Intestinal Muscle Strips Research Articles

Пригнічувальна дія загального анестетика кетаміну на транзієнти внутрішньоклітинної концентрації іонів кальцію та скорочення гладеньком’язових клітин тонкої кишки миші

The mechanisms of the negative consequences of general anaesthetics action on the nervous system have been studied in detail, but regarding smooth muscle function, such issues have not yet been sufficiently addressed. In this study, we investigated the effect of the general intravenous anaesthetic ketamine on the level of intracellular calcium in isolated ileum myocytes and the contractile activity of smooth muscle strips of the mouse small intestine. The concentration of intracellular calcium in cells was measured using the Ca2+-sensitive fluorescent dye Fura-2, and tensiometry was used to record the contractile activity of smooth muscles. It was shown that ketamine at a concentration of 100 µM significantly, by 40%, suppressed carbachol-induced contractile reactions of the ileum. The inhibitory effect correlated with the suppression of the intracellular calcium responses to carbachol in isolated smooth muscle cells after the addition of ketamine to the extracellular solution, which was by 65% on average. These results contribute to our better understanding of the possible membrane and intracellular mechanisms of the development of post-surgical intestinal motility disorders.

A comparison of effects of bisphenol A and bisphenol S on rat gut contractility in vitro after acute exposure

Background: Prevailing standard of living allows generous usage of plastic containers to store, transport, and serve edibles. A chemical named bisphenol A (BPA) leaches from plastic containers into the edibles. Ingestion of BPA contaminated food is known to alter intestinal motility in addition to detrimental effects on fetal development, fertility, behavior, cognitive functions, immunity, and metabolism. These BPA-induced ill effects on health led to marketing regulation on BPA and introduction of bisphenol S (BPS) as a safer substitute of BPA. However, BPS is yet to be evaluated for its effects on gut motility. Aims and Objectives: Therefore, this study was aimed to assess comparative effects of BPS and BPA on gut motility. Materials and Methods: In an organ bath preparation, isometric contractions were recorded from segments of dissected gastric and small intestinal muscle strips prepared from rat gut, and cumulative concentration response of BPS and BPA on in vitro gut contractility was evaluated. Results: Both BPS and BPA treatment significantly diminished basal tone, maximum contractile tension, and the contractile frequency of spontaneous contractions in gastric as well as small intestinal muscle strips. Conclusion: From the present observation, it was apparent that both BPA and BPS have similar toxicity on gastric as well as small intestinal motility. Thus, the use of BPS as a substitute of BPA needs to be more critically evaluated.

Effect of Inulin on Proteome Changes Induced by Pathogenic Lipopolysaccharide in Human Colon.

In the present study, the protective role of inulin against lipopolysaccharide (LPS)-induced oxidative stress was evaluated on human colonic mucosa using a proteomic approach. Human colonic mucosa and submucosa were sealed between two chambers, with the luminal side facing upwards and overlaid with Krebs (control), LPS or LPS+ inulin IQ solution. The solutions on the submucosal side (undernatants) were collected following 30 min of mucosal exposure. iTRAQ based analysis was used to analyze the total soluble proteomes from human colonic mucosa and submucosa treated with different undernatants. Human colonic muscle strips were exposed to the undernatants to evaluate the response to acetylcholine. Inulin exposure was able to counteract, in human colonic mucosa, the LPS-dependent alteration of some proteins involved in the intestinal contraction (myosin light chain kinase (MLCK), myosin regulatory subunit (MYL)), to reduce the up-regulation of two proteins involved in the radical-mediated oxidative stress (the DNA-apurinic or apyrimidinic site) lyase) APEX1 and the T-complex protein 1 subunit eta (CCT7) and to entail a higher level of some detoxification enzymes (the metallothionein-2 MT2A, the glutathione–S-transferase K GSTk, and two UDP- glucuronosyltransferases UGT2B4, UGT2B17). Inulin exposure was also able to prevent the LPS-dependent intestinal muscle strips contraction impairment and the mucosa glutathione level alterations. Exposure of colonic mucosa to inulin seems to prevent LPS-induced alteration in expression of some key proteins, which promote intestinal motility and inflammation, reducing the radical-mediated oxidative stress.

Targeting extra-oral bitter taste receptors modulates gastrointestinal motility with effects on satiation.

Bitter taste receptors (TAS2Rs) are present in extra-oral tissues, including gut endocrine cells. This study explored the presence and mechanism of action of TAS2R agonists on gut smooth muscle in vitro and investigated functional effects of intra-gastric administration of TAS2R agonists on gastric motility and satiation. TAS2Rs and taste signalling elements were expressed in smooth muscle tissue along the mouse gut and in human gastric smooth muscle cells (hGSMC). Bitter tastants induced concentration and region-dependent contractility changes in mouse intestinal muscle strips. Contractions induced by denatonium benzoate (DB) in gastric fundus were mediated via increases in intracellular Ca2+ release and extracellular Ca2+-influx, partially masked by a hyperpolarizing K+-efflux. Intra-gastric administration of DB in mice induced a TAS2R-dependent delay in gastric emptying. In hGSMC, bitter compounds evoked Ca2+-rises and increased ERK-phosphorylation. Healthy volunteers showed an impaired fundic relaxation in response to nutrient infusion and a decreased nutrient volume tolerance and increased satiation during an oral nutrient challenge test after intra-gastric DB administration. These findings suggest a potential role for intestinal TAS2Rs as therapeutic targets to alter gastrointestinal motility and hence to interfere with hunger signalling.

Neuropeptide S inhibits gastrointestinal motility and increases mucosal permeability through nitric oxide.

Neuropeptide S (NPS) receptor (NPSR1) polymorphisms are associated with enteral dysmotility and inflammatory bowel disease (IBD). This study investigated the role of NPS in conjunction with nitrergic mechanisms in the regulation of intestinal motility and mucosal permeability. In rats, small intestinal myoelectric activity and luminal pressure changes in small intestine and colon, along with duodenal permeability, were studied. In human intestine, NPS and NPSR1 were localized by immunostaining. Pre- and postprandial plasma NPS was measured by ELISA in healthy and active IBD humans. Effects and mechanisms of NPS were studied in human intestinal muscle strips. In rats, NPS 100-4,000 pmol·kg(-1)·min(-1) had effects on the small intestine and colon. Low doses of NPS increased myoelectric spiking (P < 0.05). Higher doses reduced spiking and prolonged the cycle length of the migrating myoelectric complex, reduced intraluminal pressures (P < 0.05-0.01), and increased permeability (P < 0.01) through NO-dependent mechanisms. In human intestine, NPS localized at myenteric nerve cell bodies and fibers. NPSR1 was confined to nerve cell bodies. Circulating NPS in humans was tenfold below the ∼0.3 nmol/l dissociation constant (Kd) of NPSR1, with no difference between healthy and IBD subjects. In human intestinal muscle strips precontracted by bethanechol, NPS 1-1,000 nmol/l induced NO-dependent muscle relaxation (P < 0.05) that was sensitive also to tetrodotoxin (P < 0.01). In conclusion, NPS inhibits motility and increases permeability in neurocrine fashion acting through NO in the myenteric plexus in rats and humans. Aberrant signaling and upregulation of NPSR1 could potentially exacerbate dysmotility and hyperpermeability by local mechanisms in gastrointestinal functional and inflammatory reactions.

Mo1313 A Rapid Initial Lag Phase of Gastric Emptying Is Associated With a Greater Degree of Obesity and Weight Gain Over Time

Background/Aim: Transient receptor potential ankyrin 1 (TRPA1) locates in the cell membrane of enterochromaffin cells (EC), and TRPA1 stimulation on EC is proposed to modulate intestinalmotility via release of serotonin (5-HT). Although a previous study has demonstrated that TRPA1 agonists produce a contractile response in isolated intestinal muscle strips in vitro, apparently contradictory results have been reported for gastrointestinal (GI) transit in vivo. The Japanese traditional medicine daikenchuto (TU-100), which is being investigated as a new drug in the US and is used for the treatment of postoperative ileus (POI) in Japan, was reported to accelerate GI transit in animals and humans. TU-100 has recently been shown to increase intestinal blood flow via stimulation of TRPA1 located on the epithelial cells of the small intestine. The present study investigates the effects of TU-100, TU-100 ingredients, and TRPA1 agonists on dysregulated intestinal transit in an established murine POI model. Methods: The POI model, which involves manipulation of colon and small intestinal transit, was evaluated by calculating the geometric center of distribution of fluorescent-labeled dextran markers. The in vitro motility of isolated mouse small intestine in an organ bath was evaluated by video imaging and intraluminal pressure was measured by a strain gauge transducer. 5-HT release from isolated small intestinal segments or a rat pancreatic endocrine cell line RIN-14B, which is supposed to have common properties with EC, was measured by ELISA and/or HPLC. The tested compounds included the well known TRPA1 agonists aryl isothiocyanate (AITC) and cinnamaldehyde (CA), as well as TU-100 and its ingredients shogaol and hydroxy-α-sanshool, which stimulate TRPA1. TRPA1 KO mice were also studied. Results: Orally administered TRPA1 agonists, TU-100, and TU100 ingredients improved small intestinal transit in the POI model. Incubation of small intestinal segments with TRPA1 agonists and TU-100 ingredients resulted in an increase in the level of 5-HT in the culture supernatant. The effects of AITC and TU-100 ingredients, but not CA, were abrogated in TRPA1 KO mice. All agents induced the release of 5-HT from RIN14B cells Conclusion: TRPA1 stimulation by TU-100 may be involved in its beneficial effect on POI. The role of TRPA1 in GI motility deserves further extensive investigation with the aim of developing a novel therapeutic strategy to treat GI motility disorders.

Mo1312 Transient Receptor Potential Ankyrin 1 (TRPA1) Agonists Improve Intestinal Transit in Murine Postoperative Ileus Model

Background/Aim: Transient receptor potential ankyrin 1 (TRPA1) locates in the cell membrane of enterochromaffin cells (EC), and TRPA1 stimulation on EC is proposed to modulate intestinalmotility via release of serotonin (5-HT). Although a previous study has demonstrated that TRPA1 agonists produce a contractile response in isolated intestinal muscle strips in vitro, apparently contradictory results have been reported for gastrointestinal (GI) transit in vivo. The Japanese traditional medicine daikenchuto (TU-100), which is being investigated as a new drug in the US and is used for the treatment of postoperative ileus (POI) in Japan, was reported to accelerate GI transit in animals and humans. TU-100 has recently been shown to increase intestinal blood flow via stimulation of TRPA1 located on the epithelial cells of the small intestine. The present study investigates the effects of TU-100, TU-100 ingredients, and TRPA1 agonists on dysregulated intestinal transit in an established murine POI model. Methods: The POI model, which involves manipulation of colon and small intestinal transit, was evaluated by calculating the geometric center of distribution of fluorescent-labeled dextran markers. The in vitro motility of isolated mouse small intestine in an organ bath was evaluated by video imaging and intraluminal pressure was measured by a strain gauge transducer. 5-HT release from isolated small intestinal segments or a rat pancreatic endocrine cell line RIN-14B, which is supposed to have common properties with EC, was measured by ELISA and/or HPLC. The tested compounds included the well known TRPA1 agonists aryl isothiocyanate (AITC) and cinnamaldehyde (CA), as well as TU-100 and its ingredients shogaol and hydroxy-α-sanshool, which stimulate TRPA1. TRPA1 KO mice were also studied. Results: Orally administered TRPA1 agonists, TU-100, and TU100 ingredients improved small intestinal transit in the POI model. Incubation of small intestinal segments with TRPA1 agonists and TU-100 ingredients resulted in an increase in the level of 5-HT in the culture supernatant. The effects of AITC and TU-100 ingredients, but not CA, were abrogated in TRPA1 KO mice. All agents induced the release of 5-HT from RIN14B cells Conclusion: TRPA1 stimulation by TU-100 may be involved in its beneficial effect on POI. The role of TRPA1 in GI motility deserves further extensive investigation with the aim of developing a novel therapeutic strategy to treat GI motility disorders.

Su1911 Neuropeptide S: Effects on Motility, Contractility and Inflammation in the Rat and Human Gastrointestinal Tract

Background: Neuropeptide S (NPS) is expressed by gastrointestinal (GI) enteroendocrine cells and macrophages in rat and man. Polymorphisms of the NPS receptor are linked to increased risk of inflammatory bowel disease as well as motor and sensory disturbances of the gut, suggesting a role for NPS in GI disorders. Further knowledge of NPS effects on motility and inflammation is needed. Methods: Studies of motility were carried out in rats with electrodes implanted in the small bowel. NPS was infused IV for 60 min and effects on myoelectrical activity were recorded. Motility effects of NPS were further studied as luminal pressure changes in anaesthetized rats where the proximal small intestine with intact blood supply was perfused with saline for motility recordings. Tissue samples were obtained from rats for evaluation of gene expression and protein elaboration of inflammatory biomarkers. Muscle strips of the human stomach, small intestine and colon were used for pharmacological analysis in organ baths of motility responses to NPS. Localization of the NPS receptor was done with fluorescent-protein tagging using Cy3-NPS. Plasma levels of NPS were measured using ELISA in 5 healthy and 14 IBD patients before and after meal. Results: In conscious rats, NPS 1 nmol kg-1min-1 increased irregular spiking, 4 nmol kg-1min-1 reduced spiking and increased the MMC cycle length (P=0.005). In anesthetized rats, NPS 0.01-1 nmol kg-1min-1 dose-dependently reduced small bowel motility (P<0.001). In rats, NPS also increased the mRNA expression of iNOS, and CXCL1 and IL-1β at the protein level. In human, fluorescent protein-tagging showed the NPS receptor primarily to be present in enolase-positive nerve fibers, but some scattered also in muscle tissue. In organ baths, NPS 1-100 nM caused TTX-dependent relaxations of the smooth muscle. We found no detectable levels of NPS in plasma of neither healthy subjects nor IBD patients. Conclusion: NPS has a smooth muscle-relaxing effect in the GI tract and dampens myoelectrical and contractile motor activity of the small bowel. This is achieved primarily through an inhibition of nerve-mediated relaxation of smooth muscle cells. Circulating NPS levels in humans are undetectable in healthy subjects and in active IBD. This implies that NPS exerts its inhibitory function by a paracrine or neurocrine mechanism in the gut. NPS seems also capable of intervening with inflammation by increasing gut cytokines and iNOS expression.

H2 S modulates duodenal motility in male rats via activating TRPV1 and K(ATP) channels.

H2 S induces vasodilatation by opening KATP channels but it may also affect other ion channels. The aim of this study was to investigate the effect of H2 S on intestinal motility in rats and its underlying mechanism. The tension of intestinal muscle strips, afferent firing of intestinal mesenteric nerves, length of duodenal smooth muscle cells and whole-cell membrane potential of dorsal root ganglion (DRG) neurons were monitored. H2 S-producing enzymes were located by immunofluorescence staining. NaHS exerted early transient excitation and late long-lasting inhibition on the intestinal contraction. The excitation was attenuated by TRPV1 antagonists capsazepine, A784168, SB-366791 and NK1 receptor antagonist L703606, while the inhibition was attenuated by glibenclamide. NaHS increased duodenal afferent nerve firing and depolarized DRG neurons. These effects were reduced by capsazepine and A784168. NaHS relaxed isolated duodenal smooth muscle cells. The KATP channels were expressed in smooth muscle cells. Cystathionine β-synthase and cystathionine γ-lyase were expressed in rat duodenal myenteric neurons. L-cysteine and S-adenosyl-L-methionine increased the contraction of duodenal muscle strips, an effect attenuated by capsazepine and L703606. NaHS induces biphasic effects on intestinal motility in rats while endogenous H2 S only exerts an excitatory effect. This transient excitatory effect might be mediated by activation of TRPV1 channels in sensory nerve terminals with the consequent release of substance P. The long-lasting inhibitory effect might be mediated by activation of KATP channels in the smooth muscle cells. These findings reveal a novel mechanism for the excitatory effect of H2 S on gastrointestinal motility.

Brain-derived neurotrophic factor enhances cholinergic contraction of longitudinal muscle of rabbit intestine via activation of phospholipase C

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of proteins best known for its role in neuronal survival, differentiation, migration, and synaptic plasticity in central and peripheral neurons. BDNF is also widely expressed in nonneuronal tissues including the gastrointestinal tract. The role of BDNF in intestinal smooth muscle contractility is not well defined. The aim of this study was to identify the role of BDNF in carbachol (CCh)- and substance P (SP)-induced contraction of intestinal longitudinal smooth muscle. BDNF, selective tropomyosin-related kinase B (TrkB) receptor agonists, and pharmacological inhibitors of signaling pathways were examined for their effects on contraction of rabbit intestinal longitudinal muscle strips induced by CCh and SP. BDNF activation of intracellular signaling pathways was examined by Western blot in homogenates of muscle strips and isolated muscle cells. One-hour preincubation with BDNF enhanced intestinal muscle contraction induced by CCh but not by SP. The selective synthetic TrkB agonists LM 22A4 and 7,8-dihydroxyflavone produced similar effects to BDNF. The Trk antagonist K-252a, a TrkB antibody but not p75NTR antibody, blocked the effect of BDNF. The enhancement of CCh-induced contraction by BDNF was blocked by the phospholipase C (PLC) antagonist U73122, but not by ERK1/2 or Akt antagonists. Direct measurement in muscle strips and isolated muscle cells showed that BDNF caused phosphorylation of TrkB receptors and PLC-γ, but not ERK1/2 or Akt. We conclude that exogenous BDNF augments the CCh-induced contraction of longitudinal muscle from rabbit intestine by activating TrkB receptors and subsequent PLC activation.

Brain‐derived neurotrophic factor augments the contraction of intestinal longitudinal muscle strips induced by Carbachol

BackgroundWe have demonstrated that intestinal longitudinal smooth muscle cells synthesize and secrete brain derived neurotrophic factor (BDNF) in response to neuropeptide transmitters. But the function of this BDNF in the gastrointestinal tract is undetermined.Aimto test the effect of BDNF on agonist‐induced contraction in intestinal longitudinal smooth muscle.Material and methodRabbit longitudinal muscle strips were prepared from the intestine. The contraction of the muscle strips was recorded in response to substance P 10–6M (SP 10–6) and Carbachol 10–5M (CCh 10–5). Muscle strips were preincubated with 10 nM exogenous BDNF and the changes in SP/CCh‐induced contraction were examined. A Trk antagonist and two Trk B specific agonists were used to investigate the role of the Trk B receptor in the effect of BDNF on contraction.ResultsBDNF significantly enhanced the contraction of longitudinal smooth muscle induced by CCh 10–5. However, contraction induced by SP 10–6 was not affected by pretreatment with BDNF. The pan Trk receptor inhibitor (K252a) inhibited the effect of BDNF on CCh‐induced contraction. Moreover, two Trk B agonists (LM 22A4 &amp; 7, 8‐dihydroxyflavone) augmented the CCh‐induced contraction of the longitudinal muscle strips.ConclusionBDNF enhances the cholinergic‐induced longitudinal muscle contraction through the activation of its cognate Trk B receptor.

Brain-derived neurotrophic factor enhances the contraction of intestinal muscle strips induced by SP and CGRP in mice

Background and aimsBrain-derived neurotrophic factor (BDNF) has been found in the intestinal tract of a variety of species. Its effects on visceral hyperalgesia have been examined to some degree, but limited studies have focused on gut motility. The aim of the present study was to investigate the effects of BDNF on gut motility of mice. MethodsLongitudinal muscle (LM) strips were prepared from mice ileum and distal colon. The motility of gut was evaluated by the contraction of LM strips, which was recorded by a polyphisograph in vitro. Firstly, the roles of substance P (SP), calcitonin gene-related peptide (CGRP), and acetylcholine (ACh) on the contraction of LM strips were clarified. Then the exogenous BDNF was administered, and the alterations of SP/CGRP/ACh-induced contractions of the muscle strips were explored. Finally, heterozygous BDNF+/− mice and antibody of TrkB were introduced to investigate the role of endogenous BDNF on the SP/CGRP/ACh-induced gut motility. Key resultsSP (10−8–10−6molL−1), CGRP (10−8–10−7molL−1) and ACh (10−8–10−6molL−1) dose-dependently caused the contraction of LM strips from ileum and distal colon, while the excitatory effect of CGRP was preceded by a transient inhibition. But 10−6molL−1 CGRP inhibited the contraction of LM strips. Pretreatment with exogenous BDNF (10−8molL−1) remarkably enhanced the contraction of LM strips induced by SP (10−9–10−7molL−1) and CGRP (10−8–10−9molL−1). However, exogenous BDNF couldn't affect the contraction induced by ACh (10−9–10−7molL−1). The excitatory effects of SP (10−8–10−6molL−1) and CGRP (10−8–10−7molL−1) on the contractions of LM strips from ileum and distal colon were significantly attenuated in BDNF+/− mice compared with those in BDNF+/+ mice, while no difference of the effects of ACh (10−8–10−6molL−1) on LM strips was observed between BDNF+/− mice and BDNF+/+ mice. The monoclonal antibody of TrkB (TrkB-Ab) dramatically attenuated the excitatory effects of SP and CGRP on the contractions of LM strips, without affecting the excitatory effects of ACh. Conclusions and inferencesThese data clarified the excitatory effects of SP, ACh and bilateral effects of CGRP on gut motility of mice and confirmed an essential role of BDNF on accelerating gut motility by enhancing the excitatory effects of SP/CGRP.

A novel CB receptor GPR55 and its ligands are involved in regulation of gut movement in rodents

This study was to investigate the effects of the novel cannabinoid receptor - G protein-coupled receptor 55 (GPR55) - and its ligands O-1602 and cannabidiol (CBD) on gastrointestinal (GI) motility in rodents. Lipopolysaccharide (LPS) was used in vivo to produce the model of septic ileus. The intestinal motility was measured by recording myoelectrical activity of jejunum in rats, and by measuring GI transit with a charcoal marker in mice, in presence of O-1602 or CBD. Inflammatory response was assessed serologically and histologically. The expression and distribution of GPR55 in the different parts of rat intestine were investigated by real-time PCR and immunohistochemistry. In vitro, the effects of the drugs on the GI movement were investigated by measuring the contraction of the intestinal muscle strips in organ bath, and the intracellular responses of the muscle cells with microelectrode technique. G protein-coupled receptor 55 was expressed in different parts of rat intestine. Lipopolysaccharide significantly inhibited the intestinal motility, increased inflammatory cytokines and GPR55 expression. Pretreatment with CBD normalized LPS-induced hypomotility and improved the inflammatory responses serologically and histologically. Both O-1602 and CBD counteracted LPS-induced disturbances of the gut contraction, but had no effect on the membrane potential of the muscle cells, while cannabinoid type 1 receptor antagonist AM251 and cannabinoid type 2 receptor antagonist AM630 increased the potential. G protein-coupled receptor 55 existed throughout the whole intestine of rats. O-1602 or CBD selectively normalized the motility disturbances. Possible mechanisms involved systemic anti-inflammation and the regulation of myoelectrical activity of the intestine.

Effect of the expression of ghrelin receptors on the postoperative underpowered small intestinal motility in rats

To investigate the effect of the expression of ghrelin receptors on the postoperative small intestine dysmotility in rat models. The effect of different concentrations of ghrelin (0, 0.01, 0.1, 0.5, 1.0 μmol/L) on the contraction of smooth muscle strips of rat small intestine in the presence or absence of carbachol was observed in vitro. End-to-side anastomosis was performed in the study group and sham controls were used. The expression of ghrelin receptors(GHS-R1a) in small intestine muscle layers was detected by immunohistochemistry and Western blot. In vitro, ghrelin enhanced the contraction of smooth muscle strips in the presence of carbachol, and the differences in contraction induced by different concentrations of ghrelin(0.1, 0.5, 1.0 μmol/L) were statistically significant [(223±18)%, (245±22)%, (264±25)%, P<0.01]. Immunohistochemistry study showed that GHS-R1a mainly located in the muscular layer of the bowel wall. The expression of GHS-R1a in the circular and longitudinal muscle was significantly weaker than that in the control group. The expression of ghrelin receptors after surgery was down-regulated in the study group, which was lower than that in the control group(0.51±0.02 vs. 0.71±0.01, P<0.01). Down regulation of ghrelin receptors in small intestine muscle layers may contribute to the occurrence of small intestine dysmotility after intestinal surgery.

Changes in muscarinic ligand binding to intestinal muscle strips produced by pre-exposure to hypotonic conditions.

The extent of the binding of [3H]propylbenzilylcholine mustard (3H-PrBCM) to muscarinic receptors in longitudinal muscle strips from guinea-pig small intestine is increased by nearly 50% when the strips are preexposed to distilled water before measurement of 3H-PrBCM binding in Krebs-Henseleit solution. The apparent rate constant for 3H-PrBCM-receptor complex formation is more than double that of intact strips. The curves for the inhibition of 3H-PrBCM binding by methylatropinium bromide in normal and treated strips are superimposable, but, in contrast, distilled water pretreatment shifts the inhibition curve for carbachol to lower concentrations by a factor of 5-6. The inhibition curve for methylfurmethide is also shifted, by a factor of approximately 4, but the effect on the curve for hexyltrimethylammonium (C6TMA) is slight. The relative inhibition produced by benzhexol in the two preparations was variable. Comparison of the rate of equilibration of benzhexol with muscarinic receptors in intact and in distilled water pretreated muscle indicates that this inconsistency is unlikely to be due to incomplete equilibration.

Daikenchuto Ameliorates Muscle Hypercontractility in a Murine T-Cell-Mediated Persistent Gut Motor Dysfunction Model

Background and Aims: Low-grade inflammation and immunological alterations are evident in functional gastrointestinal disorders such as irritable bowel syndrome (IBS). We evaluated the effects of daikenchuto (DKT), a pharmaceutical grade Japanese herbal medicine, on the hypercontractility of intestinal smooth muscle persisting after acute inflammation induced by a T-cell-activating anti-CD3 antibody (αCD3). Methods: BALB/c mice were injected with αCD3 (12.5 µg, i.p.), and DKT (2.7 g/kg) was administered orally once daily for 1 week. The contraction of isolated small intestinal muscle strips and muscle cells was examined on day 7 after αCD3 injection. The gene and protein expressions in the small intestines were evaluated by real-time PCR and multiplex immunoassays, respectively, on days 1, 3 and 7 after αCD3 injection. Results: αCD3 injection resulted in significant increases in carbachol-evoked contractility in the muscle strips and isolated smooth muscle cells on day 7. DKT ameliorated the αCD3-induced muscle hypercontractility on day 7 in both the muscle strips and smooth muscle cells. αCD3 injection rapidly up- and downregulated the mRNA and protein expressions of pro- and anti-inflammatory cytokines, respectively. Although the influence of DKT on the mRNA expressions was moderate, the protein expressions of IL-13 and IL-17 were significantly decreased. Conclusions: We observed changes in the intestinal muscle contractility in muscle strips and muscle cells following resolution of inflammation in a T-cell-mediated model of enteropathy. The observed modulation of cytokine expression and function by DKT may lead to the development of new pharmacotherapeutic strategies aimed at a wide variety of gut motor dysfunction disorders.

Developmental changes of purinergic control of intestinal motor activity during metamorphosis in the African clawed frog,Xenopus laevis

Little is known about the purinergic regulation of intestinal motor activity in amphibians. Purinergic control of intestinal motility is subject to changes during development in mammals. The aim of this study was to investigate purinergic control of intestinal smooth muscle in the amphibian Xenopus laevis and explore possible changes in this system during the developmental phase of metamorphosis. Effects of purinergic compounds on mean force and contraction frequency in intestinal circular muscle strips from prometamorphic, metamorphic, and juvenile animals were investigated. Before metamorphosis, low concentrations of ATP reduced motor activity, whereas the effects were reversed at higher concentrations. ATP-induced relaxation was not inhibited by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) but was blocked by the ecto-nucleotidase inhibitor 6-N,N-diethyl-d-beta,gamma-dibromomethylene ATP (ARL67256), indicating that an ATP-derived metabolite mediated the relaxation response at this stage. Adenosine induced relaxation before, during, and after metamorphosis, which was blocked by the A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable ATP-analog adenosine 5'-[gamma-thio]-triphosphate (ATPgammaS) and 2-methylthioATP (2-MeSATP) elicited contractions in the circular muscle strips in prometamorphic tadpoles. However, in juvenile froglets, 2-MeSATP caused relaxation, as did ATPgammaS at low concentrations. The P2Y(11)/P2X(1)-receptor antagonist NF157 antagonized the ATPgammaS-induced relaxation. The P2X-preferring agonist alpha-beta-methyleneadenosine 5'-triphosphate (alpha-beta-MeATP) evoked PPADS-sensitive increases in mean force at all stages investigated. This study demonstrates the existence of an adenosine A(1)-like receptor mediating relaxation and a P2X-like receptor mediating contraction in the X. laevis gut before, during, and after metamorphosis. Furthermore, the development of a P2Y(11)-like receptor-mediated relaxation during metamorphosis is shown.

Study on the cyclic GMP‐dependency of relaxations to endogenous and exogenous nitric oxide in the mouse gastrointestinal tract

cGMP mediates nitrergic relaxations of intestinal smooth muscle, but several studies have indicated that cGMP-independent mechanisms may also be involved. We addressed this contention by studying the effect of ODQ and ns2028, specific inhibitors of soluble guanylate cyclase, on nitrergic relaxations of the mouse gut. Mouse gastric fundus and small intestinal muscle preparations were mounted in organ baths to study relaxations to exogenous NO, NO donors and electrical field stimulation (EFS) of enteric nerves. In gastric fundus longitudinal muscle strips, ODQ and NS2028 abolished the L-nitroarginine-sensitive relaxations to EFS and the relaxations to NO and NO donors, glyceryl trinitrate (GTN), SIN-1 and sodium nitroprusside (SNP). EFS of intestinal segments and muscle strips showed L-nitroarginine-resistant relaxations, which were abolished by the purinoceptor blocker suramin. In the presence of suramin, ODQ and NS2028 abolished all relaxations to EFS in intestinal segments and strips. ODQ and NS2028 abolished the relaxations to exogenous NO and to the NO donors GTN, SIN-1 and SNP in circular and longitudinal intestinal muscle strips. Intestinal segments showed residual relaxations to NO and GTN. Our results indicate that relaxations to endogenous NO in the mouse gastric fundus and small intestine are completely dependent on cGMP. ODQ and NS2028 incompletely blocked nitrergic relaxations to exogenous NO in intact intestinal segments. However, it is unlikely that this is due to the involvement of cGMP-independent pathways because ODQ and NS2028 abolished all relaxations to endogenous and exogenous NO in intestinal muscle strips.

Angiotensin II induced contraction of rat and human small intestinal wall musculature in vitro

Angiotensin II (Ang II) is a well-known activator of smooth muscle in the vasculature but has been little explored with regard to intestinal wall muscular activity. This study investigates pharmacological properties of Ang II and expression of its receptors in small-intestinal smooth muscle from rats and humans. Isometric recordings were performed in vitro on small intestinal longitudinal muscle strips. Protein expressions of Ang II typ 1 (AT1R) and typ 2 (AT2R) receptors were assessed by Western blot. Ang II elicited concentration-dependent contractions of rat jejunal and ileal muscle preparations. The concentration-response curve (rat ileum, EC(50): 1.5 +/- 0.9 x 10(-8) M) was shifted to the right by the AT1R receptor antagonist losartan (10(-7) M) but was unaffected by the AT2R antagonist PD123319 (10(-7) M) as well as by the adrenolytic guanethidine (3 x 10(-6) M) and the anticholinergic atropine (10(-6) M). Human duodenal, jejunal and ileal longitudinal muscle preparations all contracted concentration-dependently in response to Ang II. The concentration-response curve (human jejunum, EC(50): 1.5 +/- 0.8 x 10(-8) M) was shifted to the right by losartan (10(-7) M) but was unaffected by PD123319 (10(-7) M). Both AT1R and AT2R were detected in all segments of the rat small intestinal wall musculature, whereas only AT1R was readily detectable in the human samples. Ang II elicits contractions of small-intestinal longitudinal muscle preparations from the small intestine of rats and man. The pharmacological pattern and protein expression analyses indicate mediation via the AT1R.

Glial-Derived Neurotrophic Factor Modulates Enteric Neuronal Survival and Proliferation Through Neuropeptide Y

Glial-derived neurotrophic factor (GDNF) promotes the survival and proliferation of enteric neurons. Neuropeptide Y (NPY) is an important peptide regulating gastrointestinal motility. The role of NPY on the survival and proliferation of enteric neurons is not known. We examined the effects of GDNF on the expression and release of NPY from enteric neurons and the role of NPY in promoting enteric neuronal proliferation and survival. Studies were performed in primary enteric neuronal cultures and NPY knockout mice (NPY(-/-)). GDNF-induced expression of NPY was assessed by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay. Using NPY-siRNA and NPY-Y1 receptor antagonist, we examined the role of NPY in mediating the survival and proliferation effects of GDNF. Gastrointestinal motility was assessed by measuring gastric emptying, intestinal transit, and isometric muscle recording from intestinal muscle strips. GDNF induced a significant increase in NPY messenger RNA and protein expression in primary enteric neurons and the release of NPY into the culture medium. NPY (1 mumol/L) significantly increased proliferation of neurons and reduced apoptosis. In the presence of NPY-siRNA and NPY-Y1 receptor antagonist or in enteric neurons cultured from NPY(-/-) mice, GDNF-mediated neuronal proliferation and survival was reduced. NPY increased the phosphorylation of Akt, a downstream target of the PI-3-kinase pathway. In NPY(-/-) mice, there were significantly fewer nNOS-containing enteric neurons compared with wild-type (WT) mice. NPY(-/-) mice had accelerated gastric emptying and delayed intestinal transit compared with WT mice. We demonstrate that NPY acts as an autocrine neurotrophic factor for enteric neurons.