Functional Selectivity Revealed by N-Methylation Scanning of Human Urotensin II and Related Peptides.

In accordance with their common but also divergent physiological actions, human urotensin II (1) and urotensin II-related peptide (2) could stabilize specific urotensin II receptor (UTR) conformations, thereby activating different signaling pathways, a feature referred to as biased agonism or functional selectivity. Sequential N-methylation of the amides in the conserved core sequence of 1, 2, and fragment U-II4-11 (3) shed light on structural requirements involved in their functional selectivity. Thus, 18 N-methylated UTR ligands were synthesized and their biological profiles evaluated using in vitro competition binding assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological activity diverged from that of the parent structures contingent on the location of amide methylation, indicating relevant hydrogen-bond interactions for the function of the endogenous peptides. Conformational analysis of selected N-methyl analogs indicated the importance of specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.

Structure-Activity Study of the Peptides P5U and Urantide by the Development of Analogues Containing Uncoded Amino Acids at Position†9.

Previous modifications of the peptide sequence of human urotensin-II (U-II) led to the identification of two well-known ligands: P5U and urantide. These derivatives are considered to be the most representative agonist and antagonist, respectively, at the human urotensin receptor (UT). Optimization of P5U and urantide was carried out to stabilize specific conformations that may suggest new elements for discriminating agonist versus antagonist activity. We studied novel derivatives containing uncoded amino acids. In particular, the Tyr(9) residue of both P5U and urantide was replaced with nonaromatic hydrophobic bulky residues, as well as conformationally constrained aromatic moieties to generate eight novel derivatives. These analogues further contributed to determining the influence of such residues on binding affinity for and biological activity at UT. One of these eight peptides was also investigated by NMR spectroscopy and docking studies owing to its peculiar conformational properties and mode of interaction with UT. This structure-activity study is aimed at a more thorough examination of the role of tyrosine in modulating the agonism/antagonism of human U-II.

Inner and outer portions of colonic circular muscle: ultrastructural and immunohistochemical changes in rat chronically treated with otilonium bromide.

Rat colonic circular muscle, main target of otilonium bromide (OB) spasmolytic activity, is subdivided in an inner and outer portion. Since the inner one is particularly rich in organelles involved in calcium availability (caveolae, smooth endoplasmic reticulum, mitochondria), the expression of specific markers (Caveolin-1, eNOS, calreticulin, calsequestrin) in comparison with the outer portion was investigated. The possible changes of these organelles and related markers, and of muscarinic receptors (Mr2) were then studied after OB chronic exposition. Rats were treated with 2-20 mg/kg/OB for 10 or 30 days. Proximal colon was processed by electron microscopy, immunohistochemistry, and western blot. In colon strips the stimulated contractility response to muscarinic agonist was investigated. The inner portion showed a higher expression of Caveolin-1 and Mr2, but not of eNOS, calreticulin and calsequestrin, compared to the outer portion. Chronic OB treatment caused similar ultrastructural and immunohistochemical changes in both portions. Organelles and some related markers were increased at 10 days; Mr2 expression and muscle contractility induced by methacholine was increased at 30 days. The present findings: 1) provide new information on the immunohistochemical properties of the inner portion of the circular layer that are in favour of a role it might play in colonic motility distinct from that of the outer portion; 2) demonstrate that chronically administered OB interferes with cell structures and molecules responsible for calcium handling and storage, and modifies cholinergic transmission. In conclusion, chronic OB administration in the colonic circular muscle layer directly interacts with the organelles and molecules calcium-related and with the Mr2.

Lead optimization of P5U and urantide: discovery of novel potent ligands at the urotensin-II receptor.

We have optimized 1 (P5U) and urantide, two important ligands at the h-UT receptor, designing several analogues by the exchange of the Tyr9 residue with different unnatural aromatic amino acids. This study allowed us to discover novel ligands with improved activity. In particular, the replacement of the Tyr9 residue by (pCN)Phe or (pNO2)Phe within the urantide sequence led to compounds 13 (UPG-83) and 15 (UPG-95), respectively, which showed pure antagonist activity toward UT receptor in a rat aorta bioassay. More interestingly, the replacement of the Tyr9 in 1 sequence with the Btz or the (3,4-Cl)Phe residues led to superagonists 6 (UPG-100) and 10 (UPG-92) with pEC50 values at least 1.4 log higher than that of 1, being the most potent UT agonists discovered to date. Compounds 10 and 13 showed also a good stability in a serum proteolytic assay. These ligands represent new useful tools to further characterize the urotensinergic system in human physiopathology.

New insight into the binding mode of peptides at urotensin-II receptor by Trp-constrained analogues of P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-D-Trp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp(7) residue was replaced by the highly constrained L-Tpi and D-Tpi residues. The replacement of the Trp(7) by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation-activity relationships previously reported on UT receptor ligands.

Characterization of ibodutant at NK(2) receptor in human colon.

We have characterized the pharmacological profile of the nonpeptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) through radioligand binding and contractility assays in the human colon smooth muscle. The antagonist affinity of ibodutant was evaluated through concentration-dependent inhibition curves at the [(125)I]NKA specific binding by using membranes prepared from human colon smooth muscle. In this assay the affinity of ibodutant (pKi 9.9) was compared to that of other two selective NK2 receptor antagonists, nepadutant (pKi 8.4) and saredutant (pKi 9.2). The antagonist potency of ibodutant was evaluated towards the [ßAla(8)]NKA(4-10)-mediated contractions of human colon smooth muscle strips. In this assay ibodutant (3, 10, 30 and 100 nM) induced a concentration-dependent rightward shift of the [ßAla(8)]NKA(4-10) concentration-response curves without depressing the maximal contractile effect. The analysis of the curves yielded a Schild-plot linear regression with a slope not different from unity (1.02), thus indicating a surmountable antagonist behavior. The calculated apparent antagonist potency as pKB value was 9.1. No sex related differences were observed in NK2 receptor pharmacology for [ßAla(8)]NKA(4-10) or ibodutant in colonic strips obtained from male or female patients. Reversibility experiments of tachykinin NK2 receptor blockade indicated that the inhibition of the agonist-induced contractions in preparations pre-exposed to ibodutant, and afterwards subjected to repeated washing cycles remained almost constant showing no sign of recovery during the 3h observation period. Overall, the present study indicates ibodutant as a potent tachykinin NK2 receptor antagonist in the human colon tissue, also endowed with a persistent duration of action.

Antagonist profile of ibodutant at the tachykinin NK(2) receptor in guinea pig isolated bronchi.

In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK(2) receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK(2) receptor agonist [ßAla8]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300 nM) induced a concentration-dependent rightward shift of the [ßAla8]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65-1.25), thus, indicating a surmountable behavior. The calculated apparent antagonist potency as pK(B) value was 8.31 ± 0.05. Ibodutant (0.3-100 nM) produced a concentration-dependent inhibition of the nonadrenergic-noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100 nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95 ± 4% inhibition), the calculated IC(50) value was 2.98 nM (95% c.l. 1.73-5.16 nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100 nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrates that ibodutant is a potent NK(2) receptor antagonist in guinea pig airways.

Radioligand binding characterization of the bradykinin B(2) receptor in the rabbit and pig ileal smooth muscle.

Several species-related differences have been reported in kinin B(2) receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B(2) receptor antagonist MEN16132 for the rabbit and pig B(2) receptor, and radioligand binding experiments using [(3)H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [(3)H]bradykinin binding was characterized by homologous displacement curves indicating K(d) values of 0.65 and 0.33nM in rabbit and pig, respectively. The B(2) receptor specificity of [(3)H]bradykinin binding was shown by the low affinity (>microM) displayed by agonists ([desArg(9)]bradykinin and Lys[desArg(9)]bradykinin) and antagonists [Leu(8),desArg(9)]bradykinin and Lys[Leu(8),desArg(9)]bradykinin) selective for the B(1) receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pK(i) values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and

New insight into the binding mode of peptide ligands at Urotensin-II receptor: structure-activity relationships study on P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized several analogues of P5U and urantide in which the Asp(4) residue in N-terminus position was replaced with coded and noncoded amino acids. The replacement of the Asp(4) residue by Tic led to an analogue, compound 14, more potent as antagonist (pK(B) = 8.94) compared to urantide. Furthermore, a different SAR was observed for the P5U compared to the urantide analogues. NMR and docking studies revealed a different binding mode for the agonist and antagonist ligands which could explain the observed SAR.

Pharmacological characterization of the bradykinin B2 receptor antagonist MEN16132 in rat in vitro bioassays.

The pharmacological profile of the bradykinin B(2) receptor antagonist MEN16132 at the rat B(2) receptor has been investigated and compared with that of icatibant (formerly Hoe 140). Antagonist affinity has been measured through radioligand binding experiments with membranes prepared from uterine and airway tissue. MEN16132 inhibited [(3)H]bradykinin binding with subnanomolar affinity (pK(i) values 10.4 and 10.1 in the uterus and airways, respectively), and was about 3-fold less potent than icatibant (pK(i) values 10.9 and 10.5). Antagonist potency has been estimated towards bradykinin-induced contractility of uterine and urinary bladder smooth muscle preparations. In these assays MEN16132 (pK(B): 9.7 both in uterus and bladder) was about 10-fold more potent than icatibant [pK(B): 8.8 in uterus, and pK(B) 8.0 in urinary bladder, as from Meini, S., Patacchini, R., Giuliani, S., Lazzeri, M., Turini, D., Maggi, C.A., Lecci, A., 2000a. Characterization of bradykinin B(2) receptor antagonists in human and rat urinary bladder. Eur. J. Pharmacol. 388, 177-182]. Washout experiments conducted in the uterine preparation indicated for MEN16132 (100 nM) a slower reversibility than icatibant (300 nM).Altogether present results indicate that MEN16132 displays high affinity and potency also for the rat bradykinin B(2) receptor, and thus is suitable for further investigations in pathophysiological models in this species.

Multifaceted approach to determine the antagonist molecular mechanism and interaction of ibodutant ([1-(2-phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-ethylcarbamoyl)-cyclopentyl]-amide) at the human tachykinin NK2 receptor.

Ibodutant (MEN15596, [1-(2-phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-ethylcarbamoyl)-cyclopentyl]-amide) is a tachykinin NK(2) receptor (NK(2)R) antagonist currently under phase II clinical trials for irritable bowel syndrome. This study focuses on the ibodutant pharmacodynamic profile at the human NK(2)R and compares it with two other antagonists, nepadutant (MEN11420, (cyclo-[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2beta-5beta)]) and saredutant [SR48968, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]. In functional experiments (phosphatidylinositol accumulation) in Chinese hamster ovary cells expressing the human NK(2)R, ibodutant potency measured toward concentration-response curves to neurokinin A as pK(B) was 10.6, and its antagonism mechanism was surmountable and competitive. In the same assay, antagonism equilibration and reversibility experiments of receptor blockade indicated that ibodutant quickly attains equilibrium and that reverts from receptor compartment in a slower manner. Kinetic properties of ibodutant were assessed through competitive binding kinetics experiments performed at [(3)H]nepadutant and [(3)H]saredutant binding sites. Determined K(on) and K(off) values indicated a fast association and slow dissociation rate of ibodutant at the different antagonist binding sites. Last, by radioligand binding experiments at some mutated human tachykinin NK(2)Rs, the amino acidic determinants crucial for the high affinity of ibodutant were identified at the transmembrane (TM) level: Cys167 in TM4; Ile202 and Tyr206 in TM5; Phe270, Tyr266, and Trp263 in TM6; and Tyr289 in TM7. These results indicated an extended antagonist binding pocket in the TM portion of the receptor, which is conceived crucial for TM3 and 6 arrangement and leads to G protein-coupled receptor activation. By combining this information and molecular modeling, the docking mode of ibodutant-human NK(2)R complex is proposed.

MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B2 receptor. In vitro pharmacology and molecular characterization.

The pharmacological characterization of the novel nonpeptide antagonist for the B2 receptor, namely MEN16132 (4-(S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B2 receptor has been investigated by means of competition studies at [3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B2 receptor (pKi 10.5), human lung fibroblasts (pKi 10.5), guinea pig airways (pKi 10.0), guinea pig ileum longitudinal smooth muscle (pKi 10.2), or guinea pig cultured colonic myocytes (pKi 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B2 receptor was checked at the human bradykinin B1 receptor (pKi<5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pKB 10.3) and guinea pig (colonic myocytes: pKB 10.3) B2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pKB 9.9) and guinea pig (ileum longitudinal smooth muscle: pKB 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but not much Y295W), were crucial for the high affinity of MEN16132. In conclusion, MEN16132 is a new, potent, and selective nonpeptide bradykinin B2 receptor antagonist.

Urotensin-II receptor ligands. From agonist to antagonist activity.

Urotensin II (U-II) is a disulfide bridged peptide hormone recently identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U and the compound termed urantide, which is the most potent UT receptor peptide antagonist described to date. Our previous conformational studies showed that hU-II and its analogues with agonist activity adopt a well-defined type II' beta-hairpin structure in anisotropic SDS membrane-like environment. This structural arrangement allows tight contact among the Trp7, Lys8, and Tyr9 side chains, which is fundamental to obtain full agonist activity. Here, we report an extensive SAR study on new analogues with agonist/antagonist activity on UT receptor. We investigated their biological activity and performed a conformational analysis by spectroscopic and computational methods. Our goal is to obtain a structure-based model able to explain the agonist/antagonist functional switching of these ligands.

Pharmacological investigation of hydrogen sulfide (H2S) contractile activity in rat detrusor muscle.

We have investigated the mechanism through which hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat isolated urinary bladder. Sodium hydrogen sulfide (NaHS), a donor of H2S, produced concentration-dependent contractile responses (pEC50=3.5+/-0.1) that were unaffected by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine (30 microM) and SB 366791 (10 microM) and by the N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CTX; 100 nM). In contrast, the unselective transient receptor potential (TRP) cation channels blocker ruthenium red (30 microM) almost abolished NaHS-induced contractions. Ruthenium red (30 microM) greatly reduced capsaicin-induced contractions, whereas it did not attenuate the contractile response to neurokinin A. The putative TRPV1 receptor antagonist iodo-resiniferatoxin, from 100 nM upward, produced agonist responses per se, and could not be tested against NaHS. We conclude that H2S either acts at TRPV1 receptorial sites unblocked by capsazepine or SB 366791, or stimulates a still unidentified transient receptor potential-like channel co-expressed with TRPV1 on sensory neurons.

Hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat urinary bladder.

In the rat isolated urinary bladder, NaHS (30 microm-3 mm) and capsaicin (10 nm-3 microm) produced concentration-dependent contractile responses (pEC(50)=3.5+/-0.02 and 7.1+/-0.02, respectively) undergoing dramatic tachyphylaxis. In preparations in which sensory nerves were rendered desensitized (defunctionalized) by high-capsaicin (10 microm for 15 min) pretreatment, neither capsaicin itself nor NaHS produced any motor effect. NaHS-induced contractile effects were totally prevented by the simultaneous incubation with tachykinin NK(1) (GR 82334; 10 microm) and NK(2) (nepadutant; 0.3 microm) receptor-selective antagonists. Tetrodotoxin (1 microm) only partially reduced the response to NaHS. These results provide pharmacological evidence that H(2)S stimulates capsaicin-sensitive primary afferent nerve terminals, from which tachykinins are released to produce the observed contraction by activating NK(1) and NK(2) receptors. While the molecular site of action of H(2)S remains to be investigated, our discovery may have important physiological significance since H(2)S concentrations capable of stimulating sensory nerves overlap those occurring in mammalian tissues under normal conditions.

Urantide: an ultrapotent urotensin II antagonist peptide in the rat aorta.

In this study we describe the ability of two human urotensin-II (hU-II) derivatives [Pen5,Orn8]hU-II(4-11) and [Pen5,DTrp7,Orn8]hU-II(4-11) (urantide) to block hU-II-induced contractions in the rat isolated thoracic aorta. Both compounds competitively antagonized hU-II- induced effects with pKB=7.4+/-0.06 (n=12) and pKB=8.3+/-0.09 (n=12), respectively. In contrast, neither [Pen5,Orn8]hU-II(4-11) nor urantide (1 microm each) was able to modify noradrenaline- or endothelin 1-induced contractile effects. At micromolar concentrations, [Pen5,Orn8]hU-II(4-11) produced weak (< or =25% of hU-II maximum) agonist responses in the rat aorta, whereas urantide was totally uneffective as agonist up to 1 microm. In addition, [Pen5,Orn8]hU-II(4-11) and urantide displaced [125I]urotensin II from specific binding at hU-II recombinant receptors (UT receptors) transfected into CHO/K1 cells (pKi=7.7+/-0.05, n=4 and pKi=8.3+/-0.04, n=4, respectively). To our knowledge, urantide is the most potent UT receptor antagonist so far described, and might represent a useful tool for exploring the (patho)physiological role of hU-II in the mammalian cardiovascular system.

Pharmacology of transmission to gastrointestinal muscle.

The identity of excitatory and inhibitory neurotransmitters is well established. Excitatory motor neurons synthesize and release acetylcholine and tachykinins, which act through postjunctional muscarinic M2 and M3 or tachykinin NK1 and NK2 receptors, respectively, to induce smooth muscle contraction. A residual excitatory component is mediated by ATP acting on P2X1 receptors. Conversely, inhibitory motor neurons express nitric oxide synthase and vasoactive intestinal peptide (VIP), which together with ATP, induce a coordinated muscle relaxation. The receptors involved in the inhibitory effects of ATP and VIP are unknown. Likewise, the relationships between inhibitory signals triggered by NO and those mediated by VIP need to be clarified. Recent evidence obtained using receptor knockout mice have confirmed the involvement of the above-mentioned excitatory transmitters but have revealed an unexpected complexity in the nitrergic transmission, where the effects of NO are manifested only in the presence of carbon monoxide. Interstitial cells of Cajal (ICC) are being recognized as targets of intestinal motor neurons; therefore, the signaling mechanisms are probably integrated by these cells before being transmitted to smooth muscle. Challenges in future years will be to identify the physiological role of the various excitatory and inhibitory components, and to understand the relative importance of neurotransmitter receptors expressed on ICC and smooth muscle cells.

Role of tachykinins in sephadex-induced airway hyperreactivity and inflammation in guinea pigs.

We have studied the effect of selective tachykinin NK(1) and NK(2) receptor antagonists on airway hyperreactivity to acetylcholine and increase of inflammatory cells on bronchoalveolar lavage fluid induced by sephadex beads (20 mg/kg, i.v.) in guinea pigs. Airway hyperreactivity was assessed by measuring the increase of bronchial insufflation pressure to acetylcholine (0.01-30 micromol/kg, i.v.) at 3 h (early phase) and 24 h (late phase) after sephadex administration. An increase in inflammatory cells in bronchoalveolar lavage fluid (eosinophils and macrophages) was detected at 24 h (from 11.6 x 10(6) to 49.3 x 10(6) cells) but not at 3 h from sephadex administration. Neurokinin A and substance P levels in bronchoalveolar lavage fluid showed a significant increase at 24 h (from 31.7+/-11.6 to 561+/-231 pg/ml and from 5.9+/-2.6 to 29.3+/-4.1 pg/ml for neurokinin A and substance P, respectively). At this time point, the tachykinin in bronchoalveolar lavage cellular content was depleted from 232+/-43 to 21+/-20 pg/sample and from 56.6+/-6.7 to 2+/-2 pg/sample for neurokinin A and substance P, respectively. Capsaicin pretreatment abolished the early but not the late phase of airway hyperreactivity induced by sephadex without modifying bronchoalveolar lavage total cells number and bronchoalveolar lavage levels of neurokinin A and substance P. Administration of the tachykinin NK(2) (nepadutant) and/or the NK(1) receptor antagonist (MEN 11467 or (1R,2S)-2-N[1(H)indol-3-yl-carbonyl]-1-N[N-(p-tolylacetyl)-N-(methyl)-D-3(2-naphthyl)alanyl)diaminocyclohexane)), 5 min before sephadex, prevented the early phase of airway hyperreactivity to acetylcholine but only nepadutant prevented the late phase. Nepadutant was able to abolish the early phase of airway hyperreactivity if given after sephadex administration and reduced by about 50% the increase of cell number in bronchoalveolar lavage fluid during the late phase, without affecting the levels of neurokinin A and substance P. These findings indicate an involvement of endogenous tachykinins in the genesis of airway hyperreactivity in a guinea-pig model of non-allergic asthma. Early airway hyperreactivity apparently involves release of tachykinins from capsaicin-sensitive afferent nerves acting via tachykinin NK(1)/NK(2) receptors. Late airway hyperreactivity involves tachykinins acting via tachykinin NK(2) receptors: inflammatory cells activated/recruited in response to sephadex challenge appear a likely source of tachykinins involved in the late phase of the response.