 1: a novel member of the tachykinin family V. Camarda a, A. Rizzi a, G. Calo a, R. Guerrini b, S. Salvadori b, D. Regoli a,* a
Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara, 19, 44100 Ferrara, Italy b Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, Via Fossato di Mortara, 19, 44100 Ferrara, Italy Received 14 August 2001; accepted 19 October 2001
Abstract Recently, the cloning of a novel preprotachykinin gene (PPT-C) has been reported. This gene codes for a novel peptide named hemokinin 1 (HK-1). In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. In this study, we pharmacologically characterised the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK1, the rabbit pulmonary artery (rbPA) for NK2, and rat portal vein (rPV) for NK3 receptors. In all these preparations substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) elicited concentration dependent contractions showing similar maximal effects and the following rank order of potency: SP > NKA = NKB in the rbJV, NKA > NKB H SP in the rbPA, and NKB > NKA > SP in the rPV. In those vessels HK-1 behaved as a full agonist displaying potencies similar (rbPA and rPV) or slightly higher (rbJV) than those of SP. In the rbJV, SR 140333, a selective NK1 receptor antagonist, antagonised the effects of HK-1 and SP with similar high potencies (pKB 9.3 and 9.5, respectively). Similar results were obtained with the pseudopeptide NK1 antagonist, MEN 11467 (pKB 8.8 and 8.6, respectively). Taken together, these data indicate that HK-1 behaves as a NK1 preferring receptor agonist. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Hemokinin 1; Tachykinins; NK1 receptor; Bioassay
Introduction Molecular biological studies have demonstrated the existence of three tachykinin-like peptides in mammals: the undecapeptide substance P (SP), and two decapeptides, neurokinin A (NKA) and neuro*
Corresponding author. Tel.: +39-0532-291227; fax: +39-0532-291205. E-mail address: [email protected] (D. Regoli). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 6 8 2 - X
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kinin B (NKB) [1]. The three peptides, encoded by two mammalian genes named PPT-A (SP and NKA) and PPT-B (NKB), are expressed almost exclusively in neurons and act as neurotransmitters in the central nervous system and as essential components of the nonadrenergic noncholinergic system in autonomic nerves [2]. Neurokinins, particularly SP, are released from sensory neurons by various stimuli, both in the spinal cord, where they contribute to pain transmission, and in the peripheral tissues, where they produce neurogenic inflammation [3,4]. Biological activities of neurokinins are mediated by three receptor types namely NK1, NK2 and NK3, which are expressed by a variety of target cells [5]. Recently, Zhang et al. [6] reported the cloning of a novel preprotachykinin gene (PPTC). This gene codes for a novel peptide named hemokinin 1 (HK-1). This is an undecapeptide (RSRTRQFYGLM-NH2) which shows the C-terminal pentapeptide sequence FYGLM that matches the typical tachykinin motif FXGLM. HK-1 most closely resembles SP in amino acid sequence: both have multiple cationic residues at the N-terminus and an aromatic amino acid occupying the variable X position. In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. HK-1 is able to induce plasma extravasation and to promote mast cell degranulation as SP [6]. However, this new peptide has also some peculiar properties that suggest that HK-1 might act as an autocrine factor to promote B cell survival and proliferation [6]. Aim of the present study was to pharmacologically characterize the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK1, the rabbit pulmonary artery (rbPA) for NK2, and rat portal vein (rPV) for NK3 receptors [5].
Methods Tissue preparations Male New Zealand white rabbits (1.8–2.0 Kg) and male Sprague–Dawley rats (250–300 g) were killed by stunning and exanguination. The vessels used were the rbJV or the rbPA and the rPV. Each vessel was quickly removed and cut either in helical strips about 15–20 mm long and 3 mm wide (rPV), or in rings 10 mm long (rbJV), or in open rings 4–6 mm wide (rbPA) according to Rioux et al., Gaudreau et al., and D’Orleans-Juste et al., respectively [7–9]. The endothelium was mechanically removed from all vessels by gently rubbing the internal surface with moistened filter paper. All tissues were suspended in organ baths containing oxygenated (95% O2 and 5% CO2) Krebs solution of the following composition (in mM): NaCl 118, NaHCO3 25, KCl 4.7, KHPO4 1.2, MgSO4 1.2, CaCl2 2.5, glucose 10. The solution was maintained at 37 jC and at pH 7.4. The tissues were stretched to a resting tension of 1 g for rbJV and rbPA and 0.5 g for rPV. During the equilibration period (1 h) fresh Krebs solution was applied and the tension readjusted at 20 min intervals. The contractions were converted in electrical signal by isometric transducers (GRASS FT03) and recorded by a multichannel poligraph (LINSEIS L2005). Experimental protocols Following the equilibration period, the preparations were exposed to 100 mM KCl for the rbPA, to 0.1 AM bradykinin for the rbJV and 1 AM of noradrenaline for the rPV, to assess the responsiveness of the
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preparation. About 30 min later, in the rbJV and rbPA, cumulative concentration-response curves (crc) to agonists were measured. In the rPV, crc to agonists were performed consecutively, adding to the bath different concentrations of the peptide every 20 minutes followed by washing. In a separate series of experiments in the rbJV, SP or HK-1 (both at 100 nM) were injected twice in the same tissue without washing between the first and the second injection (see tracing in Fig. 2). In the rbJV, SR 140333 (1 and 10 nM), MEN 11467 (10 and 100 nM), or their vehicle were added to the bath 20 min before measuring the crc to SP or HK-1. In the same tissue, the effects of peptidase inhibitors (captopril and thiorphan, both at 1 AM) were evaluated on the contractions elicited by SP and HK-1. Sources of chemicals and reagents Chemicals and their sources were as follows: HK-1, SP, NKA and NKB were synthesized and purified in our laboratories as previously described [10]; SR 140333 was provided by Dr X. Emonds-Alt (Sanofi, Centre de Montpellier, France) and MEN 11467 was supplied by Dr. S. Giuliani (Menarini, Florence, Italy). Captopril, thiorphan, and the other reagents were from Sigma–Aldrich (Milan, Italy). Stock solutions of peptides (1 mM) were made in saline and kept at 20 jC until use. NKB, MEN 11467 and SR 140333 were dissolved in DMSO. Data analysis All data are expressed as means F standard error of the mean (SEM) of n experiments. Data have been statistically analysed using the Student t test or the analysis of variance followed by the Dunnet test. P values less than 0.05 were considered to be significant. The pharmacological terminology used
Fig. 1. Effects of tachykinins and HK-1 in the rbJV. Left panel: Cumulative concentration-response curve to SP, NKA and NKB. Right panel: Cumulative concentration-response curve to SP and HK-1. Each point represents the mean F SEM of at least 5 experiments.
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in this paper follows IUPHAR recommendations [11]. The agonist potencies are given as pEC50, which is the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist. The Emax is the maximal effect that an agonist can elicit in a given tissue. Since both SR 140333 and MEN 11467 behaved as non competitive antagonists, their equilibrium dissociation constant KB was determined according to Kenakin [12]. In practice, a doublereciprocal plot of equieffective concentrations of agonist (A) in the absence (1/A) and in presence (1/ AV) of the antagonist (B) is constructed, and the pKB value is derived from the equation: pKB = log10[(slope-1)/B].
Results As shown in Fig. 1 (left panel), SP, NKA and NKB produced concentration-dependent contractions of the rbJV. SP was significantly more potent than NKA and NKB whereas the maximal tension (Emax) induced by the three peptides was similar (see Table 1). The novel peptide, HK-1, in the range 0.01 nM– 0.1 AM caused a concentration dependent contraction of the rbJV with a pEC50 and Emax of 8.43 and 0.92 F 0.12 g, respectively (Fig. 1, right panel). The kinetic of action of SP and HK-1 were similar: in fact, the effects of both peptides took place immediately after adding the peptide to the bath, were rapidly reversible after washing, and could be repeated in the same tissue after a 90 min interval (data not shown). In the rbPA and in the rPV SP, NKA and NKB acted as full agonists showing maximal effects of about 2 and 0.4 g, respectively (see Table 1). The rank order of potency of agonists was: NKA >
Fig. 2. Typical tracings showing the contractile responses to SP and HK-1, tested at 100 nM, in the rbJV. The two injections were performed without washing the tissues.
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Table 1 Effects of tachykinins and HK-1 in isolated tissues rbJV SP NKA NKB HK-1
rbPA
rPV
pEC50
Emax (g)
pEC50
Emax (g)
pEC50
Emax (g)
8.26 7.53 7.54 8.43
0.85 0.74 0.71 0.92
c5 7.56 6.50 c5
ND 2.14 F 0.36 1.95 F 0.42 ND
5.33 6.55 7.35 5.02
0.37 0.37 0.38 0.38
F F F F
0.12 0.05 0.12 0.12
F F F F
0.08 0.10 0.16 0.15
The data are expressed as mean F SEM of at least 5 experiments. ND: not determined since at the higher agonist concentration tested (10 AM) the crc was still incomplete.
NKB H SP in the rbPA, and NKB > NKA > SP in the rPV. In these two preparations HK-1 elicited concentration dependent contractions with potencies and maximal effects similar to those of SP (Table 1).
Fig. 3. Effects of NK1 selective antagonists vs SP and HK-1 in the rbJV. Left panels: Cumulative concentration-response curve to SP (top) and HK-1 (bottom) measured in the absence (control) or in presence of SR 140333. Right panels: Cumulative concentration-response curve to SP (top) and HK-1 (bottom) obtained in the absence (control) or in presence of MEN 11467. Each point represents the mean F SEM of at least 5 experiments. *p < 0.05 vs control according to ANOVA followed by the Dunnet test.
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Table 2 Effects of SP and HK-1 in the absence (control) and in the presence of peptidase inhibitors in the rabbit jugular vein Control SP HK-1
Captopril + Thiorphan
pEC50
Emax (g)
pEC50
Emax (g)
8.61 8.68
1.11 F 0.18 1.04 F 0.14
8.56 8.88
1.05 F 0.18 0.88 F 0.12
The data are expressed as mean F SEM of at least 4 experiments. Captopril and thiorphan were tested at 1 AM.
As shown in Fig. 2, in the rbJV, SP and HK-1 (both tested at 100 nM) induced a rapid increase in tissue tension that did not maintain the plateau and came back to the baseline in about 15–20 minutes. In these tissues, a second injection of SP or of HK-1, performed without washing the tissue, induced a contraction which was significantly reduced (about 50%) compared to that of the first injection. In these tissues, a third injection of either SP or HK-1 applied after 90 min of washing elicited an increase in tension similar to that evoked by the first injection (data not shown). In the rbJV, the responses induced by SP and HK-1 were challenged with two selective NK1 receptor antagonists, SR 140333 and MEN 11467. As shown in Fig. 3 (left panels), in the presence of SR 140333 the maximal effects induced by SP and HK-1 were reduced in a concentration dependent manner. The pKB values calculated from these data were 9.28 vs SP and 9.49 vs HK-1. In similar experiments, MEN 11467, antagonized, in a non-competitive manner, the contraction induced by SP and HK-1 showing pKB values of 8.76 and 8.60, respectively (Fig. 3, right panels). Finally, the effects of SP and HK-1 were evaluated in the rbJV in the absence and in the presence of peptidase inhibitors (captopril and thiorphan, both at 1 AM). The contractile effects of the peptides were not significantly modified in the presence of peptidase inhibitors (Table 2).
Discussion This is a classical pharmacology paper in which the profile of HK-1 was established with data obtained in isolated tissues and by applying the criteria of receptor classification recommended by Schild [13]. The major finding of the present study is the demonstration that the novel member of the TK family, HK-1, behaves, similarly to SP, as a NK1 receptor preferring agonist. Three lines of evidence support this conclusion. First, the order of potency of agonists determined in the three monoreceptor systems is similar to those reported in literature [5]. HK-1 behaves in the three tissues like the NK1 receptor preferring agonist SP being highly potent in the rbJV while showing very low pEC50 values (around 5) in the other tissues. Since the contractile effects of tachykinins in the rbJV are due exclusively to NK1 receptor activation [14], it is proposed that HK-1 is a strong stimulant of this receptor. Second, in desensitization experiments, repeating the injection of SP in rbJV tissues after a short period of time (about 15 min) caused marked desensitization to the agonist. This is in line with previous report [15] and demonstrates that the contractile effect of SP, in the rbJV, undergoes desensitization. The effect of the peptide fully recovered after 90 min and several tissue washing. Tissues desensitized with SP also displayed a reduced response to HK-1 and vice-versa tissues desensitized with HK-1 displayed a reduced response to SP. These data further suggest a common mechanism of action for SP- and HK-1-induced contractile effects in the rbJV. Third, the firmest
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indication that both SP- and HK-1-induced contraction of the rbJV are mediated by NK1 receptor activation comes from experiments with selective antagonists. In fact the selective NK1 receptor antagonists SR 140333 [16] and MEN 11467 [17] displayed similar potencies vs SP and HK-1 and acted as non competitive antagonists against the two naturally occurring agents. The body of data derived from these experiments strongly suggest that HK-1 may use the NK1 receptor as a functional target for its peripheral actions. The data present in this paper are in line with the findings by Zhang et al. [6] who demonstrated that some actions of HK-1 (i.e. survival and proliferation of pre-B cells in vitro, and development of B cell in bone marrow in vivo) could be reversed by the NK1 antagonist L-732,138 [18,19]. However, this compound was found to be more active against SP than against HK-1 whose effect were partially reversed by L-733,060 at doses that fully prevented the action of SP. This and other findings lead Zhang et al. [6] to speculate about the existence of novel NK receptor specific for HK-1. The results of in vitro assays, illustrated above, do not support the interpretation by Zhang et al. [6] and do not justify the need for another receptor. Data obtained with competitive antagonists are however needed to validate the suggestion that HK-1 is acting through the NK1 receptor. Acknowledgements We thank Dr X. Emonds-Alt form Sanofi, and Dr S. Giuliani from Menarini for providing SR 140333 and MEN 11467, respectively. This work was supported by a 60% Grant to DR from the University of Ferrara. References [1] Nakanishi S. Substance P precursor and kininogen: their structures, gene organization and regulation. Physiol Rev 1987;67:1117 – 42. [2] Barnes PJ. Neurogenic inflammation and asthma. J Asthma 1992;29:165 – 80. [3] Lembeck F, Holzer P. Naunyn-Schmiedeberg’s Arch Pharmacol 1979;310:175 – 83. [4] Otsuka M, Yanagisawa M. Pain and neurotransmitters. Cell Mol Neurobiol 1990;10:293 – 302. [5] Regoli D, Boudon A, Fauchere J. Receptors and antagonists for susbstance P and related peptides. Pharmacol Rev 1994;46:551 – 99. [6] Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1:392 – 7. [7] Rioux F, Quirion R, Leblanc MA, Regoli D, St-Pierre S. Possible interactions between neurotensin and prostaglandins in the isolated rat portal vein. Life Sci 1980;27:259 – 67. [8] Gaudreau P, Barabe J, St-Pierre S, Regoli D. Structure-activity study of kinins in vascular smooth muscles. Can J Physiol Pharmacol 1981;59:380 – 9. [9] D’Orleans-Juste P, Dion S, Drapeau G, Regoli D. Different receptors are involved in the endothelium-mediated relaxation and the smooth muscle contraction of the rabbit pulmonary artery in response to substance P and related neurokinins. Eur J Pharmacol 1986;125:37 – 44. [10] Severini C, Salvadori S, Guerrini R, Falconieri-Erspamer G, Mignogna G, Erspamer G. Parallel bioassay of 39 tachykinins on 11 smooth muscle preparations. Structure and receptor selectivity/affinity relationship. Peptides 2000;21:1587 – 95. [11] Jenkinson DH, Barnard EA, Hoyer D, Humphrey PPA, Leff P, Shankley NP. Pharmacol Rev 1995;47:255 – 66. [12] Kenanin T. Pharmacologic analysis of drug-receptor interaction. New York: Raven Press, 1993. [13] Schild HO. Receptor classification with special reference to beta adrenergic receptors. Baltimore: University Park Press, 1973.
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[14] Nantel F, Rouissi N, Rhaleb NE, Dion S, Drapeau G, Regoli D. The rabbit jugular vein is a contractile NK-1 receptor system. Eur J Pharmacol 1990;179:457 – 62. [15] Bohm SK, Grady EF, Bunnett NW. Regulatory mechanisms that modulate signalling by G-protein-coupled receptors. Biochem J 1997;322:1 – 18. [16] Emonds-Alt X, Doutremepuich JD, Heaulme M, Neliat G, Santucci V, Steinberg R, Vilain P, Bichon D, Ducoux JP, Proietto V, Van Broeck D, Soubrie´ P, Le Fur G, Brelie`re JC. In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol 1993;250:403 – 13. [17] Cirillo R, Astolfi M, Ciucci A, Palma C, Parlani M, Lopez G, Conte B, Terracciano R, Fincham CI, Sisto A, Maggi CA, Manzini S. Pharmacology of MEN 11467, a potent, selective and orally effective tachykinin NK1 antagonist. NaunynSchmiedeberg’s Arch Pharmacol 1998;358:R324. [18] MacLeod AM, Merchant KJ, Cascieri MA, Sadowski S, Ber E, Swain CJ, Baker R. N-acyl-L-tryptophan benzyl esters: potent substance P receptor antagonists. J Med Chem 1993;36:2044 – 5. [19] Cascieri MA, Macleod AM, Underwood D, Shiao LL, Ber E, Sadowski S, Yu H, Merchant KJ, Swain CJ, Strader CD, Ming Fong T. Characterization of the interaction of N-acyl-L-tryptophan benzyl ester neurokinin antagonists with the human neurokinin-1 receptor. J Biol Chem 1994;26:6587 – 891.
Pharmacological profile of hemokinin 1: a novel member of the tachykinin family V. Camarda a, A. Rizzi a, G. Calo a, R. Guerrini b, S. Salvadori b, D. Regoli a,* a
Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara, 19, 44100 Ferrara, Italy b Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, Via Fossato di Mortara, 19, 44100 Ferrara, Italy Received 14 August 2001; accepted 19 October 2001
Abstract Recently, the cloning of a novel preprotachykinin gene (PPT-C) has been reported. This gene codes for a novel peptide named hemokinin 1 (HK-1). In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. In this study, we pharmacologically characterised the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK1, the rabbit pulmonary artery (rbPA) for NK2, and rat portal vein (rPV) for NK3 receptors. In all these preparations substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) elicited concentration dependent contractions showing similar maximal effects and the following rank order of potency: SP > NKA = NKB in the rbJV, NKA > NKB H SP in the rbPA, and NKB > NKA > SP in the rPV. In those vessels HK-1 behaved as a full agonist displaying potencies similar (rbPA and rPV) or slightly higher (rbJV) than those of SP. In the rbJV, SR 140333, a selective NK1 receptor antagonist, antagonised the effects of HK-1 and SP with similar high potencies (pKB 9.3 and 9.5, respectively). Similar results were obtained with the pseudopeptide NK1 antagonist, MEN 11467 (pKB 8.8 and 8.6, respectively). Taken together, these data indicate that HK-1 behaves as a NK1 preferring receptor agonist. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Hemokinin 1; Tachykinins; NK1 receptor; Bioassay
Introduction Molecular biological studies have demonstrated the existence of three tachykinin-like peptides in mammals: the undecapeptide substance P (SP), and two decapeptides, neurokinin A (NKA) and neuro*
Corresponding author. Tel.: +39-0532-291227; fax: +39-0532-291205. E-mail address: [email protected] (D. Regoli). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 6 8 2 - X
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kinin B (NKB) [1]. The three peptides, encoded by two mammalian genes named PPT-A (SP and NKA) and PPT-B (NKB), are expressed almost exclusively in neurons and act as neurotransmitters in the central nervous system and as essential components of the nonadrenergic noncholinergic system in autonomic nerves [2]. Neurokinins, particularly SP, are released from sensory neurons by various stimuli, both in the spinal cord, where they contribute to pain transmission, and in the peripheral tissues, where they produce neurogenic inflammation [3,4]. Biological activities of neurokinins are mediated by three receptor types namely NK1, NK2 and NK3, which are expressed by a variety of target cells [5]. Recently, Zhang et al. [6] reported the cloning of a novel preprotachykinin gene (PPTC). This gene codes for a novel peptide named hemokinin 1 (HK-1). This is an undecapeptide (RSRTRQFYGLM-NH2) which shows the C-terminal pentapeptide sequence FYGLM that matches the typical tachykinin motif FXGLM. HK-1 most closely resembles SP in amino acid sequence: both have multiple cationic residues at the N-terminus and an aromatic amino acid occupying the variable X position. In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. HK-1 is able to induce plasma extravasation and to promote mast cell degranulation as SP [6]. However, this new peptide has also some peculiar properties that suggest that HK-1 might act as an autocrine factor to promote B cell survival and proliferation [6]. Aim of the present study was to pharmacologically characterize the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK1, the rabbit pulmonary artery (rbPA) for NK2, and rat portal vein (rPV) for NK3 receptors [5].
Methods Tissue preparations Male New Zealand white rabbits (1.8–2.0 Kg) and male Sprague–Dawley rats (250–300 g) were killed by stunning and exanguination. The vessels used were the rbJV or the rbPA and the rPV. Each vessel was quickly removed and cut either in helical strips about 15–20 mm long and 3 mm wide (rPV), or in rings 10 mm long (rbJV), or in open rings 4–6 mm wide (rbPA) according to Rioux et al., Gaudreau et al., and D’Orleans-Juste et al., respectively [7–9]. The endothelium was mechanically removed from all vessels by gently rubbing the internal surface with moistened filter paper. All tissues were suspended in organ baths containing oxygenated (95% O2 and 5% CO2) Krebs solution of the following composition (in mM): NaCl 118, NaHCO3 25, KCl 4.7, KHPO4 1.2, MgSO4 1.2, CaCl2 2.5, glucose 10. The solution was maintained at 37 jC and at pH 7.4. The tissues were stretched to a resting tension of 1 g for rbJV and rbPA and 0.5 g for rPV. During the equilibration period (1 h) fresh Krebs solution was applied and the tension readjusted at 20 min intervals. The contractions were converted in electrical signal by isometric transducers (GRASS FT03) and recorded by a multichannel poligraph (LINSEIS L2005). Experimental protocols Following the equilibration period, the preparations were exposed to 100 mM KCl for the rbPA, to 0.1 AM bradykinin for the rbJV and 1 AM of noradrenaline for the rPV, to assess the responsiveness of the
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preparation. About 30 min later, in the rbJV and rbPA, cumulative concentration-response curves (crc) to agonists were measured. In the rPV, crc to agonists were performed consecutively, adding to the bath different concentrations of the peptide every 20 minutes followed by washing. In a separate series of experiments in the rbJV, SP or HK-1 (both at 100 nM) were injected twice in the same tissue without washing between the first and the second injection (see tracing in Fig. 2). In the rbJV, SR 140333 (1 and 10 nM), MEN 11467 (10 and 100 nM), or their vehicle were added to the bath 20 min before measuring the crc to SP or HK-1. In the same tissue, the effects of peptidase inhibitors (captopril and thiorphan, both at 1 AM) were evaluated on the contractions elicited by SP and HK-1. Sources of chemicals and reagents Chemicals and their sources were as follows: HK-1, SP, NKA and NKB were synthesized and purified in our laboratories as previously described [10]; SR 140333 was provided by Dr X. Emonds-Alt (Sanofi, Centre de Montpellier, France) and MEN 11467 was supplied by Dr. S. Giuliani (Menarini, Florence, Italy). Captopril, thiorphan, and the other reagents were from Sigma–Aldrich (Milan, Italy). Stock solutions of peptides (1 mM) were made in saline and kept at 20 jC until use. NKB, MEN 11467 and SR 140333 were dissolved in DMSO. Data analysis All data are expressed as means F standard error of the mean (SEM) of n experiments. Data have been statistically analysed using the Student t test or the analysis of variance followed by the Dunnet test. P values less than 0.05 were considered to be significant. The pharmacological terminology used
Fig. 1. Effects of tachykinins and HK-1 in the rbJV. Left panel: Cumulative concentration-response curve to SP, NKA and NKB. Right panel: Cumulative concentration-response curve to SP and HK-1. Each point represents the mean F SEM of at least 5 experiments.
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in this paper follows IUPHAR recommendations [11]. The agonist potencies are given as pEC50, which is the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist. The Emax is the maximal effect that an agonist can elicit in a given tissue. Since both SR 140333 and MEN 11467 behaved as non competitive antagonists, their equilibrium dissociation constant KB was determined according to Kenakin [12]. In practice, a doublereciprocal plot of equieffective concentrations of agonist (A) in the absence (1/A) and in presence (1/ AV) of the antagonist (B) is constructed, and the pKB value is derived from the equation: pKB = log10[(slope-1)/B].
Results As shown in Fig. 1 (left panel), SP, NKA and NKB produced concentration-dependent contractions of the rbJV. SP was significantly more potent than NKA and NKB whereas the maximal tension (Emax) induced by the three peptides was similar (see Table 1). The novel peptide, HK-1, in the range 0.01 nM– 0.1 AM caused a concentration dependent contraction of the rbJV with a pEC50 and Emax of 8.43 and 0.92 F 0.12 g, respectively (Fig. 1, right panel). The kinetic of action of SP and HK-1 were similar: in fact, the effects of both peptides took place immediately after adding the peptide to the bath, were rapidly reversible after washing, and could be repeated in the same tissue after a 90 min interval (data not shown). In the rbPA and in the rPV SP, NKA and NKB acted as full agonists showing maximal effects of about 2 and 0.4 g, respectively (see Table 1). The rank order of potency of agonists was: NKA >
Fig. 2. Typical tracings showing the contractile responses to SP and HK-1, tested at 100 nM, in the rbJV. The two injections were performed without washing the tissues.
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Table 1 Effects of tachykinins and HK-1 in isolated tissues rbJV SP NKA NKB HK-1
rbPA
rPV
pEC50
Emax (g)
pEC50
Emax (g)
pEC50
Emax (g)
8.26 7.53 7.54 8.43
0.85 0.74 0.71 0.92
c5 7.56 6.50 c5
ND 2.14 F 0.36 1.95 F 0.42 ND
5.33 6.55 7.35 5.02
0.37 0.37 0.38 0.38
F F F F
0.12 0.05 0.12 0.12
F F F F
0.08 0.10 0.16 0.15
The data are expressed as mean F SEM of at least 5 experiments. ND: not determined since at the higher agonist concentration tested (10 AM) the crc was still incomplete.
NKB H SP in the rbPA, and NKB > NKA > SP in the rPV. In these two preparations HK-1 elicited concentration dependent contractions with potencies and maximal effects similar to those of SP (Table 1).
Fig. 3. Effects of NK1 selective antagonists vs SP and HK-1 in the rbJV. Left panels: Cumulative concentration-response curve to SP (top) and HK-1 (bottom) measured in the absence (control) or in presence of SR 140333. Right panels: Cumulative concentration-response curve to SP (top) and HK-1 (bottom) obtained in the absence (control) or in presence of MEN 11467. Each point represents the mean F SEM of at least 5 experiments. *p < 0.05 vs control according to ANOVA followed by the Dunnet test.
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Table 2 Effects of SP and HK-1 in the absence (control) and in the presence of peptidase inhibitors in the rabbit jugular vein Control SP HK-1
Captopril + Thiorphan
pEC50
Emax (g)
pEC50
Emax (g)
8.61 8.68
1.11 F 0.18 1.04 F 0.14
8.56 8.88
1.05 F 0.18 0.88 F 0.12
The data are expressed as mean F SEM of at least 4 experiments. Captopril and thiorphan were tested at 1 AM.
As shown in Fig. 2, in the rbJV, SP and HK-1 (both tested at 100 nM) induced a rapid increase in tissue tension that did not maintain the plateau and came back to the baseline in about 15–20 minutes. In these tissues, a second injection of SP or of HK-1, performed without washing the tissue, induced a contraction which was significantly reduced (about 50%) compared to that of the first injection. In these tissues, a third injection of either SP or HK-1 applied after 90 min of washing elicited an increase in tension similar to that evoked by the first injection (data not shown). In the rbJV, the responses induced by SP and HK-1 were challenged with two selective NK1 receptor antagonists, SR 140333 and MEN 11467. As shown in Fig. 3 (left panels), in the presence of SR 140333 the maximal effects induced by SP and HK-1 were reduced in a concentration dependent manner. The pKB values calculated from these data were 9.28 vs SP and 9.49 vs HK-1. In similar experiments, MEN 11467, antagonized, in a non-competitive manner, the contraction induced by SP and HK-1 showing pKB values of 8.76 and 8.60, respectively (Fig. 3, right panels). Finally, the effects of SP and HK-1 were evaluated in the rbJV in the absence and in the presence of peptidase inhibitors (captopril and thiorphan, both at 1 AM). The contractile effects of the peptides were not significantly modified in the presence of peptidase inhibitors (Table 2).
Discussion This is a classical pharmacology paper in which the profile of HK-1 was established with data obtained in isolated tissues and by applying the criteria of receptor classification recommended by Schild [13]. The major finding of the present study is the demonstration that the novel member of the TK family, HK-1, behaves, similarly to SP, as a NK1 receptor preferring agonist. Three lines of evidence support this conclusion. First, the order of potency of agonists determined in the three monoreceptor systems is similar to those reported in literature [5]. HK-1 behaves in the three tissues like the NK1 receptor preferring agonist SP being highly potent in the rbJV while showing very low pEC50 values (around 5) in the other tissues. Since the contractile effects of tachykinins in the rbJV are due exclusively to NK1 receptor activation [14], it is proposed that HK-1 is a strong stimulant of this receptor. Second, in desensitization experiments, repeating the injection of SP in rbJV tissues after a short period of time (about 15 min) caused marked desensitization to the agonist. This is in line with previous report [15] and demonstrates that the contractile effect of SP, in the rbJV, undergoes desensitization. The effect of the peptide fully recovered after 90 min and several tissue washing. Tissues desensitized with SP also displayed a reduced response to HK-1 and vice-versa tissues desensitized with HK-1 displayed a reduced response to SP. These data further suggest a common mechanism of action for SP- and HK-1-induced contractile effects in the rbJV. Third, the firmest
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indication that both SP- and HK-1-induced contraction of the rbJV are mediated by NK1 receptor activation comes from experiments with selective antagonists. In fact the selective NK1 receptor antagonists SR 140333 [16] and MEN 11467 [17] displayed similar potencies vs SP and HK-1 and acted as non competitive antagonists against the two naturally occurring agents. The body of data derived from these experiments strongly suggest that HK-1 may use the NK1 receptor as a functional target for its peripheral actions. The data present in this paper are in line with the findings by Zhang et al. [6] who demonstrated that some actions of HK-1 (i.e. survival and proliferation of pre-B cells in vitro, and development of B cell in bone marrow in vivo) could be reversed by the NK1 antagonist L-732,138 [18,19]. However, this compound was found to be more active against SP than against HK-1 whose effect were partially reversed by L-733,060 at doses that fully prevented the action of SP. This and other findings lead Zhang et al. [6] to speculate about the existence of novel NK receptor specific for HK-1. The results of in vitro assays, illustrated above, do not support the interpretation by Zhang et al. [6] and do not justify the need for another receptor. Data obtained with competitive antagonists are however needed to validate the suggestion that HK-1 is acting through the NK1 receptor. Acknowledgements We thank Dr X. Emonds-Alt form Sanofi, and Dr S. Giuliani from Menarini for providing SR 140333 and MEN 11467, respectively. This work was supported by a 60% Grant to DR from the University of Ferrara. References [1] Nakanishi S. Substance P precursor and kininogen: their structures, gene organization and regulation. Physiol Rev 1987;67:1117 – 42. [2] Barnes PJ. Neurogenic inflammation and asthma. J Asthma 1992;29:165 – 80. [3] Lembeck F, Holzer P. Naunyn-Schmiedeberg’s Arch Pharmacol 1979;310:175 – 83. [4] Otsuka M, Yanagisawa M. Pain and neurotransmitters. Cell Mol Neurobiol 1990;10:293 – 302. [5] Regoli D, Boudon A, Fauchere J. Receptors and antagonists for susbstance P and related peptides. Pharmacol Rev 1994;46:551 – 99. [6] Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1:392 – 7. [7] Rioux F, Quirion R, Leblanc MA, Regoli D, St-Pierre S. Possible interactions between neurotensin and prostaglandins in the isolated rat portal vein. Life Sci 1980;27:259 – 67. [8] Gaudreau P, Barabe J, St-Pierre S, Regoli D. Structure-activity study of kinins in vascular smooth muscles. Can J Physiol Pharmacol 1981;59:380 – 9. [9] D’Orleans-Juste P, Dion S, Drapeau G, Regoli D. Different receptors are involved in the endothelium-mediated relaxation and the smooth muscle contraction of the rabbit pulmonary artery in response to substance P and related neurokinins. Eur J Pharmacol 1986;125:37 – 44. [10] Severini C, Salvadori S, Guerrini R, Falconieri-Erspamer G, Mignogna G, Erspamer G. Parallel bioassay of 39 tachykinins on 11 smooth muscle preparations. Structure and receptor selectivity/affinity relationship. Peptides 2000;21:1587 – 95. [11] Jenkinson DH, Barnard EA, Hoyer D, Humphrey PPA, Leff P, Shankley NP. Pharmacol Rev 1995;47:255 – 66. [12] Kenanin T. Pharmacologic analysis of drug-receptor interaction. New York: Raven Press, 1993. [13] Schild HO. Receptor classification with special reference to beta adrenergic receptors. Baltimore: University Park Press, 1973.
370
V. Camarda et al. / Life Sciences 71 (2002) 363–370
[14] Nantel F, Rouissi N, Rhaleb NE, Dion S, Drapeau G, Regoli D. The rabbit jugular vein is a contractile NK-1 receptor system. Eur J Pharmacol 1990;179:457 – 62. [15] Bohm SK, Grady EF, Bunnett NW. Regulatory mechanisms that modulate signalling by G-protein-coupled receptors. Biochem J 1997;322:1 – 18. [16] Emonds-Alt X, Doutremepuich JD, Heaulme M, Neliat G, Santucci V, Steinberg R, Vilain P, Bichon D, Ducoux JP, Proietto V, Van Broeck D, Soubrie´ P, Le Fur G, Brelie`re JC. In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol 1993;250:403 – 13. [17] Cirillo R, Astolfi M, Ciucci A, Palma C, Parlani M, Lopez G, Conte B, Terracciano R, Fincham CI, Sisto A, Maggi CA, Manzini S. Pharmacology of MEN 11467, a potent, selective and orally effective tachykinin NK1 antagonist. NaunynSchmiedeberg’s Arch Pharmacol 1998;358:R324. [18] MacLeod AM, Merchant KJ, Cascieri MA, Sadowski S, Ber E, Swain CJ, Baker R. N-acyl-L-tryptophan benzyl esters: potent substance P receptor antagonists. J Med Chem 1993;36:2044 – 5. [19] Cascieri MA, Macleod AM, Underwood D, Shiao LL, Ber E, Sadowski S, Yu H, Merchant KJ, Swain CJ, Strader CD, Ming Fong T. Characterization of the interaction of N-acyl-L-tryptophan benzyl ester neurokinin antagonists with the human neurokinin-1 receptor. J Biol Chem 1994;26:6587 – 891.