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Effect of scyliorhinin I and synthetic scyliorhinin I derivatives at mammalian tachykinin NK1, NK2 and NK3 receptors
European Journal of Pharmacology, 250 (1993) 311-316 Elsevier Science Publishers B.V.
EJP 53420
Effect of scyliorhinin I and synthetic scyliorhinin I derivatives at mammalian tachykinin NK 1, NK 2 and N K 3 receptors R i c c a r d o P a t a c c h i n i *'a L a u r a Q u a r t a r a b, K r z y s z t o f R o l k a c, J o l a n t a Z b o i n s k a c, G o t f r y d K u p r y s z e w s k i c a n d C a r l o A l b e r t o Maggi a a Pharmacology and b Chemistry Departments, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131, Florence, Italy, and c Institute of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland Received 15 July 1993, revised MS received 15 September 1993, accepted 28 September 1993
The dogfish tachykinin peptide scyliorhinin I and a number of its analogues substituted in position 7 were tested in bioassays for tachykinin NK1, NK a and NK 3 receptors. Scyliorhinin I behaved as a full agonist at tachykinin NK1 receptors of the guinea-pig ileum longitudinal muscle and at NK a receptors of the rabbit pulmonary artery and hamster trachea. In these three preparations scyliorhinin I was as potent agonist as substance P methylester and neurokinin A, respectively. Evidence for activation of tachykinin NK 1 and NK 2 receptors by scyliorhinin I was obtained by using the selective tachykinin antagonists FK 888, MEN 10,376 and L 659,877. Scyliorhinin I was poorly active as an agonist at NK 3 receptors of the rat portal vein. Among scyliorhinin I analogues, [fl-(2-naphthyl)-Ala7]scyliorhinin I, [Val7]scyliorhinin I and [IleT]scyliorhinin I were 3-25 times weaker than scyliorhinin I itself at NK 1 and NK 2 receptors. [PheT]scyliorhinin I, [Phe(F)7]scyliorhinin I and [Phe(C1)7]scyliorhinin I were as potent as scyliorhinin I at NK 1 receptors in the guinea-pig ileum, while they showed 10-30 times lower affinity than scyliorhinin I for NK2 receptors. The present results are discussed in relation to the importance of position 7 in determining the potency and selectivity of scyliorhinin I analogues at tachykinin receptors. Scyliorhinin I; Tachykinin receptor; Tachykinin receptor agonist
1. Introduction Tachykinins are a family of mammalian and nonmammalian peptides which share the common Cterminal sequence Phe-Xaa-Gly-Leu-Met-NH 2. At least three distinct receptors, termed NK1, NK 2 and NK3, have been proposed to mediate the biological actions of tachykinins in mammals (Buck et al., 1984; Lee et al., 1986; Regoli et al., 1989). Scyliorhinin I and scyliorhinin II are two peptides belonging to the tachykinin family which have been isolated from dogfish gut (Conlon et al., 1986). Scyliorhinin I is a linear decapeptide, while scyliorhinin II is a naturally occurring cyclic peptide. Scyliorhinin I and scyliorhinin II share a C-terminal amino acid sequence similar to that of the mammalian tachykinins neurokinin A and neurokinin B. Binding studies have shown that scyliorhinin II binds with highest affinity to NK 3 receptors, while scyliorhinin I possesses high affinity for NK 1 and NK e receptors, and a 50-250 times reduced affinity for N K 3 receptors (Buck and
* Corresponding author. Tel. 39-55-5680350, fax 39-55-5680419.
Krstenansky, 1987; Beaujouan et al., 1988). In particular, scyliorhinin I shows the same high affinity of substance P for peripheral or central NK 1 receptors of the rat. Likewise, scyliorhinin I binds to NK z receptors expressed by hamster and rat peripheral tissues with the same high affinity shown by neurokinin A (Buck and Krstenansky, 1987; Beaujouan et al., 1988). However, nothing is known about the biological effects of scyliorhinins, apart from their ability to induce contraction of the guinea-pig ileum longitudinal muscle (ConIon et al., 1986). The aim of this study was to assess the potency and selectivity of scyliorhinin I at NK1, NK 2 and NK 3 receptors expressed by guinea-pig, rabbit, hamster and rat, in several in vitro bioassays, and to assess the specificity of this interaction by means of receptorselective antagonists. In addition, we investigated the influence exerted by the amino acid residue in position 7 of the scyliorhinin I sequence on the selectivity of scyliorhinin I towards tachykinin receptors, This position, which is occupied by Tyr in scyliorhinin I, is the variable position in the C-terminal common region of mammalian and non-mammalian tachykinins. Previous studies have shown that certain substitutions at this position in mammalian tachykinins led to a decrease of
312 agonist potency, as observed for [Ala8]substance P (Couture et al., 1979) or [Ala7]neurokinin A (Rovero et al., 1989). N-Methylation of the Phe 8 residue in substance P or Val 7 in neurokinin B increased the selectivity of the methylated analogues for tachykinin NK 3 receptors (Lavielle et al., 1988) as did the introduction of Pro in position 7 of neurokinin B (Lavielle et al., 1988). In this study we substituted the Tyr residue with Val, which is present in position 7 of both neurokinin A and neurokinin B. We also introduced Phe and lie which are present in the sequence of substance P and of the non-mammalian tachykinin eledoisin, respectively. Furthermore the amino acids /3-(2-naphthyl)Ala, Phe(F), Phe(Cl), D-Phe(F) and MePhe were introduced in position 7 of scyliorhinin I to obtain the following peptides: [ValT]scyliorhinin I, [IleT]scylior hinin I, [fl-(2-naphthyl)-Ala7]scyliorhinin I, [Phe7]scyl iorhinin I, [D-Phe(F)7]scyliorhinin I, [Phe(F)7]scyliorhinin I, [Phe(C1)7]scyliorhinin I and [MePhe7]scyliorhinin I.
being added when the effect of the preceding one had reached a steady state. 2.2. Evaluation of data
The agonist activity of each test compound was expressed as pD 2 ( - l o g ECs0 or molar concentration of peptide producing 50% of maximal effect). Antagonist potency was evaluated in terms of pK B (negative logarithm of antagonist dissociation constant), which was estimated as the mean ( + S.E.M.) of the individual values obtained with the equation: pKB= log[agonistdose ratio - 1] - log[antagonist concentration] (Kenakin, 1987; Jenkinson, 1991). 2.3. Statistical analysis
The values in the text, tables or figures are expressed as means + S.E.M. Regression analysis of log concentration-effect curves was performed by the least-squares method, considering linear such curves between 20 and 80% of the maximal response.
2. Materials and methods
2. 4. Drugs 2.1. General
Male albino New Zealand rabbits (2.5-3.0 kg), male Syrian golden hamsters (100-120 g), male albino guinea-pigs (250-300 g) and male albino rats (Wistar strain, 300-350 g) were stunned and bled. Endothelium-denuded strips of rabbit pulmonary artery, rings of hamster trachea, strips of guinea-pig ileum longitudinal muscle and rat portal veins were excised and prepared for isometric tension recording in oxygenated (96% 0 2 and 4% CO 2) normal Krebs solution in 5-ml organ baths, as described previously (Maggi et al., 1990; Mastrangelo et al., 1986; Dion et al., 1987). The activity of peptides under study was assessed at NK 2 receptors of the isolated rabbit pulmonary artery and isolated hamster trachea, two preparations bearing pharmacologically different subtypes of this receptor, preliminary termed NK2A and NK2B, respectively (Maggi et al., 1991b). The activity at NK 1 receptors was studied in the guinea-pig ileum longitudinal muscle preparation (atropine and chlorpheniramine 1/~M, indomethacin 3 /xM in the bath). The activity at NK 3 receptors was evaluated in the rat portal vein. Responses to scyliorhinin I and scyliorhinin I analogues were compared to those produced by the tachykinin agonists neurokinin A (rabbit pulmonary artery and hamster trachea), substance P methylester (guinea-pig ileum) and arginine-neurokinin B ([Arg°]neurokinin B) (rat portal vein). Concentration-response curves for the agonists were obtained in a cumulative manner, each concentration
The drugs used were: substance P methylester and neurokinin A (Peninsula, St. Helens, UK), L 659,877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)] (C.R.B., Cambridge, UK), atropine (Serva, Heidelberg, Germany), indomethacin and clorpheniramine (Sigma, St. Louis, USA), FK 888 [(N-Me)indolyl-3-carboxy-(4R)-Hyp-2Nal-N,N-(methylbenzyl)amide] (synthesized at Menarini Sud Laboratories, Pomezia, Italy, by Dr. A. Sisto), arginine neurokinin B and MEN 10,376 (H-Asp-Tyr-DTrp-VaI-D-Trp-D-Trp-Lys-NH 2) (synthesized at Menarini Laboratories, Florence, Italy, by conventional solid phase methods), scyliorhinin I (H-Ala-Lys-Phe-AspLys-Phe-Tyr-Gly-Leu-Met-NH2), [Val7]scyliorhinin I, [IleT]scyliorhinin I, [/3-(2-naphthyl)-AlaV]scyliorhinin I, [PheT]scyliorhinin I, [D-Phe(F)7]scyliorhinin I, [Phe (F)7]scyliorhinin I, [Phe(Cl)7]scyliorhinin I and [MePheT]scyliorhinin I (synthesized at the Institute of Chemistry, University of Gdansk, Gdansk, Poland, by conventional solid phase methods).
3. Results
3.1. Effect of scyliorhinin I at N K 1, N K 2 and N K 3 receptors
Scyliorhinin I behaved as a full agonist at NK 1 and NK 2 receptors, showing the same potency of the reference peptides substance P methylester and neurokinin A, respectively. At NK 1 receptors of the guinea-pig
313
ileum longitudinal muscle preparation scyliorhinin I produced a concentration-dependent contractile response (pD 2 = 8.48 + 0.12; n = 8) (fig. 1, table 1) whose maximal response averaged 105 + 3% (n = 8) of that obtained with substance P methylester (pD 2 = 8.40 + 0.11; n = 8). At N K 2 receptors of the isolated rabbit pulmonary artery and isolated hamster trachea, scyliorhinin I produced concentration-dependent contractile responses (pD 2 = 8.33 _ 0.05 and 7.34 + 0.12, respectively; n = 8 each) (fig. 1, table 1). Maximal effects averaged 99 + 2 and 1 0 1 _ 4% (n = 8 each) of those obtained with neurokinin A (pD 2 = 8.20 + 0.1 and 7.33 + 0.1 respectively; n = 8 each) in the same preparations, respectively. At NK 3 receptors of the isolated rat portal vein scyliorhinin I displayed a much weaker agonist activity as compared to that shown at NK 1 and NK 2 receptors. In this bioassay the threshold concentration of scyliorhinin I to produce a contractile response was 0.1 /zM. The contraction produced by scyliorhinin I at 0.3 /xM averaged 55 + 8% (n = 4) of that produced by [Arg°]neurokinin B at a concentration (20 nM) which corresponds to the ECs0 of this agonist at NK 3 receptors in the rat portal vein (Regoli et al., 1987). Owing to the limited amount of peptide available, a full concentration-response curve for scyliorhinin I was not made for the rat portal vein, and its apparent affinity is reported as pD 2 < 6.5 (table 1). 3.2. Effect o f scyliorhinin I analogues at N K 1, N K 2 and N K 3 receptors
The scyliorhinin I analogues [fl-(2-naphthyl)AlaT]scyliorhinin I, [ValT]scyliorhinin I, [IleT]scyliorhinin I, [PheT]scyliorhinin I, [Phe(F)7]scyliorhinin I
and [Phe(Cl)7]scyliorhinin I behaved as full agonists at NK 1 and NK 2 receptors of the guinea-pig ileum, rabbit pulmonary artery and hamster trachea preparations (table 1). [/3-(2-naphthyl)-Ala7]Scyliorhinin I, [Val7] scyliorhinin I and [IleT]scyliorhinin I were from 3 to 25 times weaker than scyliorhinin I itself at NK 1 and NK 2 receptors, each one of these analogues showed the same relative potency as scyliorhinin I in the preparations tested (table 1). On the other hand, [Phe7] scyliorhinin I, [Phe(F)7]scyliorhinin I and [Phe(C1)7] scyliorhinin I were as active as scyliorhinin I at NK 1 receptors of the guinea-pig ileum, while they showed a 10-30 times reduced potency at NK 2 receptors expressed by rabbit or hamster preparations (table 1). The concentration-response curves for [fl-(2-naphthyl)Ala7]scyliorhinin I and [PheT]scyliorhinin I, compared with those for scyliorhinin I, in the guinea-pig ileum, rabbit pulmonary artery and hamster trachea, are shown in fig. 1. The other scyliorhinin I analogues, [D-Phe(F)7]scyliorhinin I and [MePheT]scyliorhinin I, were about three orders of magnitude weaker than scyliorhinin I in producing contraction at NK 1 receptors of the guineapig ileum (table 1). In addition, the maximal responses elicited by the latter analogues in the guinea-pig ileum were lower (72 + 6 and 58 __+5%, n = 6, respectively) than that produced by scyliorhinin I. At NK 2 receptors of the rabbit pulmonary artery, [D-Phe(F)7]scyliorhinin I and [MePheT]scyliorhinin I produced very slight or no response up to 3 /zM, respectively (table 1), while in the hamster trachea both of them were inactive up to 3 /zM. At NK 3 receptors of the rat portal vein [/3-(2-naphthyl)-AlaT]scyliorhinin I, [PheT]scyliorhinin I, [Phe(F)7]scyliorhinin I and [Phe(C1)7]scyliorhinin I behaved as scyliorhinin I, producing contractile responses (up to
TABLE 1 Agonist activity of several scyliorhinin I analogues at tachykinin NKI, NK 2 and NK 3 receptors. Each pD 2 ( - l o g ECso) value in the table is the mean±S.E.M, of 6-8 determinations. R.A.: apparent affinity of each test compound for tachykinin NK1 or NK 2 receptors, relative to that shown by scyliorhinin I, obtained by the ratio: (ECs0 test compound/ECs0 scyliorhinin I) x 100, GPI: guinea-pig ileum longitudinal muscle (atropine and chlorpheniramine 1/~M, indomethacin 3/zM in the bath). RPA: endotheliumdeprived rabbit pulmonary artery. HT: hamster trachea. RPV: rat portal vein. IN = inactive up to 3 ~M concentrations. Peptide
NK t receptor
NK 2 receptor
GPI
Scyliorhinin I [fl-(2-naphthyl)-AlaT]Scyliorhinin I [Val7]Scyliorhinin I [Ile7]Scyliorhinin I [PheT]Scyliorhinin I [Phe(F)7]Scyliorhinin I [Phe(Cl)7]Scyliorhinin I [D-Phe(F)7]Scyliorhinin I [MePheT]Scyliorhinin I
NK 3 receptor
RPA
HT
RPV
pD 2
R.A.
pD 2
R.A.
pD 2
R.A.
pD 2
8.48 + 0.12 8.05 + 0.16 7.19 ± 0.12 7.05 ± 0.21 8.54-t-0.21 8.56 :t: 0.18 8.64±0.14 5.33 ± 0.14 6.06 ± 0.23
100 37 5 4 115 120 144 0.07 0.4
8.33 ± 0.05 7.64 ± 0.20 7.08 ± 0.10 6.79:1:0.12 6.98±0.20 6.88 ± 0.20 7.33±0.12 < 5.5 IN
100 20 6 3 4 3 10 < 0.1
7.34 ± 0.12 6.62 + 0.12 5.97 ± 0.16 5.57 + 0.14 5.77+0.20 5.89 + 0.20 6.01 _+0.2 IN IN
100 19 4 2 3 4 5
< 6.5 < 6.5 <6 <6 < 6.5 < 6.5 < 6.5 IN IN
314
0.3 /xM) lower than that obtained by using [Arg°]neu rokinin B (20 nM) (table 1). [ValT]Scyliorhinin I and [Ile7]scyliorhinin I, up to 1 ~ M , were unable to produce responses higher than those elicited by [Arg°]neurokinin B (20 nM), while [D-Phe(F) 7] scyliorhinin I and [MePhe7]scyliorhinin I were inactive up to 3 / x M (table 1). 3.3. Effect of tachykinin receptor selective antagonists against scyliorhinin I at N K 1 and N K 2 receptors The tachykinin NK 1 receptor-selective antagonist FK 888 (30-100 nM) (Fuji et al., 1992) and the tachykinin NK 2 receptor-selective antagonists MEN 10,376 (30-100 nM) (Maggi et al., 1991a) and L 659,877 (30-100 nM) (McKnight et al., 1991) were used to block the contractile responses produced by scyliorhinin
1 20 0
-
11
•
10
'
"
9
•
8
-
,
"
7
'
-
6
•
•
5
4
I00"
TABLE 2 Antagonist activity of F K 888, M E N 10,376 and L 659,877 towards scyliorhinin I-induced responses at tachykinin N K 1 and N K 2 receptors.
Each value in the table is mean pK B (negative logarithmof antagonist dissociationconstant)+ S.E.M. of 4 determinations.GPI: guineapig ileum longitudinalmuscle (atropine and chlorpheniramine 1 ~M, indomethacin3/xM in the bath). RPA: endothelium-deprivedrabbit pulmonary artery. HT: hamster trachea. Antagonist
F K 888 M E N 10,376 L 659,877
N K 1 receptor
N K 2 receptors
GPI
RPA
HT
8.83±0.2 8.17±0.3 8.39±0.3
I in the guinea-pig ileum, rabbit pulmonary artery and hamster trachea, respectively (15-min incubations with test antagonists in each preparation). All antagonists produced a parallel rightward shift of the concentration-response curve for scyliorhinin I in the three preparations tested. FK 888 antagonized scyliorhinin I in the guinea-pig ileum with a potency (pK B = 8.83; table 2) similar to that observed against substance P methylester (pK a = 8.81). MEN 10,376 blocked scyliorhinin I-induced effects in the rabbit pulmonary artery (pK B = 8.17; table 2) with about the same potency shown against neurokinin A (pK a = 8.08). In the hamster trachea L 659,877 was slightly more potent against scyliorhinin I (pK B = 8.39; table 2) than against neurokinin A (pK B = 7.92).
t.d Z
80-
4. Discussion
60 40 o
20 0
11
I0
9
7
6
5
4
11
6 5 10 9 8 - LOG M CONCENTRATION
4
lOO o~ Z
6O 40 20 0
Fig. 1. Concentration-response curves for scyliorhinin I (©), [/3-(2naphthyl)-Ala7]scyliorhinin I (t~) and [Phe7]scyliorhinin I ( • ) in the guinea-pig ileum longitudinal muscle preparation (upper panel), isolated rabbit pulmonary artery (middle panel) and isolated hamster trachea (lower panel). Each value is the m e a n of 6 - 8 determinations, and vertical lines show S.E.M.
The present results show that the non-mammalian peptide scyliorhinin I acts as a full receptor agonist at peripheral tachykinin NK~ receptors of the guinea-pig ileum, and at NK 2 receptors of the rabbit pulmonary artery and hamster trachea. Furthermore, scyliorhinin I displays high potency in producing agonist responses in the above preparations, comparable to that possessed by substance P methylester and neurokinin A at NK 1 and NK 2 receptors, respectively. In contrast, scyliorhinin I shows lower agonist potency at NK 3 receptor of the rat portal vein than that shown by the NK 3 receptor-selective agonist [Arg°]neurokinin B. Evidence for the interaction of scyliorhinin I with tachykinin NK 1 and NK z receptors has been provided by using potent and selective tachykinin receptor antagonists. In fact, the agonist effects produced by scyliorhinin I were blocked by FK 888 (NK 1 receptorselective antagonist; Fuji et al., 1992) in the guinea-pig ileum, by MEN 10,376 (NK 2 receptor-selective antagonist; Maggi et al., 1991a) in the rabbit pulmonary artery and by L 659,877 (NK 2 receptor-selective antagonist;
315
McKnight et al., 1991; Patacchini et al., 1991) in the hamster trachea. Furthermore, the potencies shown by the above antagonists in blocking scyliorhinin I-induced effects are very similar to those obtained against other NK 1 and NK 2 receptor-selective agonists in the same organs. Our results, obtained in functional bioassays, are in keeping with those reported previously in binding studies (Buck and Krstenansky, 1987; Beaujouan et al., 1988). In fact, the latter authors reported that scyliorhinin I possesses high affinity for NK 1 and NK 2 receptors, and 50-250 times lower affinity for NK 3 receptors. For what concerns scyliorhinin I analogues, our aim was to determine the effect of various substitutions in position 7 of the scyliorhinin I molecule on selectivity towards tachykinin receptors. First of all we tried to introduce Val, lie and Phe residues, which are present in the same position of neurokinin A and B, eleidosin and substance P sequences, respectively, lie is also present in the neurokinin A analogue [Leu3,Ile7]neurokinin A, which has been recently isolated from extracts of the brain and intestine of the European frog Rana ridibunda (Lovas et al., 1993) and shown to possess high selectivity for NK 1 receptors. The introduction of the aliphatic residues Val and lie in position 7 of scyliorhinin I did not influence the selectivity of this peptide, as [Val7]scyliorhinin I and [Ile7]scyliorhinin I showed a decrease in agonist potency of the same order of magnitude in all preparations tested. In contrast, [Phe7]scyliorhinin I maintained the same potency as scyliorhinin I for the NK~ receptor, while it was less potent at NK 2 receptors, being 22 and 37 times weaker than scyliorhinin I in the rabbit pulmonary artery and hamster trachea, respectively. This result shows that the introduction of Phe in scyliorhinin I provides the molecule with higher selectivity for the NK 1 receptor than scyliorhinin I itself. Tyr residue, which is present in position 7 of scyliorhinin I, contains an aromatic side chain substituted in the para position with an hydroxyl group. In this study we aimed to distinguish the role of the above two chemical functions in providing selectivity for tachykinin receptors to scyliorhinin I. Therefore we introduced in position 7 of scyliorhinin 1 a large aromatic residue, /3-(2-naphthyl)-Ala, and two analogues of Phe substituted at the para position with halogen atoms of different size and electronegativity, yielding [fl-(2-naphthyl)-Ala7]scyliorhinin I, [Phe(F)7] scyliorhinin I and [Phe(C1)7]scyliorhinin I. The presence of /3-(2-naphthyl)-Ala produced a general decrease in agonist potency, while [Phe(F)7]scyliorhinin I and [Phe(Cl)7]scyliorhinin I showed the same pattern of selectivity as [Phe7]scyliorhinin I. These results suggest that the electronic characteristics of the aromatic side chain play a minor role in providing the Phe analogues with potency and selectivity for tachykinin
receptors, whereas the size of the aromatic ring is more important for this class of peptides. Inversion of the chirality of Phe, with ([Phe(F)7]scyl iorhinin I being used to obtain [D-Phe(F)7]scyliorhinin I, dramatically decreased the potency of the latter analogue at all tachykinin receptors. This result is in keeping with that obtained with [D-PheS]substance P in the guinea-pig ileum preparation by Duplaa et al. (1991). The N-methylated Phe residue was introduced in scyliorhinin I to induce a strong conformational change in the molecule. This substitution, which has been shown to increase the duration of action of substance P (Eison et al., 1982), caused a marked drop of agonist potency at tachykinin receptors in all bioassays. In conclusion, our experiments provide the first functional evidence for activation of mammalian tachykinin receptors by scyliorhinin I. The present results show that scyliorhinin I is a highly potent agonist at both NK 1 and NK 2 receptors, thus confirming the high affinity for these receptors shown by scyliorhinin I in binding experiments. In addition we have developed three analogues, [Phe7]scyliorhinin I, [Phe(F)7]scyl iorhinin I and [Phe(Cl)7]scyliorhinin I, which are NK I receptor selective agonists. In view of the recently described pharmacological heterogeneity of the tachykinin NK 1 receptor (Petitet et al., 1992), these novel ligands could be useful as tools for investigation of NK 1 receptor distribution and pharmacology.
Acknowledgements This work was partially supported by the Polish Scientific Research Council-KBN (research grant 2 P 303 130 04). We want to thank Ms. Carla Sezzatini for excellent technical assistance.
References Beaujouan, J.C., M. Saffroy, F. Petitet, Y. Torrens and J. Glowinski, 1988, Neuropeptide K, scyliorhinin I and II: new tools in the tachykinin receptor field, Eur. J. Pharmacol. 151, 353. Buck, S.H. and J.L. Krstenansky, 1987, The dogfish pEptides scyliorhinin I and scyliorhinin II bind with differential selectivity to mammalian tachykinin receptors, Eur. J. Pharmacol. 144, 109. Buck, S.H., E. Burcher, C.W. Schultz, W. Lovenberg and T.L. O'Donahue, 1984, Novel pharmacology of substance K binding sites: a third type of tachykinin receptor, Science 226, 987. Conion, J.M., C.F. Deacon, L. O'Toole and L. Thim, 1986, Scyliorhinin I and II: two novel tachykinins from dogfish gut, FEBS Lett. 200, 111. Couture, R., A. Fournier, J. Magnan, S. St Pierre and D. Regoli, 1979, Structure-activity studies of substance P, Can. J. Physiol. Pharmacol. 57, 1427. Dion, S., P. D'Orleans-Juste, U. Drapeau, N.E. Rhaleb, N.E. Rouissi, C. Tousignant and D. Regoli, 1987, Characterization of neurokinin receptors in various isolated organs by the use of selective agonists, Life Sci. 41, 2269.
316 Duplaa, H., G. Chassaing, S. LavieUe, J.C. Beaujouan, Y. Torrens, M. Saffroy, J. Glowinski, P. D'Orleans-Juste, D. Regoli, A. Carruette and D. Garret, 1991, Influence of the replacement of amino acid by its t~-enantiomer in the sequence of substance P. 1. Binding and pharmacological data, Neuropeptides 19, 251. Eison, A.S., S.D. Iversen, B.E.B. Sandberg, S.P. Watson, M.R. Hanley and L.L. Iversen, 1982, Substance P analog, DiMe-C7: evidence for stability in rat brain and prolonged central action, Science 215, 188. Fuji, T., M. Murai, H. Morimoto, Y. Maeda, M. Yamaoka, D. Hagiwara, H. Miyake, N. Ikari and M. Matsuo, 1992, Pharmacological profile of a high affinity dipeptide NK-1 receptor antagonist, FK-888, Br. J. Pharmacol. 107, 785. Jenkinson, D.H., 1991, How we describe competitive antagonists: three questions of usage, Trends Pharmacol. Sci. 12, 53. Kenakin, T.P., 1987, Pharmacologic Analysis of Drug-Receptor Interaction (Raven Press, New York). Lavielle, S., G. Chassaing, O. Ploux, D. Loeuillet, J. Besseyre, S. Julien, A. Marquet, O. Convert, J.C. Beaujouan, Y. Torrens, L. Bergstrom, M. Saffroy and J. Glowinski, 1988, Analysis of tachykinin binding site interactions using constrained analogues of tachykinins, Biochem. Pharmacol. 37, 41. Lee, C.M., N.J. Campbell, B.J. Williams and L.L. Iversen,1986, Multiple tachykinin binding sites in peripheral tissue and brain, Eur. J. Pharmacol. 130, 209. Lovas, S., Y. Wang, F. O'Harte, T. Badgery-Parker, N. Chartrel, H. Vaudry, E. Burcher, D. D. Smith, R.F. Murphy and J. M. Conlon, 1993, Isolation, synthesis and binding properties of novel amphibian tachykinins, in: Peptides 1992, eds. C.H. Schneider and A.N. Eberle (ESCOM Science Publ.) p. 669. Maggi, C.A., R. Patacchini, S. Giuliani, P. Rovero, S. Dion, D. Regoli, A. Giachetti and A. Meli, 1990, Competitive antagonists discriminate between NK 2 tachykinin receptor subtypes, Br. J. Pharmacol. 100, 588.
Maggi, C.A., S. Giuliani, L. Ballati, A. Lecci, S. Manzini, R. Patacchini, A.R. Renzetti, P. Rovero, L. Quartara and A. Giachetti, 1991a, In vivo evidence for tachykininergic transmission using a new NK-2 receptor-selective antagonist, MEN 10,376, J. Pharmacol. Exp. Ther. 257, 1172. Maggi, C.A., R. Patacchini, L. Quartara, P. Rovero and P. Santicioli, 1991b, Tachykinin receptors in the guinea-pig isolated bronchi, Eur. J. Pharmacol. 197, 167. Mastrangelo, D., R. Mathison, H.J. Huggel, S. Dion, P. D'OrleansJuste, N.E. Rhaleb, G. Drapeau, P. Rovero and D. Regoli, 1986, The rat isolated portal vein: a preparation sensitive to neurokinins, particularly neurokinin B, Eur. J. Pharmacol. 134, 321. McKnight, A.T., J.J. Maguire, N.J. Elliott, A.E. Fletcher, R. Tridgett, B.J. Williams, J. Longmore and L.L. Iversen, 1991, Pharmacological specificity of novel, synthetic, cyclic peptides as antagonists at tachykinin receptors, Br. J. Pharmacol. 104, 355. Patacchini, R., M. Astolfi, L. Quartara, P. Rovero, A. Giachetti and C.A. Maggi, 1991, Further evidence for the existence of NK 2 tachykinin receptor subtypes, Br. J. Pharmacol., 104, 91. Petitet, F., M. Saffroy, Y. Torrens, S. Lavielle, G. Chassaing, D. Loeuillet, J. Glowinski and J.C. Beaujouan, 1992, Possible existence of a new tachykinin receptor subtype in the guinea-pig ileum, Peptides 13, 383. Regoli, D., G. Drapeau, S. Dion and P. D'Orleans-Juste, 1987, Receptors for neurokinins in peripheral organs, in: Substance P and Neuroldnins, eds. J.L. Henry et al. (Springer-Verlag, New York) p. 132. Regoli, D., G. Drapeau, S. Dion and P. D'Orleans-Juste, 1989, Receptors for substance P and related neurokinins, Pharmacology 38, 1. Rovero, P., V. Pestellini, N.-E. Rhaleb, S. Dion, N. Rouissi, C. Tousignant, S. T616maque, G. Drapeau and D. Regoli, 1989, Structure-activity studies of neurokinin A, Neuropeptides 13, 263.
EJP 53420
Effect of scyliorhinin I and synthetic scyliorhinin I derivatives at mammalian tachykinin NK 1, NK 2 and N K 3 receptors R i c c a r d o P a t a c c h i n i *'a L a u r a Q u a r t a r a b, K r z y s z t o f R o l k a c, J o l a n t a Z b o i n s k a c, G o t f r y d K u p r y s z e w s k i c a n d C a r l o A l b e r t o Maggi a a Pharmacology and b Chemistry Departments, A. Menarini Pharmaceuticals, Via Sette Santi 3, 50131, Florence, Italy, and c Institute of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland Received 15 July 1993, revised MS received 15 September 1993, accepted 28 September 1993
The dogfish tachykinin peptide scyliorhinin I and a number of its analogues substituted in position 7 were tested in bioassays for tachykinin NK1, NK a and NK 3 receptors. Scyliorhinin I behaved as a full agonist at tachykinin NK1 receptors of the guinea-pig ileum longitudinal muscle and at NK a receptors of the rabbit pulmonary artery and hamster trachea. In these three preparations scyliorhinin I was as potent agonist as substance P methylester and neurokinin A, respectively. Evidence for activation of tachykinin NK 1 and NK 2 receptors by scyliorhinin I was obtained by using the selective tachykinin antagonists FK 888, MEN 10,376 and L 659,877. Scyliorhinin I was poorly active as an agonist at NK 3 receptors of the rat portal vein. Among scyliorhinin I analogues, [fl-(2-naphthyl)-Ala7]scyliorhinin I, [Val7]scyliorhinin I and [IleT]scyliorhinin I were 3-25 times weaker than scyliorhinin I itself at NK 1 and NK 2 receptors. [PheT]scyliorhinin I, [Phe(F)7]scyliorhinin I and [Phe(C1)7]scyliorhinin I were as potent as scyliorhinin I at NK 1 receptors in the guinea-pig ileum, while they showed 10-30 times lower affinity than scyliorhinin I for NK2 receptors. The present results are discussed in relation to the importance of position 7 in determining the potency and selectivity of scyliorhinin I analogues at tachykinin receptors. Scyliorhinin I; Tachykinin receptor; Tachykinin receptor agonist
1. Introduction Tachykinins are a family of mammalian and nonmammalian peptides which share the common Cterminal sequence Phe-Xaa-Gly-Leu-Met-NH 2. At least three distinct receptors, termed NK1, NK 2 and NK3, have been proposed to mediate the biological actions of tachykinins in mammals (Buck et al., 1984; Lee et al., 1986; Regoli et al., 1989). Scyliorhinin I and scyliorhinin II are two peptides belonging to the tachykinin family which have been isolated from dogfish gut (Conlon et al., 1986). Scyliorhinin I is a linear decapeptide, while scyliorhinin II is a naturally occurring cyclic peptide. Scyliorhinin I and scyliorhinin II share a C-terminal amino acid sequence similar to that of the mammalian tachykinins neurokinin A and neurokinin B. Binding studies have shown that scyliorhinin II binds with highest affinity to NK 3 receptors, while scyliorhinin I possesses high affinity for NK 1 and NK e receptors, and a 50-250 times reduced affinity for N K 3 receptors (Buck and
* Corresponding author. Tel. 39-55-5680350, fax 39-55-5680419.
Krstenansky, 1987; Beaujouan et al., 1988). In particular, scyliorhinin I shows the same high affinity of substance P for peripheral or central NK 1 receptors of the rat. Likewise, scyliorhinin I binds to NK z receptors expressed by hamster and rat peripheral tissues with the same high affinity shown by neurokinin A (Buck and Krstenansky, 1987; Beaujouan et al., 1988). However, nothing is known about the biological effects of scyliorhinins, apart from their ability to induce contraction of the guinea-pig ileum longitudinal muscle (ConIon et al., 1986). The aim of this study was to assess the potency and selectivity of scyliorhinin I at NK1, NK 2 and NK 3 receptors expressed by guinea-pig, rabbit, hamster and rat, in several in vitro bioassays, and to assess the specificity of this interaction by means of receptorselective antagonists. In addition, we investigated the influence exerted by the amino acid residue in position 7 of the scyliorhinin I sequence on the selectivity of scyliorhinin I towards tachykinin receptors, This position, which is occupied by Tyr in scyliorhinin I, is the variable position in the C-terminal common region of mammalian and non-mammalian tachykinins. Previous studies have shown that certain substitutions at this position in mammalian tachykinins led to a decrease of
312 agonist potency, as observed for [Ala8]substance P (Couture et al., 1979) or [Ala7]neurokinin A (Rovero et al., 1989). N-Methylation of the Phe 8 residue in substance P or Val 7 in neurokinin B increased the selectivity of the methylated analogues for tachykinin NK 3 receptors (Lavielle et al., 1988) as did the introduction of Pro in position 7 of neurokinin B (Lavielle et al., 1988). In this study we substituted the Tyr residue with Val, which is present in position 7 of both neurokinin A and neurokinin B. We also introduced Phe and lie which are present in the sequence of substance P and of the non-mammalian tachykinin eledoisin, respectively. Furthermore the amino acids /3-(2-naphthyl)Ala, Phe(F), Phe(Cl), D-Phe(F) and MePhe were introduced in position 7 of scyliorhinin I to obtain the following peptides: [ValT]scyliorhinin I, [IleT]scylior hinin I, [fl-(2-naphthyl)-Ala7]scyliorhinin I, [Phe7]scyl iorhinin I, [D-Phe(F)7]scyliorhinin I, [Phe(F)7]scyliorhinin I, [Phe(C1)7]scyliorhinin I and [MePhe7]scyliorhinin I.
being added when the effect of the preceding one had reached a steady state. 2.2. Evaluation of data
The agonist activity of each test compound was expressed as pD 2 ( - l o g ECs0 or molar concentration of peptide producing 50% of maximal effect). Antagonist potency was evaluated in terms of pK B (negative logarithm of antagonist dissociation constant), which was estimated as the mean ( + S.E.M.) of the individual values obtained with the equation: pKB= log[agonistdose ratio - 1] - log[antagonist concentration] (Kenakin, 1987; Jenkinson, 1991). 2.3. Statistical analysis
The values in the text, tables or figures are expressed as means + S.E.M. Regression analysis of log concentration-effect curves was performed by the least-squares method, considering linear such curves between 20 and 80% of the maximal response.
2. Materials and methods
2. 4. Drugs 2.1. General
Male albino New Zealand rabbits (2.5-3.0 kg), male Syrian golden hamsters (100-120 g), male albino guinea-pigs (250-300 g) and male albino rats (Wistar strain, 300-350 g) were stunned and bled. Endothelium-denuded strips of rabbit pulmonary artery, rings of hamster trachea, strips of guinea-pig ileum longitudinal muscle and rat portal veins were excised and prepared for isometric tension recording in oxygenated (96% 0 2 and 4% CO 2) normal Krebs solution in 5-ml organ baths, as described previously (Maggi et al., 1990; Mastrangelo et al., 1986; Dion et al., 1987). The activity of peptides under study was assessed at NK 2 receptors of the isolated rabbit pulmonary artery and isolated hamster trachea, two preparations bearing pharmacologically different subtypes of this receptor, preliminary termed NK2A and NK2B, respectively (Maggi et al., 1991b). The activity at NK 1 receptors was studied in the guinea-pig ileum longitudinal muscle preparation (atropine and chlorpheniramine 1/~M, indomethacin 3 /xM in the bath). The activity at NK 3 receptors was evaluated in the rat portal vein. Responses to scyliorhinin I and scyliorhinin I analogues were compared to those produced by the tachykinin agonists neurokinin A (rabbit pulmonary artery and hamster trachea), substance P methylester (guinea-pig ileum) and arginine-neurokinin B ([Arg°]neurokinin B) (rat portal vein). Concentration-response curves for the agonists were obtained in a cumulative manner, each concentration
The drugs used were: substance P methylester and neurokinin A (Peninsula, St. Helens, UK), L 659,877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)] (C.R.B., Cambridge, UK), atropine (Serva, Heidelberg, Germany), indomethacin and clorpheniramine (Sigma, St. Louis, USA), FK 888 [(N-Me)indolyl-3-carboxy-(4R)-Hyp-2Nal-N,N-(methylbenzyl)amide] (synthesized at Menarini Sud Laboratories, Pomezia, Italy, by Dr. A. Sisto), arginine neurokinin B and MEN 10,376 (H-Asp-Tyr-DTrp-VaI-D-Trp-D-Trp-Lys-NH 2) (synthesized at Menarini Laboratories, Florence, Italy, by conventional solid phase methods), scyliorhinin I (H-Ala-Lys-Phe-AspLys-Phe-Tyr-Gly-Leu-Met-NH2), [Val7]scyliorhinin I, [IleT]scyliorhinin I, [/3-(2-naphthyl)-AlaV]scyliorhinin I, [PheT]scyliorhinin I, [D-Phe(F)7]scyliorhinin I, [Phe (F)7]scyliorhinin I, [Phe(Cl)7]scyliorhinin I and [MePheT]scyliorhinin I (synthesized at the Institute of Chemistry, University of Gdansk, Gdansk, Poland, by conventional solid phase methods).
3. Results
3.1. Effect of scyliorhinin I at N K 1, N K 2 and N K 3 receptors
Scyliorhinin I behaved as a full agonist at NK 1 and NK 2 receptors, showing the same potency of the reference peptides substance P methylester and neurokinin A, respectively. At NK 1 receptors of the guinea-pig
313
ileum longitudinal muscle preparation scyliorhinin I produced a concentration-dependent contractile response (pD 2 = 8.48 + 0.12; n = 8) (fig. 1, table 1) whose maximal response averaged 105 + 3% (n = 8) of that obtained with substance P methylester (pD 2 = 8.40 + 0.11; n = 8). At N K 2 receptors of the isolated rabbit pulmonary artery and isolated hamster trachea, scyliorhinin I produced concentration-dependent contractile responses (pD 2 = 8.33 _ 0.05 and 7.34 + 0.12, respectively; n = 8 each) (fig. 1, table 1). Maximal effects averaged 99 + 2 and 1 0 1 _ 4% (n = 8 each) of those obtained with neurokinin A (pD 2 = 8.20 + 0.1 and 7.33 + 0.1 respectively; n = 8 each) in the same preparations, respectively. At NK 3 receptors of the isolated rat portal vein scyliorhinin I displayed a much weaker agonist activity as compared to that shown at NK 1 and NK 2 receptors. In this bioassay the threshold concentration of scyliorhinin I to produce a contractile response was 0.1 /zM. The contraction produced by scyliorhinin I at 0.3 /xM averaged 55 + 8% (n = 4) of that produced by [Arg°]neurokinin B at a concentration (20 nM) which corresponds to the ECs0 of this agonist at NK 3 receptors in the rat portal vein (Regoli et al., 1987). Owing to the limited amount of peptide available, a full concentration-response curve for scyliorhinin I was not made for the rat portal vein, and its apparent affinity is reported as pD 2 < 6.5 (table 1). 3.2. Effect o f scyliorhinin I analogues at N K 1, N K 2 and N K 3 receptors
The scyliorhinin I analogues [fl-(2-naphthyl)AlaT]scyliorhinin I, [ValT]scyliorhinin I, [IleT]scyliorhinin I, [PheT]scyliorhinin I, [Phe(F)7]scyliorhinin I
and [Phe(Cl)7]scyliorhinin I behaved as full agonists at NK 1 and NK 2 receptors of the guinea-pig ileum, rabbit pulmonary artery and hamster trachea preparations (table 1). [/3-(2-naphthyl)-Ala7]Scyliorhinin I, [Val7] scyliorhinin I and [IleT]scyliorhinin I were from 3 to 25 times weaker than scyliorhinin I itself at NK 1 and NK 2 receptors, each one of these analogues showed the same relative potency as scyliorhinin I in the preparations tested (table 1). On the other hand, [Phe7] scyliorhinin I, [Phe(F)7]scyliorhinin I and [Phe(C1)7] scyliorhinin I were as active as scyliorhinin I at NK 1 receptors of the guinea-pig ileum, while they showed a 10-30 times reduced potency at NK 2 receptors expressed by rabbit or hamster preparations (table 1). The concentration-response curves for [fl-(2-naphthyl)Ala7]scyliorhinin I and [PheT]scyliorhinin I, compared with those for scyliorhinin I, in the guinea-pig ileum, rabbit pulmonary artery and hamster trachea, are shown in fig. 1. The other scyliorhinin I analogues, [D-Phe(F)7]scyliorhinin I and [MePheT]scyliorhinin I, were about three orders of magnitude weaker than scyliorhinin I in producing contraction at NK 1 receptors of the guineapig ileum (table 1). In addition, the maximal responses elicited by the latter analogues in the guinea-pig ileum were lower (72 + 6 and 58 __+5%, n = 6, respectively) than that produced by scyliorhinin I. At NK 2 receptors of the rabbit pulmonary artery, [D-Phe(F)7]scyliorhinin I and [MePheT]scyliorhinin I produced very slight or no response up to 3 /zM, respectively (table 1), while in the hamster trachea both of them were inactive up to 3 /zM. At NK 3 receptors of the rat portal vein [/3-(2-naphthyl)-AlaT]scyliorhinin I, [PheT]scyliorhinin I, [Phe(F)7]scyliorhinin I and [Phe(C1)7]scyliorhinin I behaved as scyliorhinin I, producing contractile responses (up to
TABLE 1 Agonist activity of several scyliorhinin I analogues at tachykinin NKI, NK 2 and NK 3 receptors. Each pD 2 ( - l o g ECso) value in the table is the mean±S.E.M, of 6-8 determinations. R.A.: apparent affinity of each test compound for tachykinin NK1 or NK 2 receptors, relative to that shown by scyliorhinin I, obtained by the ratio: (ECs0 test compound/ECs0 scyliorhinin I) x 100, GPI: guinea-pig ileum longitudinal muscle (atropine and chlorpheniramine 1/~M, indomethacin 3/zM in the bath). RPA: endotheliumdeprived rabbit pulmonary artery. HT: hamster trachea. RPV: rat portal vein. IN = inactive up to 3 ~M concentrations. Peptide
NK t receptor
NK 2 receptor
GPI
Scyliorhinin I [fl-(2-naphthyl)-AlaT]Scyliorhinin I [Val7]Scyliorhinin I [Ile7]Scyliorhinin I [PheT]Scyliorhinin I [Phe(F)7]Scyliorhinin I [Phe(Cl)7]Scyliorhinin I [D-Phe(F)7]Scyliorhinin I [MePheT]Scyliorhinin I
NK 3 receptor
RPA
HT
RPV
pD 2
R.A.
pD 2
R.A.
pD 2
R.A.
pD 2
8.48 + 0.12 8.05 + 0.16 7.19 ± 0.12 7.05 ± 0.21 8.54-t-0.21 8.56 :t: 0.18 8.64±0.14 5.33 ± 0.14 6.06 ± 0.23
100 37 5 4 115 120 144 0.07 0.4
8.33 ± 0.05 7.64 ± 0.20 7.08 ± 0.10 6.79:1:0.12 6.98±0.20 6.88 ± 0.20 7.33±0.12 < 5.5 IN
100 20 6 3 4 3 10 < 0.1
7.34 ± 0.12 6.62 + 0.12 5.97 ± 0.16 5.57 + 0.14 5.77+0.20 5.89 + 0.20 6.01 _+0.2 IN IN
100 19 4 2 3 4 5
< 6.5 < 6.5 <6 <6 < 6.5 < 6.5 < 6.5 IN IN
314
0.3 /xM) lower than that obtained by using [Arg°]neu rokinin B (20 nM) (table 1). [ValT]Scyliorhinin I and [Ile7]scyliorhinin I, up to 1 ~ M , were unable to produce responses higher than those elicited by [Arg°]neurokinin B (20 nM), while [D-Phe(F) 7] scyliorhinin I and [MePhe7]scyliorhinin I were inactive up to 3 / x M (table 1). 3.3. Effect of tachykinin receptor selective antagonists against scyliorhinin I at N K 1 and N K 2 receptors The tachykinin NK 1 receptor-selective antagonist FK 888 (30-100 nM) (Fuji et al., 1992) and the tachykinin NK 2 receptor-selective antagonists MEN 10,376 (30-100 nM) (Maggi et al., 1991a) and L 659,877 (30-100 nM) (McKnight et al., 1991) were used to block the contractile responses produced by scyliorhinin
1 20 0
-
11
•
10
'
"
9
•
8
-
,
"
7
'
-
6
•
•
5
4
I00"
TABLE 2 Antagonist activity of F K 888, M E N 10,376 and L 659,877 towards scyliorhinin I-induced responses at tachykinin N K 1 and N K 2 receptors.
Each value in the table is mean pK B (negative logarithmof antagonist dissociationconstant)+ S.E.M. of 4 determinations.GPI: guineapig ileum longitudinalmuscle (atropine and chlorpheniramine 1 ~M, indomethacin3/xM in the bath). RPA: endothelium-deprivedrabbit pulmonary artery. HT: hamster trachea. Antagonist
F K 888 M E N 10,376 L 659,877
N K 1 receptor
N K 2 receptors
GPI
RPA
HT
8.83±0.2 8.17±0.3 8.39±0.3
I in the guinea-pig ileum, rabbit pulmonary artery and hamster trachea, respectively (15-min incubations with test antagonists in each preparation). All antagonists produced a parallel rightward shift of the concentration-response curve for scyliorhinin I in the three preparations tested. FK 888 antagonized scyliorhinin I in the guinea-pig ileum with a potency (pK B = 8.83; table 2) similar to that observed against substance P methylester (pK a = 8.81). MEN 10,376 blocked scyliorhinin I-induced effects in the rabbit pulmonary artery (pK B = 8.17; table 2) with about the same potency shown against neurokinin A (pK a = 8.08). In the hamster trachea L 659,877 was slightly more potent against scyliorhinin I (pK B = 8.39; table 2) than against neurokinin A (pK B = 7.92).
t.d Z
80-
4. Discussion
60 40 o
20 0
11
I0
9
7
6
5
4
11
6 5 10 9 8 - LOG M CONCENTRATION
4
lOO o~ Z
6O 40 20 0
Fig. 1. Concentration-response curves for scyliorhinin I (©), [/3-(2naphthyl)-Ala7]scyliorhinin I (t~) and [Phe7]scyliorhinin I ( • ) in the guinea-pig ileum longitudinal muscle preparation (upper panel), isolated rabbit pulmonary artery (middle panel) and isolated hamster trachea (lower panel). Each value is the m e a n of 6 - 8 determinations, and vertical lines show S.E.M.
The present results show that the non-mammalian peptide scyliorhinin I acts as a full receptor agonist at peripheral tachykinin NK~ receptors of the guinea-pig ileum, and at NK 2 receptors of the rabbit pulmonary artery and hamster trachea. Furthermore, scyliorhinin I displays high potency in producing agonist responses in the above preparations, comparable to that possessed by substance P methylester and neurokinin A at NK 1 and NK 2 receptors, respectively. In contrast, scyliorhinin I shows lower agonist potency at NK 3 receptor of the rat portal vein than that shown by the NK 3 receptor-selective agonist [Arg°]neurokinin B. Evidence for the interaction of scyliorhinin I with tachykinin NK 1 and NK z receptors has been provided by using potent and selective tachykinin receptor antagonists. In fact, the agonist effects produced by scyliorhinin I were blocked by FK 888 (NK 1 receptorselective antagonist; Fuji et al., 1992) in the guinea-pig ileum, by MEN 10,376 (NK 2 receptor-selective antagonist; Maggi et al., 1991a) in the rabbit pulmonary artery and by L 659,877 (NK 2 receptor-selective antagonist;
315
McKnight et al., 1991; Patacchini et al., 1991) in the hamster trachea. Furthermore, the potencies shown by the above antagonists in blocking scyliorhinin I-induced effects are very similar to those obtained against other NK 1 and NK 2 receptor-selective agonists in the same organs. Our results, obtained in functional bioassays, are in keeping with those reported previously in binding studies (Buck and Krstenansky, 1987; Beaujouan et al., 1988). In fact, the latter authors reported that scyliorhinin I possesses high affinity for NK 1 and NK 2 receptors, and 50-250 times lower affinity for NK 3 receptors. For what concerns scyliorhinin I analogues, our aim was to determine the effect of various substitutions in position 7 of the scyliorhinin I molecule on selectivity towards tachykinin receptors. First of all we tried to introduce Val, lie and Phe residues, which are present in the same position of neurokinin A and B, eleidosin and substance P sequences, respectively, lie is also present in the neurokinin A analogue [Leu3,Ile7]neurokinin A, which has been recently isolated from extracts of the brain and intestine of the European frog Rana ridibunda (Lovas et al., 1993) and shown to possess high selectivity for NK 1 receptors. The introduction of the aliphatic residues Val and lie in position 7 of scyliorhinin I did not influence the selectivity of this peptide, as [Val7]scyliorhinin I and [Ile7]scyliorhinin I showed a decrease in agonist potency of the same order of magnitude in all preparations tested. In contrast, [Phe7]scyliorhinin I maintained the same potency as scyliorhinin I for the NK~ receptor, while it was less potent at NK 2 receptors, being 22 and 37 times weaker than scyliorhinin I in the rabbit pulmonary artery and hamster trachea, respectively. This result shows that the introduction of Phe in scyliorhinin I provides the molecule with higher selectivity for the NK 1 receptor than scyliorhinin I itself. Tyr residue, which is present in position 7 of scyliorhinin I, contains an aromatic side chain substituted in the para position with an hydroxyl group. In this study we aimed to distinguish the role of the above two chemical functions in providing selectivity for tachykinin receptors to scyliorhinin I. Therefore we introduced in position 7 of scyliorhinin 1 a large aromatic residue, /3-(2-naphthyl)-Ala, and two analogues of Phe substituted at the para position with halogen atoms of different size and electronegativity, yielding [fl-(2-naphthyl)-Ala7]scyliorhinin I, [Phe(F)7] scyliorhinin I and [Phe(C1)7]scyliorhinin I. The presence of /3-(2-naphthyl)-Ala produced a general decrease in agonist potency, while [Phe(F)7]scyliorhinin I and [Phe(Cl)7]scyliorhinin I showed the same pattern of selectivity as [Phe7]scyliorhinin I. These results suggest that the electronic characteristics of the aromatic side chain play a minor role in providing the Phe analogues with potency and selectivity for tachykinin
receptors, whereas the size of the aromatic ring is more important for this class of peptides. Inversion of the chirality of Phe, with ([Phe(F)7]scyl iorhinin I being used to obtain [D-Phe(F)7]scyliorhinin I, dramatically decreased the potency of the latter analogue at all tachykinin receptors. This result is in keeping with that obtained with [D-PheS]substance P in the guinea-pig ileum preparation by Duplaa et al. (1991). The N-methylated Phe residue was introduced in scyliorhinin I to induce a strong conformational change in the molecule. This substitution, which has been shown to increase the duration of action of substance P (Eison et al., 1982), caused a marked drop of agonist potency at tachykinin receptors in all bioassays. In conclusion, our experiments provide the first functional evidence for activation of mammalian tachykinin receptors by scyliorhinin I. The present results show that scyliorhinin I is a highly potent agonist at both NK 1 and NK 2 receptors, thus confirming the high affinity for these receptors shown by scyliorhinin I in binding experiments. In addition we have developed three analogues, [Phe7]scyliorhinin I, [Phe(F)7]scyl iorhinin I and [Phe(Cl)7]scyliorhinin I, which are NK I receptor selective agonists. In view of the recently described pharmacological heterogeneity of the tachykinin NK 1 receptor (Petitet et al., 1992), these novel ligands could be useful as tools for investigation of NK 1 receptor distribution and pharmacology.
Acknowledgements This work was partially supported by the Polish Scientific Research Council-KBN (research grant 2 P 303 130 04). We want to thank Ms. Carla Sezzatini for excellent technical assistance.
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