Capsaicin-induced Contraction Research Articles

Modifications of Capsaicin-Sensitive Neurons in Isolated Guinea Pig Ileum by [6]-Gingerol and Lafutidine

A segment of guinea pig ileum was used to confirm the hypothesis that [6]-gingerol and lafutidine interact with capsaicin-sensitive neurons. Addition of 30 and 100 μM [6]-gingerol (a pungent constituent of ginger) induced contraction of the ileum immediately. Like capsaicin, [6]-gingerol-induced contraction was inhibited by antagonists of the vanilloid receptor (capsazepine and ruthenium red), tetrodotoxin, and atropine. Treatment with [6]-gingerol up to 0.3 μM, which alone had no effect, enhanced 3 μM capsaicin-induced contraction, but greater than 3 μM [6]-gingerol significantly inhibited capsaicin-induced contraction. Treatment with lafutidine (a new type of antagonist of the histamine H2 receptor), which was suggested to interact with capsaicin-sensitive neurons in vivo, also showed both stimulatory and inhibitory effects on capsaicin-induced contraction depending on the concentrations. Lafutidine alone had no effect. The enhanced contraction induced by capsaicin in the [6]-gingerol- or lafutidine-treated ileum was also inhibited by antagonists of the vanilloid receptor, tetrodotoxin, and atropine. Capsaicin and [6]-gingerol, but not lafutidine, at 30 μM stimulated [3H]choline release from the prelabeled slices of the ileum. These findings suggest that [6]-gingerol and lafutidine act on capsaicin-sensitive cholinergic neurons and modulate the contraction in isolated guinea pig ileum.

Descending monoaminergic pathways projecting to the spinal defecation center enhance colorectal motility in rats.

The central regulating mechanisms of defecation, especially roles of the spinal defecation center, are still unclear. We have shown that monoamines including norepinephrine, dopamine, and serotonin injected into the spinal defecation center cause propulsive contractions of the colorectum. These monoamines are the main neurotransmitters of descending pain inhibitory pathways. Therefore, we hypothesized that noxious stimuli in the colorectum would activate the descending monoaminergic pathways projecting to the spinal defecation center and that subsequently released endogenous monoamine neurotransmitters would enhance colorectal motility. Colorectal motility was measured in rats anesthetized with α-chloralose and ketamine. As a noxious stimulus, capsaicin was administered into the colorectal lumen. To interrupt neuronal transmission in the spinal defecation center, antagonists of norepinephrine, dopamine, and/or serotonin receptors were injected intrathecally at the L6-S1 spinal level, where the spinal defecation center is located. Intraluminal administration of capsaicin, acting on the transient receptor potential vanilloid 1 channel, caused transient propulsive contractions. The effect of capsaicin was abolished by surgical severing of the pelvic nerves or thoracic spinal transection at the T4 level. Capsaicin-induced contractions were blocked by preinjection of D2-like dopamine receptor and 5-hydroxytryptamine subtype 2 and 3 receptor antagonists into the spinal defecation center. We demonstrated that intraluminally administered capsaicin causes propulsive colorectal motility through reflex pathways involving the spinal and supraspinal defecation centers. Our results provide evidence that descending monoaminergic neurons are activated by noxious stimulation to the colorectum, leading to facilitation of colorectal motility. NEW & NOTEWORTHY The present study demonstrates that noxious stimuli in the colorectum activates the descending monoaminergic pathways projecting to the spinal defecation center and that subsequently released endogenous monoamine neurotransmitters, serotonin and dopamine, enhance colorectal motility. Our findings provide a possible explanation of the concurrent appearance of abdominal pain and bowel disorder in irritable bowel syndrome patients. Thus the present study may provide new insights into understanding of mechanisms of colorectal dysfunction involving the central nervous system.

TRPV1 alterations in urinary bladder dysfunction in a rat model of STZ-induced diabetes

AimsMore than half of diabetic patients experience voiding disorder termed diabetic urinary bladder dysfunction (DBD). Here we have investigated how the alterations in transient receptor potential vanilloid 1 (TRPV1) ion channel expressed in bladder-innervating afferents may contribute to DBD pathogenesis. Main methodsThe rat model of streptozotocin (STZ)-induced diabetes was used. The functional profile of TRPV1 in retrogradely labeled afferent, bladder-innervating dorsal root ganglia (DRG) neurons was examined using patch clamp. The level of TRPV1 transcripts in DRG was assessed with qRT-PCR. TRPV1-dependent component of detrusor smooth muscle (DSM) contractions was studied with muscle strip tensiometry. Key findingsTRPV1-mediated current (ITRPV1) was increased in diabetic animals vs. controls by 42%. The expression of Trpv1 gene was found to be 63% higher in STZ-treated rats compared to controls, consistent with the respective electrophysiological data. Surprisingly, capsaicin-induced contractions of DSM were found to be 3-to-10-fold weaker in diabetic group depending on concentration of the agonist (100nM to 10μM). SignificanceOur findings suggest the dual role of TRPV1 in DBD. On the one hand, the increase of its functional expression may enhance micturition reflex arc functioning. On the other hand, at the local level, the decrease of TRPV1-dependent contractions may contribute to organ decompensation.

Contribution of sensory nerves to LPS-induced hyperresponsiveness of human isolated bronchi

Bacterial lipopolysaccharide (LPS) can induce bronchial hyperresponsiveness (BHR), but the underlying mechanisms remain to be determined. Here, the possible contribution of sensory nerves to LPS-induced BHR was examined in human isolated bronchi to pharmacologically identify the mechanisms underlying this phenomenon. Human isolated bronchial tone was induced by electrical field stimulation (EFS). The responses of airways to LPS, with or without capsaicin desensitization or thiorphan treatment were studied and the transient receptor potential vanilloid type 1 (TRPV1) expression was assessed. We performed similar experiments in the presence of a TRPV1 or a neurokinin (NK) 2 receptor antagonist using SB366791 and GR159897, respectively. LPS increased (≃2.3-fold, P<0.001) the contraction induced by EFS, compared to control tissues. Acute administration of capsaicin enhanced (≃2.3-fold, P<0.001) the EFS-mediated contraction, but did not potentiate the effect of LPS. Thiorphan increased (≃1.3-fold, P<0.05) the contractile response of LPS treated tissues and, at lower frequencies, it enhanced (≃1.7-fold, P<0.001) the capsaicin-induced contraction. In capsaicin-desensitized bronchi, LPS did not modify (P>0.05) the EFS contractile response, nor after treatment with thiorphan. Capsaicin desensitization reduced (≃0.4-fold, P<0.001) the LPS-induced BHR. SB366791 and GR159897 prevented the LPS-induced BHR and the release of NKA. LPS increased (+85.3±9.5%, P<0.01) the surface membrane expression of TRPV1 in parasympathetic ganglia. Our results demonstrate the involvement of capsaicin-sensitive sensory nerves and neutral endopeptidases in LPS-induced BHR of the human bronchi, associated with an upregulation of TRPV1 and release of NKA.

Role of the intraluminal contents and the continuity of intrinsic neurons in intracolonic capsaicin-induced contraction and defecation in dogs

We, herein, examined the role of the intraluminal contents and continuity of colonic intrinsic neurons in intracolonic capsaicin-induced enhancement of colonic motility and defecation. Five beagle dogs were equipped with three strain gauge force transducers throughout the colon. The colonic contractile activity in response to intracolonic capsaicin was studied in intact dogs, dogs after colonic cleansing and dogs with transection/re-anastomosis (T/R) between the proximal and middle colon. The effects of intravenous yohimbine, a α2 adrenergic antagonist, on the colonic motility and defecation were also studied in the same models. In intact dogs, capsaicin (10mg) and yohimbine (2mg/kg) immediately induced contractions throughout the colon, with defecation occurring in all experiments. In dogs after colonic cleansing and T/R, the capsaicin (10mg)-induced enhancement of colonic motility was decreased in the middle and distal colon, and capsaicin-induced defecation was observed in 0-20% of experiments (p<0.05 compared to intact dogs). The effect of yohimbine (2mg/kg) in inducing colonic contractions was unaltered after colonic cleansing and T/R; in contrast, yohimbine-induced defecation was not observed after colonic cleansing, but was unchanged after T/R. The continuity of the colonic intrinsic nerves as well as the intraluminal contents appear to play an important role in the colonic motor response to intracolonic capsaicin.

1645 DIFFERENTIAL EFFECTS OF BOTULINUM NEUROTOXIN-A (BONT/A) ON BLADDER CONTRACTILE RESPONSES TO ACTIVATION OF EFFERENT NERVES, SMOOTH MUSCLES AND AFFERENT NERVES IN RATS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Neurogenic Voiding Dysfunction1 Apr 20121645 DIFFERENTIAL EFFECTS OF BOTULINUM NEUROTOXIN-A (BONT/A) ON BLADDER CONTRACTILE RESPONSES TO ACTIVATION OF EFFERENT NERVES, SMOOTH MUSCLES AND AFFERENT NERVES IN RATS Ryosuke Takahashi, Takakazu Yunoki, Seiji Naito, and Naoki Yoshimura Ryosuke TakahashiRyosuke Takahashi Pittsburgh, PA More articles by this author , Takakazu YunokiTakakazu Yunoki Fukuoka, Japan More articles by this author , Seiji NaitoSeiji Naito Fukuoka, Japan More articles by this author , and Naoki YoshimuraNaoki Yoshimura Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1477AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES BoNT/A, an effective therapeutic agent for overactive bladder, reportedly has inhibitory effects not only on acetylcholine exocytosis from efferent nerve terminals, but also on transmitter release from afferent nerve terminals. We also showed that BoNT/A inhibits voltage-gated Ca2+ channels (VGCC) in rat and human detrusor smooth muscles using patch-clamp techniques (AUA 2009). We therefore compared the effects of BoNT/A on rat bladder smooth muscle contractions induced by activation of efferent nerves, afferent nerves and VGCC on smooth muscle to examine the relative importance of these three inhibitory mechanisms. METHODS Bladder strips without urothelium were obtained from female SD rats. These strips were incubated for 3 h at 37°C in Krebs solution with different concentrations of BoNT/A (0.3-100 nM) and tension development was measured in an organ bath. To examine the effect of BoNT/A on neurotransmitter release from efferent nerve terminals and afferent nerve terminals and on VGCC of smooth muscle, changes in electrical field stimulation (EFS: 20V; 0.5-64 Hz), 1 μM capsaicin and 70 mM KCl-induced contractions were evaluated, respectively. In addition, the contractile response to carbachol (CCh) was also examined. RESULTS The significant inhibitory effect of BoNT/A on KCl-induced contractions was observed at a concentration of 3nM or higher, and the maximal inhibition at 100 nM was 30% compared to control strips. EFS-induced contractions were significantly inhibited by the incubation with BoNT/A of 10 nM or higher, and the maximal inhibition at 100 nM was 70% compared to control strips. The pretreatment of 1μM atropine plus 10μM αβMet-ATP inhibited EFS-induced contractions to the same level as in BoNT/A treatment. Capsaicin-induced contractions were inhibited by 30% with overnight incubation of 100 nM BoTN/A, but not with 3 h incubation. The contractile responses to CCh were not altered by the incubation with BoNT/A. CONCLUSIONS The order of inhibitory potency of BoNT/A is efferent nerve terminals > L-type VGCC on smooth muscles > afferent nerve terminals. In addition, BoNT/A can inhibit both acetylcholine and ATP release from efferent nerve terminals. Thus, BoNT/A has multiple inhibitory mechanisms affecting bladder function, which would enhance the efficacy of BoNT/A treatment for overactive bladder. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e665 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryosuke Takahashi Pittsburgh, PA More articles by this author Takakazu Yunoki Fukuoka, Japan More articles by this author Seiji Naito Fukuoka, Japan More articles by this author Naoki Yoshimura Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Use and limitations of three TRPV-1 receptor antagonists on smooth muscles of animals and man: A vote for BCTC

Specificity of the effect is a crucial factor in using antagonists for detecting the physiological/pathophysiological roles of receptors. Here we examined the capsaicin receptor antagonist effects of three commercially-available substances, capsazepine, iodo-resiniferatoxin (I-RTX) and BCTC, on isolated smooth muscle preparations, including the human intestine. Care was taken to observe possible non-specific effects, to find out safe and effective concentrations. Capsazepine appeared to have a low margin of safety. I-RTX (up to 1μM) specifically inhibited capsaicin-induced contractions in the guinea-pig ileum and urinary bladder. I-RTX showed agonist activity on the rat urinary bladder. BCTC (1μM) abolished the contractile effects of capsaicin (1 or 2μM) on all preparations tested (guinea-pig ileum, bladder, trachea, as well as rat and mouse bladder), and on the guinea-pig renal pelvis, where it failed to influence capsaicin-sensitive, sensory neuron-mediated positive inotropy in response to field stimulation. On human intestinal preparations BCTC prevented the relaxant effect of capsaicin. It is concluded that of the three antagonists tested BCTC seems the safest one for inhibiting TRPV-1 receptors. The effect of capsazepine may be complicated by non-specific inhibition of smooth muscle contractility and that of I-RTX by agonist activity. The “local efferent” function of capsaicin-sensitive sensory neurons is not influenced by BCTC, as shown by the results obtained in the renal pelvis. In conclusion, of the TRPV-1 receptor antagonists studied, BCTC (1μM) seems the most reliable in isolated organ experiments. This substance is also effective in the human intestine.

TRPV1 expressing extrinsic primary sensory neurons play a protective role in mouse oxazolone-induced colitis

TRPV1 expressing sensory neurons which have been considered to be largely associated with neurogenic inflammation were chemically denervated by capsaicin treatment in neonatal mice. However, neonatal capsaicin treatment aggravated mouse oxazolone-induced colitis, and did not affect the expression of calcitonin gene-related peptide (CGRP)- or substance P-immunoreactive nerve fibers in the colon. Meanwhile, the capsaicin-induced contraction was absent in the colon of neonatal capsaicin treatment mouse. These results suggest a protective role of TRPV1 expressing extrinsic sensory neurons in oxazolone-induced colitis and the involvement of some neurotransmitter other than CGRP and substance P in the pathogenesis of the colitis.

Analysis of the mechanisms underlying the contractile response induced by the hydroalcoholic extract of Phyllanthus urinaria in the guinea-pig urinary bladder in-vitro.

The hydroalcoholic extract of Phyllanthus urinaria (Euphorbiaceae), substance P and substance P methyl ester all caused graded contractions in the guinea-pig urinary bladder. Responses to hydroalcoholic extract and substance P were markedly inhibited in calcium-free Krebs solution, this effect being reversed by reintroduction of calcium in the medium. The contraction in response to hydroalcoholic extract was unaffected by atropine, propranolol, prazosin, yohimbine, tetrodotoxin, w-conotoxin, nicardipine, HOE 140, guanethidine, staurosporine, phorbol ester or indomethacin, excluding the involvement of nervous mediated responses, or action via cholinergic, adrenergic, kinins, cyclo-oxygenase metabolites, protein kinase C or activation of L or N-type calcium channels. The selective NK1 tachykinin antagonist (FK 888), but not NK2 (SR 48968) antagonized substance P-induced contraction, but both drugs failed to effect Phyllanthus urinaria-induced contraction. Prolonged desensitization of guinea pig urinary bladder with capsaicin (10 microM) or preincubation of guinea-pig urinary bladder with capsazepine did not affect contraction caused by hydroalcoholic extract. Ruthenium red almost completely abolished capsaicin-induced contraction, but had no effect on hydroalcoholic extract-mediated contraction. Substance P and the hydroalcoholic extract caused marked potentiation of the twitch response in the preparations field stimulated. The facilitatory effect of substance P, but not that of hydroalcoholic extract, was prevented by the NK1 (FK 888), but not by NK2 (SR 48968) antagonist. We concluded that contraction induced by hydroalcoholic extract of Phyllanthus urinaria in the guinea pig urinary bladder involves direct action on smooth muscle and relies on the mobilization of extracellular calcium influx unrelated to activation of L- and N-type calcium channels or activation of protein kinase C mechanisms. In addition contraction caused by the hydroalcoholic extract of Phyllanthus urinaria in guinea-pig urinary bladder does not involve the activation of tachykinin or vanilloid receptors.

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.

Enhancement of capsaicin-induced contraction of guinea-pig tracheal smooth muscle by vanadate.

Contractions of guinea-pig isolated tracheal smooth muscle by submaximal capsaicin (0.1 microM) were enhanced by 43% (P less than 0.01) after vanadate (10 microM for 10 min) preincubation. Contractile responses to acetylcholine (0.1-100 microM), histamine (1-100 microM) or substance P (0.01-1 microM) were, in contrast, not affected by prior vanadate exposure. It is suggested that tachykinin release from capsaicin-sensitive afferent nerve endings within the airways was enhanced by vanadate while airway smooth muscle reactivity remained unchanged.

Inhibitory effect of Iboga-type indole alkaloids on capsaicin-induced contraction in isolated mouse rectum

Voacanga africana (Apocynaceae) is used as an anti-diarrheal medicine in West Africa. In the present study, we investigated the effect of an extract of V. africana and its constituents on smooth muscle contraction induced by capsaicin in mouse rectum, where transient receptor potential vanilloid type 1 (TRPV1)-immunoreactive fibers are abundant. Methanol and alkaloid extracts of the root bark of V. africana were found to inhibit capsaicin-induced contraction in a dose-dependent manner (30-300 μg/ml). Major constituents isolated from the alkaloid extract were then studied for their effects on the capsaicin-induced contraction. The main active constituents were found to be Iboga-type alkaloids, including voacangine (1), 3-oxovoacangine (2), voacristine (3), and (7α)-voacangine hydroxyindolenine (4). The voacangine concentration dependently (3-100 μM) inhibited the capsaicin-induced contraction. The capsaicin-induced contraction was almost completely inhibited by the TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC). On the other hand, the Iboga-type alkaloids did not inhibit the contractions induced by 3 μM acetylcholine and 300 μM nicotine. These results suggest that Iboga-type alkaloids isolated from V. africana inhibit capsaicin-induced contraction in the mouse rectum, possibly via the inhibition of a TRPV1-mediated pathway. This inhibition may be involved in the anti-diarrheal effect of V. africana.

T1668 Mast Cell Proteases Stimulate Neurogenic and Non-Neurogenic Mucosal Chloride Secretion in Guinea Pig Distal Ileum

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

T1667 Canonical Transient Receptor Potential Channels (TRPC Channels) in Regulation of Mucosal Chloride Secretion in Guinea Pig Ileum

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.

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 μM) inhibits capsaicin (0.03 μM), allyl isothiocyanate (100 μM) and cinnamaldehyde (300 μM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 μM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10–1000 μM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [ 3H]-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 μM) or indomethacin (1 μM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 μM) or suramin (300 μM). The exposure of urinary bladders to allyl isothiocyanate (100 μM) 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.

Pharmacological investigation of hydrogen sulfide (H 2S) contractile activity in rat detrusor muscle

We have investigated the mechanism through which hydrogen sulfide (H 2S) stimulates capsaicin-sensitive primary afferent neurons in the rat isolated urinary bladder. Sodium hydrogen sulfide (NaHS), a donor of H 2S, produced concentration-dependent contractile responses (pEC 50=3.5±0.1) that were unaffected by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine (30 μM) and SB 366791 (10 μM) and by the N-type Ca 2+ channel blocker omega-conotoxin GVIA (ω-CTX; 100 nM). In contrast, the unselective transient receptor potential (TRP) cation channels blocker ruthenium red (30 μM) almost abolished NaHS-induced contractions. Ruthenium red (30 μM) greatly reduced capsaicin-induced contractions, whereas it did not attenuate the contractile response to neurokinin A. The putative TRPV1 receptor antagonist iodo-resiniferatoxin, from 100 nM upward, produced agonist responses per se, and could not be tested against NaHS. We conclude that H 2S either acts at TRPV1 receptorial sites unblocked by capsazepine or SB 366791, or stimulates a still unidentified transient receptor potential-like channel co-expressed with TRPV1 on sensory neurons.

Peripheral activity of a new NK1 receptor antagonist TAK-637 in the gastrointestinal tract.

Pathways controlling gastrointestinal function involve the activation of neurokinin NK1 receptors by substance P (SP) under normal and pathological conditions. Our aim was to pharmacologically characterize the effect of a nonpeptide NK1 receptor antagonist TAK-637 [(aR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g] [1,7]naphthyridine-6,13-dione] and determine key mechanisms of TAK-637 action in the gastrointestinal tract. Experiments were performed using intestinal preparations isolated from the guinea pig. The selective agonists of NK1 receptors, [Sar9,Met(O2)11]-SP and GR 73632 [H2N-(CH2)4-CO-Phe-Phe-Pro-NMe-Leu-Met-NH2], induced contractions in colonic longitudinal muscle pretreated with atropine. TAK-637 (1-100 nM) caused a rightward shift of the concentration-response curves showing nanomolar affinity against [Sar9,Met(O2)11]-SP (Kb = 4.7 nM) and GR 73632 (K(b) = 1.8 nM). This antagonist effect remained unchanged by tetrodotoxin. Furthermore, neither the contractions of colonic circular muscle induced by selective activation of NK2 receptors by GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) nor the responses of taenia coli induced by the selective NK3 receptor agonist senktide were affected by TAK-637 (100 nM). Studies of electrically induced neurogenic contractions showed that TAK-637 had no effect on cholinergic responses to single-pulse (0.5 ms) stimulation or stimulation with increasing frequency (1-16 Hz, 0.5 ms, 5-s train duration). In contrast, TAK-637 significantly reduced nonadrenergic, noncholinergic contractions of colonic longitudinal muscle evoked at frequencies of 8 to 16 Hz and prevented the development of capsaicin-induced contractions in isolated segments of terminal ileum. Our results indicate that TAK-637 is a selective antagonist of smooth muscle NK(1) receptors that activate intestinal muscle contraction. Additionally TAK-637 inhibits neuronal NK1 receptors involved in the "local" motor response to stimulation of capsaicin-sensitive primary afferents.

Evidence for the involvement of ATP, but not of VIP/PACAP or nitric oxide, in the excitatory effect of capsaicin in the small intestine

The contractile effect of capsaicin in the guinea-pig small intestine involves an activation of enteric cholinergic neurons. Our present data show that the P 2 purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 30 μM) significantly reduces the contractile response to capsaicin (2 μM) in the presence, but not in the absence, of the tachykinin receptor antagonists [O-Pro 9, (Spiro-γ-lactam)Leu 10, Trp 11]physalaemin (1–11) (GR 82334; 3 μM) and ( S)-( N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidine-4-yl)- N-methylacetamide (SR 142801; 100 nM) (for blocking tachykinin NK 1 and NK 3 receptors, respectively). PPADS (30 μM) fails to influence submaximal cholinergic contractions evoked by cholecystokinin octapeptide (CCK-8; 2–3 nM) or senktide (1 nM), or the direct smooth muscle-contracting effect of histamine (100–200 nM). A higher concentration (300 μM) of PPADS is also without effect against the stimulatory action of cholecystokinin octapeptide. This means that PPADS can probably be safely used as a purinoceptor antagonist in intestinal preparations. The putative pituitary adenylate cyclase activating peptide (PACAP) receptor antagonist PACAP-(6–38) (3 μM) significantly reduces the contractile effect of PACAP-(1–38) (10 nM) and abolishes that of vasoactive intestinal polypeptide (VIP; 10 nM). PACAP-(6–38) (3 μM) fails to influence the effect of capsaicin (2 μM) both in the absence and in the presence of tachykinin receptor antagonists. The nitric oxide (NO) synthase inhibitor N G-nitro- l-arginine ( l-NOARG; 100 μM) also fails to inhibit the capsaicin-induced motor response. We conclude that an endogenous ligand of PPADS-sensitive P 2 purinoceptors (possibly ATP), but not a VIP/PACAP-like peptide or NO, is involved in the nontachykininergic activation of cholinergic neurons in the course of the capsaicin-induced contraction.

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 82 334 (3 μM) for neurokinin-1 receptors, MEN 10 627 (3 μM; ileum) or MEN 11 420 (1 μM; oesophagus) for neurokinin-2 receptors and SR 142 801 (0.1 μM) for neurokinin-3 receptors. In the ileum, the peak contraction evoked by capsaicin (2 μM) 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 82 334 plus SR 142 801 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 82 334 plus SR 142 801 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 μM, respectively) also reduced the contractile effect of capsaicin. In the oesophagus, capsaicin (1 μM) induced biphasic contractions which were strongly inhibited by atropine (1 μM) or capsaicin pretreatment (1 μM 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 82 334, MEN 11 420 and SR 142 801 were 7.5±0.8% and 9.1±2.2%, respectively ( n=6, P<0.001 and 0.05, respectively). Tetrodotoxin (0.5 μM) 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.

Neurokinin A-LI release after antigen challenge in guinea-pig bronchial tubes: influence of histamine and bradykinin.

1. Our aim was to determine if antigen challenge stimulates sensory nerves and provokes the release of tachykinins. The involvement of histamine and bradykinin was studied by using specific receptor antagonists. Capsaicin-induced responses were also examined. Experiments were performed in vitro on tracheal and bronchial preparations from ovalbumin-sensitized guinea-pigs. 2. Characterization of ovalbumin-induced contraction, with regard to histamine and bradykinin, was carried out on airway ring preparations in the presence of phosphoramidon. The histamine H1 receptor antagonist pyrilamine reduced allergen-induced bronchial contractions by about 30%, whereas the bradykinin B2 receptor antagonist icatibant (Hoe 140) did not significantly affect the response. Combined treatment with pyrilamine (1 microM) and icatibant (0.1 microM) reduced the contractions by about 80%, indicating a synergistic inhibitory action. Tracheal preparations were not significantly affected by treatments, neither were capsaicin-induced contractions. 3. To study the outflow of tachykinins, we used a perfused bronchial-tube preparation, allowing simultaneous measurement of smooth muscle tension and mediator release. Neurokinin A-like immunoreactivity (NKA-LI) and substance P-like immunoreactivity (SP-LI) were determined by radioimmunoassay. 4. The results of the perfusion study showed an increased outflow of NKA-LI into the perfusate in response to ovalbumin (127% of basal) challenge. SP-LI determined in some of the samples showed a much lower amount (40 to 70 times lower) of SP-LI than NKA-LI. Treatment with icatibant and pyrilamine, separately and in combination, significantly reduced the ovalbumin-induced NKA-LI outflow by 38%, 26% and 22%, respectively. 5. Capsaicin-induced outflow (124% of basal) was not significantly affected by treatments (icatibant 121%, pyrilamine 107% and combined treatment 111% of basal). However, when pyrilamine was present the increased outflow was not statistically significant. 6. In conclusion, we found that allergen provocation of guinea-pig bronchi caused an increased outflow of NKA-LI that was reduced by treatment with both pyrilamine and icatibant. These findings demonstrate that the allergen-induced release of histamine and bradykinin stimulate sensory nerves and thereby increase outflow of tachykinins that contribute to the allergic reaction.