Effects of selective tachykinin-receptor antagonists on tachykinin-induced airway mucus secretion in the rat

Neuropeptides (1999) 33 (1), 55–61 © 1999 Harcourt Brace & Co. Ltd

Effects of selective tachykininreceptor antagonists on tachykinininduced airway mucus secretion in the rat U. Wagne r, H.-C. Fehmann, D. Bredenbröke r, D. Klübe r, A. Lange,.Pvon Wichert Department of Internal Medicine, Philipps-Universit y, Marburg/Lahn, Germany

Summar yTachykinins like substance P (SP), neurokinin A (NKA), neurokinin B (NKB) di fferentially stimulate airway mucus secretion with the following rank order of potency in rat trachea: SP>NKA>NKB. These di fferential actions are , NK2 and NK3. In this study we most likely due to di fferent affinities to the tachykinin receptors, termed neurokinin (NK) 1 characterized the receptor subtype responsible for the differential secretagogue effects in rat trachea by means of selective receptor antagonists and receptor agonists. SR 140333 [NK1-antagonist] completely inhibited SP action (283,2 ±921, 12%→84,53±4, 09%; P<0,01) and P<0,01) and NKB significantly reduced the e ffects of NKA (179,08±17,34%→118,86±6,7%; (171,89±5,75%→109,5±4,11%; P<0,01). SR 48968 [NK2-antagonist] did not a ffect SP action, but reduced the effects of NKA and NKB. SR 142801 [NK3-antagonist] did not change any e ffect of SP, NKA or NKB. [Sar9]SP (NK1-agonist) caused strong dose-dependent secretagogue ffects e similar to SP, [ βAla8]NKA (NK2-agonist) showed only slight and 7 [Pro ]NKB (NK3-agonist) no effects. The present data suggest that the secretagogue fefects elicited by tachykinins in rat trachea are mediated via NK 1 receptors.

INTRODUCTION Airway mucus secretion and smooth muscle tone are controlledby complex networks of neural, neuroendocrine and paracrine systems.1,2 Among these chame nisms the non -adrenergic non -cholinergic nervous system NANC) ( plays an important role representing a neuronal network. It consists of heterogenous neural structures communicating by a variety of peptidergic mediators.This heterogenous collection of fibers has diverse connections to the central nervous system and

Received 18 November 1998 Accepted 10 February 1999 Correspondence to : Ulrich Wagner MD, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Philipps-Universit y, Baldingerstr aβe, D-35033 Marburg/Lahn, German y. Tel: 49 6421 28 3691; Fax: 49 6421 28 8987; E-mail: wagnerul@maile r.uni-marburg.de

overlapping connections to the upper ower and respiral tory tract and regulatesnyma airway functions via afferent and ferent ef pathways. 3 Among other neuropeptides of the NANC systems of animal and human airways ch taykinins are of outstanding importance. Tachykinins (TKs) resemble a group of peptides with a common -terminal C sequence of amino acids X- (PheGly-Leu-Met), but dif ferent aminoterminal sequences. SP, neurokinin NKA) A ( and neurokininNKB) B ( are mem2,4 bers of this peptide ly fami . SP stimulates mucus secretion from submucosal glands in airways more ly potent 5–9 than the otherchta ykinins NKA andNKB. Moreover, we found inhibitory fects ef of Galanin and somatostatin 8,9 on tachykinin -induced mucus secretion. The tachykinins mediate their fects efby three distinct receptors:NK 1, NK 2 andNK 3 receptors. These receptors recognize the ta chykinins withferent dif affinities. They belong to the superfami ly of G protein -coupled receptors with 55

56 Wagner et al.

seven putative transmembrane spanning segments. Their activation results in a rise in cytosolic calcium ion (Ca2+) concentration mediated by the phosphatidylinositol pathway.10 In the past few years a number of potent and selective antagonists has been developed for NK1, NK2 and NK3 receptors.11 Concerning the secretagogue effects in rat trachea we found the following rank order of potency: SP>NKA>NKB. These differential potencies are most likely due to the involvement of different affinities to tachykinin receptors. Therefore, the purpose of this study was to determine the receptor subtype responsible for the differential actions of tachykinins on macromolecule secretion in rat trachea by means of a panel of selective NK1, NK2 and NK3 receptor antagonists and receptor agonists. MATERIAL AND METHODS Materials Rat substance P, NKA; NKB and the selective agonists [Sar9]SP [NK1], [βAla8]NKA [NK2] and [Pro7]NKB [NK3] were purchased from Bachem (Heidelberg, Germany). The three non-peptid NK receptor antagonists SR 140333, SR 48968 and SR 142801 were gifts from Dr X. Emonds-Alt and Dr. J.C. Breliere, Sanofi research centre, Montpellier, France. Animals Male Sprague-Dawley rats (Zentralinstitut für Versuchstierzucht, Hannover, Germany) with an average body weight of 400 g were kept in a light and temperature controlled room. They had free access to water and a rat standard diet (Altromin, Lage, Germany). Studies on tracheal mucus secretion Male Sprague-Dawley rats with an average body weight of 400 g were anaesthesized with pentobarbital (Nembutal) using doses of 70 mg/kg intraperitoneally and sacrificed by excision of the tracheae (cranially from the larynx, caudally to the bifurcation) through a ventral collar midline incision and median sternotomy. The trachea was transferred to an organ bath filled with Medium M-199 in Earle’s balanced salt solution (Gibco, Eggenstein, Germany), equilibrated with 95% oxygen and 5% carbon dioxide. The trachea was opened along the posterior membrane and mounted between the two halves of an Ussing chamber. The pH was adjusted to 7.41 at 37°C. To the solution bathing the submucosal side 50 µCi Na235SO4 (Amersham, Braunschweig, Germany) were added and allowed to equilibrate with the tissues for the duration of the experiment. After 2 h, release of bound 35SO4 reaches steady state rates of secretion Neuropeptides (1999) 33(1), 55–61

(=basal secretion) to the luminal (=mucosal) side. The perfusate bathing the luminal side (7 ml, according to the volume of the perfusion device) was collected at 15 min intervals. The perfusate portions of the luminal side were collected in cellulose dialysis tubing (Serva, Heidelberg, Germany; 12,000–14,000 D molecular mass cutoff) which retains secreted molecules with molecular masses over 12.000–14.000 Daltons and dialyzed against distilled water containing unlabeled SO4 to remove unincorporated 35 SO4, and sodium azide (10 mg/l) to prevent bacterial degradation. Dialysis was complete when the radioactivity of the dialysis fluid 3 h after the last change was equal to the radioactivity of water used for dialysis. The radioactivity of the samples was then determined using a szintillation counter. The count rates of the labeled macromolecules reflect the mucus secretion rates. This Ussing-chamber-method separating submucosal and mucosal solutions is well established to measure macromolecular secretion of glands and epithelium of tracheal preparations. It offers the advantage that the radiolabelled precursor can equilibrate with the submucosal side of the tissue, during which time materials secreted into the lumen can be collected. The method depends on an intact epithelial diffusion barrier, which effectively avoids free diffusion of the precursor. According to our own studies and literature, in the choice of suitable precursors sulfate being present on many of the carbohydrate side chains as a terminal residue has great advantages over other precursors (e.g. labelled amino acids or sugars), especially because it is not metabolized.8,9,12–14 Thus, the output of labelled mucins can be assessed. The used dialysis with distilled water containing excess unlabeled SO4 effectively removes ionically bound unincorporated 35 SO4, so that an unintentional study of a mere SO4-transport can be excluded. Study design Previous studies characterized the effects of SP, NKA and NKB on mucus secretion in rat trachea.8,9 SP, NKA and NKB showed dose-dependent secretagogue effects with maximum stimulation at 1 µM. 1) In different sets of experiments the effects of the selective agonists [Sar9]SP [NK1], [βAla8]NKA [NK2] and [Pro7]NKB [NK3] (each 1 nM–1 µM) were tested. 1.1) Subsequent to the determination of basal airway mucus secretion (four fractions every 15 min) [Sar9]SP (n=5) was applied to the submucosal side in rising concentrations (1 nM–1 µM) at 60 min intervals. At the end of each experiment luminal stimulation with 1 mM acetylcholine was performed as quality control of the bioassay system. 1.2) [βAla8]NKA (n=5) and 1.3.) [Pro7]NKB (n=5): The same procedure as mentioned above was followed. © 1999 Harcourt Brace & Co. Ltd

Effects of selective tachykinin-receptor antagonists 57

Because the maximum effects of SP, NKA and NKB as well as the selective agonists [Sar9]SP, [βAla8]NKA and [Pro7]NKB were seen at 1 µM this concentration was used in all following experiments. At the end of each experiment acetylcholine (ACh) 1 mM was added to the luminal side to assure viability of the organ culture (maximum secretion). 2) Following four collections for the assessment of basal secretion SP 1 µM was given to the luminal side alone (n=6). Subsequent to an interval of four additional collections the NK-1 antagonist SR 140333 1 µM was given to the luminal side. Following four further collections SP 1 µM/SR 140333 1 µM were given to the luminal side. At the end (after four additional collections) ACh 1 mM was added to the luminal side to assure viability. 3) Using the same design as in 2) SP and the NK2receptor antagonist SR 48968 (1 µM) (n=5) were tested. 4) Using the same design as in 2) SP and the NK3receptor antagonist SR 142801 (1 µM) (n=6) were tested. 5) Following four collections for the assessment of basal secretion NKA 1 µM was given to the luminal side alone (n=5). Subsequent to an interval of four additional collections the NK-1 antagonist SR 140333 1 µM was given to the luminal side. Following four further collections NKA 1 µM/SR 140333 1 µM were given to the luminal side. At the end (after four additional collections) ACh 1 mM was added to the luminal side to assure viability. 6) Using the same design as in 5) NKA and the NK2receptor antagonist SR 48968 (1 µM) (n=5) were tested.

7) Using the same design as in 5) NKA and the NK3receptor antagonist SR 142801 (1 µM) (n=8) were tested. 8) Following four collections for the assessment of basal secretion NKB 1 µM was given to the luminal side alone (n=8). Subsequent to an interval of four additional collections the NK-1 antagonist SR 140333 1 µM was given to the luminal side. Following four further collections NKB 1 µM/SR 140333 1 µM were given to the luminal side. At the end (after four additional collections) ACh 1 mM was added to the luminal side to assure viability. 9) Using the same design as in 8) NKB and the NK2receptor antagonist SR 48968 (1 µM) (n=8) were tested. 10) Using the same design as in 8) NKB and the NK3receptor antagonist SR 142801 (1 µM) (n=8) were tested. Statistics The effects of the drugs are presented relatively to the basal secretion (as a percentage of basal secretion). Scattering of the values is expressed as standard error of the mean (SEM). Statistical analysis of the data was performed by use of student’s t-test for unpaired samples. Table 1 shows published affinities of the used peptides as tachykinin receptors. RESULTS First, we characterized the effects of SP, NKA and NKB on rat tracheal mucus secretion.8,9 These tachykinins displayed dose-dependent secretagogue effects as shown in Table 2 and Figures 1A & B.

Table 1 Affinities of SP, NKA, NKB, the tachykinin receptor agonists [Sar9]SP, [β-Ala]NKA, [Pro7]NKB and the tachykinin receptor antagonists SR 140333, SR 48968, SR 142801 to NK-receptors Peptide

NK1-Recpetor

SP NKA NKB [Sar9]SP

EC50 100 pM in guinea pig ileum15

[β-Ala]NKA [Pro7]NKB SR 140333

NK3 Receptor

EC50 1,2 nM in guinea pig ileum15 pD2 (rabbit iris sphincter) 9,75±0,0925 EC50 3,8 nM16 EC50 83 pM in guinea pig ileum15 EC50 1,7 µM in mucus secretion of ferret trachea17 EC50 10 nM in guinea pig ileum15 pD2 (rabbit iris sphincter) 8,56±0,0925 Ki 0,74 nM16 Ki: 0,02 nM in rat cortical member., 0,01 nM in IM9 human cells18 IC50 of Sar-SP antagonism: 1,6 nM16 pD2 9,65–10,1619 pA2 in rat 7,6–8,2 and pIC50 8,4–9,520 pKB (anti-bronchoconstrictor activity in guinea pigs) 9,57±0,221 pA2 in human bronchus contraction 9,40±0,1922 pA2 in tissues of the rat 9,4–9,623 pA2 in tissues of rabbit, hamster and rat 8,3–9,624

SR 48968

SR 142801

NK2-Receptor

Inactive15

© 1999 Harcourt Brace & Co. Ltd

EC50> 10 µM in rat NK3 receptor26

pA2 in rat portal vein 7,0; Ki 15 nM27 Ki 11±0,5 nM in rat NK3 receptors26 Ki 0,21±0,03 nM in human NK3 receptor28 pKB 7,49 rat portal vein29 pKB 3,2 nM, guinea pig ileum30

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58 Wagner et al.

Table 2

Effects of SP, NkA NKB [Sar9]SP, [βAla8]NKA and [Pro7]NKB on mucus secretion from isolated rat trachea [% of basal] [Sar9]SP

1 nM 10 nM 100 nM 1 µM

Fig. 1A

[βAla8]NKA

118±11 141±6 157±14 262±28

96±9 108±8 126±6 99±8

[Pro7]NKB

SP

NKA

98±6 115±8 97±5 110±17

120±7 154±19 223±36 276±16

106±3 116±2 124±5 185±9

Effects of tachykinins on mucus secretion.

Then we studied the actions of specific tachykinin receptor agonists on airway mucus secretion. [Sar9]SP, a NK1 receptor agonist stimulated macromolecule output comparable to SP. [βAla8]NKA, a NK2 receptor agonist, showed a weak effect; increasing mucus secretion only slightly at 100 nM (126±6) and being ineffective at higher concentrations. [Pro7]NKB, a NK3 receptor agonist, failed to elicit statistically significant changes in native tracheae (Table 2, Figs 1A & B). Finally, we applied subtype-specific tachykinin receptor-antagonists. SR 140333, an antagonist of NK1 receptor (Fig. 2) completely inhibited the stimulatory action of SP (283,29±21,12%→84,53±4,09%; P<0,01) and also strongly reduced the effects of NKA (179,08±17,34%→118,86±6,7%; P<0,01) and NKB (171,89±5,75%→109,50±4,11%; P<0,01). SR 48968, an NK2 receptor antagonist (Fig. 3), significantly reduced the effects of NKA (179,08±17,34%→113,55±12,39%; P<0,01) and weakly that of NKB (171,89±5,75%→135,80±21,39%; P=0,066), while the action of SP (283,29±21,12%→280,12±21,18) remained unchanged. Antagonism of the NK3 receptor by SR 142801 (Fig. 4) did not influence the effects of SP (283,29±21,12%→288,58±27,54) or NKA (179,08± 17,34%→187,58±27,81%). SR 142801 only slightly reduced the effects of NKB (171,89± 5,75%→142,35±9,02%; P=0,05). None of the NK receptor antagonists tested (SR 140333, SR 48968 and SR 142801) had any stimulatory Neuropeptides (1999) 33(1), 55–61

Fig. 1B

NKB 103±2 104±3 120±7 175±14

Effects of NK receptor agonists on mucus secretion.

or inhibitory effect on basal mucus secretion when given alone. DISCUSSION Tachykinins (TKs) are a family of peptides which share in common the C-terminal sequence Phe-X-Gly-Leu-MetNH2. They have been recognized as neurotransmitters in mammals and are widely distributed in both the central and peripheral nervous system. They are released from sensory nerves and exert a variety of biological actions through activation of specific membrane receptors termed neurokinin[NK]1, NK2 and NK3 receptors.4 According to the concept of neurogenic inflammation, a process assumed to play an important role in the pathophysiology of asthma, much attention is paid to a better understanding of these actions in the airways.31 Tachykinins are potent stimulators of airway mucus secretion. As we demonstrated previously the rank order of potency is SP > NKA > NKB.8,9 These findings are consistent with the results of other authors, who found that SP enhances submucosal gland secretion in animal and human airways.3,7,31 The NK1 receptor is operationally defined as the mediator of the biological activities encoded by the C-terminal sequence of TKs for which SP is a more potent agonist than NKA od NKB. The receptor has been isolated from rat brain and salivary glands, from human IM9 lymphoblast cell line and lung, from guinea pig uterus © 1999 Harcourt Brace & Co. Ltd

Effects of selective tachykinin-receptor antagonists 59

Fig. 2

Effects of NK1 receptor-antagonist.

Fig. 3

Effects of NK2 receptor-antagonist.

Fig. 4

Effects of NK3 receptor-antagonist.

and mouse genoma. Two identical receptors have been cloned from human tissues. The cloned human and rat NK1 receptors show a high degree (about 95%) of homology: only 21 of 407 amino acid residues differ between the two species with the majority of variant positions being localized at the N- and C-terminal ends of the © 1999 Harcourt Brace & Co. Ltd

receptor protein. Because of its wide distrubution in mammalian tissues the NK1 receptor mediates effects like vasodilation, increase of vascular permeability stimulation of salivary and airway secretion and contraction of smooth muscles.24 The NK2 receptor is defined as the mediator of the biological actions encoded by the C-terminal sequence of TKs for which NKA is a more potent agonist than SP or NKB. NK2 receptors are widely distributed in the peripheral nervous system, especially in the smooth muscle of the respiratory, gastrointestinal and urinary tract, where they mediate the spasmogenic action of endogenous TKs released by nerve stimulation. Besides, they are involved in the facilitation of transmitter release and excitation as well as stimulation of certain cells like guinea pig alveolar macrophages.24 The NK3 receptor is defined as the mediator of those biological actions encoded by the C-terminal sequence of TKs for which NKB is a more potent agonist than NKA or SP. The NK3 receptor has been cloned from rat and human species. Both receptors show 88% identity with seven variant positions.32 In contrast to NK1 and NK2 receptors the NK3 receptors are abundantly present in central nervous tissue, whereas they are poorely expressed in peripheral tissues. The kind of expression in airways seems unclear thus far. The focus of the present study was a detailed characterization of the receptors mediating these secretagogue effects. We can draw several conclusions from our data: this study repeats our previous data demonstrating potent stimulatory effects of the tachykinins on airway mucus secretion. We suggest that SP acts through the NK1 receptor. NKA and NKB use both NK1 and NK2 receptors. We do not find a role for the NK3 receptor in mediating airway mucus secretion. Indeed, the expression of the NK1 and NK2 receptors in the respiratory tract has been shown before, whereas the expression of NK3 receptors remains unclear. Different functional tests investigated the involvement of tachykinins in airway dysfunctions.33 TKs are obviously involved in airway hyperresponsiveness with a predominating role of NK2 receptors, for NK2 receptorantagonists were able to prevent antigen-induced airway hyperresponsiveness in the guinea-pig, whereas the NK1antagonist was without effect concerning microvascular leakage NK1 receptors seem to be mainly involved as: it was shown that SR 140333 markedly reduced SP-enhanced potentiation of microvascular leakage induced by histamine, whereas SR 48968 did not.34 In guinea-pigs, however, SR 48968 inhibited NKA-induced microvascular leakage, so that, in this species a role for NK2 receptors cannot be excluded.35 An involvement of TKs seems likely with cough. Cough induced by capsaicin in guinea pig could be antagonized by SR 48968 Neuropeptides (1999) 33(1), 55–61

60 Wagner et al.

approximately 150 times stronger than by codeine, whereby the effect of SR 48968 could not be antagonized by naloxone.36 Regarding the effect of NK1 antagonists results are unclear. Most studies could not show inhibitory actions so that the NK1 receptor seems to play minor or neglegable roles in this regard.37 Applying to the actual view data from the literature suggest a preponderating role of NK1 versus NK2 receptors mediating airway hyperresponsiveness in guinea pigs.33 On the other hand, only NK2 receptors seem to be responsible for he mediation of bronchoconstriction. Moreover, in allergic rhinitis, nasal obstruction is mainly mediated by NK1 receptors, whereas albumin leakage and recruitment of inflammatory cells probably involve NK1 and NK2 receptors.38 Additional recent findings, however, demonstrated that the NK3 receptor antagonist SR 142801 markedly reduced the bronchial hyperresponsiveness and increased microvascular leakage after exposure of guinea-pigs to SP.39 With regard to mucus secretion in rat airways, our own data clearly demonstrates that the secretagogue effects of TKs are mediated via NK1 receptors, because the NK1 receptor antagonist SR 140333 completely inhibited the effects of SP, NKA and NKB. An involvement of NK2 receptors seems unlikely, because the NK2 receptor antagonist SR 48968 did not affect the action of SP and because the NK2 receptor agonist [βAla8]NKA did not elicit significant effects. The NK3 receptor antagonist SR 142801 was also without any remarkable effect indicating that the NK3 receptor is not involved in mediating TK-induced secretagogue effects. This suggestion was also supported by the use of NK receptor specific agonists. Thus, [Sar9]SP (NK1-agonist) mimicked the dose-response correlation of SP. [βAla8]NKA, an NK2 agonist, failed to elicit remarkable effects in contrast to NKA so that the latter is supposed to act via NK1 agonistic side-effects. The NK3 receptor agonist [Pro 7]NKB as well as the NK3 receptor antagonist SR 142801 produced no major effects in favour of the hypothesis that the NK3 receptor is not involved in the mediation of secretagogue effects. In conclusion, these data confirm an important role of the tachykinins in airway mucus secretion. Specific NK1 receptor antagonists could be useful in the treatment of respiratory disorders with mucus hypersecretion.

ACKNOWLEDGEMENTS This study was supported by the Deutsche Forschungsgemeinschaft, Wa 844/2–1 and Wa 844/3–1. We would like to thank Dr X. Emonds-Alt and Dr J.C. Breliere, Sanofi research centre, Montpellier, France, for

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kindly providing us with the NK receptor antagonists and Heike Priebe for her invaluable technical assistance. REFERENCES 1. Wagner U, Fehmann H-C, Bredenbröker D et al. Amylin stimulates airway secretion in the rat. Res Exp Med 1993; 193: 347–352. 2. Piedimonte G. Tachykinin petides, receptors, and peptidases in airway disease. Experimental Lung Research 1995; 21: 809–834. 3. Baraniuk JN, Kaliner MA. Neuropeptides in the upper and lower respiratory tract. Immunology and Allergy Clinics of North America 1990; 10: 383–407. 4. Casale TB. Neuropeptides and the lung. J Allergy Clin Immunol 1991; 88: 1–16. 5. Coles SJ, Neill KH, Reid LM. Potent stimulation of glycoprotein secretion in canine trachea by substance P. J Appl Physiol 1984; 57: 1323–1327. 6. Rogers DF, Aursudkij B, Barnes PJ. Effects of TK’s on mucus secretion in human bronchi in vitro. Eur J Pharmacol 1989; 174: 283–286. 7. Gentry S Tachykinin receptors mediating airway macromolecular secretion. Life sciences 1991; 48: 1609–1618. 8. Wagner U, Fehmann B, Bredenbröker D et al. Galanin and somatostatin inhibition of substance P-induced airway mucus secretion in the rat. Neuropeptides 1995; 28: 59–64. 9. Wagner U, Fehmann B, Bredenbröker D, Yu F, Barth PJ, von Wichert P. Galanin and somatostatin inhibition of neurokinin A and B induced airway mucus secretion in the rat. Life Sciences 1995; 57: 283–289. 10. Guard S, Watson SP. Tachykinin receptor types: classification and membrane signalling mechanisms. Neurochem Int 1991; 18: 149–165. 11. Maggi CA. The mammalian tachykinin receptors. Gen Pharmac 1995; 26: 911–944. 12. Borson DB, Gashi AA, Nadel JA. Methods for studying secretions from airways. In: Methods in bronchial mucology. Braga, PC, Allegra, L. eds 1st ed. New York: Raven Press. 1988: 303. 13. Wagner U, Fehmann H-C, Göke B et al. CGRP stimulates airway mucus secretion in rats exposed to NO2. Eur Respir J 1993; 6 (Suppl. 17): 473s. 14. Wagner U, von Wichert P. Control of mucus secretion in airways. Respiration 1991; 58: 1–8. 15. Maggi CA, Patacchini R, Meini S et al. Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea pig ileum and proximal colon. Br J Pharmacol 1994; 112: 150–160. 16. Oury-Donat F, Levevre LA, Thurneyssen O et al. SR 140333, a novel, selective, and potent nonpeptide antagonist of the NK1 tachykinin receptor: characterization on the U373MG cell line. J Neurochem 1994; 62: 1399–1407. 17. Meini S, Mak JC, Rohde JA, Rogers DF. Tachykinin control of ferret airways: mucus secretion, bronchoconstriction and receptor mapping. Neuropeptides 1993; 24: 81–89. 18. Jung M, Calassi R, Maruani J et al. Neuropharmacological characterization of SR 140333, a nonpeptide antagonist of NK1 receptors. Neuropharmacology 1994; 33: 167–179. 19. Emonds-Alt X, Doutremepuich JD, Heaulme M, et al. In vitro and in vivo biological activities of SR 140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol 1993; 250: 403–413.

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