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Antagonists for the neurokinin NK-3 receptor evaluated in selective receptor systems
Regulatory Peptides, 31 (1990) 125-135
125
Elsevier REGPEP 00972
Antagonists for the neurokinin NK-3 receptor evaluated in selective receptor systems G. Drapeau, N. Rouissi, F. Nantel, N.-E. Rhaleb, C. Tousignant and D. Regoli Department of Pharmacology, Medical School, University of Sherbrooke, Sherbrooke (Canada)
(Received 26 June 1990; revised version received and accepted 23 August 1990) Key words: Neurokinin B; Receptor; Antagonist, Vascular smooth muscle
Summary Four isolated vessels that are monoreceptor systems for neurokinins, the dog carotid artery and rabbit jugular vein (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3), were used to compare the activities of selective neurokinin agonists and evaluate the affinities of new NK-3 antagonists. Chemical modifications in the partial sequences NKA (4-10) and NKB (4-10), particularly the replacement ofVal 7 with an aromatic residue (Tyr, MePhe or Trp) and the extension of the peptide backbone in position 8, obtained with fl-Ala, led to compounds that maintain weak agonistic activities on the NK-1 and NK-2, and some of them also on NK-3 receptors but exert potent antagonism against NKB on the NK-3 receptor of the rat portal vein. Antagonistic affinity is the highest when Trp is used in position 7 of [fl-Alas ]-NKA (4-10) and MePhe in position 7 of [fl-Ala8 ]-NKB (4-10). Antagonism is selective for NKB or [MePhe 7 ]-NKB, and appears to be specific, since the most active compound [Trp 7,fl-Ala8 ]-NKA (4-10) is inactive against bradykinin on the rabbit jugular vein (B z receptor), against SP on the rabbit jugular vein (NK-1 receptor), against desArg9-bradykinin on the rabbit aorta (B 1 receptor), and against angiotensin II and histamine (AT and H receptors, respectively) in the rabbit aorta. The new NK-3 receptor antagonists described in the present study provide useful tools for neurokinin receptor characterization and for determining the roles of neurokinins in physiopathology.
Correspondence: D. Regoli,Department of Physiologyand Pharmacology,Medical School, Universityof
Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
126
Introduction
The neurokinins are a family of mammalian peptides sharing the C-terminal sequence Phe-X-Gly-Leu-Met-NH2. Three of these peptides, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) have been identified and shown to exert various biological effects in the central nervous system and in peripheral organs [12,15,23]. Neurokinin B (neurokinin r, neuromedin K) is a decapeptide first isolated from the porcine spinal cord by Kimura et al. [ 11 ] and shown to be a powerful neuromodulator of the parasympathetic nerve endings in the guinea-pig isolated ileum [12] and a stimulant of the micturition reflex in the anaesthetized rat [ 16]. Neurokinin B, along with the other two neurokinins, was shown to act on several (at least three) receptor types [9,10]. The receptors were pharmacologically characterized by the use of neurokinins, tachykinins and neurokinin fragments [21,22], as well as by synthetic selective analogues of the three neurokinins receptors [5,13,22,23]. These studies also brought to identification of vascular preparations that contain one single neurokinin receptor type. Of these, the rat portal vein has been extensively used to characterize NK-3 receptors of which neurokinin B is the most potent of the naturally occurring agonists [ 10,18,22-25]. To date, only weak and possibly non-selective NK-3 antagonists have been reported [29]. Still, such compounds are instrumental for NK-3 receptor identification and characterization. The principal aim of the present study was to provide a careful pharmacological analysis of new NK-3 receptor antagonists derived from the sequence of NKA (4-10) or NKB (4-10). Some new compounds were found to be fairly potent and selective antagonists for the NK-3 receptor.
Materials and Methods
The experiments described below were carried out on tissues taken from rats (Albino Sprague-Dawley, 200-300 g), rabbits (Albino, New Zealand, 1.0-1.5 kg) and mongrel dogs (10-20 kg) of either sex. The animals were killed by stunning and exsanguination, except the dogs, which were previously anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally). The following vessels were used: the rat portal vein, the rabbit jugular vein, the rabbit pulmonary artery, the dog carotid artery and the rabbit aorta: the vessels were carefully taken out of the animal and plunged in Krebs physiological buffer of the following composition (in mM): NaCI: 117.5; KCI: 4.7; K2HzPO4: 1.18; MgSO 4 • 7H20: 1.18; CaC12 • 6H20 2.5; NaHCO3: 25.0; 2.5 and D-glucose: 5.5. Helicoidal rings of the rat portal vein were obtained by the method of Rioux et al. [26 ]. Rings of rabbit pulmonary artery without endothelium were prepared according tO D'Orl6ans-Juste et al. [4] and, rings of dog carotid artery were dissected with extreme care to preserve the endothelium as recommended by Furchgott and Zawadski [ 7 ] and they were cut along the horizontal axis as described before [4]. Strips of the rabbit jugular vein and rabbit aorta were prepared according to Gaudreau et al. [8] and Regoli et al. [20], respectively. The tissues were suspended in 10ml organ baths containing warm (37°C),
127
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128 oxygenated (95 ~o 02, 5 ~o C02) Krebs solution. For the dog carotid artery, the solution contained indomethacin (2.8.10- 6 M) to avoid the release of endogenous prostaglandins and EDTA 5.95.10 - 5 M) to prevent the oxidation ofnoradrenaline (2.8.10 - 8 M) which was used to contract the tissues [2]. The tissues were submitted to a tension of 0.5 g (the rat portal vein and rabbit jugular vein), 1.0 g (the rabbit pulmonary artery) and 2.0 g (the dog carotid artery): they were allowed to stabilize for 60-90 min during which time they were washed repeatedly and the tension readjusted every 15 min. Changes of tension were measured with Grass isometric transducer (FT O3C) and displayed on Grass polygraph model 7D.
Experimental protocols The neurokinins; SP, NKA and NKB, induce concentration-dependent contractions of the rat portal vein, rabbit jugular vein and rabbit pulmonary artery and relaxation on the dog carotid artery previously contracted with noradrenaline (2.8.10-8 M). The antagonists were tested against NKB or [MePhe 7 ]-NKB on the rat portal vein, against NKA on the rabbit pulmonary artery and against SP on the rabbit jugular vein and dog carotid artery. The antagonist apparent affinities are expressed in terms of pA 2 (the negative log of the concentration of antagonist that reduces the effect of a double concentration of agonist to that of a single, according to Schild [28] and those of the agonists in terms of pD 2, the negative log of the concentration of agonist that produces 50~o of the maximal response according to Ari~ns et al. [1].
Peptides and other agents All peptides were prepared in our laboratory by the solid-phase method as described by Drapeau et al. [6]. Their primary structure is given in Table I. The peptides were purified by high pressure liquid chromatography (HPLC) and their structures were confirmed by fast atom bombardment (FAB) mass spectrometry. Indomethacin, histamine, noradrenaline and ascorbic acid were obtained from Sigma (St. Louis) and EDTA from Fisher (Montreal). Concentrated solutions of peptides were prepared in distilled water and stored at - 20 °C until used. Ascorbic acid (5 ~ ) was added to the concentrated solutions of NA to prevent oxidation of the amine.
Results
Concentration-response curves measured with neurokinins and related peptides in the four vascular preparations that contain a single neurokinin receptor type are shown in Fig. 1. Relaxations of the dog carotid artery previously contracted with noradrenaline are brought about by very small concentrations (ED50 around 10- 10 M) of SP and of the selective NK-1 agonist (Ac.[Argr,Sar9,Met(O2)ll]-SP (6-11)), while the other selective compounds, namely [fl-AIa8]-NKA (4-10) and [MePheT]-NKB are active only at much higher concentrations (10 - 7 M). Similar results are observed in the rabbit jugular vein, a pure NK-1 preparation that has been identified and characterized recently [ 19]. In the rabbit pulmonary artery, the NK-2 receptor system, NKA and the selective
129 Rabbit jugular vein
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Fig. 1. Concentration-response curves obtained with substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and some selective agonists, SeE NK-1 (Ac.[Arg6,Sar9,Met(O2)ll]SP (6-11), Sel. NK-2 ([fl-AlaS]NKA (4-10) and Sel. NK-3 ([MePhe7]NKB)) on four isolated vessels: (a) the dog carotid artery that relaxes in response to neurokinins and (b) the rabbit jugular vein, rabbit pulmonary artery and rat portal vein that are stimulated by neurokinins to contract. Points are means and vertical bars the standard errors of the mean of at least 6 determinations. Abscissa: molar concentration of agonist. Ordinate: biological effect in percent of the maximum effect obtainable with the reference compound, SP (dog carotid artery and rabbit jugular vein), NKA (rabbit pulmonary artery) and NKB (rat portal vein). N K - 2 agonists are quite active, while [ M e P h e 7 ] - N K B and Ac.[Arg6,Sar9,Met(02)ll ] SP (6-11) are completely inactive. O n the rat portal vein, the N K - 3 receptor system, the most potent c o m p o u n d is the selective N K - 3 agonist, [ M e P h e 7 ] - N K B and N K B itself, while [fl-Ala 8 ] - N K A (4-10) is very weak and Ac.[Arg6,Sar9,Met(O2) 11 ]SP (6-11) is completely inactive. Results obtained with six putative N K - 3 antagonists are summarized in Table II. The c o m p o u n d s are all analogues o f the sequences [fl-AlaS]-NKA (4-10) and [fl-Ala8] N K B (4-10) two heptapeptides which differ only by the residue in position 5 (Ser in N K A (4-10) and Phe in N K B (4-10)). Both these natural sequences act as agonists in all preparations (see p D 2 values in Table II), but when the Gly in position 8 is replaced by a B-Ala and Val in position 7 is replaced by an aromatic residue, either Tyr, Phe, MePhe or Trp, the c o m p o u n d s become fairly selective N K - 3 antagonists, pA2 values of 7.0 are observed with [Tyr 7, fl-Ala s ] - N K A (4-10) (compound 3) on the NK-3
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131 TABLE III Pharmacological activity( p A 2 ) a of tWONK-3 antagonists against NKB and [MePheT]-NKBon the rat portal vein Peptide
[TrpT,fl-AlaS]-NKA(4-10) [MePheV,fl-AlaS]-NKB(4-10)
Agonist NKB
[MePheT]-NKB
7.46 7.26
7.46 7.26
Log of the concentrationof antagonist that reduces the effectof a double dose of agonist to that of a single one.
a pA2: _
system (rat portal vein) while the compound is an agonist in the other three tissues and also in the rat portal vein when applied at very high concentrations. Elimination of the Met residue in position 10 (compound 4) leads to a decrease of affinity and selectivity, since [Tyr7,fl-AIa8]-NKA (4-9) has a pA 2 value of only 5.46 on the rat portal vein (NK-3) and of 5.85 on the rabbit pulmonary artery (NK-2). When Phe is used instead of Tyr in position 7, as in [Phe 7, fl-Alas ]-NKA (4-10) the compound maintains a good antagonistic activity (pA 2 value of 6.92) but still shows some agonistic effect on the rat portal vein. However, [Phe 7, fl-Ala8 ]-NKA (4-10) is not selective for the NK-3 receptor, because it acts also as an antagonist on the NK-2 site of the rabbit pulmonary artery, where it shows a pA 2 of 7.04. When Trp is used to replace Val 7, an increase of antagonist affinity on the rat portal vein is observed (pA 2 value of 7.5) and the compound is a weak agonist on the dog carotid artery and the rabbit pulmonary artery. Indeed, [TrpT,fl-Ala8 ]-NKA (4-10) shows a good antagonistic activity both against NKB and the selective NK-3 agonist [MePheT]-NKB on the rat portal vein (Table lII). [TrpT,fl-Ala8]-NKA (4-10) stimulates the rat portal vein only when applied at high concentrations (pD 2 = 5.73) which are almost two log units higher than those needed for antagonism. Analogues of the heptapeptide NKB (4-10) were also prepared and the results are shown in Table II. Both compounds, [TyrT, fl-AIa8]-NKB ( 4 - 1 0 ) a n d [MePheT, flAla 8 ]-NKB (4-10) act as antagonists on the rat portal vein and as agonists on the three other preparations. The antagonist affinity of the second compound (number 8) is fairly good (pA 2 = 7.26) and is comparable to that of the best antagonist of the first series. Moreover, [MePheT, fl-Ala8]-NKB (4-10) is less active as an agonist than [Trp7,flAla8]-NKA (4-10) on the rabbit pulmonary artery and the dog carotid artery [MePhe7,fl-Ala8 ]-NKB (4-10) shows some agonistic effects (partial agonist on the rat portal vein) when applied at high concentrations, similar t o [ T r p 7, fl-Ala8 ]-NKA (4-10).
Specificity of [TrpT,~-AlaS]-NKA (4-10)for the NK-3 receptor In order to determine if [TrpT,/~-AlaS]-NKA (4-10), the most potent antagonist identified in the present study, is specific for neurokinin receptors, particularly the NK-3 type, the contractile effects of other peptides, bradykinin, angiotensin or the nonpeptide agent histamine were measured in the absence and in presence of compound 6. As shown in Table IV, compound 6 did not modify the contractile responses of the rabbit
132 TABLE IV Absence of effects of [Trp 7, fl-AlaS]-NKA (4-10) (1.15.10- 5 M) on the contractions (expressed in mm) of the rabbit jugular vein and the rabbit aorta in respone to various stimulants Tissue Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit
jugular jugular jugular jugular jugular aorta aorta
vein vein vein vein vein
Agonist
Control
+ antagonist
BK (8.0 nM) Histamine (22.5 #M) ATII (8.2 nM) SP (6.5 nM) [MePhe7]-NKB (2.5 #M) desArggBK (93 nM) A T . (8.2 nM)
18.0 22.8 12.8 20.8 17.5 16.0 32.7
18.0 21.7 12.2 18.0 17.0 21.7 29.0
+ 2.9 a + 2.5 + 1.1 _+ 3.3 + 1.5 + 1.5 _+ 1.8
+_ 2.2 + 3.1 + 1.5 + 2.2 + 2.6 + 1.2 + 2.7
a values are means + standard errors of at least 6 determinations.
jugular vein to bradykinin, histamine, angiotensin II, SP and [MePhe 7 ]-NKB, as well as the contractions of the rabbit aorta after stimulation with desArg9-BK (DBK) or angiotensin II. These findings indicate that compound 6 is specific for the neurokinin receptors on which it might act as antagonist (NK-3) or as a weak agonist (NK-2, NK-1). pA 2 value were measured for [Trp 7, fl-Ala8 ]-NKB (4-10) against NKB and the selective NK-3 agonist, [MePhe 7 ]-NKB (Table III), but differences pA2-pA m could not be evaluated because of the agonistic activity of [TrpT,fl-Ala8 ]-NKA (4-10) at high doses.
Discussion
It has been well established that neurokinins act on three different receptors [9] and selective agonists for each receptor have been obtained with modifications of either the Gly (which occupies position 9 of substance P) or the preceeding residue (Phe in SP and Val in NKA and NKB). Thus, replacement of Gly by Sar brought to selective NK-1 and the substitution of Gly by fl-Ala to selective NK-2 agonists while selective NK-3 compounds were obtained by replacing Val with MePhe [5,24]. It is conceivable that the use of N-methyl-amino acids (MePhe, Sar) or the elongation of the peptide backbone by one carbon atom (obtained with ~-Ala) brings to alternative conformers of the natural sequences that might be energetically more favorable to occupation and activation of one of the three receptor sites. The discovery and further improvement of NK-1, NK-2 and NK-3 selective agonists has been achieved after the identification of isolated organs whose responses to neurokinins depend entirely from the activation of a single receptor site [22,23 ]. Recently, a contractile preparation, the rabbit jugular vein [19], has been identified and might replace the dog carotid artery which has several disadvantages and limitations, as discussed by Nantel et al. [ 19]. The selectivity of these organs for one or the other neurokinin and their selective agonists is illustrated in Fig. 1, by comparing the concentration-response curves obtained with the neurokinins and the neurokinin receptor
133 selective agonists. From these results it is evident that the receptor of the rabbit pulmonary artery and the rat portal vein are extremely selective while the two NK-1 receptor systems maintain good sensitivity for NKA, NKB [24] as well as for the NK-2 and the NK-3 selective agonists, when these compounds are applied at high concentrations. This suggests that exclusive selectivity for the NK-1 receptor site might be more difficult to obtain than for the other sites. In an attempt to find NK-2 or NK-3 receptor antagonists, the sequences NKA (4-10), which has been shown [22] to be very active on the NK-2 site and has been made selective for this site by replacing Gly with fl-Ala [27] was further modified. From previous studies [5] it is known that Val is important for NK-2 but not for NK-3 receptor activation, since it can be replaced advantageously by MePhe. Therefore, Gly was replaced with fl-Ala and Val with an aromatic residue, either Tyr, Phe, MePhe or Trp. The results reported above indicate that Trp is the best substitution: the presence of a large aromatic residue instead of Val not only reduces the affinity for the NK-2 and NK-1 sites by more than two log units but also leads to a potent antagonist of the NK-3 receptor. A similar effect was obtained by using the sequence NKB (4-10) in which Gly was replaced by fl-Ala and Val with the N-methylated aromatic residue (MePhe). The compound shows a similar pharmacological spectrum as [TIp 7, fl-Alas ]-NKA (4-10). Both compounds are weak agonists on the NK-3 receptor when applied at concentrations 100 to 500 times higher than those needed for NK-3 antagonism. Although the molecular mechanisms involved in the activation of neurokinin receptors are still unknown, it is believed that agonists are able to induce conformational changes in the membraneous domains of receptor molecules and such changes lead to activation of the intracellular loop (the third loop) which, according to Lefkowitz et al. [ 14], interacts with G proteins. The present results suggest that Valine may play an important role in this process and its replacement with an aromatic residue (Trp or MePhe) brings about a conformational change, perhaps energetically disfavorable, for receptor activation and leads to NK-3 antagonism: when Val is replaced by Phe, the compound acts as antagonist on both NK-3 and NK-2. This observation is interesting in view of further design of selective NK-2 receptor antagonists.
Acknowledgements The authors acknowledge the secretarial work of Mrs. C. Th6berge and the technical assistance of M. Boussougou, R. Laprise and M. Battistini. Work presented in this paper was performed with the financial support of the Medical Research Council of Canada (M.R.C.C.) and the Quebec Heart and Shock Foundation. G. Drapeau is a fellow of the Fonds de la Recherche en Sant6 du Qu6bec, F. Nantel is a student of the Georges Ph6nix Foundation, C. Tousignant is a student of the (M.R.C.C.), N.-E. Rhaleb is a student of the Canadian Heart Foundation and D. Regoli is a Career Investigator of the M.R.C.C.
134
References 1 Ariens, E.J., Simonis, A.M. and Van Rossum, J.M., Drug-receptor interaction: Interaction of one or more drugs with one receptor system, In E.J. Ari~ns (Ed.), Molecular Pharmacology, Academic Press, New York, 1964, pp. 119-286. 2 Couture, R., Gaudreau, P., St-Pierre, S. and Regoli, D., The dog common carotid artery: a sensitive bioassay for studying vasodilatator effects of substance P and kinins, Can. J. Physiol. Pharmaeol., 58 (1980) 1234-1244. 3 Dion, S., D'Orl6ans-Juste, P., Drapeau, G., Rhaleb, N.E., Rouissi, N., Tousignant, C. and Regoli, D., Characterization of neurokinin receptors in various isolated organs by the use of selective agonists, Life Sci., 41 (1987) 2269-2278. 4 D'Orl6ans-Juste, P., Dion, S., Drapeau, G. and 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., 125 (1986) 37-44. 5 Drapeau, G., D'Orl6ans-Juste, P., Dion, S., Rhaleb, N.E., Rouissi, N. and Regoli, D., Selective agonists for substance P and neurokinins, Neuropeptides, 10 (1987) 43-54. 6 Drapeau, G., Rovero, P., D'Orl6ans-Juste, P., Dion, S., Rhaleb, N.-E. and Regoli, D., Mammalian neurokinins: Structure-activity study of the N- and C-terminals. In J. L. Henry, R. Couture, A. C. Cuello, G. Pelletier, R. Quirion, D. Regoli (Eds.), Substance P and Neurokinins, Springer, New York, 1987, pp. 132-134. 7 Furchgott, R. F. and Z awadski, J.V., The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature, 288 (1980) 373-376. 8 Gaudreau, P., St-Pierre, S. and Regoli, D., Pharmacological studies ofkinins on venous smooth muscles, Can. J. Physiol. Pharmacol., 59 (1981) 371-379. 9 Henry, J.L., Couture, R., Cuello, A.C., Pelletier, G., Quirion, R. and Regoli, D., Substance P and Neurokinins, Springer, New York, 1987, pp. 17-18. 10 Iversen, L.L., Foster, A.C., Watling, K.J., McKnight, A.T., Williams, B.J. and Lee, C.M., Multiple receptor binding sites for tachykinins. In J. L. Henry, R. Couture, A.C. Cuello, G. Pelletier, R. Quirion, D. Regoli (Eds.), Substance P and Neurokinins, Springer, New York, 1987, pp. 40-43. 11 Kimura, S., Okada, M., Sugita, Y., Kanazauki, I. and Munekata, E., Novel neuropeptides, Neurokinin ~t and fl, isolated from porcine spinal cord, Proc. Jpn. Acad. Ser., B59 (1983) 101-104. 12 Laufer, R., Wormser, U., Friedman, Z.Y., Gilon, C., Chorev, M. and Selinger, Z., Neurokinin B is a preferred agonist for a neuronal substance P receptor and its action is antagonized by enkephalin, Proc. Nat. Acad. Sci. USA, 82 (1985) 7444-7448. 13 Laufer, R., Gilon, C., Chorev, M. and Selinger, Z., Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand, J. Biol. Chem., 262 (1986) 10257-10263. 14 Lefkowitz, R.J. and Caron, M.G., Adrenergic receptors: models for study of receptors coupled to guanine nucleotide regulatory proteins, J. Biol. Chem., 263 (1988) 4993-4996. 15 Maggi, C.A., Santicioli, P., Giuliani, S., Regoli, D. and Meli, A., Activation of micturition reflex by substance P and substance K: indirect evidence for the existence of multiple tachykinin receptors in the rat urinary bladder, J. Pharmacol. Exp. Ther., 238 (1986) 259-266. 16 Maggi, C.A., Giuliani, S., Santicioli, P., Abelli, L., Regoli, D. and Meli, A., Further studies on the mechanism responsible for the neurokinin induced activation of micturition reflex in rats: evidence for the involvement of the capsaicin-sensitive bladder mechanoreceptors, Eur. J. Pharmacol., 136 (1987) 189-206. 17 Maggi, C.A., Giuliani, S., Santicioli, P., Regoli, D. and Meli, A., Peripheral effects of neurokinins: functional evidence for the existence of multiple receptors, J. Auton. Pharmacol., 7 (1987) 11-32. 18 Mastrangelo, D., Mathison, R., Huggel, H.J., Dion, S., D'Orl6ans-Juste, P., Rhaleb, N. E., Drapeau, G., Rovero, P. and Regoli, R., The rat isolated portal vein: a preparation sensitive to neurokinins, particularly neurokinin B., Eur. J. Pharmacol., 134 (1987) 321-326. 19 Nantel, G., Rouissi, N., Rhaleb, N.E., Dion, S., Drapeau, G. and Regoli, D., The rabbit jugular vein is a contractile NK-1 receptor system, Eur. J. Pharmacol., 179 (1990) 457-462. 20 Regoli, D., Barabe, J. & Park, W.K. (1977) Receptors for bradykinin in rabbit aorta, Can. J. Physiol., 55 (1977) 855-867.
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125
Elsevier REGPEP 00972
Antagonists for the neurokinin NK-3 receptor evaluated in selective receptor systems G. Drapeau, N. Rouissi, F. Nantel, N.-E. Rhaleb, C. Tousignant and D. Regoli Department of Pharmacology, Medical School, University of Sherbrooke, Sherbrooke (Canada)
(Received 26 June 1990; revised version received and accepted 23 August 1990) Key words: Neurokinin B; Receptor; Antagonist, Vascular smooth muscle
Summary Four isolated vessels that are monoreceptor systems for neurokinins, the dog carotid artery and rabbit jugular vein (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3), were used to compare the activities of selective neurokinin agonists and evaluate the affinities of new NK-3 antagonists. Chemical modifications in the partial sequences NKA (4-10) and NKB (4-10), particularly the replacement ofVal 7 with an aromatic residue (Tyr, MePhe or Trp) and the extension of the peptide backbone in position 8, obtained with fl-Ala, led to compounds that maintain weak agonistic activities on the NK-1 and NK-2, and some of them also on NK-3 receptors but exert potent antagonism against NKB on the NK-3 receptor of the rat portal vein. Antagonistic affinity is the highest when Trp is used in position 7 of [fl-Alas ]-NKA (4-10) and MePhe in position 7 of [fl-Ala8 ]-NKB (4-10). Antagonism is selective for NKB or [MePhe 7 ]-NKB, and appears to be specific, since the most active compound [Trp 7,fl-Ala8 ]-NKA (4-10) is inactive against bradykinin on the rabbit jugular vein (B z receptor), against SP on the rabbit jugular vein (NK-1 receptor), against desArg9-bradykinin on the rabbit aorta (B 1 receptor), and against angiotensin II and histamine (AT and H receptors, respectively) in the rabbit aorta. The new NK-3 receptor antagonists described in the present study provide useful tools for neurokinin receptor characterization and for determining the roles of neurokinins in physiopathology.
Correspondence: D. Regoli,Department of Physiologyand Pharmacology,Medical School, Universityof
Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
126
Introduction
The neurokinins are a family of mammalian peptides sharing the C-terminal sequence Phe-X-Gly-Leu-Met-NH2. Three of these peptides, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) have been identified and shown to exert various biological effects in the central nervous system and in peripheral organs [12,15,23]. Neurokinin B (neurokinin r, neuromedin K) is a decapeptide first isolated from the porcine spinal cord by Kimura et al. [ 11 ] and shown to be a powerful neuromodulator of the parasympathetic nerve endings in the guinea-pig isolated ileum [12] and a stimulant of the micturition reflex in the anaesthetized rat [ 16]. Neurokinin B, along with the other two neurokinins, was shown to act on several (at least three) receptor types [9,10]. The receptors were pharmacologically characterized by the use of neurokinins, tachykinins and neurokinin fragments [21,22], as well as by synthetic selective analogues of the three neurokinins receptors [5,13,22,23]. These studies also brought to identification of vascular preparations that contain one single neurokinin receptor type. Of these, the rat portal vein has been extensively used to characterize NK-3 receptors of which neurokinin B is the most potent of the naturally occurring agonists [ 10,18,22-25]. To date, only weak and possibly non-selective NK-3 antagonists have been reported [29]. Still, such compounds are instrumental for NK-3 receptor identification and characterization. The principal aim of the present study was to provide a careful pharmacological analysis of new NK-3 receptor antagonists derived from the sequence of NKA (4-10) or NKB (4-10). Some new compounds were found to be fairly potent and selective antagonists for the NK-3 receptor.
Materials and Methods
The experiments described below were carried out on tissues taken from rats (Albino Sprague-Dawley, 200-300 g), rabbits (Albino, New Zealand, 1.0-1.5 kg) and mongrel dogs (10-20 kg) of either sex. The animals were killed by stunning and exsanguination, except the dogs, which were previously anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally). The following vessels were used: the rat portal vein, the rabbit jugular vein, the rabbit pulmonary artery, the dog carotid artery and the rabbit aorta: the vessels were carefully taken out of the animal and plunged in Krebs physiological buffer of the following composition (in mM): NaCI: 117.5; KCI: 4.7; K2HzPO4: 1.18; MgSO 4 • 7H20: 1.18; CaC12 • 6H20 2.5; NaHCO3: 25.0; 2.5 and D-glucose: 5.5. Helicoidal rings of the rat portal vein were obtained by the method of Rioux et al. [26 ]. Rings of rabbit pulmonary artery without endothelium were prepared according tO D'Orl6ans-Juste et al. [4] and, rings of dog carotid artery were dissected with extreme care to preserve the endothelium as recommended by Furchgott and Zawadski [ 7 ] and they were cut along the horizontal axis as described before [4]. Strips of the rabbit jugular vein and rabbit aorta were prepared according to Gaudreau et al. [8] and Regoli et al. [20], respectively. The tissues were suspended in 10ml organ baths containing warm (37°C),
127
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128 oxygenated (95 ~o 02, 5 ~o C02) Krebs solution. For the dog carotid artery, the solution contained indomethacin (2.8.10- 6 M) to avoid the release of endogenous prostaglandins and EDTA 5.95.10 - 5 M) to prevent the oxidation ofnoradrenaline (2.8.10 - 8 M) which was used to contract the tissues [2]. The tissues were submitted to a tension of 0.5 g (the rat portal vein and rabbit jugular vein), 1.0 g (the rabbit pulmonary artery) and 2.0 g (the dog carotid artery): they were allowed to stabilize for 60-90 min during which time they were washed repeatedly and the tension readjusted every 15 min. Changes of tension were measured with Grass isometric transducer (FT O3C) and displayed on Grass polygraph model 7D.
Experimental protocols The neurokinins; SP, NKA and NKB, induce concentration-dependent contractions of the rat portal vein, rabbit jugular vein and rabbit pulmonary artery and relaxation on the dog carotid artery previously contracted with noradrenaline (2.8.10-8 M). The antagonists were tested against NKB or [MePhe 7 ]-NKB on the rat portal vein, against NKA on the rabbit pulmonary artery and against SP on the rabbit jugular vein and dog carotid artery. The antagonist apparent affinities are expressed in terms of pA 2 (the negative log of the concentration of antagonist that reduces the effect of a double concentration of agonist to that of a single, according to Schild [28] and those of the agonists in terms of pD 2, the negative log of the concentration of agonist that produces 50~o of the maximal response according to Ari~ns et al. [1].
Peptides and other agents All peptides were prepared in our laboratory by the solid-phase method as described by Drapeau et al. [6]. Their primary structure is given in Table I. The peptides were purified by high pressure liquid chromatography (HPLC) and their structures were confirmed by fast atom bombardment (FAB) mass spectrometry. Indomethacin, histamine, noradrenaline and ascorbic acid were obtained from Sigma (St. Louis) and EDTA from Fisher (Montreal). Concentrated solutions of peptides were prepared in distilled water and stored at - 20 °C until used. Ascorbic acid (5 ~ ) was added to the concentrated solutions of NA to prevent oxidation of the amine.
Results
Concentration-response curves measured with neurokinins and related peptides in the four vascular preparations that contain a single neurokinin receptor type are shown in Fig. 1. Relaxations of the dog carotid artery previously contracted with noradrenaline are brought about by very small concentrations (ED50 around 10- 10 M) of SP and of the selective NK-1 agonist (Ac.[Argr,Sar9,Met(O2)ll]-SP (6-11)), while the other selective compounds, namely [fl-AIa8]-NKA (4-10) and [MePheT]-NKB are active only at much higher concentrations (10 - 7 M). Similar results are observed in the rabbit jugular vein, a pure NK-1 preparation that has been identified and characterized recently [ 19]. In the rabbit pulmonary artery, the NK-2 receptor system, NKA and the selective
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Fig. 1. Concentration-response curves obtained with substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and some selective agonists, SeE NK-1 (Ac.[Arg6,Sar9,Met(O2)ll]SP (6-11), Sel. NK-2 ([fl-AlaS]NKA (4-10) and Sel. NK-3 ([MePhe7]NKB)) on four isolated vessels: (a) the dog carotid artery that relaxes in response to neurokinins and (b) the rabbit jugular vein, rabbit pulmonary artery and rat portal vein that are stimulated by neurokinins to contract. Points are means and vertical bars the standard errors of the mean of at least 6 determinations. Abscissa: molar concentration of agonist. Ordinate: biological effect in percent of the maximum effect obtainable with the reference compound, SP (dog carotid artery and rabbit jugular vein), NKA (rabbit pulmonary artery) and NKB (rat portal vein). N K - 2 agonists are quite active, while [ M e P h e 7 ] - N K B and Ac.[Arg6,Sar9,Met(02)ll ] SP (6-11) are completely inactive. O n the rat portal vein, the N K - 3 receptor system, the most potent c o m p o u n d is the selective N K - 3 agonist, [ M e P h e 7 ] - N K B and N K B itself, while [fl-Ala 8 ] - N K A (4-10) is very weak and Ac.[Arg6,Sar9,Met(O2) 11 ]SP (6-11) is completely inactive. Results obtained with six putative N K - 3 antagonists are summarized in Table II. The c o m p o u n d s are all analogues o f the sequences [fl-AlaS]-NKA (4-10) and [fl-Ala8] N K B (4-10) two heptapeptides which differ only by the residue in position 5 (Ser in N K A (4-10) and Phe in N K B (4-10)). Both these natural sequences act as agonists in all preparations (see p D 2 values in Table II), but when the Gly in position 8 is replaced by a B-Ala and Val in position 7 is replaced by an aromatic residue, either Tyr, Phe, MePhe or Trp, the c o m p o u n d s become fairly selective N K - 3 antagonists, pA2 values of 7.0 are observed with [Tyr 7, fl-Ala s ] - N K A (4-10) (compound 3) on the NK-3
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131 TABLE III Pharmacological activity( p A 2 ) a of tWONK-3 antagonists against NKB and [MePheT]-NKBon the rat portal vein Peptide
[TrpT,fl-AlaS]-NKA(4-10) [MePheV,fl-AlaS]-NKB(4-10)
Agonist NKB
[MePheT]-NKB
7.46 7.26
7.46 7.26
Log of the concentrationof antagonist that reduces the effectof a double dose of agonist to that of a single one.
a pA2: _
system (rat portal vein) while the compound is an agonist in the other three tissues and also in the rat portal vein when applied at very high concentrations. Elimination of the Met residue in position 10 (compound 4) leads to a decrease of affinity and selectivity, since [Tyr7,fl-AIa8]-NKA (4-9) has a pA 2 value of only 5.46 on the rat portal vein (NK-3) and of 5.85 on the rabbit pulmonary artery (NK-2). When Phe is used instead of Tyr in position 7, as in [Phe 7, fl-Alas ]-NKA (4-10) the compound maintains a good antagonistic activity (pA 2 value of 6.92) but still shows some agonistic effect on the rat portal vein. However, [Phe 7, fl-Ala8 ]-NKA (4-10) is not selective for the NK-3 receptor, because it acts also as an antagonist on the NK-2 site of the rabbit pulmonary artery, where it shows a pA 2 of 7.04. When Trp is used to replace Val 7, an increase of antagonist affinity on the rat portal vein is observed (pA 2 value of 7.5) and the compound is a weak agonist on the dog carotid artery and the rabbit pulmonary artery. Indeed, [TrpT,fl-Ala8 ]-NKA (4-10) shows a good antagonistic activity both against NKB and the selective NK-3 agonist [MePheT]-NKB on the rat portal vein (Table lII). [TrpT,fl-Ala8]-NKA (4-10) stimulates the rat portal vein only when applied at high concentrations (pD 2 = 5.73) which are almost two log units higher than those needed for antagonism. Analogues of the heptapeptide NKB (4-10) were also prepared and the results are shown in Table II. Both compounds, [TyrT, fl-AIa8]-NKB ( 4 - 1 0 ) a n d [MePheT, flAla 8 ]-NKB (4-10) act as antagonists on the rat portal vein and as agonists on the three other preparations. The antagonist affinity of the second compound (number 8) is fairly good (pA 2 = 7.26) and is comparable to that of the best antagonist of the first series. Moreover, [MePheT, fl-Ala8]-NKB (4-10) is less active as an agonist than [Trp7,flAla8]-NKA (4-10) on the rabbit pulmonary artery and the dog carotid artery [MePhe7,fl-Ala8 ]-NKB (4-10) shows some agonistic effects (partial agonist on the rat portal vein) when applied at high concentrations, similar t o [ T r p 7, fl-Ala8 ]-NKA (4-10).
Specificity of [TrpT,~-AlaS]-NKA (4-10)for the NK-3 receptor In order to determine if [TrpT,/~-AlaS]-NKA (4-10), the most potent antagonist identified in the present study, is specific for neurokinin receptors, particularly the NK-3 type, the contractile effects of other peptides, bradykinin, angiotensin or the nonpeptide agent histamine were measured in the absence and in presence of compound 6. As shown in Table IV, compound 6 did not modify the contractile responses of the rabbit
132 TABLE IV Absence of effects of [Trp 7, fl-AlaS]-NKA (4-10) (1.15.10- 5 M) on the contractions (expressed in mm) of the rabbit jugular vein and the rabbit aorta in respone to various stimulants Tissue Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit
jugular jugular jugular jugular jugular aorta aorta
vein vein vein vein vein
Agonist
Control
+ antagonist
BK (8.0 nM) Histamine (22.5 #M) ATII (8.2 nM) SP (6.5 nM) [MePhe7]-NKB (2.5 #M) desArggBK (93 nM) A T . (8.2 nM)
18.0 22.8 12.8 20.8 17.5 16.0 32.7
18.0 21.7 12.2 18.0 17.0 21.7 29.0
+ 2.9 a + 2.5 + 1.1 _+ 3.3 + 1.5 + 1.5 _+ 1.8
+_ 2.2 + 3.1 + 1.5 + 2.2 + 2.6 + 1.2 + 2.7
a values are means + standard errors of at least 6 determinations.
jugular vein to bradykinin, histamine, angiotensin II, SP and [MePhe 7 ]-NKB, as well as the contractions of the rabbit aorta after stimulation with desArg9-BK (DBK) or angiotensin II. These findings indicate that compound 6 is specific for the neurokinin receptors on which it might act as antagonist (NK-3) or as a weak agonist (NK-2, NK-1). pA 2 value were measured for [Trp 7, fl-Ala8 ]-NKB (4-10) against NKB and the selective NK-3 agonist, [MePhe 7 ]-NKB (Table III), but differences pA2-pA m could not be evaluated because of the agonistic activity of [TrpT,fl-Ala8 ]-NKA (4-10) at high doses.
Discussion
It has been well established that neurokinins act on three different receptors [9] and selective agonists for each receptor have been obtained with modifications of either the Gly (which occupies position 9 of substance P) or the preceeding residue (Phe in SP and Val in NKA and NKB). Thus, replacement of Gly by Sar brought to selective NK-1 and the substitution of Gly by fl-Ala to selective NK-2 agonists while selective NK-3 compounds were obtained by replacing Val with MePhe [5,24]. It is conceivable that the use of N-methyl-amino acids (MePhe, Sar) or the elongation of the peptide backbone by one carbon atom (obtained with ~-Ala) brings to alternative conformers of the natural sequences that might be energetically more favorable to occupation and activation of one of the three receptor sites. The discovery and further improvement of NK-1, NK-2 and NK-3 selective agonists has been achieved after the identification of isolated organs whose responses to neurokinins depend entirely from the activation of a single receptor site [22,23 ]. Recently, a contractile preparation, the rabbit jugular vein [19], has been identified and might replace the dog carotid artery which has several disadvantages and limitations, as discussed by Nantel et al. [ 19]. The selectivity of these organs for one or the other neurokinin and their selective agonists is illustrated in Fig. 1, by comparing the concentration-response curves obtained with the neurokinins and the neurokinin receptor
133 selective agonists. From these results it is evident that the receptor of the rabbit pulmonary artery and the rat portal vein are extremely selective while the two NK-1 receptor systems maintain good sensitivity for NKA, NKB [24] as well as for the NK-2 and the NK-3 selective agonists, when these compounds are applied at high concentrations. This suggests that exclusive selectivity for the NK-1 receptor site might be more difficult to obtain than for the other sites. In an attempt to find NK-2 or NK-3 receptor antagonists, the sequences NKA (4-10), which has been shown [22] to be very active on the NK-2 site and has been made selective for this site by replacing Gly with fl-Ala [27] was further modified. From previous studies [5] it is known that Val is important for NK-2 but not for NK-3 receptor activation, since it can be replaced advantageously by MePhe. Therefore, Gly was replaced with fl-Ala and Val with an aromatic residue, either Tyr, Phe, MePhe or Trp. The results reported above indicate that Trp is the best substitution: the presence of a large aromatic residue instead of Val not only reduces the affinity for the NK-2 and NK-1 sites by more than two log units but also leads to a potent antagonist of the NK-3 receptor. A similar effect was obtained by using the sequence NKB (4-10) in which Gly was replaced by fl-Ala and Val with the N-methylated aromatic residue (MePhe). The compound shows a similar pharmacological spectrum as [TIp 7, fl-Alas ]-NKA (4-10). Both compounds are weak agonists on the NK-3 receptor when applied at concentrations 100 to 500 times higher than those needed for NK-3 antagonism. Although the molecular mechanisms involved in the activation of neurokinin receptors are still unknown, it is believed that agonists are able to induce conformational changes in the membraneous domains of receptor molecules and such changes lead to activation of the intracellular loop (the third loop) which, according to Lefkowitz et al. [ 14], interacts with G proteins. The present results suggest that Valine may play an important role in this process and its replacement with an aromatic residue (Trp or MePhe) brings about a conformational change, perhaps energetically disfavorable, for receptor activation and leads to NK-3 antagonism: when Val is replaced by Phe, the compound acts as antagonist on both NK-3 and NK-2. This observation is interesting in view of further design of selective NK-2 receptor antagonists.
Acknowledgements The authors acknowledge the secretarial work of Mrs. C. Th6berge and the technical assistance of M. Boussougou, R. Laprise and M. Battistini. Work presented in this paper was performed with the financial support of the Medical Research Council of Canada (M.R.C.C.) and the Quebec Heart and Shock Foundation. G. Drapeau is a fellow of the Fonds de la Recherche en Sant6 du Qu6bec, F. Nantel is a student of the Georges Ph6nix Foundation, C. Tousignant is a student of the (M.R.C.C.), N.-E. Rhaleb is a student of the Canadian Heart Foundation and D. Regoli is a Career Investigator of the M.R.C.C.
134
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