Human Colonic Muscle Research Articles

Different responses of the blockade of the P2Y1 receptor with BPTU in human and porcine intestinal tissues and in cell cultures.

Gastrointestinal smooth muscle relaxation is accomplished by activation of P2Y1 receptors, therefore this receptor plays an important role in regulation of gut motility. Recently, BPTU was developed as a negative allosteric modulator of the P2Y1 receptor. Accordingly, the aim of this study was to assess the effect of BPTU on purinergic neurotransmission in pig and human gastrointestinal tissues. Ca2+ imaging in tSA201 cells that express the human P2Y1 receptor, organ bath and microelectrodes in tissues were used to evaluate the effects of BPTU on purinergic responses. BPTU concentration dependently (0.1 and 1µmolL-1 ) inhibited the rise in intracellular Ca2+ evoked by ADP in tSA201 cells. In the pig small intestine, 30µmolL-1 BPTU reduced the fast inhibitory junction potential by 80%. Smooth muscle relaxations induced by electrical field stimulation were reduced both in pig ileum (EC50 =6µmolL-1 ) and colon (EC50 =35µmolL-1 ), but high concentrations of BPTU (up to 100µmolL-1 ) had no effect on human colonic muscle. MRS2500 (1µmolL-1 ) abolished all responses. Finally, 10µmolL-1 ADPβS inhibited spontaneous motility and this was partially reversed by 30µmolL-1 BPTU in pig, but not human colonic tissue and abolished by MRS2500 (1µmolL-1 ). BPTU blocks purinergic responses elicited via P2Y1 receptors in cell cultures and in pig gastrointestinal tissue. However, the concentrations needed are higher in pig tissue compared to cell cultures and BPTU was ineffective in human colonic tissue.

Opposite effects of dopamine on the mechanical activity of circular and longitudinal muscle of human colon.

Because dopamine (DA) has gained increasing evidence as modulator of gut motility, we aimed to characterize dopaminergic response in human colon, evaluating function and distribution of dopamine receptors in circular vs longitudinal muscle strips. Mechanical responses to DA and dopaminergic agonists on slow phasic contractions and on basal tone were examined in vitro as changes in isometric tension. RT-PCR was used to reveal the distribution of dopaminergic receptors. In spontaneous active circular muscle, DA induced an increase in the amplitude of slow phasic contractions and of the basal tone, via activation of D1-like receptors. DA contractile responses were insensitive to neural blockers or to atropine and inhibited by phospholipase C (PLC) pathway inhibitors. In precontracted circular muscle strips, DA, at the higher concentrations tested, caused a relaxant response via activation of D2-like receptors. In the longitudinal muscle, DA caused only muscular relaxation due to activation of D2-like receptors. DA relaxant responses were insensitive to neural blockers or to nitric oxide synthase inhibitor and reduced by a wide-spectrum K+ channel blockers. Transcripts encoding for all the dopaminergic receptor subtypes was observed in both circular and longitudinal preparations. Dopamine is able to modulate contractile activity of the human colon. In the circular muscle layer, DA induces mainly muscular contraction activating non-neural D1-like receptors, coupled to PLC/IP3 pathway. In the longitudinal muscle layer, DA induces muscular relaxation acting on non-neural D2-like receptors leading to the increase in K+ conductance.

Increased Tone of the Human Colon Muscle by Bisacodyl In Vitro.

Background/AimsAlthough bisacodyl is a widely administered laxative, its underlying mechanism of action remains generally unknown. This study focuses on investigating the effects of bisacodyl on the human colon muscle contraction, and elucidating its mechanism of action.MethodsSigmoid colon muscle strips (20 longitudinal and 18 circular muscles) were obtained from 20 subjects who underwent colectomy for colon cancer. Isometric force measurements were calculated in response to electrical field stimulation (EFS, 0.3 milliseconds in trains of 10 Hz for 20 seconds, 150 V). Peak and nadir (tone) during and after EFS, were measured in a controlled state, and after sequential addition of bisacodyl (1 μM), atropine (1 μM), N-nitro-L-arginine (L-NNA, 100 μM), MRS2500 (1 μM), and tetrodotoxin (TTX, 1 μM) to the organ bath.ResultsTransient phasic contractions were observed during EFS, and after cessation of EFS. In the longitudinal muscles, nadir during EFS, and tone after EFS, significantly increased after addition of bisacodyl, and persisted after sequential addition of atropine, L-NNA, MRS2500, and TTX, indicating a direct action of bisacodyl on the smooth muscle. In the second experiment, pretreatment of TTX abolished EFS-induced phasic contractions. Although no phasic contraction was produced after perfusion of bisacodyl, tone was increased, thereby supporting evidence of a direct mechanism of action of bisacodyl on the colon smooth muscle.ConclusionsBisacodyl increases the tone of longitudinal muscle in the human sigmoid colon through a direct action on the smooth muscle. Further study is warranted to investigate the neural mechanism of action of bisacodyl.

Inhibition effect of Bifidobacterium longum, Lactobacillus acidophilus, Streptococcus thermophilus and Enterococcus faecalis and their related products on human colonic smooth muscle in vitro.

ObjectiveTo investigate the effects of four strains, generally used in clinic, including Bifidobacterium longum, Lactobacillus acidophilus, Streptococcus thermophilus and Enterococcus faecalis, and their related products on human colonic smooth muscle in vitro.MethodsHuman colonic circular muscle strips obtained from disease-free margins of resected segments from 25 patients with colorectal cancer were isometrically examined in a constant-temperature organ bath and exposed to different concentrations of living bacteria, sonicated cell fractions and cell-free supernatant (CFS). The area under the curve (AUC) representing the contractility of smooth muscle strips was calculated.Results(1) The four living probiotics inhibited the contractility of human colonic muscle strips only at high concentration (1010 CFUs/mL, all P<0.05). (2) The sonicated cell fractions from the four probiotics obviously inhibited human colonic smooth muscle strips in a dose-dependent manner (P<0.01). (3) The CFS from the four probiotics also inhibited colonic smooth muscle strips in a dose-dependent manner (all P<0.05). (4) The inhibition effect of CFS from Streptococcus thermophilus and Enterococcus faecalis decreased obviously when pretreated with NG-nitro-L-arginine (L-NNA, 10−5 mol/L) (P<0.05), but not the Bifidobacterium longum and Lactobacillus acidophilus (P>0.05).ConclusionFour common probiotics related products, including the sonicated cell fractions and the CFS, obviously inhibited human colonic smooth muscles strips contraction in a dose-dependent manner. Only high concentration living probiotics (1010 CFUs/mL) can inhibit the colonic smooth muscles strips contraction. The NO pathway may be partly involved in the inhibitory effect of CFS from Streptococcus thermophilus and Enterococcus faecalis.

Modelling Human Colonic Smooth Muscle Cell Electrophysiology.

The colon is a digestive organ that is subject to a wide range of motility disorders. However, our understanding of the etiology of these disorders is far from complete. In this study, a quantitative single cell model has been developed to describe the electrical behaviour of a human colonic smooth muscle cell (hCSMC). This model includes the pertinent ionic channels and intracellular calcium homoeostasis. These components are believed to contribute significantly to the electrical response of the hCSMC during a slow wave. The major ion channels were constructed based on published data recorded from isolated human colonic myocytes. The whole cell model is able to reproduce experimentally recorded slow waves from human colonic muscles. This represents the first biophysically-detailed model of a hCSMC and provides a means to better understand colonic disorders.

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.

Supernatants of irritable bowel syndrome mucosal biopsies impair human colonic smooth muscle contractility.

Changes in intestinal motility are likely to contribute to irritable bowel syndrome (IBS) pathophysiology. The aim of the study was to investigate the effects of IBS mucosal supernatants on human colonic muscle contractility. Supernatants were obtained from biopsies of 18 IBS patients-nine with constipation (IBS-C) and nine with diarrhea-predominant IBS (IBS-D)-and nine asymptomatic subjects, used as controls. Colonic circular smooth muscle strips or isolated cells (SMC) were exposed to control or IBS supernatants. Spontaneous phasic contractions on strips and morphofunctional parameters on cells were evaluated in basal conditions and in response to acetylcholine (Ach). Incubation with IBS supernatants was also conducted in the presence of antagonists and inhibitors (namely histamine, protease and prostaglandin antagonists, nuclear factor-kappa B inhibitor, catalase, NADPH oxidase inhibitor, and the cAMP- and/or cGMP-cyclase inhibitors). Exposure to IBS-C and IBS-D supernatants induced a significant reduction in basal tone and Ach-elicited contraction of muscle strips and a significant shortening and impairment of Ach contraction of SMCs. The NADPH oxidase inhibitor prevented the effect of supernatants, while the protease antagonist only IBS-C effect. No effect was observed with the other antagonists and inhibitors. Dilution of IBS-D supernatants partially restored the effects only on SMCs, whereas dilution of IBS-C supernatants significantly reverted the effects on muscle strips and Ach-elicited response on SMC. Supernatants from mucosal biopsies of IBS patients reduce colonic contractility. The observed impairment was concentration dependent, likely occurring through intracellular oxidative stress damage, involving different neuromotor mechanisms depending on the IBS subtype.

Effects of pro-inflammatory cytokines, lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE2, PGF2α) or their corresponding ethanolamides (PGE2-EA or PGF2α-EA) over 48h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10−5M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10−4M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE2 transiently increased Caco-2 monolayer permeability at 24h, while LPS (10μg/ml) increased permeability over 24–48h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE2 transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.

Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon.

Background & AimsHirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells.MethodsCells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2′-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed.ResultsENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia.ConclusionsNC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.

Ranolazine inhibits voltage-gated mechanosensitive sodium channels in human colon circular smooth muscle cells.

Human jejunum smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs) express the SCN5A-encoded voltage-gated, mechanosensitive sodium channel NaV1.5. NaV1.5 contributes to small bowel excitability, and NaV1.5 inhibitor ranolazine produces constipation by an unknown mechanism. We aimed to determine the presence and molecular identity of Na(+) current in the human colon smooth muscle and to examine the effects of ranolazine on Na(+) current, mechanosensitivity, and smooth muscle contractility. Inward currents were recorded by whole cell voltage clamp from freshly dissociated human colon SMCs at rest and with shear stress. SCN5A mRNA and NaV1.5 protein were examined by RT-PCR and Western blots, respectively. Ascending human colon strip contractility was examined in a muscle bath preparation. SCN5A mRNA and NaV1.5 protein were identified in human colon circular muscle. Freshly dissociated human colon SMCs had Na(+) currents (-1.36 ± 0.36 pA/pF), shear stress increased Na(+) peaks by 17.8 ± 1.8% and accelerated the time to peak activation by 0.7 ± 0.3 ms. Ranolazine (50 μM) blocked peak Na(+) current by 43.2 ± 9.3% and inhibited shear sensitivity by 25.2 ± 3.2%. In human ascending colon strips, ranolazine decreased resting tension (31%), reduced the frequency of spontaneous events (68%), and decreased the response to smooth muscle electrical field stimulation (61%). In conclusion, SCN5A-encoded NaV1.5 is found in human colonic circular smooth muscle. Ranolazine blocks both peak amplitude and mechanosensitivity of Na(+) current in human colon SMCs and decreases contractility of human colon muscle strips. Our data provide a likely mechanistic explanation for constipation induced by ranolazine.

Neuropeptide S Reduces Gut Motility in Rats and Humans

The actions of NPS on intestinal motility and duodenal mucosal barrier function in anaesthetized rat in vivo and human circular muscle strips in vitro were elucidated in this study.A segment of the proximal duodenum or descending colon with an intact blood supply of male Sprague‐Dawley rat was perfused in situ. The effects of intravenous (i.v.) administration of neuropeptide S (NPS) and NG‐nitro‐L‐arginine methyl ester (L‐NAME) were investigated on intestinal motility and duodenal mucosal paracellular permeability. The influence of NPS on ethanol‐induced changes of duodenal mucosal bicarbonate secretion (DBS) was studied. Normal human circular colonic muscle strips were studied in organ bath. Tension changes were measured after NPS administration, NPS challenge with L‐NAME, tetrodotoxin (TTX) or stimulated with EFS. Values are mean ± SEM, compared by 1‐ or 2‐way ANOVA or paired t test when appropriate.NPS decreases both small intestinal and colonic motility but increases duodenal paracellular permeability. Pre‐treating animals with the non‐selective NOS inhibitor L‐NAME abolished the effect of NPS on duodenal motility but not on permeability. NPS reduces ethanol‐induced increases in DBS. In the human colonic smooth muscle strip, NPS dampened contractility with or without electrical field stimulation, which abolished by administration of L‐NAME and TTX.NPS inhibits gastrointestinal motility in animal and man, most likely via a neural nitric oxide (NO) release. To this end, NPS increases paracellular permeability may represent an early step of inflammatory response.

Antioxidant and DNA Damage Protective Effects of Asparagus racemosus in Human Colon and Mice Muscle Cells

Introduction: The present study was designed to investigate the in vitro antioxidant and macromolecule damage protective effects of Asparagus racemosus water (AWE) and methanolic (AME) fractions of roots. Methods: The in vitro antioxidant activity of AWE/AME was estimated by free radical scavenging assays. The DNA damage of HT29 and C2C12 cells was analyzed by comet assay. The plasmid DNA damage and protein oxidation were carried out by agarose gel electrophoresis and SDS-PAGE analysis respectively, where as lipid peroxidation was performed by TBARS assay. Results: Both the extracts showed scavenging activity with IC 50 values of 417.4 ± 19.5 / 298 ± 13.5, 381 ± 18.2 / 235 ± 11.9, 54.8 ± 2.95 / 31.6 ± 1.52, 28.9 ± 1.73 / 19.7 ± 1.55 µg/mL for DPPH, metal chelating, ABTS and Nitric oxide scavenging activities respectively. Similarly the methanolic extract showed more potent reducing power and total antioxidant activities over water fraction. The AME showed 56.8% and 41.2% protection against H 2 O 2 (Hydrogen peroxide) induced DNA damage of HT29 human colon cells and C2C12 murine myoblasts. The extract also showed protection against H 2 O 2 induced plasmid DNA damge, AAPH induced protein oxidation of bovine serum albumin (BSA) and lipid peroxidation of rat hepatic tissue. Conclusion: Over all this study showed remarkable antioxidant and macromolecule damage protective effects of A.racemosus. The observed biological properties may be attributed to the high content phenols and flavonoids in the methanolic extract A. racemosus over water extract.

Effects of menthol on circular smooth muscle of human colon: Analysis of the mechanism of action

Menthol is the major constituent of peppermint oil, an herbal preparation commonly used to treat nausea, spasms during colonoscopy and irritable bowel disease. The mechanism responsible for its spasmolytic action remains unclear. The aims of this study were to investigate the effects induced by menthol on the human distal colon mechanical activity in vitro and to analyze the mechanism of action. The spontaneous or evoked-contractions of the circular smooth muscle were recorded using vertical organ bath. Menthol (0.1mM–30mM) reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions without affecting the frequency and the resting basal tone. The inhibitory effect was not affected by 5-benzyloxytryptamine (1μM), a transient receptor potential-melastatin8 channel antagonist, or tetrodotoxin (1μM), a neural blocker, or 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (10µM), inhibitor of nitric oxide (NO)-sensitive soluble guanylyl cyclase, or tetraethylammonium (10mM), a blocker of potassium (K+)-channels. On the contrary, nifedipine (3nM), a voltage-activated L-type Ca2+ channel blocker, significantly reduced the inhibitory menthol actions. Menthol also reduced in a concentration-dependent manner the contractile responses caused by exogenous application of Ca2+ (75–375μM) in a Ca2+-free solution, or induced by potassium chloride (KCl; 40mM). Moreover menthol (1–3mM) strongly reduced the electrical field stimulation (EFS)-evoked atropine-sensitive contractions and the carbachol-contractile responses. The present results suggest that menthol induces spasmolytic effects in human colon circular muscle inhibiting directly the gastrointestinal smooth muscle contractility, through the block of Ca2+ influx through sarcolemma L-type Ca2+ channels.

Antioxidant activity of inulin and its role in the prevention of human colonic muscle cell impairment induced by lipopolysaccharide mucosal exposure.

BackgroundFructans, such as inulin, are dietary fibers which stimulate gastro-intestinal (GI) function acting as prebiotics. Lipopolysaccharide (LPS) impairs GI motility, through production of reactive oxygen species. The antioxidant activity of various fructans was tested and the protective effect of inulin on colonic smooth muscle cell (SMC) impairment, induced by exposure of human mucosa to LPS, was assessed in an ex vivo experimental model.MethodsThe antioxidant capacity of fructans was measured in an in vitro system that simulates cooking and digestion processes. Human colonic mucosa and submucosa, obtained from disease-free margins of resected segments for cancer, were sealed between two chambers, with the mucosal side facing upwards with Krebs solution with or without purified LPS from a pathogenic strain of Escherichia coli (O111:B4) and inulin (Frutafit IQ), and the submucosal side facing downwards into Krebs solution. The solutions on the submucosal side were collected following mucosal exposure to Krebs in the absence (N-undernatant) or presence of LPS (LPS-undernatant) or LPS+inulin (LPS+INU-undernatant). Undernatants were tested for their antioxidant activity and the effects on SMCs contractility. Inulin protective effects on mucosa and submucosa layers were assessed measuring the protein oxidation level in the experimental conditions analyzed.ResultsAntioxidant activity of inulin, which was significantly higher compared to simple sugars, remained unaltered despite cooking and digestion processes. Inulin protected the mucosal and submucosal layers against protein oxidation. Following exposure to LPS-undernatant, a significant decrease in maximal acetylcholine (Ach)-induced contraction was observed when compared to the contraction induced in cells incubated with the N-undernatant (4±1% vs 25±5% respectively, P<0.005) and this effect was completely prevented by pre-incubation of LPS with Inulin (35±5%).ConclusionsInulin protects the human colon mucosa from LPS-induced damage and this effect appears to be related to the protective effect of inulin against LPS-induced oxidative stress.

Experimental evidence and mathematical modeling of thermal effects on human colonic smooth muscle contractility.

It has been shown, in animal models, that gastrointestinal tract (GIT) motility is influenced by temperature; nevertheless, the basic mechanism governing thermal GIT smooth muscle responses has not been fully investigated. Studies based on physiologically tuned mathematical models have predicted that thermal inhomogeneity may induce an electrochemical destabilization of peristaltic activity. In the present study, the effect of thermal cooling on human colonic muscle strip (HCMS) contractility was studied. HCMSs were obtained from disease-free margins of resected segments for cancer. After removal of the mucosa and serosa layers, strips were mounted in separate chambers. After 30 min, spontaneous contractions developed, which were measured using force displacement transducers. Temperature was changed every hour (37, 34, and 31°C). The effect of cooling was analyzed on mean contractile activity, oscillation amplitude, frequency, and contraction to ACh (10(-5) M). At 37°C, HCMSs developed a stable phasic contraction (~0.02 Hz) with a significant ACh-elicited mean contractile response (31% and 22% compared with baseline in the circular and longitudinal axis, respectively). At a lower bath temperature, higher mean contractile amplitude was observed, and it increased in the presence of ACh (78% and 43% higher than the basal tone in the circular and longitudinal axis, respectively, at 31°C). A simplified thermochemomechanical model was tuned on experimental data characterizing the stress state coupling the intracellular Ca(2+) concentration to tissue temperature. In conclusion, acute thermal cooling affects colonic muscular function. Further studies are needed to establish the exact mechanisms involved to better understand clinical consequences of hypothermia on intestinal contractile activity.

Su1489 Antioxidant Activity of Inulin and Its Ability to Prevent Human Colonic Muscle Cell Impairment Induced by Lipopolysaccharide Mucosal Exposure

Su1489 Antioxidant Activity of Inulin and Its Ability to Prevent Human Colonic Muscle Cell Impairment Induced by Lipopolysaccharide Mucosal Exposure

Myogenic effect of SP-1f and SP-1h two novel β3-adrenoceptor (β3-AR) agonists in human colonic circular smooth muscle

The effect of two novel β3-adrenoceptor (β3-AR) agonists SP-1f and SP-1h on human colon circular smooth muscle contractility and β3-AR mRNA expression have been determined. β3-AR is ascertained co-participates to the control of the gut motility. Isometric tension on human colon muscle strips was measured in response to increasing concentrations of SP-1f, SP-1h and (−)-isoprenaline, alone and in the presence of Betaxolol, ICI 11,855 and SR 59230A (β1-, β2- and β3-AR antagonists, respectively). (−)-Isoprenaline concentration-dependently relaxed circular muscle strips with an EC50=0.32±0.06μM. Such an effect was antagonized either by the contemporaneously presence of Betaxolol and ICI 11,855 [(−)-isoprenaline EC50=1.75±0.35μM, pKB=7.88±0.10] or by Betaxolol, ICI 11,855 and SR 59230A [(−)-isoprenaline EC50=3.49±0.38μM, pKB=8.51±0.14]. Besides, SP-1f and SP-1h concentration-dependently relaxed circular muscle strips with an EC50=0.35±0.07μM and 0.45±0.12μM, respectively. These values remained unchanged by blocking the β1- and β2-AR. The presence of SR 59230A antagonized the relaxing effect of SP-1f (EC50=3.51±0.94μM, pKB=8.93±0.16) and did not modify the SP-1h relaxing potency. In colon circular smooth muscle and in mucosa, β3-AR mRNA expression levels were found to be 0.39±0.70 and 0.26±0.12 (P<0.05), respectively. Such results provide further evidence of the β3-adrenoceptor functional role in the human colon and the crucial contribution of SP-1f to the control of the gut dysmotility.

Lactobacillus rhamnosus protects human colonic muscle from pathogen lipopolysaccharide‐induced damage

Lactobacillus species might positively affect gastrointestinal motility. These Gram-positive bacteria bind Toll-like receptor 2 (TLR2) that elicits anti-inflammatory activity and exerts protective effects on damage induced by lipopolysaccharide (LPS). Whether such effect occurs in gastrointestinal smooth muscle has not been established yet. Aim of this study was to characterize the effects of Lactobacillus rhamnosus GG (LGG) and of supernatants harvested from LGG cultures on human colonic smooth muscle and to explore their protective activity against LPS-induced myogenic morpho-functional alterations. The effects of LGG (ATCC 53103 strain) and of supernatants have been tested on both human colonic smooth muscle strips and isolated cells in the absence or presence of LPS obtained from a pathogenic strain of Escherichia coli. Their effects on myogenic morpho-functional properties, on LPS-induced NFκB activation, and on cytokine production have been evaluated. Toll-like receptor 2 expression has been analyzed by qPCR and flow cytometry. Lactobacillus rhamnosus GG exerted negligible transient effects per se whereas it was capable of activating an intrinsic myogenic response counteracting LPS-induced alterations. In particular, both LGG and supernatants significantly reduced the LPS-induced morpho-functional alterations of muscle cells, i.e. cell shortening and inhibition of contractile response. They also hindered LPS-induced pro-inflammatory effects by decreasing pro-inflammatory transcription factor NFκB activation and pro-inflammatory cytokine IL-6 secretion, and restored the secretion levels of anti-inflammatory cytokine IL10. Taken together these data demonstrate that LGG protects human colonic smooth muscle from LPS-induced myogenic damage and might be beneficial on intestinal motor disorders due to bacterial infection.

Sa2061 Peri-Capillary Interstitial Cells of Cajal in the Human Colon Muscle Layers

Sa2061 Peri-Capillary Interstitial Cells of Cajal in the Human Colon Muscle Layers

Characterization of Vabicaserin (SCA-136), a Selective 5-Hydroxytryptamine 2C Receptor Agonist

The 5-hydroxytryptamine 2C (5-HT(2C)) receptor subtype has received considerable attention as a target for drug discovery, having been implicated in a wide variety of disorders. Here, we describe the in vitro pharmacological profile of the novel 5-HT(2C) receptor-selective agonist vabicaserin [(-)-4,5,6,7,9,9a,10,11,12,12a-decahydrocyclopenta[c] [1,4]diazepino[6,7,1-ij]quinoline hydrochloride] (SCA-136), including a comprehensive strategy to assess 5-HT(2B) receptor selectivity using diverse preparations and assays of receptor activation. Vabicaserin displaced (125)I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT(2C) receptor sites in Chinese hamster ovary cell membranes with a K(i) value of 3 nM and was >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT(2B) receptor subtype using [(3)H]5HT was 14 nM. Vabicaserin was a potent and full agonist (EC(50), 8 nM; E(max), 100%) in stimulating 5-HT(2C) receptor-coupled calcium mobilization and exhibited 5-HT(2A) receptor antagonism and 5-HT(2B) antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. In rat stomach fundus and human colonic longitudinal muscle endogenously expressing 5-HT(2B) receptors, vabicaserin failed to induce a 5-HT(2B) receptor-dependent contraction and produced a rightward shift of the 5-HT and α-methyl-5-HT concentration-response curves in these preparations, respectively, consistent with 5-HT(2B) competitive antagonism. Likewise, vabicaserin failed to induce a 5-HT(2B) receptor-mediated contraction in arteries from deoxycorticosterone acetate-salt-treated rats, a model of hypersensitized 5-HT(2B) receptor function, and produced a rightward shift in the 5-HT-induced response that was consistent with 5-HT(2B) receptor antagonism. In summary, vabicaserin is a novel, potent, and selective 5-HT(2C) receptor agonist.