Bronchodilator activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a potent, long-acting, and selective muscarinic M3 receptor antagonist.

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t(½) = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [(3)H]CHF5407 dissociated quickly from hM2 receptors (t(½) = 31 min), whereas [(3)H]tiotropium dissociated slowly from both hM3 (t(½) = 163 min) and hM2 receptor (t(½) = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC(50) = 9.0-9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED(50) value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

Influence of lipophilicity on the biological activity of cyclic pseudopeptide NK-2 receptor antagonists.

A series of cyclic pseudopeptides of the formula cyclo(Leu psi[CH2NH]Xaa-Gln-Trp-Phe-beta Ala), where Xaa represents the residue of an alpha-amino acid, has been synthesized in order to establish the role of the Xaa side chain for tachykinin NK-2 receptor antagonist activity. Syntheses have been carried out in solid phase with either Fmoc or Boc strategy. The antagonist potency on NK-2 receptors in the hamster isolated trachea (HT) and the rabbit isolated pulmonary artery (RPA) bioassays increases with Xaa lipophilicity; cyclo(Leu psi[CH2NH]Cha-Gln-Trp-Phe-beta Ala) and cyclo(Leu psi[CH2NH]Asp(NHBzl)-Gln-Trp-Phe-beta Ala) resulted in being the two most active antagonists (pA2 = 9.06 and 9.26 on HT, respectively). A significant linear correlation was found between pA2 values determined in HT and RPA bioassays and capacity factors measured in reversed phase HPLC. The comparison between the biological activities of cyclic hexapeptides containing or not containing the aminomethylene moiety proved the crucial role of the pseudopeptide bond for determining high antagonist potency at the NK-2 receptor.

Tachykinin receptors are involved in the "local efferent" motor response to capsaicin in the guinea-pig small intestine and oesophagus.

The sensory neuron stimulant drug capsaicin stimulates primary afferent nerve endings in the guinea-pig small intestine, which in turn activate myenteric cholinergic neurons by an unknown mechanism. The tachykinins substance P and neurokinin A are present in primary afferent neurons. This study was performed to assess the possible involvement of endogenous tachykinins acting via neurokinin-1, neurokinin-2 and neurokinin-3 receptors in the contractile effect of capsaicin in the isolated guinea-pig ileum and oesophagus by using the receptor-specific antagonists GR 82334 (3 microM) for neurokinin-1 receptors, MEN 10627 (3 microM; ileum) or MEN 11420 (1 microM; oesophagus) for neurokinin-2 receptors and SR 142801 (0.1 microM) for neurokinin-3 receptors. In the ileum, the peak contraction evoked by capsaicin (2 microM) was not reduced when tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors were blocked separately, whereas an inhibition of neurokinin-3 receptors diminished the area under the curve of the capsaicin response. A combined blockade of neurokinin-1 and neurokinin-3 receptors significantly depressed the effect of capsaicin; the amplitude of the contractile response was 53.3+/-3.7% of the maximal longitudinal spasm in control preparations, whereas in the presence of GR 82334 plus SR 142801 it reached only 27.6+/-5% (P<0.001, Kruskal-Wallis test; n=9 and 10, respectively). Also, the area under the curve of the contractile response to capsaicin was more than 85% lower in the group of preparations treated with GR 82334 plus SR 142801 than in the control group (P<0.001). Including a neurokinin-2 blocker in the combination did not produce any further inhibition. A concomitant tachyphylaxis to substance P (natural neurokinin-1 receptor stimulant) and the neurokinin-3 receptor agonist senktide (5 and 1 microM, respectively) also reduced the contractile effect of capsaicin. In the oesophagus, capsaicin (1 microM) induced biphasic contractions which were strongly inhibited by atropine (1 microM) or capsaicin pretreatment (1 microM for 10 min). Here again, a blockade of tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors separately failed to inhibit the response to capsaicin, whereas a combined blockade of any two tachykinin receptors caused a partial inhibition. The reduction of the contractile effect of capsaicin was strongest when all three tachykinin receptors were blocked. In seven control preparations, peaks for the first and second phases of contraction reached 35.3+/-3.7% and 20+/-3.2% of maximal longitudinal spasm; the corresponding values in the presence of a combination of GR 82334, MEN 11420 and SR 142801 were 7.5+/-0.8% and 9.1+/-2.2%, respectively (n=6, P<0.001 and 0.05, respectively). Tetrodotoxin (0.5 microM) practically abolished the contractile effect of capsaicin in both tissues studied. It is concluded that an interplay of neuronal tachykinin neurokinin-1 and neurokinin-3 receptors (ileum) and neurokinin-1, neurokinin-2 and neurokinin-3 receptors (oesophagus) is involved in the contractile action of capsaicin, probably in mediating excitation of myenteric neurons by tachykinins released from primary afferents. In both tissues, there also seems to be a non-tachykininergic component of the capsaicin-induced contraction.

Low pH-induced release of calcitonin gene-related peptide from capsaicin-sensitive sensory nerves: mechanism of action and biological response.

Protons can release in a Ca(2+)-dependent manner, calcitonin gene-related peptide (CGRP)-like immunoreactivity from peripheral endings of capsaicin-sensitive afferents. Here we have studied the mechanism by which proton promotes CGRP-like immunoreactivity release and whether the neuropeptide released might exert a biological action. In muscle slices of guinea-pig urinary bladder high pH (pH 8 or 9) media neither enhanced CGRP-like immunoreactivity outflow nor affected the capsaicin-evoked CGRP-like immunoreactivity release. The CGRP-like immunoreactivity release evoked by superfusion with pH 5 medium was not affected by tetrodotoxin (0.3 microM) indomethacin (10 microM) or the protein kinase C inhibitor H-7 (30 microM). However, it was reduced by 35% in the presence of the voltage-sensitive Ca(2+)-channel antagonists nifedipine (1 microM) and omega-conotoxin (0.1 microM) and by 80% in presence of the capsaicin "antagonist" Ruthenium Red (10 microM). The CGRP-like immunoreactivity release by capsaicin (10 microM) was reduced by 80% in the presence of Ruthenium Red, and not affected by voltage-sensitive Ca(2+)-channel blockers, while that evoked by 80 mM K+ was decreased by 82% in the presence of nifedipine and omega-conotoxin. The Ca(2+)-channel agonist Bay K 8644 enhanced the high K(+)-evoked CGRP-like immunoreactivity release but not that induced by capsaicin or pH 5 medium. Exposure to pH 6 solution of one half of the neck of guinea-pig urinary bladder induced a slowly developing inhibition of electrically evoked contractions, that was absent in the half pre-exposed in vitro to a desensitizing dose of capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)

Similarities and differences in the action of resiniferatoxin and capsaicin on central and peripheral endings of primary sensory neurons.

We have compared the ability of capsaicin and resiniferatoxin, a natural diterpene present in the latex of plants of the Euphorbia family to excite and desensitize capsaicin-sensitive primary afferents in a variety of models. Both capsaicin and resiniferatoxin inhibited the twitch contractions of the rat isolated vas deferens and prevented, in a concentration-related manner, the effect of a subsequent challenge with 1 microM capsaicin (desensitization). Resiniferatoxin was 1000-10,000 times more potent than capsaicin in both cases. The time course of action of resiniferatoxin was much slower than that of capsaicin, suggesting a slower penetration rate in the tissue. The action of resiniferatoxin was blocked by Ruthenium Red, a proposed antagonist at the cation channel coupled to the capsaicin receptor. Both capsaicin and resiniferatoxin produced a contraction of the rat isolated urinary bladder. Resiniferatoxin was about as potent as capsaicin in this assay although it was 500-1000 times more potent than capsaicin in desensitizing the primary afferents to a subsequent challenge with capsaicin itself. Resiniferatoxin did not affect motility in the isolated vasa deferentia or urinary bladder from capsaicin-pretreated rats. After topical application onto the rat urinary bladder both resiniferatoxin (10 nM) and capsaicin (10 microM) increased bladder capacity as assessed in a volume-evoked micturition reflex model in rats without affecting micturition contraction. Intrarterial injection of resiniferatoxin or capsaicin in the ear of anesthetized rabbits evoked a systemic depressor reflex due to activation of paravascular nociceptors, resiniferatoxin being about three times more potent than capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional differences in the motor response to capsaicin in the guinea-pig urinary bladder: relative role of pre- and postjunctional factors related to neuropeptide-containing sensory nerves.

Capsaicin induced a contraction of isolated strips from the guinea-pig urinary bladder which was more evident in the dome than in the neck and inhibited contractions induced by field stimulation, particularly in the neck. Both responses exhibited prompt desensitization and were tetrodotoxin-resistant, suggesting a specific action on transmitter release from sensory nerve terminals. Indeed, the contractile response in the dome was prevented by a substance P antagonist while the inhibitory response in the neck was prevented by immunoblockade with anticalcitonin gene-related peptide (CGRP) serum. Substance P produced a contraction of the guinea-pig bladder, being about 5 times more potent in the dome than in the neck, while CGRP inhibited the evoked contractions, being about 8 times more potent in the neck than in the dome. Further, the maximal effect of CGRP in the neck was almost double that in the dome. Substance P- and CGRP-like immunoreactivity were detected in both the dome and the neck with no regional differences for each peptide. CGRP-like immunoreactivity was 6.3 and 7.9 times higher than substance P-like immunoreactivity in the dome and the neck, respectively. Exposure to capsaicin evoked release of both substance P- and CGRP-like immunoreactivity from the dome and the neck. Peak CGRP-like immunoreactivity released by capsaicin was 12.3 and 8 times greater than substance P-like immunoreactivity in the dome and the neck, respectively. For each peptide, no difference was found in peak release in the dome vs neck. Total substance P-like immunoreactivity released from the neck was 25% lower than that released from the dome. The ability of CGRP to stimulate accumulation of 3',5' cyclic adenosine monophosphate in membranes prepared from the bladder muscle was greater in preparations from the neck than from the dome. These findings indicate that postjunctional mechanisms (type and number of receptors for sensory neuropeptides, coupling with second messengers) are a major determinant of the type of motor responses consequent of the release of sensory neuropeptides from capsaicin-sensitive nerves.

Functional, biochemical and anatomical changes in the rat urinary bladder induced by perigangliar injection of colchicine.

The aim of this study was to assess the effect of blocking the axonal transport of sensory neuropeptides, by local injection of colchicine at pelvic ganglia level, on the sensory and efferent functions mediated by capsaicin-sensitive primary afferent neurons innervating the rat urinary bladder. Bilateral injection of colchicine in the prostatic tissue underneath the pelvic ganglia of male rats induced a time-dependent reduction (maximal at 72 h, 100% reduction) of the in vitro contraction of the bladder strips induced by capsaicin (1 microM). The response to electrical field stimulation was also reduced, although to a lesser extent. The direct contractions induced by substance P (100 nM) or KCl (80 mM) were not affected by colchicine pretreatment. In vivo, perigangliar injection of colchicine (72 h before) greatly increased bladder capacity, and reduced the amplitude of micturition contractions and micturition frequency. Capsaicin-induced plasma protein extravasation was abolished in the urinary bladder and reduced in the distal, but not the proximal ureter of colchicine-treated rats. Topical application of capsaicin onto the urinary bladder or onto the stomach induced a cardiovascular pressor reflex in urethane-anaesthetized, spinalized rats. Colchicine pretreatment reduced (by about 50%) the pressor response elicited by chemonociceptive stimulation of the bladder but not that arising from the stomach. Colchicine pretreatment did not produce overt changes of nerve profiles immunoreactive for calcitonin gene-related peptide- or tachykinin-like material in the rat urinary bladder. A more intense staining of nerve fibres positive for calcitonin-gene related peptide-like immunoreactivity and tachykinin-like immunoreactivity was observed in pelvic ganglia of colchicine-pretreated rats. No changes were detected in the dorsal horns of spinal cord segments where pelvic bladder afferents project (L6-S1). Colchicine pretreatment reduced, but did not abolish, bladder levels of substance P-, neurokinin A-, calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity. However, vasoactive intestinal peptide-like immunoreactivity levels were not changed. The capsaicin-evoked (1 microM) release of calcitonin gene-related peptide was abolished in capsaicin as well as in colchicine-pretreated animals. The present findings demonstrate that local treatment of pelvic ganglia with colchicine totally eliminates the "efferent" functions of capsaicin-sensitive afferent nerves in the urinary bladder. Although reduced, tissue levels of sensory neuropeptides are not completely depleted, thus indicating the existence of a releasable versus non-releasable pool. The chemically induced blockade of axoplasmic transport also induces a limited impairment of the sensory function of capsaicin-sensitive afferents, and of the parasympathetic efferent system.

Motor response of the human isolated colon to capsaicin and its relationship to release of vasoactive intestinal polypeptide.

The aim of this study was to obtain indirect evidence of the presence of capsaicin-sensitive afferents in the human colon by studying the motor response to capsaicin of longitudinal strips from the human isolated taenia coli in parallel to the ability of capsaicin or KCl to induce peptide release from the human superfused colon. Capsaicin (1 microM) evoked a relaxation of the taenia, approaching 60-80% of the response to isoprenaline. Tachykinins evoked contractions of the taenia, while calcitonin gene-related peptide induced a relaxation. Neither tachyphylaxis to calcitonin gene-related peptide nor preincubation with an anti-calcitonin gene-related peptide serum did block the response to capsaicin which was also unaffected by tetrodotoxin, apamin, naloxone or an anti-galanin serum. Vasoactive intestinal polypeptide produced a concentration-dependent tetrodotoxin-resistant relaxation which was shifted rightward in the presence of anti-vasoactive intestinal polypeptide serum. The anti-vasoactive intestinal polypeptide serum reduced the response to capsaicin and application of capsaicin prevented the ability of anti-vasoactive intestinal polypeptide serum to block exogenous vasoactive intestinal polypeptide. Capsaicin (1 microM) evoked a significant release of vasoactive intestinal polypeptide-like immunoreactivity from the superfused muscle but not mucosa of the human colon. A significant vasoactive intestinal polypeptide-like immunoreactivity release was also observed in response to KCl (80 mM). KCl but not capsaicin evoked a significant release of neurokinin A-like immunoreactivity from colonic muscle and mucosa. No significant release of either substance P-, neuropeptide Y-, galanin- or calcitonin gene-related peptide-like immunoreactivity was detected in response to capsaicin or KCl although detectable levels of each peptide were evident in tissue extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Tachykinin NK1 receptor in the guinea-pig isolated proximal urethra: characterization by receptor selective agonists and antagonists.

1. The tachykinin receptor mediating contraction of the guinea-pig isolated proximal urethra has been characterized by use of receptor selective agonists and antagonists. All experiments were performed in the presence of peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each) in order to reduce peptide degradation. 2. The natural tachykinins, substance P and neurokinin A produced a concentration-dependent contraction of rings of the proximal urethra which approached the same maximum (about 50% of the response to 80 mM KCl). Substance P (EC50 155 nM) was slightly (3.6 times) more potent than neurokinin A (EC50 560 nM). 3. The tachykinin NK1 receptor selective agonist, [Sar9]substance P sulphone (EC50 62 nM), was slightly more potent than substance P and produced the same maximal response of natural tachykinins. The NK2 receptor selective agonist, [beta Ala8] neurokinin A(4-10), was active only at microM concentrations and its maximal effect did not exceed 20% of that to substance P or neurokinin A. The NK3 receptor selective agonist, senktide, was ineffective up to 30 microM. 4. The response to [Sar9]substance P sulphone was antagonized in a competitive manner by either (+/-)-CP 96,345 (pA2 7.75, slope - 1.10) or GR 82,334 (pA2 7.31, slope - 1.26), which are selective NK1 receptor antagonists, while it was unaffected (up to 10 microM) by MEN 10,376, a selective NK2 receptor antagonist. 5. The response to 10 microM [beta Ala8]neurokinin A (4-10) was abolished by either 0.2 microM (+/-)-CP 96,345 or 1 microM GR 82,334, suggesting the involvement of NK1 receptors.6. Electrical field stimulation (5 and 10 Hz, 0.25 ms, 100 V, trains of 5 s duration) produced tetrodotoxin-sensitive phasic contractions of the urethra which were abolished by atropine plus phentolamine (3 microM each). Capsaicin (1 microM) produced a small transient contraction of the urethra which was abolished by ( )-CP 96,345 (0.1 microM). ( )-CP 96,345 did not modify the response to electrical field stimulation.7. We conclude that tachykinin NK, receptors are the main if not the only mediators of the contractile response of guinea-pig proximal urethra to peptides of this family and that this preparation is useful for assessing the affinities of various ligands for the NK, receptor. Endogenous tachykinins released from peripheral endings of capsaicin-sensitive primary afferents produce urethral contraction by activating NK, receptors.

Tachykinin NK1 receptor subtypes in the rat urinary bladder.

1. Following the recent proposal that the selective agonist septide, ([pGlu6,Pro9]SP(6-11)), acts on a novel tachykinin receptor distinct from the 'classical' NK1 receptor, the aim of the study was to investigate the possible heterogeneity of tachykinin NK1 receptors in the rat urinary bladder. 2. The synthetic tachykinin receptor agonists, septide (pD2 7.87) and [Sar9]substance P (SP) sulphone (pD2 7.64) produced concentration-dependent contractions of the rat isolated urinary bladder. 3. The NK1 receptor antagonists GR82,334, (+/-)-CP96,345, and RP67,580 competitively antagonized (slopes of Schild plot not significantly different from unity) the response to septide with the rank order of potency (pKB values in parentheses): RP 67,580 (7.57) > GR 82,334 (7.01) > (+/-)-CP 96,345 (6.80). The same antagonists were significantly less potent when tested against [Sar9]SP sulphone, while maintaining the same rank order of potency: RP 67,580 (7.00) > GR 82,334 (5.93) > (+/-)-CP 96,345 (< 6). The antagonists did not affect the concentration-response curve to bombesin. 4. To exclude the involvement of the NK2 receptor, a second series of experiments was performed in the presence of the potent nonpeptide NK2 receptor antagonist, SR 48,968. SR 48,968 (1 microM) produced a rightward shift of the concentration-response curve to the NK2 receptor selective agonist, [beta Ala8]neurokinin A (NKA) (4-10). SR 48,968 did not significantly modify the response to SP, NKA, neurokinin B (NKB), neuropeptide K (NPK), neuropeptide gamma (NP gamma), SP(4-11), SP(6-11), septide or [Sar9]SP sulphone. 5. In the absence or presence of SR 48,968, RP 67,580 antagonized in a competitive manner the response to septide, [Sar9]SP sulphone, SP(4-11) and SP(6-11): pKB values obtained in the absence and presence of SR 48,968 were not significantly different for any of these four agonists.6. RP 67,580 antagonized the response to SP and NKA both in the absence and presence of SR 48,968.In both cases, the slopes of the Schild plots were significantly different from unity. Mean dose-ratios produced by RP 67,580 in the presence of SR 48,968 were larger than those measured without NK2receptor blockade for both SP and NKA.7. RP 67,580 (3 MicroM) did not antagonize the response to NKB in the absence of SR 48,968. In the presence of SR 48,968, RP 67,580 acted as a competitive antagonist of NKB-induced contractions with apKB value (7.63) not significantly different from that measured towards septide. In the present of SR48,968, RP 67,580, GR 82,334 and (+/-)-CP 96,345 antagonized the response to NKB with a rank order of potency identical to that measured towards septide or [Sar9]SP sulphone.8. In the absence of SR 48,968, RP 67,580 (3 MicroM) produced a small shift of the concentration-response curve to neuropeptide K and was ineffective toward neuropeptide T. In the presence of SR 48,968 a clear shift of the curve to both agonists was observed.9. These findings are compatible with the idea that a septide-sensitive tachykinin receptor may exist in the rat urinary bladder. The septide-sensitive receptor is recognized by NK1 receptor antagonists with higher affinity than the 'classical' NK1 receptor recognized by [Sar9]SP sulphone. Our data suggest that NKB, after NK2 receptor blockade, is a more suitable ligand than SP for activation of the 'septidesensitive'receptor. While the final proof for the existence of possible NK1 receptor subtypes must await confirmation at the molecular level, the present findings provide strong pharmacological evidence that either NK, receptor subtypes or a novel type of tachykinin receptor exist in the rat urinary bladder.

Structure-activity studies on endothelin (16-21), the C-terminal hexapeptide of the endothelins, in the guinea-pig bronchus.

1. We describe the results of a structure-activity study in which the C-terminal hexapeptide of the endothelins, endothelin (16-21), is compared with shorter fragments; hexapeptides bearing amino-acid substitutions and the corresponding C-terminal fragments of sarafotoxins. The guinea-pig bronchus was used in this study since it is the most sensitive preparation for endothelin (16-21) thus far developed. 2. The biological results obtained with endothelin (16-21) and analogues demonstrate that the contractile activity of the C-terminal hexapeptide of endothelin on the guinea-pig bronchus depends on quite close structural requirements, strongly suggestive of a receptor interaction. The following features appear to be essential for the biological activity: (a) the C-terminal free carboxylic function; (b) the L-configuration of Trp-21; (c) the beta-carboxylic function of Asp-18; (d) the presence of Leu-17 and (e) the imidazole moiety of His-16. 3. The hexapeptide corresponding to the C-terminal portion of sarafotoxin, sarafotoxin (16-21), was devoid of biological activity. This behaviour might be related to the proposed existence of more than one receptor for the endothelin/sarafotoxin family in the guinea-pig bronchus.

Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct.

1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK1, NK2 and NK3 receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle. 2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK1 and NK3 receptor-selective agonists. 3. Among the natural tachykinins neurokinin B (EC50 = 3.2 nM; 95% c.l. = 2.0-5.1; n = 4) was the most potent, being about 40 and 25 fold more potent than substance P (EC50 = 121.6 nM; 95% c.l. = 94-157; P < 0.01; n = 4) and neurokinin A (EC50 = 83.4 nM; 95% c.l. = 62-112; P < 0.01; n = 4), respectively. Among the synthetic analogues the NK3 receptor-selective agonist senktide (EC50 = 1.1 nM; 95% c.l. = 0.7-1.8; n = 8) was the most potent, being about 120, 110 and 20 fold more potent than [Sar9]substance P sulfone (NK1 receptor-selective) (EC50 = 130.4 nM; 95% c.l. = 99-172; P < 0.01; n = 8), [beta Ala8]NKA (4-10) (NK2 receptor-selective) (EC50 = 120.1 nM; 95% c.l. = 95-151; P < 0.01; n = 8) and septide (NK1 receptor-selective) (EC50 = 22.6 nM; 95% c.l. = 18-28; P < 0.01; n = 8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar Emax, averaging about 50% of that produced by KCl (80 mM). 4. Atropine (1 microM) did not affect the responses to either NK1 or NK2 receptor-selective agonists, whereas it reduced the Emax of senktide by about 50%, without affecting its potency (EC50). Tetrodotoxin (1 microM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK1 and NK2 receptor-selective antagonists GR 82334 and MEN 11420 (1 microM each), respectively. 5. GR 82334 (1 microM) blocked with apparent competitive kinetics septide- (apparent pKB = 7.46 +/- 0.10; n = 5) and [Sar9]substance P sulfone- (apparent pKB = 6.80 +/- 0.04; n = 4) induced contractions. MEN 11420 (30-300 nM), a novel potent NK2 receptor antagonist, potently antagonized [beta Ala8]NKA (4-10), with competitive kinetics (pKB = 8.25 +/- 0.08; n = 12: Schild plot slope = -0.90; 95% c.l. = -1.4; -0.35). The NK3 receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (Emax inhibited by 64%). None of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration-response curve to methacholine (0.1-300 microM). 6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK1, NK2 and NK3 receptors. The responses obtained by activating NK1 and NK2 receptors are atropine-resistant. The contraction obtained by stimulating NK3 receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK1/NK2 receptors. The role of the NK3 receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.

Tachykinin NK1 and NK2 receptor antagonists and atropine-resistant ascending excitatory reflex to the circular muscle of the guinea-pig ileum.

1. The aim of this study was to investigate the effect of various antagonists, selective for the tachykinin NK1 or NK2 receptor, on the atropine-resistant ascending excitatory reflex (AER) to the circular muscle of the guinea-pig ileum elicited by radial stretch (balloon distension) or electrical field stimulation. 2. Submaximal and maximal atropine- (1 microM) resistant AER elicited by balloon distension averaged about 40-50% and 70-90% of maximal circular spasm to 80 mM KCl, respectively. The NK1 receptor antagonist, (+/)-CP 96,345 (1 microM) inhibited both maximal and submaximal AER. FK 888 (1-3 microM) inhibited submaximal AER only. RP 67,580 (1 microM) was ineffective. The NK2 receptor antagonist, GR 94,800, inhibited both maximal and submaximal AER at all concentrations tested (0.1-3.0 microM), while SR 48,968 was effective only at 1.0 microM. The NK2 receptor antagonists, MEN 10,376 and MEN 10,573 inhibited both submaximal and maximal AER at 10 and 1.0 microM, respectively. 3. In other experiments, an NK1 receptor antagonist, (+/-)-CP 96,345 or FK 888 (1.0 microM in each case) was administered first and the effect of GR 94,800 (1.0 microM) on the residual AER response was determined; or GR 94,800 was administered first and the effect of (+/-)-CP 96,345 or FK 888 was determined. The results of these experiments indicated an additive effect produced by the combined treatment with NK1 and NK2 receptor antagonists. 4. Electrical field stimulation (10 Hz for 0.5 s, 10-20 V, 0.15-0.3 ms pulse width) with electrodes placed at 1.4-1.8 cm anal to the recording site, produced ascending contractions which were almost abolished by 10 MicroM hexamethonium (electrically-evoked AER). In the presence of apamin (0.1 MicroM) and N0-nitro-L-arginine (30 MicroM) these contractions were reproducible over 10 consecutive stimulation cycles.GR 94,800 (1 MicroM) and FK 888 (1 MicroM) both produced a partial inhibition of the electrically-evoked AER and their combined administration produced an inhibitory effect which was larger than that induced by each antagonist alone.5. FK 888 (1-3 MicroM), GR 94,800 (1-3 MicroM), MEN 10,573 (1 MicroM) and MEN 10,376 (10 MicroM) did not significantly affect the atropine-sensitive twitch contractions produced by electrical field stimulation of the guinea-pig ileum longitudinal muscle-myenteric plexus preparation, which were abolished by 10-30 MicroM procaine, 1 MicroM tetrodotoxin or 1 MicroM atropine. (+/-)-CP 96,345 (1 MicroM) and SR 48,968 (1 ILM)produced 12% and 27% inhibition of cholinergic twitches in the longitudinal muscle of the ileum,respectively.6. We conclude that both NK1 and NK2 receptors mediate the atropine-resistant AER to the circular muscle of the ileum. NK2 receptor activation plays a more important role than NK1 receptor activation in the AER evoked by radial stretch. Since a consistent fraction of the distension- and electrically evoked atropine-resistant AER persists in the presence of combined NK1 and NK2 receptor blockade,the existence of a third excitatory transmitter to the circular muscle of the ileum, in addition to acetylcholine and tachykinins, is suggested.

Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission.

1. The possible involvement of tachykinins (TKs) in the contraction produced by capsaicin in the rat isolated urinary bladder was addressed on the hypothesis that co-release of substance P (SP) and neurokinin A (NKA) occurs from sensory nerve terminals. 2. A low concentration of SP (30 nM) produced a rapid contraction which faded to baseline within 10 min. A low concentration of NKA (10 nM) produced a slowly developing contraction which was still evident at 10 min. Capsaicin (1 microM) produced a rapid phasic response and a tonic response (late response to capsaicin). Co-administration of SP and NKA mimicked the response to capsaicin more than each TK alone. 3. Fading of the response to SP was not caused by receptor desensitization and was partially prevented by peptidase inhibitors. 4. Spantide (3 microM) selectively antagonized the SP-induced contraction while L-659,877 (3-10 microM) or MEN 10,376 (10-30 microM) which are NK2 receptor selective antagonists selectively blocked the response to NKA. Co-administration of spantide and L-659,877 inhibited the response to both SP and NKA by an amount not greater than that produced by each antagonist alone. 5. Spantide selectively reduced the peak response to capsaicin, while leaving the late response unaffected. L-659,877 (3 microM) and MEN 10,376 (10 microM) selectively inhibited the late response to capsaicin while, at higher concentrations, also reduced the peak response to capsaicin. Co-administration of spantide and L-659,877 reduced the peak response to capsaicin more than that produced by each antagonist alone. 6. Bombesin (10 nM) produced a tonic contraction similar to that induced by NKA. The response to bombesin was not affected by spantide, L-659,877 or MEN 10,376. 7 P2. purinoceptor desensitization by repeated administration of alpha,betal-methylene ATP depressed the twitch response to electrical stimulation of postganglionic nerves but did not affect the peak or the late response to capsaicin. 8. We conclude that multiple TKs are coreleased by capsaicin in the rat bladder and mediate the capsaicin-induced contraction by activating both NKI and NK2 receptors. Endogenous TK with preferential affinity for the NK, receptor (putatively SP) are selectively involved in the peak response to capsaicin while endogenous TK with preferential affinity for the NK2 receptor (putatively NKA) are selectively involved in the late response to capsaicin and partly contribute to the peak response. These findings provide pharmacological evidence for tachykinin-mediated cotransmission in the rat urinary bladder. ATP is unlikely to be involved in the efferent function of capsaicin-sensitive sensory nerves in the rat bladder.

Tachykininergic transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of NK2 receptors.

1. The effect of newly developed, receptor-selective tachykinin antagonists (GR 71,251 for NK1 receptors, MEN 10,376 and L 659,877 for NK2 receptors) on noncholinergic transmission to the circular muscle of the guinea-pig ileum has been investigated. 2. In circular muscle strips of the ileum, electrical field stimulation in the presence of atropine (2 microM) and apamin (0.1 microM) evoked a complex motor response. The tonic primary contraction in this response was reduced by GR 71,251 (10 microM) and MEN 10,376 (3-10 microM) but not by L 659,877 (up to 10 microM). The presence of apamin was necessary in this experimental arrangement to unmask an atropine-resistant primary contraction, sensitive to tachykinin antagonists. The motor response was abolished by tetrodotoxin. 3. In circular strips of the ileum GR 71,251 (10 microM) inhibited the tonic contraction produced by [Sar9] substance P sulphone, a selective NK1 receptor agonist but not that produced by [beta Ala8] neurokinin A (4-10), a selective NK2 receptor agonist. By contrast, MEN 10,376 antagonized the effect of the NK2 agonist while leaving the response to the NK1 agonist unaffected. 4. In whole segments of the ileum, distension of the gut wall by an intraluminal balloon placed at about 1 cm from the point of recording of mechanical activity of the circular muscle produced atropine-sensitive phasic contractions (ascending enteric reflex). In the presence of atropine (2 microM), a noncholinergic response was elicited, which required larger volumes of distension that the cholinergic one. The atropine-resistant ascending enteric reflex was enhanced by apamin (0.1 microM) and abolished by tetrodotoxin, either in the presence or absence of apamin.5. MEN 10,376 (3-lOmicroM) inhibited the atropine-resistant ascending enteric reflex in the presence of apamin while GR 71,251 or L 659,877 (10 microM each) were ineffective. MEN 10,376 inhibited the atropine-resistant ascending enteric reflex to a larger extent in the absence than in the presence of apamin and also slightly inhibited the ascending enteric reflex in the absence of atropine.6. These findings provide evidence for an involvement of NK2 tachykinin receptors in excitatory transmission to the circular muscle of the guinea-pig ileum. NK2 receptors are also involved in the physiological-like circular muscle activation produced by stimulation of intramural neuronal pathways which subserve the atropine-resistant ascending enteric reflex.

Facilitatory effects of selective agonists for tachykinin receptors on cholinergic neurotransmission: evidence for species differences.

1. Exogenous tachykinins modulate cholinergic neurotransmission in rabbit and guinea-pig airways. We have investigated the effect of selective tachykinin receptor agonists and antagonists on cholinergic neurotransmission evoked by electrical field stimulation (EFS) of bronchial rings in rabbit, guinea-pig and human airways in vitro to assess which type of tachykinin receptor is mediating this facilitatory effect. 2. Bronchial rings were set up for isometric tension recording. Contractile responses to EFS (60 V, 0.4 ms, 2 Hz for 10 s every min) and exogenous acetylcholine (ACh) were obtained and the effects of selective tachykinin agonists and antagonists were investigated. 3. In rabbit bronchi the endogenous tachykinins, substance P (SP) and neurokinin A (NKA) (10 nM) potentiated cholinergic responses to EFS (by 287.6 +/- 121%, P < 0.01 and 181.4 +/- 56.5%, P < 0.001 respectively). 4. The NK1 receptor selective agonist, [Sar9]SP sulphone (10 nM) evoked a maximal facilitatory action on cholinergic responses of 334.9 +/- 63% (P < 0.01) (pD2 = 8.5 +/- 0.06) an effect which was blocked by the selective NK1-receptor antagonist, CP 96,345 (100 nM) (P < 0.05) but not by the NK2 receptor antagonist, MEN 10,376 (100 nM). The NK2 receptor selective agonist, [beta Ala8]NKA(4-10) (10 nM), produced a maximum enhancement of 278 +/- 83.5% (P < 0.01) (pD2 = 8.7 +/- 0.1) an effect which was blocked by MEN 10,376 (100 nM) (P < 0.05) and not by CP 96,345. [MePhe7]NKB, an NK3 receptor selective agonist was without effect. 5. The rank order of potency of NK2 receptor antagonists against enhancement of cholinergic responses by [Beta Ala8]NKA(4-10) was MEN 10,376> L 659,877> R 396. This pattern together with the observation of the full agonist activity of MDL 28,564 indicates that the NK2 receptors in the rabbit bronchus are similar to those which are present in the rabbit pulmonary artery.6. Neither [Sar9]SP sulphone (5 nM) nor [Beta Ala8]NKA(4- 10) (1 nM) had any effect on contractile responses to ACh (10 MicroM) suggesting a pre-junctional mechanism of action.7. By contrast, in guinea-pig bronchi only the NK1-receptor agonist [Sar9]SP sulphone (3 nM) was effective in enhancing cholinergic neurotransmission but the effect was relatively small (maximal enhancement 25.7 +/- 5.5%, P<0.01). In human bronchial rings all the selective neurokinin agonists were without effect on cholinergic neurotransmission.8. These results suggest that tachykinins may play an important role in modulating cholinergic neurotransmission in rabbit (via NK1 and NK2 receptors) and guinea-pig airways (via NK1 receptor) but have no demonstrable effect on human airways

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family. The response to activation of NK3-receptors is totally neurogenic and partially mediated by endogenous acetylcholine, the response to activation of NK1-receptors is partly neurogenic and largely myogenic and the response to activation of NK2-receptors is totally myogenic.

Differential effects of BQ-123 against endothelin-1 and endothelin-3 on the rat vas deferens: evidence for an atypical endothelin receptor.

1. Endothelin-1 and endothelin-3 enhanced concentration-dependently the rat vas deferens twitch response to electrical stimulation, endothelin-1 being three times more potent. Sarafotoxin S6c was at least 200 times less active than endothelin-1. 2. The response to endothelin was antagonized in a competitive manner by the supposedly selective ETA receptor antagonist, BQ-123 (pA2:7.0 +/- 0.1). In contrast, the endothelin-1 concentration-response curve was only shifted two fold in the presence of 10 microM BQ-123, while no effect was observed at 1 microM. 3. This evidence suggests the rat vas deferens contains an endothelin receptor not conforming to the ETA/ETB receptor subtype classification so far proposed.

Multiple mechanisms in the motor responses of the guinea-pig isolated urinary bladder to bradykinin.

1. Bradykinin (1 nm-1 microM) produced a contraction of bladder strips excised from the dome of the guinea-pig urinary bladder, an effect which was greatly enhanced by removal of the mucosal layer or by thiorphan (10 microM). All subsequent experiments were performed in mucosa-free strips and in the presence of thiorphan. 2. In carbachol (5 microM)-contracted strips, bradykinin produced a concentration (1 nm-1 microM)-dependent transient relaxation. 3. Kallidin was slightly more potent than bradykinin in producing a contraction and a relaxation of the carbachol-induced tone. By contrast, [des-Arg9]-bradykinin, a selective B1 receptor agonist was barely effective up to 1 microM. 4. The contractile response to bradykinin was: (a) unaffected by either tetrodotoxin (1 microM), in vitro capsaicin desensitization (10 microM for 30 min) or apamin (0.1 microM); (b) antagonized by indomethacin (5 microM), the prostaglandin receptor antagonist SC-19220 (100 microM) or the B2 receptor antagonist [D-Arg0, Hyp3, Thi5,8, Phe7]-bradykinin (10 micron) and (c) almost abolished by nifedipine (1 microM). 5. The antagonism of the contractile response to bradykinin produced by indomethacin and SC-19220 was non-additive while that produced by indomethacin and the B2 receptor antagonist was additive. 6. The relaxant response to bradykinin was unaffected by tetrodotoxin, in vitro capsaicin desensitization or indomethacin but antagonized in a competitive manner by the B2 receptor antagonist. Further, this response was abolished by apamin (0.1 microM) but unaffected by glibenclamide (1 microM). 7. Bradykinin (10 microM) produced a consistent release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) but not substance P-LI from the guinea-pig bladder muscle. CGRP-LI release by bradykinin was greatly reduced in bladders exposed to indomethacin. [des-Arg9]-bradykinin (10 microM) was ineffective. 8. We conclude that: (a) bradykinin-induced contraction involves activation of both B2 receptors and prostanoid synthesis, via distinct mechanisms which act by inducing calcium influx via nifedipine-sensitive channels; (b) bradykinin-induced relaxation involves activation of B2 receptors and opening of apamin-sensitive potassium channels; (c) bradykinin stimulates sensory nerves in this tissue largely via prostanoid production.

Multiple sources of calcium for contraction of the human urinary bladder muscle.

1. KCl, carbachol, neurokinin A and endothelin produced concentration-dependent contractions of mucosa-free muscle strips from the dome of the human urinary bladder. The maximal response to carbachol or neurokinin A exceeded that to KCl, while the maximal response to endothelin approached that to KCl. 2. Nifedipine (1 microM) abolished the response to KCl, reduced the response to carbachol or neurokinin A but had no effect on the response to endothelin. Bay K 8644 (1 microM) markedly potentiated the response to KCl but had little or no effect on the response produced by the other stimulants. 3. Superfusion of the strips with a nominally calcium (Ca)-free medium containing EDTA (1 mM) for 30 min markedly reduced the response to carbachol, neurokinin A and endothelin, although a small response was still evident at high concentrations. Likewise, after a prolonged (60 min) superfusion of the strips with a high K (80 mM) Ca-free medium plus EDTA (1 mM) these three agonists still produced a small contractile response. 4. The nifedipine (1 microM) resistant response to carbachol, neurokinin A or endothelin was markedly depressed by LaCl3 (1 mM). In contrast, the nifedipine-(1 microM) resistant response to carbachol was not modified by NiCl2 (0.1 mM) or omega-conotoxin (0.1 microM). 5. Caffeine produced divergent effects depending upon the temperature of incubation: a relaxation at 37 degrees C and a concentration-dependent (2.5-20 mM) contraction at 25 degrees C. The latter was markedly inhibited by procaine (3 mM) but unaffected by nifedipine (1 microM). 6. After a prolonged (60 min) superfusion with a high K, Ca-free medium containing EDTA the response to carbachol (100 microM) was abolished by previous exposure to procaine (3 mM). Conversely, the response to endothelin (1 microM) was unaffected by procaine. The response to endothelin in these experimental conditions was also resistant to LaCl3 (1 mM). 7. These findings indicate that multiple sources of Ca are mobilized for contraction of the human bladder muscle by different stimulants. Dihydropyridine- and voltage-sensitive Ca channels provide the major if not the sole source of Ca for the response to KCl, play some role in the response to muscarinic (carbachol) or NK-2 tachykinin receptor stimulation but are not involved in the response to endothelin. Carbachol, neurokinin A and endothelin all mobilize a Ca pool (either extracellular or located at membrane level) which is LaCl3-sensitive but nifedipine-resistant. Neither T- nor N-type channels appear to be involved in the response to carbachol. In addition, these agents mobilize a tightly bound Ca pool independently from membrane depolarization. This latter pool is probably a procaine-sensitive intracellular source of activator Ca mobilized by caffeine and carbachol. The failure of procaine to prevent the response to endothelin in high K, Ca-free medium raises the possibility that this peptide mobilizes an intracellular source of activator Ca, distinct from the caffeine- and carbachol-sensitive pool.