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In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neurons had been achieved by incubating the tissue with capsaicin (10 microM) for 15 min. At immunohistochemistry, tachykinin- and CGRP-immunoreactivities (TK-IR/CGRP-IR) were detected in varicose nerve fibers throughout the common bile duct, while TK-IR cell bodies were observed in the terminal portion (ampulla) only. In vivo pretreatment with capsaicin (50 mg/kg; 6-7 days before) decreased the number of CGRP-IR nerves, whereas the TK-IR neural network was apparently unchanged. In conclusion, our data provide functional evidence for the presence of capsaicin-sensitive primary afferent nerve endings in the guinea-pig terminal biliary tract, whose stimulation by capsaicin or EFS produces the release of tachykinins and CGRP. In addition, morphological evidence is provided that the bulk of TK-IR material in the biliary tract is contained in intrinsic neuronal elements, while CGRP in this tissue is of extrinsic origin only. Tachykinins, probably released in small amounts by capsaicin, act by activating receptors of the NK1, NK2 and NK3 type, most probably located on intrinsic cholinergic neurons, which in turn release ACh to produce the final excitatory motor response. The contractile response to capsaicin obtained in the presence of the three tachykinin receptor antagonists could be due to the co-released CGRP and/or to other unknown neurotransmitters. CGRP produces either indirect excitatory or direct inhibitory responses by stimulation of CGRP2 and CGRP1 receptors, respectively.

We have characterized the contractile responses produced by stimulation of the tachykinin NK2 receptor in the hamster urinary bladder in vitro and in vivo. In isolated bladder strips, neurokinin A (NKA, pD2 7.40, Emax 71% of the response to 80 mM KCl) and the synthetic tachykinin NK2 receptor selective agonist [betaAla8]NKA(4-10) (pD2 7.48, Emax 77% of the response to KCl) both induced a concentration-dependent contraction, whereas the tachykinin NK1 and NK3 receptor selective agonists, [Sar9]substance P sulfone and senktide, respectively, produced a negligible contractile effect. The bicyclic peptide antagonists MEN 11420 and MEN 10627 behaved as competitive antagonists of the response to [betaAla8]NKA(4-10) with apparent pK(B) values of 9.3 and 9.7, respectively. Comparable apparent pK(B) values were estimated against NKA (pK(B) 9.2 and 9.4 for MEN 11420 and MEN 10627, respectively). Under isovolumetric recording of the intravesical pressure, the nicotinic receptor agonist DMPP (0.6 micromol/kg i.v.) produced a phasic contraction of the hamster bladder in vivo that was abolished by hexamethonium (110 micromol/kg i.v.) or by surgical ablation of pelvic ganglia. In vivo [betaAla8]NKA(4-10) (10 nmol/kg i.v.) induced a tonic-type sustained bladder contraction with superimposed high frequency and small amplitude (12 mmHg) phasic contractions and, in about 70% of cases examined, a few high amplitude (20 mmHg) phasic contractions. Hexamethonium abolished the high amplitude phasic contractions, indicating their reflex origin. In animals subjected to the ablation of pelvic ganglia, the urinary bladder response to [betaAla8]NKA(4-10) was comparable to that observed after administration of hexamethonium. Moreover, hexamethonium did not affect the contractile responses to [betaAla8]NKA(4-10) in ganglionectomized animals. MEN 10627 and MEN 11420 produced a dose-dependent and long-lasting inhibition of the contractile response to [betaAla8]NKA(4-10): the least effective doses of the two antagonists were 30 and 3 nmol/kg i.v. for MEN 10627 and MEN 11420, respectively. An almost complete and long-lasting inhibition of the response to the agonist was produced at doses of 10 and 100 nmol/kg i.v. of MEN 11420 and MEN 10627. In urethane-anaesthetized hamsters the non-stop intravesical infusion of saline (50 microl/min) produced repetitive micturition cycles which were abolished by hexamethonium (110 micromol/kg i.v.) or by surgical removal of the pelvic ganglia. MEN 11420 (100 nmol/kg) had no significant effect on the volume-evoked micturition reflex in anaesthetized hamsters. In conclusion, the hamster urinary bladder is a suitable preparation for studying the action of tachykinin NK2 receptor antagonists in vivo: in this species, the stimulation of tachykinin NK2 receptors induces bladder contractions. Blockade of tachykinin NK2 receptors does not appreciably modify the volume-evoked micturition reflex in this species.

This study investigates the role of tachykinin NK1 and NK2 receptors in motor responses induced by the intravesical instillation of capsaicin in urethane-anaesthetized rats. SR 140,333 (1 micromol/kg, i.v.), a nonpeptide NK1 receptor antagonist, abolished urinary bladder contractions induced by the selective NK1 receptor agonist [Sar9]SP-sulfone (0.1-100 nmol/kg, i.v.) without affecting those induced by the NK2 receptor agonist [betaAla8]NKA(4-10). MEN 11,420 (100 nmol/kg, i.v.), a cyclic peptide NK2 receptor antagonist, abolished bladder contractions induced by [betaAla8]NKA(4-10) (0.3-300 nmol/kg, i.v.) without modifying those induced by [Sar9]SP-sulfone. Intravesical instillation of capsaicin (6 nmol/0.6 ml/rat) produced a motor response consisting in a primary contraction followed by a series of high amplitude phasic contractions. The intravesical instillation of saline (0.6 ml/rat) produced a primary contraction of lower amplitude with respect to that induced by capsaicin and the total area under the curve was also lower in saline-instilled rats, however the number and the amplitude of phasic contractions was similar to that induced by capsaicin. MEN 11,420 (100 nmol/kg, i.v.) did not modify motor responses induced by the intravesical administration of saline. In contrast, in capsaicin-instilled rats, MEN 11,420 (100 nmol/kg, i.v.) reduced the primary contraction, the area under the curve and also the number of phasic contractions. SR 140,333 (1 micromol/kg, i.v.) reduced the primary contraction but not other parameters. The combination of SR 140,333 (1 micromol/kg, i.v.) and MEN 11,420 (100 nmol/kg, i.v.) produced an additive inhibitory effect on the primary contraction but not a further inhibition on other parameters with respect to that observed with MEN 11,420 alone. In hexamethonium (110 micromol/kg, i.v.)-pretreated animals the intravesical instillation of capsaicin produced a tonic contraction having greater amplitude and area than that induced by saline. MEN 11,420, but not SR 140,333, significantly reduced the bladder response to capsaicin in hexamethonium-pretreated rats. Again, the combined administration of MEN 11,420 and SR 140,333 did not produce further inhibitory effect in comparison to MEN 11,420 alone. It is concluded that the motor responses induced by the intravesical instillation of capsaicin are mediated by the activation of peripheral tachykinin NK2 receptors.

The effect of Hoe 140, a potent bradykinin B2 receptor antagonist, on the micturition reflex and detrusor hyperreflexia induced by chemical cystitis has been investigated in anaesthetized rats. Hoe 140 (1–100 nmol/kg i. v.) produced a dose-dependent blockade of the contraction of the rat urinary bladder induced by i. v. administration of bradykinin (100 nmol/kg) without affecting the response produced by the selective tachykinin NK-1 receptor agonist, [Sar9] substance P (SP) sulfone (1 nmol/kg i. v.). At doses which produce selective and long-lasting blockade of bradykinin receptors in the urinary bladder, Hoe 140 did not modify urodynamic parameters in normal rats. Intravesical instillation of xylene in female rats decreased bladder capacity and increased micturition frequency. These effects also occurred in rats pretreated with capsaicin as adults. Hoe 140 did not modify xylene-induced cystitis. Intraperitoneal administration of cyclophosphamide (150 mg/kg, 48 h before) decreased bladder capacity and increased micturition frequency. These effects of cyclophosphamide were abolished in rats pretreated with capsaicin as adults. Hoe 140 increased bladder capacity and decreased micturition frequency in rats pretreated with cyclophosphamide. Addition of bradykinin (10 µmol/l) to the medium in the superfused rat urinary bladder preparation evoked a prompt increase in the outflow of calcitonin gene-related peptide like immunoreactivity (CGRP-LI). Hoe 140 (3 µmol/l) inhibited (by about 50%) the CGRP-LI outflow stimulated by bradykinin. These findings demonstrate the participation of bradykinin, through 132 receptors, in the genesis of detrusor hyperreflexia during cyclophosphamide-induced cystitis. Capsaicin-sensitive primary afferent neurons are a likely target for Hoe 140 action in this model of experimental cystitis, as exemplified by its ability to prevent CGRP-LI outflow by bradykinin.

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.

In isolated strips of the hamster urinary bladder the selective tachykinin NK2 receptor agonist [betaAla8]NKA(4-10) induced a concentration-dependent (1 nM-10 microM) contraction (EC50 104 nM) associated with significant release of prostaglandin E2 (PGE2, 50+/-17 pg/mg tissue). In mucosa-free bladder strips [betaAla8]NKA(4-10) was as potent as in the presence of mucosa (EC50 46 nM), although the evoked PGE2 release was significantly less than in controls (6+/-1.7 pg/mg tissue). Dexketoprofen (10 microM) produced a significant but limited rightward shift of the concentration/response curve to [betaAla8]NKA(4-10) both in the presence and absence of the mucosal layer: the EC50 for [betaAla8]NKA(4-10) was increased five- and threefold, respectively. The evoked PGE2 release was abolished in both cases. The selective tachykinin NK2 receptor antagonist, nepadutant (10 nM-1 microM) produced a concentration-dependent and even inhibition of both contraction and PGE2 release induced by [betaAla8]NKA(4-10). The L-type calcium channel blocker, nifedipine (1 microM) and the non-selective cationic channel blocker SKF 96365 (30 microM) both inhibited the contractile response to [betaAla8]NKA(4-10) (89+/-2 and 83+/-2% inhibition, respectively). The evoked PGE2 release was not affected by nifedipine but was almost abolished by SKF 96365. Arachidonic acid (100 microM) induced a contractile response (5.9+/-0.7 mN) associated with a large production of PGE2 (383+/-78 pg/mg tissue). The contractile response to arachidonic acid was inhibited by both nifedipine (1 microM) and SKF 96365 (30 microM) (83+/-5 and 79+/-3% inhibition, respectively). The PGE2 production induced by arachidonic acid was markedly inhibited by SKF 96365 only (about 94% inhibition). Exogenous PGE2 contracted hamster bladder strips in the presence of dexketoprofen (EC50 1 microM) and strongly potentiated the contractile response to a submaximal concentration of [betaAla8]NKA(4-10). In anaesthetized hamsters the administration of [betaAla8]NKA(4-10) (10 nmol/kg, i.v.) produced a contractile response of the urinary bladder (13+/-0.4 mmHg) that was inhibited partly by dexketoprofen (25+/-3 and 35+/-4% inhibition for 0.2 and 2 mg/kg, i.v. dexketoprofen, respectively). We conclude that activation of tachykinin NK2 receptors determines prostanoid synthesis/release in the hamster urinary bladder and that this effect is largely ascribable to structures present in the bladder mucosa. Prostanoids generated following NK2 receptor activation amplify the direct contractile effect of NK2 receptor agonists. This latter response is largely due to activation of L-type calcium channels (nifedipine-sensitive) although this source of calcium apparently is not essential for activation of prostanoid synthesis.

Chemical stimulation of primary afferent nerves in the rat urinary bladder in vivo with topical capsaicin (1 microg in 50 microl saline) determines a dual motor response, consisting of a contractile effect mediated by tachykinins released from sensory nerves in the bladder wall and a transient activation of a bladder-to-bladder micturition reflex organized at the supraspinal level (chemoceptive micturition reflex). Both responses undergo complete desensitization upon repeated applications of capsaicin. The i.v. administration of the novel neuropeptide nociceptin (100 nmol/kg) produced a long-lasting protection from capsaicin desensitization of afferent nerves which mediate the chemoceptive micturition reflex. In fact a chemoceptive micturition reflex could be repeatedly evoked by topical capsaicin in nociceptin-pretreated rats. In sharp contrast, nociceptin did not influence the development of desensitization of the local response to capsaicin, corresponding to the 'efferent' function of capsaicin-sensitive afferent neurons. These results suggest that the afferent and 'efferent' function of capsaicin-sensitive primary afferent neurons (CSPANs) in the rat bladder are differentiated by nociceptin. Alternative mechanisms underlying this phenomenon are discussed.

The study aimed to establish the possible role of tachykinins as mediators of atropine-resistant reflex contractions evoked by balloon distension in the proximal duodenum of urethane-anesthetized, guanethidine (34 mumol/kg s.c.)-pretreated rats. Distension of the balloon with a small amount (0.2-0.3 ml) of saline induced the appearance of phasic rhythmic contractions (about 11 mmHg in amplitude) which were promptly suppressed by either atropine (3 mumol/kg i.v.) or hexamethonium (28 mumol/kg i.v.). Despite the continuous i.v. infusion of atropine (2 mumol/h), low-amplitude rhythmic phasic contractions recovered, which were promptly suppressed by hexamethonium, to indicate the involvement of an atropine-resistant excitatory reflex. The amplitude of these atropine-resistant contractions was increased to about 4-5 mmHg by further distension of the balloon (0.4-0.6 ml) : under these conditions, the atropine-resistant contractions undergo a progressive fading. The fading was prevented by i.v. administration of the nitric oxide (NO) synthase inhibitor, L-nitroarginine methyl ester (L-NAME, 55 mumol/h), to provide a suitable baseline (amplitude of contractions was 7-8 mmHg) for studying the effect of tachykinin receptor antagonists. I.v. administration of the selective tachykinin NK2 receptor antagonists, MEN 10,627 (10-100 nmol/kg) and SR 48968 (100-300 nmol/kg) or of the selective NK1 antagonist SR 140333 (100 nmol/kg), at doses which do not affect the duodenal contractions induced by acetylcholine (5.5 mumol/kg i.v.), produced a prompt and long lasting suppression of the atropine-resistant reflex duodenal contractions produced by balloon distension in urethane-anesthetized rats, whilst SR-48965 (300 nmol/kg), the enantiomer of SR-48968 devoid, of NK2 receptor blocking activity, was without effect. I.v. administration of the selective NK1 receptor agonists [Sar9] substance P sulfone and septide or of the NK2 receptor selective agonist, [beta Ala8] neurokinin A(4-10) produced dose-dependent contractions of the duodenum. SR 140333 (100 nmol/kg i.v.) selectively antagonized the duodenal contractions produced by [Sar9] substance P sulfone and septide without affecting those produced by [beta Ala8] neurokinin A(4-10). On the other hand, MEN 10,627 (30-100 nmol/kg i.v.) and SR 48968 (100-300 nmol/kg i.v.) but not SR 48965 (300 nmol/kg i.v.) antagonized, at a comparable extent, duodenal contractions induced by both the selective NK2 and NK1 receptor agonists. We conclude that endogenous tachykinins are involved in mediating atropine-resistant reflex contractions evoked by distension of the rat duodenum in vivo: both NK1 and NK2 receptors are activated by endogenous ligands to produce NANC contractions of rat duodenum in vivo. However, the contractile response to i.v. administered NK1 receptor agonists, [Sar9] substance P sulfone and septide, may involve the release of mediators producing smooth muscle contraction via NK2 receptors.