Intestinal Smooth Muscle Contractility Research Articles

Inhibitory effect and underlying mechanism of essential oil of Prangos ferulacea Lindl (L.) on spontaneous and induced uterine contractions in non-pregnant rats

Evidence suggests the use of natural compounds as support in the management of uterine contractility disorders. We recently demonstrated that the essential oil of Apiacea Prangos ferulacea (L.) (Prangoil) modulates intestinal smooth muscle contractility. Thus, we aimed to evaluate if Prangoil could also affect the contractility of uterine muscle in non-pregnant rat and to investigate the related action mechanism/s. The effects of the aromatic monoterpenes, β-ocimene and carvacrol, constituents of Prangoil, were also evaluated. Spontaneous contractions and contraction-induced by K+-depolarization and oxytocin in rat uterus were recorded in vitro, using organ bath technique. Prangoil reduced the amplitude of spontaneous contractions as well as responses to KCl and oxytocin. β-ocimene and carvacrol matched oil inhibitory effects. Prangoil effects were not affected by nitrergic and adenylyl cyclase inhibitors or non-specific potassium channel blocker, but they were reduced by nifedipine, L-type calcium channel inhibitor, or 2-aminoethoxydiphenylborate (2-APB), membrane-permeant inositol 1,4,5-triphosphate receptor inhibitor. The response to β-ocimene was reduced by nifedipine and by 2-APB (20 μM), whilst carvacrol inhibitory effect was attenuated only by nifedipine. In conclusion, Prangoil, and its components, β-ocimene and carvacrol, reduced spontaneous and KCl or oxytocin-induced contractions of rat myometrium, mainly modulating extracellular Ca2+ influx through L-Type channels and Ca2+ release from the intracellular store. Further studies could contribute to evaluate the potential use of Prangoil against disorders characterized by abnormal uterine contractions.

Effect of agomelatine on intestinal smooth muscle contractility in rats

Objective: The purpose of the present study was to investigate in vitro the effect of agomelatine, an analog of melatonin, on KCl-stimulated contractions in duodenum, jejunum, ileum, and colon in rats. Material and methods: A total of 7 adult Wistar Albino female rats were used in the study. After the rats were decapitated without anesthesia, duodenum, jejunum, ileum, and colon strips were prepared and placed in the organ bath. Agomelatine was applied in doses of 100, 200, and 1000 μM to the strips that were contracted with KCl (80 mM). Then, 1000 μM agomelatine was applied after preliminary treatment of melatonin receptor antagonist luzindole (1 μM) to investigate the action mechanism of agomelatine on intestinal smooth muscles. The records were analyzed before and after each dose, and frequency, peak to peak (P-P) and area values were compared. Results: Agomelatine inhibited the KCI- induced contractions in duodenum, jejunum, ileum, and colon strips in a dose-dependent manner. Depending on the agomelatine application, the frequency, “P-P”, and the area values under the curve decreased at statistically significant levels (p<0.05). However, the effect of agomelatine decreased at significant levels as a result of luzindole treatment (p<0.05). Conclusion: The dose-dependent inhibitor effect of agomelatine on KCl-stimulated contractions in the rat intestinal smooth muscle was shown for the first time in this study. Agomelatine was found to affect the smooth muscles of the intestines through melatonin receptors. This result suggests that agomelatine might have therapeutic potential in diseases in which gastrointestinal motility is impaired.

Nimodipine inhibits intestinal and aortic smooth muscle contraction by regulating Ca2+-activated Cl− channels

Nimodipine is a clinically used dihydropyridine L-type calcium channel antagonist that effectively inhibits transmembrane Ca2+ influx following the depolarization of smooth muscle cells, but the detailed effect on smooth muscle contraction is not fully understood. Ca2+-activated Cl− channels (CaCCs) in vascular smooth muscle cells (VSMCs) may regulate vascular contractility. We found that nimodipine can inhibit transmembrane protein 16A (TMEM16A) activity in a concentration-dependent manner by cell-based fluorescence-quenching assay and short-circuit current analysis, with an IC50 value of ~5 μM. Short-circuit current analysis also showed that nimodipine prevented Ca2+-activated Cl− current in both HT-29 cells and mouse colonic epithelia accompanied by significantly decreased cytoplasmic Ca2+ concentrations. In the absence of extracellular Ca2+, nimodipine still exhibited an inhibitory effect on TMEM16A/CaCCs. Additionally, the application of nimodipine to CFTR-expressing FRT cells and mouse colonic mucosa resulted in mild activation of CFTR-mediated Cl− currents. Nimodipine inhibited basolateral CCh-activated K+ channel activity with no effect on Na+/K+-ATPase activity. Evaluation of intestinal smooth muscle contraction showed that nimodipine inhibits intestinal smooth muscle contractility and frequency, with an activity pattern that was similar to that of non-specific inhibitors of CaCCs. In aortic smooth muscle, the expression of TMEM16A in thoracic aorta is higher than that in abdominal aorta, corresponding to stronger maximum contractility in thoracic aorta smooth muscle stimulated by phenylephrine (PE) and Eact. Nimodipine completely inhibited the contraction of aortic smooth muscle stimulated by Eact, and partially inhibited the contraction stimulated by PE. In summary, the results indicate that nimodipine effectively inhibits TMEM16A/CaCCs by reduction transmembrane Ca2+ influx and directly interacting with TMEM16A, explaining the mechanisms of nimodipine relaxation of intestinal and aortic smooth muscle contraction and providing new targets for pharmacological applications.

Plumbagin Prevents Secretory Diarrhea by Inhibiting CaCC and CFTR Channel Activities.

Secretory diarrhea, which primarily originates through intestinal pathogens and viruses, is a health burden in many regions worldwide. Enterocyte Cl− channels, as the final step in enterotoxin-induced fluid secretion, constitute an attractive class of targets for diarrhea therapy. Chloride channel inhibitors have become a new class of candidates for antisecretion and anti-intestinal motility agents. In the present study, we identified plumbagin as a transmembrane protein 16A (TMEM16A) inhibitor in a cell-based fluorescence-quenching assay, and the IC50 value was ∼12.46 µM. Short-circuit current measurements showed that plumbagin reversibly inhibited the Eact-induced Cl− current on the apical side of TMEM16A-transfected Fischer rat thyroid (FRT) cells with no significant effect on cytoplasmic Ca2+ signaling. Notably, plumbagin also inhibited the activity of intestinal epithelial calcium-activated chloride channel (CaCC) and cystic fibrosis transmembrane conductance regulator (CFTR) in both HT-29 cells and mouse colons, but had no effects on the activity of the Na+-K+ ATPase or K+ channels. In in vivo experiments, the administration of plumbagin reduced both Escherichia coli heat-stable enterotoxin (STa)- and cholera toxin (CT)-induced intestinal fluid secretion. In neonatal mouse models of CT- and rotavirus infection-induced diarrhea, 0.4 µg plumbagin inhibited secretory diarrhea by >40% and 50%, respectively, without affecting intestinal epithelial integrity or the rotaviral infection. In addition, plumbagin exerted inhibitory effects on the vasoactive intestinal peptide (VIP)-, prostaglandin E2 (PGE2)-, and 5-hydroxytryptamine (5-HT)-stimulated Cl− currents. In the evaluations of intestinal motility, plumbagin significantly delayed intestinal motility and inhibited intestinal smooth muscle contractility without an evident impact on contractive frequency. Collectively, our results indicate that plumbagin inhibits both Ca2+- and cAMP-activated Cl− channels, accounting for the mechanisms of plumbagin inhibition of chloride secretion and intestinal motility. Thus, plumbagin can be a lead compound in the treatment of CT-induced, Traveler’s, and rotaviral diarrhea, as well as other types of secretory diarrhea that result from excessive intestinal fluid secretion and increased intestinal peristalsis.

Altered Gut Motility in Mice Lacking Neuropilin 2 in Smooth Muscle

Introduction/ObjectivesProfound changes in gut motility and colonic smooth muscle contraction are observed in inflammatory bowel disease states such as colitis. However, the mechanisms that contribute to altered motility and contractility under these circumstances are incompletely defined. Recent data from our group have identified the smooth muscle of the gastrointestinal tract as a major site of expression of neuropilin 2 (Nrp2) in vivo. Nrp2 and the related molecule neuropilin 1 are 130–140 kDa transmembrane receptors for the class 3 semaphorin (SEMA3) family of axonal guidance regulators and for members of the vascular endothelial growth factor (VEGF) family that regulate angiogenesis. Based on recent data from our group implicating Nrp2 as a novel regulator of bladder smooth muscle contractility, the objective of this study was to determine the functional significance of Nrp2 in gastrointestinal smooth muscle.MethodsTwo mouse models were used to explore a role for Nrp2 in gastrointestinal motility: (i) SM22alpha‐Cre; Nrp2 fl/fl mice to enable inducible smooth muscle‐specific deletion of Nrp2 in vivo under controlled conditions and (ii) wild type Balb/c mice exposed to dextran sodium sulfate (DSS) to evoke acute colitis. Gut tissues were subjected to isometric tension testing to characterize contractility. Gut transit was evaluated by charcoal meal assay. Nrp2 levels were assessed by immunohistochemical staining and immunoblot analysis of tissues and of primary colonic smooth muscle cells exposed to inflammatory cytokines.ResultsSmooth muscle‐specific deletion of Nrp2 led to an increase in force generation of colonic rings, upon exposure to electrical field stimulation or treatment with the cholinergic agonist, carbachol. Nrp2 deletion also led to elevated gut transit as assessed by charcoal meal assay. In response to colitis, Nrp2 levels were found to decrease in the distal colon, in parallel with elevated contractility of distal colon rings. In addition, treatment of primary human colonic smooth muscle cells with IL‐4, representative of the TH2 inflammatory milieu associated with DSS colitis, decreased Nrp2 levels while treatment with TNF‐a, characteristic of the TH1 response in colitis increased Nrp2 levels.ConclusionsThese observations suggest that dynamic alterations in Nrp2 under conditions of inflammation may underlie altered contractility of intestinal smooth muscle, resulting in dysmotility. Moreover, these findings suggest that Nrp2 may represent a novel therapeutic target for restoration of normal gut motility.Support or Funding InformationHarvard Digestive Diseases Center P&F Grant (RMA); R01 DK104641 (RMA, DRB, MPS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interferon-gamma depresses human intestinal smooth muscle cell contractility: Relevance to inflammatory gut motility disturbances

Aim/backgroundIn addition to absorptive disturbances, inflammatory bowel diseases (IBD) perturb normal contractility of intestinal smooth muscle. Such motility disturbances may reflect both nervous alterations and increased abundance of cytokines (e.g. IL-1β, TNF-α, and IFN-γ), which impair normal intestinal smooth muscle structure and function. In a previous study, we reported that IL-1β decreased mesenteric muscular contractility, consistent with cytokine-mediated changes in contraction present in IBD. Here, we considered the impact of pro-inflammatory cytokines on human intestinal smooth muscle cell (HISMC) contractility in vitro, which might provide a method for evaluating treatments for IBD. Materials and methodsWe used an in vitro tonic contraction assay to study how HISMC contractility was affected by cell density, serum, and cytokines (IL-1β, TNF-α, and IFN-γ). MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) and wound healing analyses were also used to measure cell proliferation and migration in HISMC in response to IFN-γ. Key findingsWe found that IFN-γ (but not IL-1β or TNF-α) significantly depressed HISMC tonic contractility over six days. IFN-γ also decreased HISMC proliferation, migration, and smooth muscle F-actin expression in a dose-dependent manner (studied at 4 days). SignificanceOur studies indicate that IFN-γ dose and time-dependently reduces normal HISMC contractility, motility and proliferation which may contribute to dysmotility observed in GI inflammatory disorders and that IFN-γ therapeutics might restore normal HISMC contractility impaired in IBD.

Sourcebook update: intestinal smooth muscle contractility and autonomic control.

This laboratory practical requires first-year students to anticipate the effects of drugs active at cholinergic and adrenergic receptors on gut motility in order to design experiments during an authentic inquiry exercise. Rather than specifying a strict sequence of drug additions that aim to provide ideal demonstrations of pharmacological and physiological antagonism, I have instead designed switches into the drugs provided and set students, working in small teams, the task of identifying the switched drugs, an inquiry activity. To extend the teamwork aspect, laboratory reports were submitted by the student teams rather than individual students. Staff observed that discussions within the teams were stimulated by the inquiry-led nature of the practical. The quality of the laboratory reports submitted by teams were substantially improved over the individual reports submitted in previous years. (Students previously worked in teams, but simply followed a list of prescribed experiments and wrote individual reports.) Although, in conversation, teams of students had an improved understanding of the regulation of gut motility by the parasympathetic and sympathetic divisions of the autonomic nervous system and could readily distinguish between pharmacological and functional antagonism, no attempt was made to evaluate learning because the revision was triggered by the observed effect of a technical error and was not otherwise planned. It is likely that laboratory practicals, in general, would benefit from inclusion of inquiry.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.

Autonomic modification of intestinal smooth muscle contractility.

Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe this spontaneous activity and its modification by agents associated with parasympathetic and sympathetic nerve activity. A section of the rabbit small intestine is suspended in an organ bath, and the use of a pressure transducer and data-acquisition software allows the measurement of tension generated by the smooth muscle of intestinal walls. The application of the parasympathetic neurotransmitter ACh at varying concentrations allows students to observe an increase in intestinal smooth muscle tone with increasing concentrations of this muscarinic receptor agonist. Construction of a concentration-effect curve allows students to calculate an EC50 value for ACh and consider some basic concepts surrounding receptor occupancy and activation. Application of the hormone epinephrine to the precontracted intestine allows students to observe the inhibitory effects associated with sympathetic nerve activation. Introduction of the drug atropine to the preparation before a maximal concentration of ACh is applied allows students to observe the inhibitory effect of a competitive antagonist on the physiological response to a receptor agonist. The final experiment involves the observation of the depolarizing effect of K(+) on smooth muscle. Students are also invited to consider why the drugs atropine, codeine, loperamide, and botulinum toxin have medicinal uses in the management of gastrointestinal problems.

Protective effects of proanthocyanidins on intestinal smooth muscle contractility in rats with intestinal ischemia-reperfusion injury

Protective effects of proanthocyanidins on intestinal smooth muscle contractility in rats with intestinal ischemia-reperfusion injury

Alterations of colonic contractility in an interleukin-10 knockout mouse model of inflammatory bowel disease.

Background/AimsInflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10−/−) mice.MethodsPrepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining.ResultsThe spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10−/− mice compared to IL-10+/+ mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10−/− mice compared to IL-10+/+ mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10−/− mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10−/− mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased.ConclusionsThese results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon.

ERK and p38MAPK pathways regulate myosin light chain phosphatase and contribute to Ca2+ sensitization of intestinal smooth muscle contraction

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase (ERK) and p38MAPK, are known regulators of smooth muscle contractility. The contraction of smooth muscle is mainly regulated by the phosphorylation of regulatory light chains of myosin II (LC20), which is driven by the balance between myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We hypothesized that one possible mechanism for MAPK-dependent modulation of intestinal smooth muscle contractility is via the regulation of MLCP activity. Contractile responses to carbachol (CCh) and effects of MAPK inhibitors on CCh-induced contractions were assessed with isolated rat ileal longitudinal smooth muscle strips. Biochemical assessments of MLCP activity and myosin phosphatse targeting subunit (MYPT1) and CPI-17 phosphorylations were completed. Treatment of ileal smooth muscle with PD98059 (10 μM; MEK inhibitor) or SB203580 (10 μM; p38MAPK inhibitor) significantly inhibited CCh-induced contractile force. Decreased MLCP activity was observed during sustained contractions induced by CCh; the MLCP activity was recovered by treatment with PD98059 and SB203580. However, MYPT1 (Thr697 and Thr855) and CPI-17 (Thr38) phosphorylations were not affected. Application of ML-7 (MLCK inhibitor) during CCh-induced sustained contraction elicited an MLCP-dependent relaxation, the rate of which was accelerated by application of PD98059 and SB203580 with proportional changes in LC20 phosphorylation levels but not MYPT1 phosphorylation (Thr697 or Thr855). ERK and p38MAPK contribute to CCh-induced sustained contraction in a LC20 phosphorylation dependent manner. Moreover, both kinases inhibit MLCP activity possibly by a novel mechanism.

In a non‐human primate model, aging disrupts the neural control of intestinal smooth muscle contractility in a region‐specific manner

BackgroundIncidences of gastrointestinal (GI) motility disorders increase with age. However, there is a paucity of knowledge about the aging mechanisms leading to GI dysmotility. Motility in the GI tract is a function of smooth muscle contractility, which is modulated in part by the enteric nervous system (ENS). Evidence suggests that aging impairs the ENS, thus we tested the hypothesis that senescence in the GI tract precipitates abnormalities in smooth muscle and neurally mediated contractility in a region-specific manner.MethodsJejunal and colonic circular muscle strips were isolated from young (4–10 years) and old (18+ years) baboons. Myogenic responses were investigated using potassium chloride (KCl) and carbachol (CCh). Neurally mediated contractile responses were evoked by electrical field stimulation (EFS) and were recorded in the absence and presence of atropine (1 μM) or NG-Nitro-l-arginine methyl ester (l-NAME; 100 μM).Key ResultsThe myogenic responses to KCl in the jejunum and colon were unaffected by age. In the colon, but not the jejunum, CCh-induced contractile responses were reduced in aged animals. Compared to young baboons, there was enhanced EFS-induced contractility of old baboon jejunal smooth muscle in contrast to the reduced contractility in the colon. The effect of atropine on the EFS response was lower in aged colonic tissue, suggesting reduced participation of acetylcholine. In aged jejunal tissue, higher contractile responses to EFS were found to be due to reduced nitregic inhibition.Conclusions & InferencesThese findings provide key evidence for the importance of intestinal smooth muscle and ENS senescence in age-associated GI motility disorders.

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.

In vitro effects of lidocaine on contractility of circular and longitudinal equine intestinal smooth muscle

The purpose of the study was to compare the contractility-enhancing effects of lidocaine in equine jejunal circular (CSM) and longitudinal smooth muscle (LSM) in vitro. In previous studies, more pronounced effects of lidocaine were observed in ischaemia–reperfusion (IR) injured smooth muscle. Therefore in this study, effects were examined in both non-injured control tissues and tissues challenged by a defined, artificial IR injury. Isometric contractile performance of CSM and LSM, assessed by frequency (F), amplitude (A) and mean active force (MAF) of contractions, was defined as contractility.LSM featured lower basic contractility compared to CSM. Lidocaine provoked contractility-enhancing effects in both smooth muscle layers, but except for F at high lidocaine concentrations, contractility of LSM remained lower throughout the trial. Additionally, higher lidocaine concentrations were required to cause significant effects in LSM. No differences were observed in contractility of control and IR injured smooth muscle, but higher lidocaine concentrations were needed to provoke effects in IR injured smooth muscle. In contrast to CSM, contractility of LSM did not decrease at comparably high lidocaine concentrations.Differences in basic contractility of CSM and LSM might be explained by physiologically lower activity of LSM per se or by a thinner LSM layer with fewer smooth muscle cells taking part in contractions. The smaller thickness of the LSM layer may also contribute to persisting discrepancies in contractility following lidocaine application. Additionally, variations in lidocaine concentrations necessary for inducing significant effects could result from differences in the molecular structure of CSM and LSM cells.

A Cav1.2 Ca2+ channel mutation that reduces intestinal smooth muscle contractility

Ca2+ entry through Cav1.2 L‐type voltage‐gated Ca2+ channels is a Ca2+ source for intestinal smooth muscle contraction. We examined Cav1.2 in a Timothy Syndrome (TS) mouse with a TS2 G406R mutation and a neo cassette suppressor of Cav1.2 expression. Isometric force was measured in rings of proximal colon from TS2 positive (TS2NEO) and negative (LM) littermate mice. Spontaneous contractile activity occurred in all tissues, but treatment with Nω‐nitro‐L‐arginine (10−3 M, L‐NA), a nitric oxide synthase antagonist, caused rhythmic contractions (LM, 1.37±0.62 vs. 0.02±0.16 A(area under response curve)/min/g tissue, untreated, n=3). Electric field stimulation (EFS, 0.5–20 Hz) produced frequency‐dependent tension in all tissues, suggesting the release of multiple neurotransmitters. An increase in tension was seen when stimulation was stopped, which was significantly less in TS2NEO mice (5 Hz, LM, 0.24±0.05; TS2NEO, 0.11±0.04 g tension/g tissue, n=5). Tissues relaxed when stimulated by 20 Hz EFS, but contracted at 20 Hz if pre‐exposed to L‐NA, and tension was significantly less in TS2NEO mice (LM, 5.44±1.08; TS2NEO, 1.61±0.44 A/min/g, n=5). L‐NA‐induced tension was blocked by the muscarinic antagonist, atropine (10−3 M), suggesting nitronergic and cholinergic control of muscle activity. These data show that mutations associated with TS result in reduced contractile activity. (DOD AR080107, NIH R21HL088058)

Biphasic Regulation of Myosin Light Chain Phosphorylation by p21-activated Kinase Modulates Intestinal Smooth Muscle Contractility

Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

IBS and the role of otilonium bromide

Awareness of the seriousness of irritable bowel disorder (IBS) remains low among clinicians. In this review, we summarize the current knowledge of IBS and highlight the major personal, economic, and social burden of the disease, and the importance of adequate treatment of what is still often viewed as a trivial disorder. In fact, IBS is a major reason for referral. It is crucial that the varied pathophysiologies of this complex heterogeneous disease are understood in order to be able to treat both the presenting symptoms (pain, bloating, flatulence, abnormal defecation, diarrhea, constipation) and the underlying disorder effectively. Low-grade inflammatory and immune activation has been observed, but the precise triggers and mechanisms, and the relevance to symptom generation, remain to be established. IBS patients require different treatment strategies according to the pattern, severity, frequency, and symptoms. While initial therapy traditionally targets the most bothersome symptom, long-term therapy aims at maintaining symptom control and preventing recurrence. In addition to dietary/lifestyle interventions and psychosocial strategies, a wide range of pharmacologic therapies are approved for use in IBS depending on the symptoms reported. Musculotropic spasmolytics, which act directly on intestinal smooth muscle contractility, such as otilonium bromide, are effective, particularly in the relief of abdominal pain and bloating, and are well tolerated in IBS. THE OBIS TRIAL: The recent large placebo-controlled Otilonium Bromide in Irritable Bowel Syndrome study demonstrated the superiority of otilonium bromide versus placebo not only in the reduction of pain and bloating, but also in protection from relapse due to the long-lasting effect.

Modulation of gastrointestinal function by MuDelta, a mixed µ opioid receptor agonist/ µ opioid receptor antagonist

Loperamide is a selective µ opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating µ opioid receptor agonism with δ opioid receptor antagonism, by combining reference compounds or using a novel compound ('MuDelta'), could normalize GI motility without constipation. MuDelta was characterized in vitro as a potent µ opioid receptor agonist and high-affinity δ opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic δ opioid receptor immunoreactivity was quantified. δ Opioid receptor antagonism opposed µ opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated δ opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model. MuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.

Inhibitory effects of daidzein on intestinal motility in normal and high contractile states

Context: Daidzein is a naturally occurring compound and has various health benefits. However, its effects on intestinal smooth muscle contractility remain unknown.Aims: The present study was to characterize the effects of daidzein on the contractility of isolated jejunal smooth muscle and its underlying mechanisms.Methods: Ex vivo assay was selected as the major method to determine the effects of daidzein on the contractility of isolated jejunal smooth muscle fragment (JSMF).Results: Daidzein (5–160 µmol/L) inhibited the contractility of JSMF in normal contractile state and in a dose-dependent manner. Daidzein also inhibited the contractility of JSMF induced by ACh, histamine, erythromycin and high Ca2+, respectively, and decreased charcoal propulsion in the small intestine in vivo. The inhibitory effects of daidzein were partially blocked by phentolamine or propranolol and were abolished in the presence of varapamil or at Ca2+-free assay condition. However, the inhibitory effects of daidzein on jejunal contraction were not significantly influenced by nitric oxide (NO) synthase inhibitor L-NG-nitro-arginine (l-NNA). Daidzein was also found to directly inhibit the phosphorylation and Mg2+-ATPase activity of smooth muscle myosin.Discussion and Conclusion: The results implicated that α- and β-adrenergic receptors were involved in the inhibitory effects produced by daidzein rather than via NO pathway. As a phytoestrogen, daidzein has shown its potential value in relieving the hypercontractility of small intestine.