Inhibitory effect of capsaicin on the ascending pathway of the guinea-pig ileum and antagonism of this effect by ruthenium red.

A segment of guinea-pig ileum, which was continuous with a strip of longitudinal muscle-myenteric plexus (LM-MP) at the anal end, was used to examine the effect of capsaicin on ascending excitatory pathways. Electrical field stimulation of the LM-MP caused an ascending contraction of the segment. After initially causing contraction capsaicin (3 microM) inhibited the ascending contraction. This inhibitory effect of capsaicin exhibited rapid desensitization and was abolished after extrinsic (mesenteric) denervation. Desensitization to calcitonin gene-related peptide (CGRP) prevented the capsaicin-induced inhibition without affecting the ascending contraction. Neither naloxone nor alpha- and beta-adrenoceptor antagonists affected the capsaicin-induced inhibition. CGRP (25 nM) also inhibited the ascending contraction, mimicking the inhibition induced by capsaicin. Ruthenium red (0.1-3 microM) antagonized the capsaicin-induced inhibition in a concentration-related manner, but did not affect the CGRP-induced inhibition. These findings suggest that the inhibitory effect of capsaicin on the ascending pathways might be mediated via the release of CGRP from extrinsic nerve terminals, and that the site of the antagonism of the action of capsaicin by ruthenium red is prejunctional.     

Extrinsic origin of the capsaicin-sensitive innervation of rat duodenum: possible involvement of calcitonin gene-related peptide (CGRP) in the capsaicin-induced activation of intramural non-adrenergic non-cholinergic neurons

Capsaicin produces a concentration-related relaxation of the longitudinal muscle of the rat isolated duodenum in the presence of atropine (3 microM) plus guanethidine (3 microM). This effect of capsaicin is partly (about 40%) antagonized by tetrodotoxin (1.0 microM) suggesting the involvement of intramural non-adrenergic non-cholinergic (NANC) neurons. The capsaicin-induced relaxations are unaffected by previous bilateral vagotomy or removal of the inferior mesenteric ganglion but are completely prevented by removal of the coeliac ganglia plus the superior mesenteric ganglion (72 h before). Acute duodenal denervation did not modify the response to capsaicin. Unlike various neuropeptides (substance P, kassinin, neurokinin A, cholecystokinin octapeptide, somatostatin, vasoactive intestinal polypeptide) only the calcitonin gene-related peptide (CGRP) closely mimicked, both qualitatively and quantitatively, the capsaicin-induced relaxations. The CGRP-induced relaxations were unaffected by hexamethonium and partly reduced (about 40%) by tetrodotoxin. In preparations desensitized to adenosine-triphosphate (ATP) a putative NANC inhibitory neurotransmitter of the rat duodenum, the effects of CGRP were reduced (about 30%) as compared to controls. After ATP-desensitization tetrodotoxin did not produce any further reduction of the CGRP-induced relaxations suggesting the involvement of endogenous ATP in the neuronal (tetrodotoxin-sensitive) component of the CGRP-induced relaxations. Either ATP- or CGRP-desensitization reduced (about 50 and 65% respectively) the amplitude of the capsaicin-induced relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Several neuropeptides determine the visceromotor response to capsaicin in the guinea-pig isolated ileal longitudinal muscle

Capsaicin (1 microM) produced, after an initial contraction, a depression of the field stimulation-induced contraction of the guinea-pig isolated ileal longitudinal muscle. Both effects exhibited prompt desensitization, indicating the involvement of a specific action on sensory nerves. The initial contraction was inhibited by [D-Pro4,D-Trp7,9,Phe11]SP-(4-11), a substance P (SP) antagonist, which did not affect the inhibitory component of the response. Incubation of the strips with antiCGRP (CGRP = calcitonin gene-related peptide) serum did not modify the amplitude of the capsaicin-induced contraction but inhibited the twitch depression induced by capsaicin. AntiCGRP serum blocked the effects of exogenous CGRP but not the inhibitory response induced by baclofen. These findings provide evidence that the release of several neuropeptides from sensory nerves determines the visceromotor response to capsaicin in this preparation. In particular, a CGRP-like peptide could be responsible for the inhibitory phase which follows the initial contraction which is due to release of SP and/or related peptides.     

GABAA receptor-mediated positive inotropism in guinea-pig isolated left atria: evidence for the involvement of capsaicin-sensitive nerves.

1. Isolated left atria from reserpine-pretreated guinea-pigs, electrically driven (3 Hz) in the presence of atropine (1 microM), phentolamine (0.3 microM) and propranolol (1 microM), responded to a train of stimuli (10 Hz for 2.5s) with a delayed neurogenic positive inotropic response which was insensitive to hexamethonium (10 microM) but abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to calcitonin gene-related peptide (CGRP). 2. In these experimental conditions, gamma-aminobutyric acid (GABA) produced a concentration-related (10 microM-1 mM) positive inotropic response similar to that produced by electrical field stimulation. The effect of GABA was competitively antagonized by bicuculline methiodide (10 microM), a GABAA receptor antagonist. 3. The selective GABAA receptor agonists, muscimol and homotaurine mimicked the positive inotropic effect of GABA while baclofen, the selective GABAB receptor agonist, did not. 4. The action of GABA (1 mM) was abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to CGRP, while it was unaffected by hexamethonium. In contrast, the inotropic response to CGRP was unaffected by tetrodotoxin, omega-conotoxin, bicuculline methiodide, hexamethonium or in vitro capsaicin desensitization, but was abolished by CGRP desensitization. 5. In the spontaneously beating guinea-pig right atrium, GABA (1 microM) produced a small and transient positive chronotropic effect that was no longer observed after in vitro desensitization with capsaicin (1 microM). 6. In the guinea-pig isolated perfused heart from reserpine-pretreated animals (with atropine, phentolamine and propranolol in the perfusion medium), GABA (1 microM) produced a transient tachycardia and a small increase in coronary flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visceromotor responses to calcitonin gene-related peptide (CGRP) in the rat lower urinary tract: evidence for a transmitter role in the capsaicin-sensitive nerves of the ureter

Either intra-arterial or topical administration of calcitonin gene-related peptide (CGRP) had little effect on motility of the urinary bladder in urethane-anaesthetized rats. Only a high concentration (50 microM) of topical CGRP activated the micturition reflex and potentiated the response to exogenous substance P (SP). In the isolated rat bladder CGRP had inconsistent effects on spontaneous or field-stimulated contractions. CGRP neither produced any significant plasma extravasation (Evans blue leakage) in the rat lower urinary tract, nor potentiated the response to exogenous SP. CGRP inhibited motility in the rat isolated proximal urethra and ureters and counteracted the contractile response to neurokinins. An inhibitory effect of capsaicin on stimulated motility of the urethra was observed in all preparations and a small contractile response was evident in about 40% of cases. Lack of desensitization to the action of CGRP prevented the study of its interaction with capsaicin. The inhibitory effect of CGRP in the ureter exhibited a specific desensitization: if the preparations were pre-exposed to exogenous CGRP, the inhibition of motility produced by antidromic activation of the capsaicin-sensitive nerve terminals (field stimulation) as well as the response to capsaicin (1 microM) was prevented but the inhibitory response to isoprenaline was unaffected. These findings indicate that CGRP is able to influence markedly the motility of the rat lower urinary tract, but exhibits marked regional differences in its action. Endogenous CGRP could be the inhibitory transmitter which, when released from capsaicin-sensitive fibers, participate in the control of ureteral motility.     

Cadmium chloride induces contractions of the rat isolated urinary bladder by activation of capsaicin-sensitive sensory nerves

Cadmium chloride (CdCl2)(30 microM-1 mM) produced a concentration-related contraction of the rat isolated urinary bladder which was abolished by tetrodotoxin (1 microM) or extrinsic bladder denervation (72 h before). Complete cross-desensitization was observed between the contractile response to Cd and capsaicin, indicating that, at the peripheral level, this inorganic calcium channel blocker can activate the 'efferent' function of capsaicin-sensitive sensory nerves.     

Bronchodilatation by tachykinins and capsaicin in the mouse main bronchus.

1. The effect of sensory neuropeptides and capsaicin on basal and stimulated tone of mouse bronchial smooth muscle has been evaluated. 2. In basal conditions neither sensory neuropeptides (substance P, neurokinin A or calcitonin gene-related peptide (CGRP) nor capsaicin exerted any contractile effects. However, when a tonic contraction was induced with carbachol (1 microM) a prompt relaxation was induced by substance P (1- 100 nM) and by neurokinin A (1- 100 nM), with substance P being more potent. A second application of substance P was without effect. CGRP (10 nM) produced only a very small and erratic relaxation. Relaxation was also induced by capsaicin (1 microM), and this response could be evoked only once in each preparation. In 4 out of 6 preparations a cross-desensitization between substance P and capsaicin was observed. 3. The selective NK1 tachykinin agonist, [Pro9]-SP sulphone (1 microM), exerted potent bronchodilator actions on carbachol-contracted mouse bronchial preparations. In contrast, neither [beta Ala8]-NKA (4-10) nor [MePhe7]-NKB (both at a concentration of 1 microM), selective synthetic agonists for NK2 and NK3 receptors, exerted significant relaxant effects. Furthermore, the selective NK1 tachykinin antagonist, (+/-)-CP 96,345 (1 microM), abolished substance P (1 nM)- but not isoprenaline (0.1 microM)-induced relaxations. 4. Application of electrical field stimulation (EFS) (20 Hz, supramaximal voltage, 0.5 ms for 10 s) to carbachol-contracted preparations evoked a transient contraction followed by a relaxation. The tetrodotoxin-sensitive slow component of this relaxation was reduced following capsaicin desensitization. 5. In the presence of indomethacin (5 microM) the relaxation induced by substance P, capsaicin or EFS was suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Involvement of endogenous tachykinins and CGRP in the motor responses produced by capsaicin in the guinea-pig common bile duct

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.     

Capsaicin-induced relaxation in the rat isolated external urethral sphincter: characterization of the vanilloid receptor and mediation by CGRP.

1. The potential role of capsaicin-sensitive nerves in the relaxation of the rat external urethral sphincter (REUS) was evaluated by demonstrating the existence of specific vanilloid (capsaicin) receptors and by investigating the sensory neurotransmitter(s) putatively involved in this relaxation. 2. Capsaicin (1 microM) relaxed REUS strips precontracted with noradrenaline (NA) (0.1 mM). This effect underwent desensitization and it was absent in preparations taken from adult capsaicin-pretreated rats. 3. Capsaicin-induced relaxation of NA-precontracted REUS was mimicked by calcitonin gene-related peptide (CGRP, 0.3-10 microM), but not by substance P (1 microM), vasoactive intestinal polypeptide (VIP, 1 microM), alpha-beta methylene ATP (10 microM), gamma-aminobutyric acid (GABA, 3 mM) or galanin (1 microM). A cross-tachyphylaxis between capsaicin (1 microM) and CGRP (1 microM) was observed. Both capsaicin and CGRP-induced relaxation were partially antagonized by the proposed CGRP antagonist, CGRP (8-37) (10 microM). 4. Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 s every 5 min) of REUS evoked a contraction characterized by a largely adrenergic slowly developing tonic contraction with superimposed fast twitches due to the striated component of the strips. Both capsaicin (1 microM) and CGRP (0.01-1 microM) produced an almost complete inhibition of EFS-induced tonic contraction. A cross-tachyphylaxis between capsaicin and CGRP was observed. Furthermore, these inhibitory actions were unaffected by CGRP (8-37) (10 microM). 5. [3H]-resiniferatoxin displayed specific, saturable binding to rat urethral membranes. Data were consistent with a single site with a Kd of 105 pM and a Bmax of 40 fmol mg-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of reserpine on sympathetic, purinergic neurotransmission in the isolated mesenteric artery of the dog: a pharmacological study.

Electrical transmural stimulation evoked a transient contraction in the isolated mesenteric artery of the dog. This contraction was abolished by guanethidine or tetrodotoxin and was partially inhibited by prazosin. Noradrenaline was competitively antagonized by prazosin. Similarly, in the reserpine-treated artery, electrical transmural stimulation produced a transient contraction which was abolished by guanethidine or tetrodotoxin. However, prazosin failed to inhibit this contraction. The contraction to noradrenaline was not significantly different from the response it produced in control vessels. Tyramine (10(-5) M), which acts on sympathetic nerves to release noradrenaline, evoked a tonic contraction in the untreated artery. This contraction was abolished or markedly attenuated by prazosin or guanethidine. The response was not observed in the reserpine-treated artery, indicating that reserpine had depleted the nerves of noradrenaline. In the control vessel alpha,beta-methylene-ATP produced a transient contraction which was followed by a complete relaxation to the basal level. This contractile response was not significantly different in the presence of guanethidine or prazosin or in the reserpine-treated artery. After desensitization of the vessel to alpha,beta-methylene ATP (5 X 10(-6) M) the prazosin-resistant contractions induced by electrical transmural stimulation were abolished both in reserpine-treated and untreated arteries. Also the contractile responses to ATP and alpha-beta-methylene-ATP were abolished but the responses to tyramine (control vessels), noradrenaline and KCl were not affected. 8-Phenyltheophylline (10(-5) M) showed no inhibitory effect on the contractile responses to electrical transmural stimulation, tyramine, ATP or alpha,beta-methylene-ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens.

1. The ability of capsazepine, a recently developed capsaicin receptor antagonist, to prevent the effects of capsaicin on the rat isolated urinary bladder (contraction) and vas deferens (inhibition of electrically-evoked twitches) was compared to that of ruthenium red, a dye which behaves as a functional antagonist of capsaicin. 2. In the rat bladder, capsazepine (3-30 microM) produced a concentration-dependent rightward shift of the curve to capsaicin without any significant depression of the maximal response to the agonist. By contrast, ruthenium red (10-30 microM) produced a non-competitive type of antagonism, characterized by marked depression of the maximal response attainable. Similar findings were obtained in the rat isolated vas deferens in which capsazepine (10 microM) produced a rightward shift of the curve to capsaicin while ruthenium red (3 microM) depressed the maximal response to the agonist. 3. At the concentrations used to block the effect of capsaicin, neither capsazepine nor ruthenium red affected the contractile response of the rat urinary bladder produced by either neurokinin A or electrical field stimulation or the twitch inhibition produced by rat alpha-calcitonin gene-related peptide (alpha CGRP) in the vas deferens. 4. These findings provide additional evidence that both capsazepine and ruthenium red are valuable tools for exploration of the function of capsaicin-sensitive primary afferent neurones. The antagonism of the action of capsaicin by capsazepine is entirely consistent with the proposed interaction of this substance with a vanilloid receptor located on primary afferents, while the action of ruthenium red apparently involves a more complex, non-competitive antagonism.     

The effect of calcium free medium and nifedipine on the release of substance P-like immunoreactivity and contractions induced by capsaicin in the isolated guinea-pig and rat bladder

1. Capsaicin produced a prompt release of substance P-like immunoreactivity (SP-LI) from superfused mucosa-free muscle strips excised from the guinea-pig urinary bladder. A second application of capsaicin had no further effect, indicating desensitization. 2. Neither tetrodotoxin (1 microM) or nifedipine (10 microM) had any inhibitory effect on SP-LI release by capsaicin nor influenced the establishment of the desensitized state. Nifedipine produced per se some SP-LI release. 3. SP-LI release by capsaicin was abolished by incubation in a Calcium(Ca)-free medium containing EDTA (1.0 mM) which also afforded a partial protection toward desensitization. A lower EDTA concentration (0.1 mM) did not suppress SP-LI release by capsaicin but still inhibited desensitization. 4. When the concentration of CaCl2 in the medium was lowered to 1/10-1/100 of that present in normal Krebs solution, capsaicin still evoked a marked SP-LI release and desensitization occurred. In a nominally Ca free medium (maximal Ca concentration due to impurities was 6.7 microM) SP-LI release was still observed and desensitization was incomplete. 5. In a nominally Ca free medium, removal of Mg ions enhanced the SP-LI release induced by capsaicin and enhanced desensitization. 6. In functional studies, nifedipine greatly reduced or abolished the capsaicin- or SP-induced contraction of the rat or guinea-pig isolated bladder but did not prevent desensitization. Likewise, SP-LI depletion in the rat bladder following systemic capsaicin desensitization was not prevented by nifedipine pretreatment. On the other hand, the protective action of Ca free media (containing EDTA) was confirmed in organ bath studies (guinea-pig bladder). 7. These findings indicate that: (a) the requirements of extracellular calcium for activation of neuropeptide release from sensory nerves by capsaicin are very low; (b) both excitation of sensory fibers (SP-LI release) and desensitization are dependent upon the presence of extracellular calcium and (c) L-type voltage-sensitive Ca channels are not likely to be involved in the actions of capsaicin on sensory nerve terminals.     

Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder

TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10-100 microM) inhibits capsaicin (0.03 microM), allyl isothiocyanate (100 microM) and cinnamaldehyde (300 microM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 microM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10-1000 microM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [(3)H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca(2+) influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1 microM) or indomethacin (1 microM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 microM) or suramin (300 microM). The exposure of urinary bladders to allyl isothiocyanate (100 microM) causes an increase in the prostaglandin E(2) and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca(2+) influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E(2). Thus, TRPA1 appears to exert an important role in urinary bladder function.     

Adenosine-modulation of cholinergic and non-adrenergic non-cholinergic neurotransmission in the rabbit iris sphincter.

The characteristics of smooth muscle responses to transmural nerve stimulation in the rabbit iris sphincter were examined. Transmural stimulation elicited a composite contractile response that could be divided in two phases. Atropine abolished the phase I contraction and inhibited the phase II contraction. The atropine-resistant component of the phase II contraction which was unaltered by sympathetic denervation, was mimicked by substance P and abolished by capsaicin. Adenosine inhibited the phase I contraction. The adenosine analogue L-N6-phenylisopropyladenosine (L-PIA) was more potent than 5'-N-ethylcarboxamideadenosine (NECA) in mimicking this adenosine effect. By contrast, adenosine enhanced the phase II contraction in non-pretreated preparations, as well as the atropine-resistant capsaicin-sensitive part of this contraction. Here, NECA was more potent than L-PIA. Adenosine, NECA, L-PIA and D-PIA also enhanced the atropine-sensitive component of the phase II contraction, as well as the contractile response to exogenous acetylcholine or carbachol, but not to exogenous substance P. In this respect, L-PIA was the most powerful adenosine analogue with at least 10 fold higher potency than D-PIA. The adenosine antagonist 8-p-sulphophenyltheophylline enhanced the phase I contraction and decreased the capsaicin-sensitive non-adrenergic non-cholinergic component of the phase II contraction. We conclude that adenosine inhibited the nerve-induced cholinergic twitch (phase I) responses by action at prejunctional A1-receptors. Furthermore, adenosine enhanced the phase II contractile responses via postjunctional enhancement of the cholinergic transmission by action at A1-receptors, and via enhancement of the non-adrenergic non-cholinergic transmission by action at presumably prejunctional A2 receptors.     

Albaconol from the mushroom Albatrellus confluens induces contraction and desensitization in guinea pig trachea

The contraction and desensitization induced by albaconol and the influence of capsazepine, capsaicin and extracellular Ca2+ were investigated to see whether the actions were mediated via a specific VR receptor in guinea pig trachea spiral strips in vitro. Both albaconol and capsaicin were contractors of tracheal smooth muscle, but albaconol was not so potent as capsaicin, with -log (M) EC50 values of 4.23 +/- 0.18 (n = 10) and 7.33 +/- 0.21 (n = 10) respectively. 2.5 microM capsazepine competitively antagonized the contractile response to albaconol and capsaicin. Albaconol increased the contraction induced by a low dose of capsaicin (10(-10) to 10(-9) M), but non-competitively antagonized the contraction induced by a high dose of capsaicin (10(-8) to 10(-3) M). Either albaconol (1 or 100 mM) or capsaicin (3 or 10 microM) was able to desensitize the isolated guinea pig bronchi to subsequent addition of albaconol. Capsazepine (5.0 microM) significantly prevented the desensitization induced by either albaconol (1 or 100mM) or capsaicin (3 or 10 microM). Extracellular Ca2+ was essential for albaconol to induce excitation, but it did not affect albaconol- or capsaicin-induced desensitization. In summary, the results from the present study suggest that albaconol induces contraction and desensitization of guinea pig trachea in vitro as a partial agonist for VR.     

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional evidence for the existence of a capsaicin-sensitive innervation in the rat urinary bladder

Capsaicin (0.03-3 microM) induces contractions of the rat isolated bladder which are unaffected by either atropine (3 microM) or tetrodotoxin (0.5 microM). In the presence of capsaicin (0.1 microM) an enhancement of field stimulation-induced contractions was observed. Capsaicin-desensitization did not modify the height of these. The neurogenic nature of the capsaicin-induced contractions was proved by the observation that 'chronic' (48 h) denervation prevented, while 'acute' (2 h) denervation did not modify the effect of capsaicin. Denervated bladders maintained their responsiveness to acetylcholine but not to field stimulation. Isolated bladders from rat pups (1-2 days old) did not respond to capsaicin while strong contractile responses to acetylcholine or field stimulation were obtained in these preparations. In bladders from two week old animals, capsaicin produced similar contractions to those observed in preparations from adult animals. The bladders from rats receiving a high dose of capsaicin (50 mg kg-1 s.c.) at birth were heavier than those of their age-matched, vehicle-treated controls. Isolated bladders from 2 month old animals pretreated with capsaicin at birth were unresponsive to capsaicin while responsiveness to acetylcholine, substance P or field stimulation was unaffected compared with that of vehicle-treated controls. These experiments provide evidence that a capsaicin-sensitive innervation exists in the rat urinary bladder which undergoes a postnatal development at end organ level.     

The bronchoconstrictive action of evodiamine, an indoloquinazoline alkaloid isolated from the fruits of Evodia rutaecarpa, on guinea-pig isolated bronchus: possible involvement on vanilloid receptors

Evodiamine, a constituent of Evodiae Fructus (Evodia rutaecarpa Benth., Rutaceae), produced a bronchial contraction that is resistant to atropine and abolished by pretreatment with a mixture of the NK1 and NK2 receptor antagonists. Contractile responses to evodiamine were examined in guinea-pig isolated bronchus and compared with those to capsaicin. Both compounds evoked bronchial contraction in a concentration-dependent manner. Maximal contractions for evodiamine and capsaicin were observed at concentrations of 3 microM and 1 microM, respectively. Capsazepine (10 microM), an established antagonist of vanilloid receptor (capsaicin receptor), competitively inhibited the bronchial contraction evoked by evodiamine, suggesting that evodiamine activated vanilloid receptors. Evodiamine (3 microM) and capsaicin (1 microM) produced complete crossed tachyphylaxis. Both compounds desensitized tissues to subsequent additions of either evodiamine or capsaicin. These results suggest that the evodiamine-induced contractile response of the bronchus could be attributed to the resultant tachykinin release from sensory neurons by binding of evodiamine to vanilloid receptors. Rutaecarpine, which belongs to the same indoloquinazoline-type alkaloid as evodiamine, showed neither bronchoconstrictive, desensitizing effects nor vanilloid antagonistic effects at all the concentrations examined (up to 200 microM).