Gender-related differential effect of tachykinin NK2 receptor-mediated visceral hyperalgesia in guinea pig colon.

BACKGROUND AND PURPOSE: The tachykinin NK2 receptor antagonist ibodutant is under Phase III clinical investigation to treat female patients with irritable bowel syndrome. The aim of this study was to investigate the NK2 receptor-related gender specificity in a model of colitis. EXPERIMENTAL APPROACH: Colitis was induced by rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 0.5 mL, 30 mg·mL(-1) in 30% ethanol) in female and male guinea pigs. Electromyographic recording of the responses to colorectal distension (CRD) was made 3 days later. Ibodutant (0.33 , 0.65, 1.9 and 6.5 mg·kg(-1) ) was given s.c., 30 min before CRD. Release of neurokinin A and substance P from isolated mucosal and smooth muscle tissues following treatment with KCl (80 mM) or capsaicin (10 µM) was measured by EIA. Plasma pharmacokinetics of ibodutant following a single s.c. administration (0.73 or 2.1 mg·kg(-1) ) were measured over 24 h. KEY RESULTS: Ibodutant did not affect abdominal contractions in control animals. After TNBS-induced colitis, ibodutant prevented the increased visceral hypersensitivity to CRD in females, at lower doses than in males. Ibodutant pharmacokinetics did not differ between females and males. Tachykinins release was greater in smooth muscle than in mucosal samples. Capsaicin-stimulated release of tachykinins from inflamed mucosal samples from females was significantly lower than in males. CONCLUSIONS AND IMPLICATIONS: Ibodutant prevented abdominal nociception in a model of visceral hypersensitivity in guinea pigs with a greater efficacy in females than in males. Our results highlight a gender-related difference in colonic visceral hypersensitivity and mucosal nerve activation.

Chronic treatment with otilonium bromide induces changes in L-type Ca²âº channel, tachykinins, and nitric oxide synthase expression in rat colon muscle coat.

BACKGROUND: Otilonium bromide (OB) is a quaternary ammonium derivative used for the treatment of intestinal hypermotility and is endowed with neurokinin2 receptor (NK2r) antagonist and Ca²âº channel blocker properties. Therefore, the possibility that OB might play a role in the neurokinin receptor/Substance-P/nitric oxide (NKr/SP/NO) circuit was investigated after chronic exposition to the drug. METHODS: Rats were treated with OB 2-20 mg kg⁻¹ for 10 and 30 days. In the proximal colon, the expression and distribution of muscle NOsynthase 1 (NOS1), NK1r, NK2r, SP and Cav 1.2 subunit (for L-type Ca²âº channel) and the spontaneous activity and stimulated responses to NK1r and NK2r agonists were investigated. KEY RESULTS: Immunohistochemistry showed a redistribution of NK1r and L-type Ca²âº channel in muscle cells with no change of NK2r at 30 days, a significant increase in muscle NOS1 expression at 10 days and a significant decrease in the SP content early in the ganglia and later in the intramuscular nerve fibers. Functional studies showed no change in spontaneous activity but a significant increase in maximal contraction induced by NK1r agonist. CONCLUSIONS & INFERENCES: Chronic exposition to OB significantly affects the NKr/SP/NO circuit. The progressive decrease in SP-expression might be the consequence of the persistent presence of OB, the increase of NOS1 expression in muscle cells at 10 days in an attempt to guarantee an adequate NO production, and, at 30 days, the redistribution of the L-type Ca²âº channel and NK1r as a sign to compensate the drug channel block by re-cycling both of them. The physiological data suggest NK1r hypersensitivity.

Effect of otilonium bromide and ibodutant on the internalization of the NK2 receptor in human colon.

BACKGROUND: The present aim was to study the modulation of NK2 receptor internalization by two compounds, the spasmolytic otilonium bromide (OB) endowed with NK2 receptor antagonistic properties and the selective NK2 receptor antagonist ibodutant. METHODS: Full-thickness human colonic segments were incubated in the presence of OB (0.1-10 µmol L(-1)) or ibodutant (0.001-0.1 µmol L(-1)), with or without the NK2 receptor selective agonist [ßAla8]NKA(4-10) and then fixed in 4% paraformaldehyde. Cryosections were processed for NK2 receptor immunohistochemical revelation. Quantitative analysis evaluated the number of the smooth muscle cells that had internalized the NK2 receptor. KEY RESULTS: Immunohistochemistry revealed that in basal condition, the NK2 receptor was internalized in about 23% of total smooth muscle cells. The exposure to the selective NK2 receptor agonist induced internalization of the receptor in more than 77% of the cells. Previous exposure to both OB or ibodutant, either alone or in the presence of the agonist, concentration-dependently reduced the number of the cells with the internalized receptor. CONCLUSIONS & INFERENCES: Both OB and ibodutant antagonize the internalization of the NK2 receptor in the human colon. As NK2 receptors are the predominant receptor mediating spasmogenic activity of tachykinins on enteric smooth muscle, we hypothesize that the antagonistic activity found for both OB and ibodutant should play a specific therapeutic role in gut diseases characterized by hypermotility.

Comparative antagonist pharmacology at the native mouse bradykinin B2 receptor: radioligand binding and smooth muscle contractility studies.

BACKGROUND AND PURPOSE: The aim was to characterize the recently discovered non-peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists. EXPERIMENTAL APPROACH: [3H]-BK binding experiments used mouse lung and ileum tissue membranes and antagonist potency was measured in the isolated ileum contractility assay. KEY RESULTS: Two BK binding sites resulted from saturation and homologous competition experiments. A role for the B1 receptor was excluded because of the poor affinity of B1 receptor ligands (pIC50<5). MEN16132, and the other reference antagonists, inhibited only one portion of BK specific binding, and the rank order of potency was (pIC50): Icatibant (lung 10.7; ileum 10.2)=MEN11270 (lung 10.4; ileum 9.9)=MEN16132 (lung 10.5; ileum 9.9).>LF16-0687 (lung 8.9; ileum 8.8)>FR173657 (lung 8.6; ileum 8.2). BK homologous curves performed with lung membranes after treatment with the antagonist MEN16132 or Icatibant (10 nM) displayed only the low affinity site. The functional antagonism by MEN16132 (pA2 9.4) and Icatibant (pA2 9.1), towards BK (control EC50 6.1 nM) induced ileum contractions, was concentration-dependent and surmountable, but the Schild plot slope was less than unity. CONCLUSIONS AND IMPLICATIONS: In mouse tissue, radiolabelled BK recognizes two binding sites and B2 receptor antagonists can compete only for the higher affinity one. The pharmacological profile of the novel non-peptide antagonist MEN16132 indicates that it exhibits subnanomolar affinity and potency for the mouse B2 receptor and is suitable for further characterization in in vivo pathophysiological models.

Characterization of kinin receptors in human cultured detrusor smooth muscle cells.

BACKGROUND AND PURPOSE: Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor. EXPERIMENTAL APPROACH: Human detrusor cells from biopsies were isolated and maintained in culture. B(1) and B(2) kinin receptors were characterized by means of radioligand and functional experiments (PI accumulation and PGE(2) release). KEY RESULTS: [(3)H]-[desArg(9)]-Lys-BK and [(3)H]-BK saturation studies indicated receptor density (B(max)) and K (d) values of 19 or 113 fmol mg(-1), and 0.16 or 0.11 nM for the B(1) or B(2) receptors, respectively. Inhibition binding studies indicated the selectivity of the B(1) receptor antagonist [desArg(9)Leu(8)]-Lys-BK and of the B(2) receptor antagonists Icatibant and MEN16132. [DesArg(9)]-Lys-BK and BK induced PI accumulation with an EC(50) of 1.6 and 1.4 nM and different maximal responses (E(max) of [desArg(9)]-Lys-BK was 10% of BK). BK also induced prostaglandin E(2) release (EC(50) 2.3 nM), whereas no response was detected with the B(1) receptor agonist. The incubation of detrusor smooth muscle cells with interleukin 1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1)) induced a time-dependent increase in radioligand-specific binding, which was greater for the B(1) than for the B(2) receptor. CONCLUSIONS AND IMPLICATIONS: Human detrusor smooth muscle cells in culture retain kinin receptors, and represent a suitable model to investigate the mechanisms and changes that occur under chronic inflammatory conditions.

Urodynamic effects induced by intravesical capsaicin in rats and hamsters.

This study compared the effect of acute intravesical capsaicin administration on transvesical cystometries in urethane-anesthetized rats and hamsters, and aimed to assess whether sensory neuropeptides (tachykinins; calcitonin gene-related peptide, CGRP) play a role in the urodynamic effects of capsaicin in these species. The following urodynamic parameters were evaluated: the mean micturition interval (MI), the pressure threshold for micturition (PT), and the mean amplitude of micturition contractions (MAC). Two concentrations of capsaicin (10 and 100 microM) were evaluated in both species. Here, we demonstrate that 10-microM capsaicin decreased the PT in both rats and hamsters, and 100-microM capsaicin decreased the PT in hamsters and decreased the MI in both species. In addition, 100-microM capsaicin increased the MAC in rats but decreased the MAC in hamsters. Administration of CGRP (10 nmol kg(-1) , i.v.) significantly decreased both MAC and PT in hamsters only, while capsaicin-induced desensitization of neuropeptide-containing afferents antagonized the urodynamic effects of intravesical capsaicin. In addition, administration of the tachykinin NK2 receptor antagonist, Nepadutant (100 nmol kg(-1), i.v.), reduced the effects of capsaicin (100 microM) only in rats. These results indicate that capsaicin induces bladder hyperactivity in both rats and hamsters, but the urodynamic characteristics of this hyperactivity markedly differ in these two species. The differences observed may be due to differential expression of sensory neuropeptides in capsaicin-sensitive bladder afferents or neuropeptide receptors in smooth muscle cells and in nerve fibers.

The role of sensory neuropeptides in motor innervation of the hamster isolated urinary bladder.

In this study we have characterized the role of sensory fibers and of the sensory peptides, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), on the contractile responses evoked by single pulse electrical field stimulation (EFS) in the hamster urinary bladder. EFS of the hamster isolated urinary bladder produced twitch contractions which were unaffected by atropine but abolished by tetrodotoxin. The P2 purinoreceptor antagonist PPADS (30 microM) inhibited twitches by 66+/-4% on its own and by 78+/-3% in the presence of atropine. The selective tachykinin NK2 receptor antagonist nepadutant produced a slight but consistent reduction of twitch amplitude (-21+/-3%) at 1 microM. Addition of nepadutant to atropine and PPADS did not further increase their inhibitory effect. The application of hCGRP (10-300 nM) produced a concentration-dependent inhibition of twitches (Emax -38+/-3%, EC50=12 nM) and a small reduction of tone (0.5+/-0.09 mN). Similar effects were obtained with capsaicin (0.1-10 microM) which inhibited EFS-evoked contractions with an EC50 of 100.0 nM and a maximal effect of 34+/-4% inhibition at 1 microM. Under submaximal parameters of stimulation NKA (10 nM) increased the amplitude of twitches by 45+/-6% and produced a concentration-dependent tonic contraction (EC50=55.9 nM). The CGRP1 receptor subtype antagonist, hCGRP(8-37), increased by 29+/-8% the EFS-evoked contractions and significantly reduced the response to 0.1 microM CGRP. Capsaicin (10 microM) increased both CGRP-LI and NKA-LI release from superfused slices of hamster urinary bladder by about sixfold and by about 70%, over baseline, respectively. A second application of capsaicin was ineffective, indicating a complete desensitization of sensory nerve efferent function. In the hamster urinary bladder the sensory neuropeptides NKA and CGRP are co-released by sensory fibers after stimulation either by EFS or capsaicin. However, the role of CGRP appears functionally predominant.

Differences in electromechanical coupling between bradykinin and the nonpeptide kinin B2 receptor agonist, FR 190997, in the circular muscle of guinea-pig colon.

We have compared the effect of bradykinin (BK) and the nonpeptide kinin B2 receptor agonist, FR 190997, in producing changes in membrane potential and tension in the circular muscle of guinea-pig colon by the sucrose gap technique. In the presence of atropine (1 microM), S-ketoprofen (3 microM) and apamin (0.1 microM), BK (1 microM for 20 s) induced a transient depolarization of the membrane with superimposed action potentials (spikes) and transient contraction. Nifedipine (1 microM) eliminated the spikes and markedly inhibited the BK-induced contractions. FR 190997 (3-10 microM for 20 s) induced a slowly developing sustained small depolarization associated with a slowly developing and sustained contraction but, contrary to BK, FR 190997 was unable to trigger spikes. Nifedipine had no effect on depolarization and contraction induced by FR 190997. In the presence of 1 microM nifedipine, the combined application of a blocker of receptor-operated cation channels, SKF 96365 (50 microM for 30 min), and of an inhibitor of sarcoplasmic reticulum calcium pump, cyclopiazonic acid (CPA 10 microM for 30 min), reduced the BK-induced depolarization and contraction by about 45%-60%. The same treatment induced about 40% reduction of the sustained contraction induced by FR 190997, whereas the concomitant depolarization was not significantly affected. The tyrosine kinase inhibitor genistein (40 microM for 20 min) had no effect on the BK- and FR 190997-induced depolarization and contraction in the presence of nifedipine. In radioligand binding experiments performed in membranes of colonic smooth muscle cells, both agonists displaced the [3H]BK specific binding with a pIC50 of 9.6 and 8.5 for BK and FR 190997, respectively. These findings indicate a substantial qualitative difference in mechanisms of excitation contraction coupling activated by BK and FR 190997 via B2 receptors in guinea-pig colon.

Tachykinin-mediated effect of nociceptin in the rat urinary bladder in vivo.

The application of nociceptin (5-50 nmol/rat) onto the serosa in the urinary bladder of urethane-anaesthetized rats, with the intravesical volume kept below threshold for activation of the micturition reflex, induced a low amplitude tonic contraction (local, i.e., resistant to ganglionectomy) with high amplitude phasic contractions (reflex, i.e., abolished by ganglionectomy) superimposed. The pharmacology of the local contraction was studied in animals with acute bilateral ablation in the pelvic ganglia: the combined administration of tachykinin NK(1) (S)1-¿2-[3-(3, 4-dichlorophenyl)-1-(3-isopropoxyphenyl-acetyl)-piperidin-3-yl]eth yl¿-4-phenyl-1-azoniabicyclo[2.2.2.]octane chloride (SR 140333) and NK(2) c¿[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta++ +)¿ (MEN 11420) receptor antagonists (given at doses of 1+0.1 micromol/kg, intravenous (i.v.), respectively) abolished the local bladder contraction induced by topical nociceptin (50 nmol/rat). These results indicate that the topical application of nociceptin onto the bladder evokes a tachykinin-mediated contraction.

Effect of 18beta-glycyrrhetinic acid on electromechanical coupling in the guinea-pig renal pelvis and ureter.

We have tested the effect of the gap junction inhibitor, 18beta-glycyrrhetinic acid (18betaGA) on electromechanical coupling in the guinea-pig renal pelvis and ureter by the sucrose gap technique. In the ureter 18betaGA (3 - 30 microM) produced a concentration-dependent inhibition of the spike component of the action potential (AP) and reduced contraction evoked by electrical stimulation. Neurokinin A (NKA) produced a slow depolarization with superimposed APs and phasic contractions of the ureter. 18betaGA (30 microM) markedly inhibited the depolarization and APs evoked by NKA. However the contractile response was more sustained in the presence than in the absence of 18betaGA. At 100 microM, 18betaGA inhibited the mechanical responses to NKA. KCl (80 mM) produced APs and phasic contractions followed by sustained depolarization and tonic contraction. At 30 microM 18betaGA markedly inhibited the KCl-evoked APs and phasic contractions without affecting the sustained responses. At 100 microM 18betaGA inhibited the tonic contraction to KCl. In the renal pelvis 18betaGA (30 microM) inhibited the amplitude of pacemaker potentials and accompanying contractions and induced the appearance of low-amplitude APs not associated with contraction. We conclude that, up to 30 microM, the action of 18betaGA is consistent with an inhibition of cell-to-cell electrical coupling via gap junctions. The single-unit character of smooth muscles in the guinea-pig upper urinary tract is partly converted to a multi-unit pattern. At high concentrations 18betaGA possesses non specific effects which limit its usefulness as a tool for studying the role of gap junctions in smooth muscles. British Journal of Pharmacology (2000) 129, 163 - 169

Antimuscarinic, calcium channel blocker and tachykinin NK2 receptor antagonist actions of otilonium bromide in the circular muscle of guinea-pig colon.

We have analyzed, by the sucrose gap method, the action of otilonium bromide, a quaternary ammonium derivative in use for the symptomatic therapy of irritable bowel syndrome, on the electrical and mechanical responses initiated by different stimuli in the circular muscle of the guinea-pig proximal colon. Otilonium bromide produced a concentration-dependent inhibition of membrane depolarization (IC50 4.1 microM), action potentials (APs) and contraction (IC50 3.7 microM) produced by the muscarinic receptor agonist, methacholine. It also produced a concentration-dependent inhibition of APs and accompanying contraction (IC50 31 microM) produced by KCl (30 mM), and had a biphasic effect on the cholinergic excitatory junction potential (e.j.p.) produced by single pulse electrical field stimulation: at low concentrations (0.1-0.3 microM) otilonium bromide enhanced the e.j.p. and, at higher concentrations (IC50 22 microM and 16 microM toward depolarization and contraction), produced a concentration-dependent inhibition. Otilonium bromide eliminated the APs superimposed on the depolarization induced by the tachykinin NK1 receptor agonist, [Sar9]substance P-sulphone and suppressed the corresponding contraction (IC50 43 microM) but had little effect on the sustained membrane depolarization induced by this agonist. On the other hand, otilonium bromide produced a similar inhibitory effect on both membrane depolarization and contraction (IC50 38 microM and 45 microM, respectively) induced by the tachykinin NK2 receptor agonist [betaAla8]neurokinin A (4-10). When tested in the presence of nifedipine (1 microM), otilonium bromide had no effect on the membrane depolarization induced by [Sar9]substance P-sulphone but inhibited in a concentration-dependent manner the depolarization induced by [betaAla8]neurokinin A (4-10) (IC50 41 microM). In contrast, the blocker of receptor-operated cation channels, SKF 96365, inhibited with similar potency the depolarization induced by both [Sar9]substance P-sulphone and [betaAla8]neurokinin A (4-10) (IC50 60 microM and 54 microM, respectively). In radioligand binding experiments otilonium bromide produced a concentration-dependent inhibition of the binding of both an agonist ([125I]neurokinin A, Ki 7.2 microM) and an antagonist ([3H]SR 48968, Ki 2.2 microM) to membranes of Chinese hamster ovary cells transfected with the human tachykinin NK2 receptor. In conclusion, the present findings demonstrate that, in the microM range of concentrations, otilonium bromide acts as a muscarinic and tachykinin NK2 receptor antagonist and as a calcium channel blocker. The latter property is likely to account for its ability to suppress contraction initiated by the tachykinin NK1 receptor agonist. Therefore multiple mechanisms of action account for the ability of otilonium bromide to reduce stimulated motility of intestinal smooth muscle.

Excitatory motor and electrical effects produced by tachykinins in the human and guinea-pig isolated ureter and guinea-pig renal pelvis.

1. In isolated tissue experiments, neurokinin A (NKA) produced concentration-dependent contraction of human and guinea-pig ureter (pD2 = 6.7 and 7.2, respectively); an effect greatly reduced (>80% inhibition) by the tachykinin NK2 receptor-selective antagonist MEN 11420 (0.1 microM). The tachykinin NK1 and NK3 receptor agonists septide and senktide, respectively, were ineffective. 2. Electrical field stimulation (EFS) of the guinea-pig isolated renal pelvis produced an inotropic response blocked by MEN 11420 (0.01-1 microM). In the same preparation MEN 11420 (0.1 microM) blocked (apparent pK(B) = 8.2) the potentiation of spontaneous motor activity produced by the NK2 receptor-selective agonist [betaAla8]NKA(4-10). 3. In sucrose-gap experiments, EFS evoked action potentials (APs) accompanied by phasic contractions of human and guinea-pig ureter, which were unaffected by tetrodotoxin or MEN 11420 (3 microM), but were blocked by nifedipine (1-10 microM). NKA (1-3 microM) produced a slow membrane depolarization with superimposed APs and a tonic contraction with superimposed phasic contractions. NKA prolonged the duration of EFS-evoked APs and potentiated the accompanying contractions. MEN 11420 completely prevented the responses to NKA in both the human and guinea-pig ureter. 4. Nifedipine (1-10 microM) suppressed the NKA-evoked APs and phasic contractions in both human and guinea-pig ureter, and slightly reduced the membrane depolarization induced by NKA. A tonic-type contraction of the human ureter in response to NKA persisted in the presence of nifedipine. 5. In conclusion, tachykinins produce smooth muscle excitation in both human and guinea-pig ureter by stimulating receptors of the NK2 type only. NK2 receptor activation depolarizes the membrane to trigger the firing of APs from latent pacemakers.

Pharmacokinetics of the bicyclic peptide tachykinin NK2 receptor antagonist MEN 11420 (nepadutant) in rats.

The pharmacokinetics of MEN 11420 [nepadutant, c[[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta++ +)]], a potent glycosylated analogue of the selective, bicyclic peptide, tachykinin NK2 receptor antagonist MEN 10627 [c[(Met-Asp-Trp-Phe-Dpr-Leu)c(2beta-5beta)]], were studied in rats after different routes of administration. The plasma concentration profile for MEN 11420 after iv administration (1 mg/kg) was compared with that for the parent compound MEN 10627. The mean plasma half-life (44 min) and AUC value (285 micrograms.min/ml) for MEN 11420 were almost 3-fold greater than those for MEN 10627, and the systemic clearance was reduced to one third. The absolute bioavailability of MEN 11420 after intranasal (1 mg/kg) or ip (1 mg/kg) administration was virtually complete. However, bioavailability was only approximately 5% after intrarectal treatment (5 mg/kg) and was too low to be quantified (<3%) after sublingual (1 mg/kg) or oral (10 mg/kg) doses. The urinary excretion of unchanged compound, after an iv dose of 1 mg/kg, was approximately 34% of the dose for MEN 11420 but was <2% for MEN 10627. This is in agreement with in vitro data showing that MEN 11420 is more resistant to hydrolytic and oxidative metabolism than is MEN 10627. It is concluded that the hydrophilic modification of MEN 10627 to produce MEN 11420 resulted in marked improvement in the pharmacokinetic and metabolic characteristics of the peptide.

Characterization of the antibronchoconstrictor activity of MEN 11420, a tachykinin NK2 receptor antagonist, in guinea-pigs.

We have investigated the antibronchoconstrictor activity of a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist, MEN 11420 c¿[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta++ +)¿, as compared to MEN 10627 c[(Met-Asp-Trp-Phe-Dpr-Leu)c(2beta-5beta)] and to the nonpeptide antagonist SR 48968 ((S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2-3,4-dichlorophenyl)bu tyl] benzamide. In the guinea-pig isolated bronchus MEN 11420 (pK(B) 8.40+/-0.07) and MEN 10627 (pK(B) 8.67+/-0.09) competitively antagonized the contraction induced by the tachykinin NK2 receptor agonist, [betaAla8]neurokinin A-(4-10). SR 48968 showed an apparent pK(B) of 9.57+/-0.2. The atropine-resistant response to electrical stimulation was reduced in a concentration-dependent manner by MEN 11420, MEN 10627 and SR 48968. In urethane-anaesthetized guinea-pigs, MEN 11420 produced a dose-dependent inhibition of bronchoconstriction induced by [betaAla8]neurokinin A-(4-10). Comparable inhibitory effects were observed after i.v. administration of SR 48968 and MEN 10627. Bilateral electrical stimulation of the vagi (20 Hz for 20 s) induced a bronchoconstriction that was dose-dependently inhibited by i.v. MEN 11420, SR 48968 and MEN 10627. MEN 11420 was also effective in inhibiting the capsaicin (20 nmol/kg i.v.)-induced bronchoconstriction. MEN 11420 (1.1 micromol/kg i.v.) showed a longer plasma half-life and a greater area under the plasma concentration-time curve value (AUC) than those of MEN 10627. These findings indicate that MEN 11420 is a potent and selective antagonist of the tachykinin NK2 receptor in guinea-pig airways with a long duration of action.

Bladder distension and activation of the efferent function of sensory fibres: similarities with the effect of capsaicin.

1. The effects of the tachykinin NK2 receptor antagonist MEN 11420 (100 nmol kg(-1), i.v.) and isoprenaline (400 nmol kg(-1), i.v.) were compared in a model of distension-induced bladder activity in isovolumetric conditions. MEN 11420 induced a relaxation of the basal tone of the urinary bladder that was dependent on the volume of the viscus: the effect was absent at low volumes (0.2 and 0.5 ml) and it was maximal at high volumes of distension (1 and 2 ml), approaching about 60% of the isoprenaline-induced relaxation. The relaxant effect of isoprenaline was always evident at all volumes of distension. 2. Tetrodotoxin (1-100 microM, intravesically applied) abolished distension-evoked micturition contractions, but did not prevent the relaxant effect of MEN 11420- or isoprenaline on the bladder tone. 3. The cyclo-oxygenase inhibitor S-ketoprofen (0.5 micromol kg(-1), i.v.) produced a marked decrease of the bladder tone and a concomitant reduction of bladder motility at 1 ml volume of distension. At 2 ml of distension, S-ketoprofen still decreased the minimal pressure but had no significant effect on other parameters of vesical motility. In S-ketoprofen-pretreated rats, the relaxant effect of MEN 11420 was significant at 2 but not at 1 ml of distension, and that of isoprenaline was reduced by 50% at both 1 and 2 ml. 4. Ruthenium red (10 micromol kg(-1), i.v.) had no effect at a low volume of distension (0.2 ml) or at highest volume (2 ml) but decreased the basal tone and the frequency of bladder contractions at 1 ml of distension. In ruthenium red-pretreated rats, MEN 11420 failed to decrease bladder tone at 1 ml, whereas at 2 ml the effect of MEN 11420 was not different from that observed in controls (43 vs 60% of isoprenaline-induced relaxation, respectively). 5. At both 1 and 2 ml of distension, capsaicin pretreatment (164 micromol kg(-1), s.c. 5 days before) reduced the frequency of micturition contractions but had no effect on the bladder tone. Capsaicin pretreatment prevented the relaxant effect of MEN 11420 on the bladder tone both at 1 and at 2 ml of distension. 6. It is concluded that the release of tachykinins from capsaicin-sensitive afferent nerves induced by bladder distension is resistant to tetrodotoxin and to prostaglandin synthesis inhibition. Tachykinins modulate the vesical tone by acting through NK2 receptors.

Evidence for the involvement of multiple mechanisms in the excitatory action of bradykinin in the circular muscle of guinea-pig colon.

We have investigated, by using the sucrose gap technique, the mechanisms of the excitatory action of bradykinin in the circular muscle of the guinea-pig proximal colon. In the presence of atropine (1 microM) and S-ketoprofen (3 microM), the application of bradykinin (1 microM for 20 s) produced complex changes in membrane potential and muscle tension. The prevailing response was a small hyperpolarization followed by a slowly developing depolarization and a tonic contraction. The selective B2 receptor antagonist, HOE 140 (0.3 microM) blocked the responses to bradykinin (1 microM) while tetrodotoxin (0.3 microM) had no affect. The selective B1 receptor agonist, [des-Arg9]bradykinin (1 microM) did not affect the electrical or mechanical activities of the circular muscle. Apamin (0.1 microM) blocked the transient hyperpolarization and potentiated the bradykinin-induced depolarization and contraction. In the presence of apamin, nifedipine (1 microM) blocked spikes (when present) and the phasic contraction while leaving the tonic contraction unaffected. The excitatory action of bradykinin was further investigated in the presence of atropine (1 microM), S-ketoprofen (3 microM), apamin (0.1 microM) and nifedipine (1 microM). The depolarization but not the contraction induced by bradykinin was reduced by about 30% in low-Na+ (25 mM) but not in low Cl- (9.7 mM) Krebs solution. The depolarization and contraction evoked by bradykinin were reduced (by about 30 and 75%, respectively) in Ca2+-free (2 min) Krebs solution. The blocker of the sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (CPA, 10 microM) reduced the nifedipine-resistant depolarization and contraction induced by bradykinin by about 40 and 60%, respectively. The inhibitor of receptor-operated cation channels, SKF 96365 (50 microM) reduced the nifedipine-resistant bradykinin-induced depolarization and contraction by about 40 and 30%, respectively, whereas the inhibitor of Ca2+-dependent chloride channels, niflumic acid (100 microM) was without effect. The inhibitory effect of SKF 96365 (50 microM) and CPA (10 microM) was additive: in the presence of both drugs the bradykinin-induced depolarization and contraction were reduced by about 70-80%. The protein kinase C inhibitor, GF 109203x (10 microM) did not affect the nifedipine-resistant bradykinin-induced depolarization and contraction. At a concentration of 30 microM, GF 109203x reduced the bradykinin-induced contraction by about 50% while leaving the bradykinin-induced depolarization unaffected. The KCl (40 mM)-induced contraction was significantly reduced (by about 30%) by GF 109203x (30 microM). The present findings indicate that, in the presence of apamin and nifedipine, the bradykinin-induced contraction of circular muscle of the guinea-pig colon is due to the influx of extracellular Ca2+ via non-selective cation channels and, in part, to the release of Ca2+ from a loosely bound internal store. Intracellular Ca2+ facilitates the bradykinin-induced depolarization, a response which does not involve a protein kinase C-dependent mechanism.

Depolarization evoked co-release of tachykinins from enteric nerves in the guinea-pig proximal colon.

The aim of this study was to assess at which extent an even co-release of the tachykinins, substance P (SP) and neurokinin A (NKA), occurs from enteric neurons/nerves of the guinea-pig proximal colon during graded depolarization. In this preparation, a sharply diverging NK1/NK2 receptor pattern of tachykininergic co-transmission has been observed in physiological studies. The experiments were performed in capsaicin-pretreated (10 microM for 15 min) mucosa-free smooth muscle of guinea-pig proximal colon, to exclude the mucosa and the peripheral endings of primary afferent nerves as possible sources of released tachykinins. The content of extractable tachykinins was measured as SP- and NKA-like immunoreactivities (-LI) by radioimmunoassay. Chromatographic characterization of aqueous acetic acid extracts showed one peak of SP-LI corresponding to authentic SP, whereas there were multiple peaks of NKA-LI, the major one co-eluting with authentic NKA. An increased outflow of both SP- and NKA-LI was evenly produced in a concentration-dependent manner when the preparations were superfused with a high potassium (K) medium in which NaCl had been replaced with equimolar amounts (20-100 mM) of KCl. The high K-evoked release of SP- and NKA-LI was dependent upon the presence of extracellular calcium and was inhibited by about 50% in the presence of the N-type voltage-dependent calcium channel blocker, omega-conotoxin GVIA (0.1 microM). Omega-conotoxin MVIIC (1 microM), a non-selective blocker of N-, P- and Q-type voltage-dependent calcium channels, likewise produced about 40% inhibition of evoked release of both peptides. No evidence for a role of L-type channels in tachykinin release was obtained, since the addition of nifedipine (1 microM) or Bay K8644 (1 microM) did not significantly affect the response to high K. Neither NK1 receptor agonist (septide, 0.1 microM) or antagonist (SR 140333, 10 nM) nor NK2 receptor agonists ([betaAla8]NKA(4-10) and GR 64349, 0.1 microM each) or antagonist (SR 48968, 10 nM) did affect the high K-evoked release of tachykinins. We conclude that SP and NKA are evenly co-released in response to graded depolarization of enteric nerves in the guinea-pig colon. Therefore, the specialization of tachykininergic transmission observed in functional studies does not originate at the prejunctional level. The co-release of tachykinins involves the influx of extracellular calcium via N-type but not L-type calcium channels. No evidence for the presence of NK1 or NK2 autoreceptors affecting tachykinin release from enteric neurons was obtained.

MEN 11420, a potent and selective tachykinin NK2 receptor antagonist in the guinea-pig and human colon.

We have characterized the action of the novel, water-soluble, tachykinin NK2 receptor antagonist MEN 11420 ([Asn(2-AcNH-beta-D-Glc)-Asp-Trp-Phe-Dap-Leu] c(2 beta-5 beta)) on the circular muscle of the guinea-pig and human colon in vitro and on the guinea-pig colon in vivo. In organ bath experiments on guinea-pig colon MEN 11420 produced a concentration-dependent rightward shift of the concentration-response curve to the NK2 receptor selective agonist, [beta Ala8]neurokinin A (NKA) (4-10) with a pKB value of 8.1. Up to 1 microM MEN 11420 had no effect on the concentration-response curve to methacholine, to the NK1 receptor selective agonist, [Sar9]substance P (SP) sulfone, to the NK3 receptor selective agonist, senktide, or on the response to exogenous SP. The response to exogenous NKA was inhibited, although the shift of the concentration-response curve to NKA produced by MEN 11420 at 1 microM (dose ratio 5.3) was much smaller than that produced against [beta Ala8]NKA (4-10) (dose ratio 102), presumably because NKA also stimulates NK1 receptors at relatively low concentrations. In sucrose gap, MEN 11420 concentration-dependently inhibited both depolarization (IC50 0.34 microM) and contraction (IC50 = 0.32 microM) produced by [beta Ala8]NKA (4-10) (0.3 microM for 10 s) in the guinea-pig colon without affecting the corresponding responses produced by [Sar9]SP sulfone. When similar experiments were performed in the circular muscle of the human colon MEN 11420 concentration-dependently inhibited both depolarization and contraction induced by [beta Ala8]NKA(4-10) with IC50s of 99 and 75 nM, respectively. MEN 11420 (1 microM) had no effect on the nonadrenergic noncholinergic (NANC) depolarization and contraction produced by a short period of electrical field stimulation (EFS, 10 Hz for 1 s) in the guinea-pig colon and selectively inhibited the sustained component of depolarization produced during a prolonged period of EFS (3 Hz for 3 min), without affecting the concomitant depolarization. Nifedipine (1 microM) eliminated the NANC contraction to a short period of EFS and the phasic contraction in response to a prolonged period of EFS. MEN 11420 (1 microM) abolished the nifedipine-resistant NANC contraction produced by prolonged period of electrical field stimulation (EFS, 3 Hz for 3 min). All electrical and mechanical NANC responses to EFS which were resistant to MEN 11420, either in the absence or presence of nifedipine, were abolished by the subsequent application of the NK1 receptor antagonist, SR 140333 (1 microM). Up to 3 microM, MEN 11420 had no significant effect on the cholinergic excitatory junction potential or the NANC inhibitory junction potential evoked by single pulse EFS, nor did it affect membrane conductance. In urethane-anaesthetized guinea-pigs MEN 11420 (10-100 nmol/kg i.v.) produced a dose-dependent and long lasting (> 3 h) inhibition of the contractile response (15 +/- 2 mmHg) of the proximal colon induced by [beta Ala8]NKA (4-10) (3 nmol/kg i.v.). MEN 11420 (300 nmol/kg i.v.) did not affect the contraction produced by [Sar9]SP sulfone. MEN 11420 (300 nmol/kg) produced a limited (Emax about 40% inhibition) and transient (recovery within 60 min) inhibition of the atropine- and hexamethonium-sensitive phasic contractions of the proximal colon induced by threshold distension of a colonic balloon. On the other hand, MEN 11420 (10-300 nmol/kg i.v.) produced a dose-dependent complete and prolonged (> 2 h from administration) inhibition of the atropine-resistant and hexamethonium-sensitive phasic contraction induced by suprathreshold distension of the colonic balloon. We conclude that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist devoid of significant inhibitory activity toward excitatory transmission mediated via tachykinin NK1 or muscarinic receptors. The present findings indicate that SP and NKA are likely involved in the preferential activation of NK1 and NK2 receptors during tachykininergi

Tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitatory neuromuscular transmission in the human ileum.

Tachykinin NK1 and NK2 receptor selective antagonists and agonists were used to study excitatory non-adrenergic non-cholinergic (NANC) transmission in circular muscle strips from human ileum by the sucrose-gap method. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), apamin (0.1 microM), and N omega-nitro-L-arginine (L-NOARG, 30 microM), electrical field simulation (EFS) produced a NANC inhibitory junction potential (i.j.p.) followed by NANC excitatory junction potential (e.j.p.) with superimposed action potentials and contraction of the circular muscle of human ileum. The selective tachykinin NK1 receptor antagonist, GR 82334 (0.1-3 microM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. (IC50 = 0.21 microM) and contraction (IC50 = 0.21 microM). The selective tachykinin NK2 receptor antagonist, MEN 10627 (0.01-1 microM), likewise produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. (IC50 = 0.07 microM) and contraction (IC50 = 0.03 microM). Either antagonist was more effective in inhibiting the mechanical than the electrical response to EFS. Neither GR 82334 nor MEN 10627 had any effect on the apamin- and L-NOARG-resistant NANC i.j.p. Activation of the NK1 or NK2 receptors by the selective receptor agonists, [Sar9]substance P (SP) sulfone and [beta Ala8]neurokinin A (NKA) (4-10), respectively (0.3 microM for 20 s each), produced depolarization with superimposed action potentials and contractions. GR 82334 selectively inhibited the responses to [Sar9]]SP sulfone, without affecting the responses to [beta Ala8]NKA (4-10). MEN 10627 inhibited the responses to [beta Ala8]NKA (4-10), without affecting the responses to [Sar9]SP sulfone. We conclude that both tachykinin NK1 and NK2 receptors co-operate in producing NANC excitation and contraction of the circular muscle in human ileum.