Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea-pig ileum and proximal colon.

1. The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea-pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6-11) (septide) and [beta Ala8]NKA(4-10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2. The natural agonists, SP and NKA, produced concentration-dependent contraction in both preparations. EC50 values were 100 pM and 5 nM for SP, 1.2 nM and 19 nM for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). 3. Synthetic NK1 and NK2 receptor agonists produced concentration-dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pM, 36 pM and 10 nM in the ileum, 8 nM, 0.7 nM and 12 nM in the colon for [Sar9]SP sulphone, septide and [beta Ala8]NKA-(4-10), respectively. The pseudopeptide derivative of NKA(4-10), MDL 28,564 behaved as a full or near-to-full agonist in both preparations, its EC50s being 474 nM and 55 nM in the ileum and colon, respectively. 4. Nifedipine (1 microM) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [beta Ala8]NKA(4-10) was abolished in the ileum and largely unaffected in the colon. 5. The NK1 receptor antagonists, (+/-)-CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [beta Ala8]NKA(4-10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6. The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [beta Ala8]NKA(4-10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7. MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [beta Ala8]NKA(4-10) in the ileum and colon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitation and contraction in the circular muscle of guinea-pig duodenum.

1. In the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), apamin (0.1 microM) and L-nitroarginine (L-NOARG, 30 microM), electrical field simulation (EFS) produced a nonadrenergic, noncholinergic (NANC) excitatory junctional potential (e.j.p.), action potentials and contraction of the circular muscle of the guinea-pig proximal duodenum, recorded by the single sucrose gap technique. 2. The selective tachykinin (TK) NK1 receptor antagonist, GR 82,334 (30 nM-3 microM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. Similarly, the selective NK2 receptor antagonists, MEN 10,627 (30 nM-3 microM) and GR 94,800 (100 nM-10 microM), both produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. and contraction. GR 82,334 inhibited the electrical and mechanical NANC responses to EFS in an almost parallel manner, while MEN 10,627 and GR 94,800 were more effective in inhibiting the mechanical than the electrical response to EFS. 3. Activation of the NK1 or NK2 receptor by the selective agonists, [Sar9]substance P (SP) sulphone and [beta Ala8]neurokinin A (NKA) (4-10), respectively (0.3 microM each), produced depolarization, action potentials and contractions. GR 82,334 selectively inhibited the responses to [Sar9]SP sulphone, without affecting the responses to [beta Ala8]NKA (4-10). MEN 10,627 and GR 94,800 inhibited or abolished the responses to [beta Ala8]NKA (4-10), without affecting the responses to [Sar9]SP sulphone. 4. Nifedipine (1 microM) abolished the action potentials and contraction produced either by EFS or by the TK receptor agonists [Sar9]SP sulphone or [beta Ala8]NKA (4-10). 5. In the presence of nifedipine, the NANC e.j.p. produced by EFS was biphasic: in the majority of strips tested (21 out of 29) an early fast phase of depolarization was followed by a second slow component. The combined administration of GR 82,334 and GR 94,800 (3 microM each) reduced both components, the slow phase being inhibited to a greater extent than the fast phase. 6. The P2 purinoreceptor antagonist, suramin (100 microM) reduced the fast phase of the e.j.p. produced by EFS in the presence of nifedipine, without affecting the slow phase. The combined administration of suramin, GR 82,334 and GR 94,800 produced a nearly complete blockade of the e.j.p. produced by EFS in the presence of nifedipine. 7. When tested in the absence of apamin and L-NOARG, EFS induced a NANC inhibitory junction potential (i.j.p.) followed by an e.j.p., and the selective P2Y receptor agonist, adenosine-5'-O-(2-thiodiphosphate) (ADP beta S, 10 microM), produced membrane hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of cyclopiazonic acid on contractions produced by tachykinin NK1 and NK2 receptor agonists in the circular muscle of guinea-pig colon

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK₁ and NK₂ receptor agonists, [Sar⁹]substance P (SP) sulfone and[βAla⁸]neurokinin A (NKA) (4–10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 μm ) and indomethacin (10 μm ). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nm ) was selected: [Sar⁹]SP sulfone (10 nm ) produced a biphasic contraction, the two amplitudes averaging 75 ± 2 and 43 ± 3% of the maximal response to KCl (80 mm ) at 1 and 15 min from application of the agonist, respectively. CPA (3 μm for 60 min) slightly reduced the phasic response to [Sar⁹]SP sulfone (16 ± 4% inhibition) and markedly suppressed the tonic component (89 ± 3% inhibition). 3 The contraction produced by [βAla⁸]NKA (4–10) (10 nm ) was more sustained than that induced by the NK₁ receptor agonist: it averaged 69 ± 5 and 73 ± 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [βAla⁸]NKA (4–10) (18 ± 7 and 21 ± 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK₁ and NK₂ receptor antagonists (SR 140333 and MEN 10627, respectively, 1 μm each) and of l -nitroarginine (100 μm ), KCl (40 mm ) produced a distinct phasic and tonic contraction which was suppressed by 1 mm nifedipine. CPA (3 μm ) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 μm nifedipine, the response to [βAla⁸]NKA (4–10) was slightly depressed (32 ± 6% inhibition) in its early component only, while the response to [Sar⁹]SP sulfone was abolished. CPA produced a slight inhibition (15 ± 9 and 33 ± 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [βAla⁸]NKA (4–10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [βAla⁸]NKA (4–10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar⁹]SP sulfone (0.1 μm for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 μm ) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar⁹]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK₁ receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK₂ receptor-mediated contractile responses.     

Occurrence and pharmacological characterization of four human tachykinin NK2 receptor variants

Tachykinin NK(2) receptor antagonists are potentially beneficial in treating various disorders including irritable bowel syndrome, urinary incontinence, depression and anxiety. The current study evaluates the frequency of single nucleotide polymorphisms (SNPs) in the human NK(2) receptor gene (TACR2). In addition, the potency of the endogenous peptide agonist neurokinin A (NKA), and the small molecule antagonists saredutant (NK(2)-selective) and ZD6021 (pan-NK antagonist) at the various NK(2) receptor protein variants were determined. The TACR2 gene was sequenced from 37 individuals. Two amino acid changing SNPs encoding the NK(2) receptor variants Ile23Thr and Arg375His were found. The frequency of the four possible protein variants differed between populations. Site-directed mutagenesis was performed introducing either SNP or both SNPs into the TACR2 gene and the constructs were transfected into CHO cells. NKA-evoked increases in intracellular Ca(2+) were monitored by FLIPR. The potency of saredutant and ZD6021 was evaluated by their ability to inhibit NKA-induced increases in intracellular Ca(2+). NKA evoked increases in intracellular Ca(2+) with a potency ranging between 1 and 5nM in CHO cells expressing the different constructs. Saredutant and ZD6021 blocked NKA-evoked increases in intracellular Ca(2+) with pK(b) values ranging between 8.8-9.3 and 7.9-8.7, respectively. The current study demonstrates that polymorphisms leading to the Ile23Thr and Arg375His amino acid exchanges are highly prevalent in the human TACR2 gene. These polymorphisms however do not appear to affect the potency of the endogenous agonist NKA or the small molecule antagonists saredutant and ZD6021 with respect to intracellular Ca(2+) signalling.     

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 μM), action potentials (APs) and contraction (IC₅₀ 3.7 μM) produced by the muscarinic receptor agonist, methacholine. It also produced a concentration-dependent inhibition of APs and accompanying contraction (IC₅₀ 31 μM) 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 μM) otilonium bromide enhanced the e.j.p. and, at higher concentrations (IC₅₀ 22 μM and 16 μM 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 μM) 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 μM and 45 μM, respectively) induced by the tachykinin NK₂ receptor agonist [βAla⁸]neurokinin A (4–10). When tested in the presence of nifedipine (1 μM), 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 [βAla⁸]neurokinin A (4–10) (IC₅₀ 41 μM). 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 [βAla⁸]neurokinin A (4–10) (IC₅₀ 60 μM and 54 μM, respectively). In radioligand binding experiments otilonium bromide produced a concentration-dependent inhibition of the binding of both an agonist ([¹²⁵I]neurokinin A, K _i 7.2 μM) and an antagonist ([³H]SR 48968, K _i 2.2 μM) to membranes of Chinese hamster ovary cells transfected with the human tachykinin NK₂ receptor. In conclusion, the present findings demonstrate that, in the μM 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. Received: 27 November 1998 / Accepted: 2 February 1999     

Human tachykinin NK2 receptor: a comparative study of the colon and urinary bladder

1. The present study compared the binding and functional characteristics of tachykinin NK2 receptors in human detrusor muscle with those in human colon circular muscle. 2. In radioligand binding studies, similar KD values were observed for tachykinin NK2 receptor radioligands [125I]-neurokinin (NK) A, [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10,]NKA(4-10) and [3H]-SR48968 in both human colon circular muscle (0.28-1.1 nmol/L) and human bladder detrusor (0.49-0.91 nmol/L), suggesting binding was primarily to tachykinin NK2 receptors. Receptor capacity (Bmax) was greater in colon compared with detrusor muscle. 3. In functional studies of isolated smooth muscle contraction, there was an excellent positive correlation between human bladder detrusor and colon circular muscle with respect to in vitro contractile potency (r = 0.97) and maximum responses (r = 0.98) to tachykinins, selective tachykinin receptor ligands and l-Ala-substituted NKA(4-10) analogues. 4. Species differences between the human and rat tachykinin NK2 receptors were apparent as observed by a low correlation for potency (r = 0.77) and efficacy (r = 0.32) of l-Ala-substituted analogues in isolated smooth muscle contractile studies. 5. Minor differences observed in the affinity and potency of NK2 receptor agonists between colon and bladder are dependent on the tissue of interest, the receptor-effector coupling and the presence of other tachykinin receptors. Overall, the NK2 receptors of human colon and urinary bladder smooth muscle appear pharmacologically identical.     

Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon.

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bidirectional regulation of human colonic smooth muscle contractility by tachykinin NK(2) receptors

In this study, we attempted to clarify the mechanism of tachykinin-induced motor response in isolated smooth muscle preparations of the human colon. Fresh specimens of normal colon were obtained from patients suffering from colonic cancer. Using mucosa-free smooth muscle strips, smooth muscle tension with circular direction was monitored isometrically. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) produced marked contraction. All of these contractions were inhibited by saredutant, a selective NK(2)-R antagonist, but not by CP122721, a selective NK(1)-R antagonist or talnetant, a selective NK(3)-R antagonist. βAla(8)-NKA(4-10) induced concentration-dependent contraction similar to NKA, but Sar(9)-Met(11)-SP and Met-Phe(7)-NKB did not cause marked contraction. Colonic contraction induced by βAla(8)-NKA(4-10) was completely blocked by saredutant, but not by atropine. Tetrodotoxin or N(G)-nitro-L-arginine methyl ester pretreatment significantly enhanced βAla(8)-NKA(4-10)-induced contraction. Immunohistochemical analysis showed that the NK(2)-R was expressed on the smooth muscle layers and myenteric plexus where it was also co-expressed with neuronal nitric oxide synthase in the myenteric plexus. These results suggest that the NK(2)-R is a major contributor to tachykinin-induced smooth muscle contraction in human colon and that the NK(2)-R-mediated response consists of an excitatory component via direct action on the smooth muscle and an inhibitory component possibly via nitric oxide neurons.     

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-β-D-Glc)-Asp-Trp-Phe-Dap-Leu] c(2β–5β)) 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, [βAla⁸]neurokinin A (NKA) (4–10) with a pK_B value of 8.1. Up to 1 μM 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 μM (dose ratio 5.3) was much smaller than that produced against [βAla⁸]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 μM) and contraction (IC₅₀ = 0.32 μM) produced by [βAla⁸]NKA (4–10) (0.3 μM 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 [βAla⁸]NKA(4– 10) with IC_50s of 99 and 75 nM, respectively. MEN 11420 (1 μM) 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 μM) 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 μM) 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 μM). Up to 3 μM, 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 [βAla⁸]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 tachykininergic transmission, although NKA can act as an effective NK₁ receptor ligand. Received: 24 March 1997 / Accepted: 9 June 1997     

The human tachykinin NK1 (short form) and tachykinin NK4 receptor: a reappraisal

Excessive secretion of placental neurokinin B into the circulation during the third trimester of pregnancy is seen in women with preeclampsia. To determine a role for neurokinin B, we have used a number of different animal models to ascertain the expression of the three tachykinin receptors (NK1--both short and long forms, NK2 and NK3) and the putative human tachykinin NK4 receptor in the placenta. Human and rat placenta express all three classical tachykinin receptors. However, we failed to reveal the expression of the short tachykinin NK1 receptor or the tachykinin NK4 receptor in any of 24 human tissues examined including the placenta. We conclude that the proposed short form of the tachykinin NK1 receptor is a truncated genomic clone and that the human tachykinin NK4 receptor is in fact, the guinea pig tachykinin NK3 receptor.     

In vitro characterization of tachykinin NK2-receptors modulating motor responses of human colonic muscle strips

1. Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose-dependently contracted by neurokinin A (EC₅₀, 4.9 nM), the tachykinin NK₂-receptor selective agonist [β-Ala⁸]neurokinin A (4–10) ([β-Ala⁸]NKA (4–10)) (EC₅₀, 5.0 nM), neurokinin B (EC₅₀, 5.3 nM) and substance P (EC₅₀, 160 nM), but not by the tachykinin NK₁-receptor selective agonist [Sar⁹Met(O₂)¹¹] substance P, or the NK₃-receptor selective agonists, senktide and [MePhe⁷] neurokinin B. No regional differences between transverse and distal colon were observed in response to [β-Ala⁸]NKA (4–10).
2. Atropine (1 μM) and tetrodotoxin (1 μM) did not significantly inhibit responses to [β-Ala⁸]NKA (4–10), neurokinin A, substance P or neurokinin B.
3. The newly developed non-peptide antagonists for tachykinin NK₂-receptors SR 48968, SR 144190 and its N-demethyl (SR 144743) and N,N-demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [β-Ala⁸]NKA (4–10), with similar potency (Schild plot pA₂ values 9.4, 9.4 and 9.3, slope=1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept∼9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co-incubation of SR 48968 (10, 100 nM) and SR 144782 (10 nM) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pK_B) was 8.2. In addition, SR 144782 (0.1 μM) antagonized responses to neurokinin A, substance P and neurokinin B, with pK_B consistent with its affinity for tachykinin NK₂-receptors. The potent and selective NK₁ and NK₃-receptor antagonists, SR 140333 and SR 142801 (both 0.1 μM), failed to inhibit contractions induced by SP or NKB.
4. In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non-neuronal tachykinin NK₂-receptors, but not tachykinin NK₁- or NK₃-receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK₂-receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK₂-receptor might well account for the complexity of the observed interaction.

     

Functional characterization of tachykinin NK1 receptors in the mouse uterus

1. Contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of oestrogen-treated mice. 2. In the presence of thiorphan (3 microM), captopril (10 microM), and bestatin (10 microM), substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-related contractions of uterine preparations. The order of potency was SP > or =NKA>NKB. 3. Neither atropine (0.1 microM) nor l-NOLA (100 microM), nor indomethacin (10 microM) alone or in combination with either ranitidine (10 microM) or mepyramine (10 microM), affected responses to SP. These findings indicate that SP actions are not mediated or modulated through the release of acetylcholine, nitric oxide, prostanoids or histamine. 4. In the presence of peptidase inhibitors, the tachykinin NK(1) receptor-selective agonist [Sar(9)Met(O(2))(11)]SP, produced a concentration-dependent contractile effect. The tachykinin NK(2) and NK(3) receptor-selective agonists [Lys(5)MeLeu(9)Nle(10)]NKA(4-10) and [MePhe(7)]NKB were relatively inactive. The potencies of SP analogues in which Glu replaced Gln(5) and/or Gln(6) were similar to that of SP. 5. The tachykinin NK(1) receptor-selective antagonist, SR140333 (10 nM), alone or combined with the tachykinin NK(2) receptor-selective antagonist, SR48968 (10 nM), shifted log concentration curves to SP, NKA and NKB to the right. SR140333 (10 nM) reduced the effect of [Sar(9)Met(O(2))(11)]SP. SR48968 did not affect responses to SP or [Sar(9)Met(O(2))(11)]SP, but reduced the effect of higher concentrations of NKA and shifted the log concentration-response curve to NKB to the right. The tachykinin NK(3) receptor-selective antagonist, SR 142801 (0.3 microM), had little effect on responses to SP and NKB. 6. We conclude that the tachykinin NK(1) receptor mediates contractile effects of SP, NKA and NKB and [Sar(9)Met(O(2))(11)]SP in myometrium from the oestrogen-primed mouse. The tachykinin NK(2) receptor may also participate in the responses to NKA and NKB.     

Responses of rat spinal neurons to distension of inflamed colon: role of tachykinin NK2 receptors

Tachykinin NK2 receptors are implicated in nociception and the control of intestinal motility. Here we examined their involvement in responses of spinal lumbosacral neurons with colon input to distension of normal or inflamed colon in anesthetized rats. The responses of single neurons to colorectal distension (5-80 mmHg), to electrical stimulation of the pelvic nerve (bypassing sensory receptors) and to somatic stimulation were characterized. The effect of cumulative doses of an NK2 receptor antagonist, MEN 11420 (10-1000 microg kg(-1) IV), on responses to these stimuli was tested in control conditions (n=6), or 45 min after intracolonic instillation of acetic acid (n=6). After colonic inflammation, neuronal responses to colorectal distension and pelvic nerve stimulation were significantly greater. MEN 11420 dose-dependently inhibited the enhanced responses to colorectal distension after inflammation (ID50=402+/-14 microg kg(-1)), but had no significant effect on responses to pelvic nerve stimulation or distension of the normal colon, suggesting a peripheral action selective for the inflamed colon. We conclude that MEN 11420 possesses peripheral anti-hyperalgesic effects on neuronal responses to colorectal distension. These results provide a neurophysiological basis for a possible use of tachykinin NK2 receptor antagonists in treating abdominal pain in irritable bowel syndrome patients.     

Characterization of the tachykinin NK2 receptor in the human bronchus: influence of amastatin-sensitive metabolic pathways.

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a ''NK2A'' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A.     

Small molecule antagonists of the tachykinin NK 2 receptor

The NK(2) receptor (member of the tachykinin receptor family) is mainly located in the smooth muscle of the urinary, respiratory and gastrointestinal tracts, with limited presence in the CNS. This has raised interest in tachykinin NK(2) receptor antagonists for the treatment of urological disorders, asthma. This review outlines progress done after 1998 in the field of NK(2) small molecule antagonists, both acting on the NK(1)/NK(2), NK(2)/NK(3), NK(1)/NK(2)/NK(3) receptors and selective for the NK(2) one.     

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further evidence for the existence of NK2 tachykinin receptor subtypes.

1. We have evaluated the biological activity of a number of neurokinin A (4-10), (NKA (4-10)) analogues in the endothelium-deprived rabbit isolated pulmonary artery (RPA) and hamster isolated trachea (HT), two tissues rich in different NK2 receptor subtypes. 2. MDL 28,564, a pseudopeptide selective for NK2 receptor sites, behaved as a full agonist in the RPA, while in the HT it competitively antagonized NKA or [beta Ala8]-NKA (4-10) contractile effects. 3. The peculiar behaviour of MDL 28,564 in the RPA and HT may be explained neither by a difference in receptor reserve between the two organs (the reserve being three times greater in RPA than in the HT) nor by a different affinity for the two receptor subtypes (identical dissociation constants, pKA or pKB, calculated in the RPA and in the HT). On the other hand, MDL 28,564 displayed a very different intrinsic efficacy for the two receptor subtypes. 4. The novel peptides MEN 10,295 ([Trp7, beta Ala8]-NKA-(4-10)) and MEN 10,296 ([Tyr5, Trp7, beta Ala8]-NKA-(4-10] behaved as weaker agonists than MDL 28,564 in the RPA, but retained appreciable agonist activity also in the HT. 5. The novel peptides: MEN 10,282 ([Tyr5, D-Trp6,8, Trp9, Arg10]-NKA-(4-10], MEN 10,449 ([diI-Try5, D-Trp6,8,9, Arg10]-NKA-(4-10] and the cyclic hexapeptide L 659,877 (cyclo [Leu-Met-Gln-Trp-Phe-Gly]) behaved as competitive antagonists against NKA contractile effects both in the RPA and HT. MEN 10,282 and MEN 10,449 were unable to distinguish between the NK2 receptor subtypes, having almost the same affinity in the two organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and pharmacological characterization of a functional tachykinin NK3 receptor cloned from the rabbit iris sphincter muscle

1. A functional tachykinin NK3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues. 2. Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK3 receptors respectively. 3. In CHO-K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>NKA>/=SP to displace [125I]-[MePhe7]-NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]-NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7 - 0.8), suggesting the possibility of multiple affinity states of the receptor. 4. Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]-[MePhe7]-NKB was also observed in the ciliary processes using autoradiography. 5. Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and 'atypical' NK3 receptor subtypes previously reported.     

Molecular and pharmacological characterization of the murine tachykinin NK(3) receptor

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.