Contraction Of Longitudinal Smooth Muscle Research Articles

Embryonic Development of Motility: Lessons from the Chicken.

I outline here the development of intestinal motility in the chicken embryo. The first contractile events are circular smooth muscle driven calcium waves (E6), that gain a clock-like regularity when interstitial cells of Cajal become electrically active (E14). Soon after longitudinal smooth muscle contractions become prominent (E14), the enteric nervous system starts controlling motility (E16) by coupling the longitudinal and circular contractions via inhibitory neurotransmission. It gives rise to circular-longitudinal antagonism, to the migrating motor complex, and to the polarized ascending contraction-descending relaxation pressure response known as the "law of the intestine". The kinetics of gut development in the chicken appears to follow faithfully that of humans by simply converting embryonic days of chicken development into embryonic weeks of human development.

On Structure-Function Relationships in the Female Human Urethra: A Finite Element Model Approach.

Remarkably little is known about urethral striated and smooth muscle and vascular plexus contributions to maintaining continence or initiating micturition. We therefore developed a 3-D, multiphysics, finite element model, based on sequential MR images from a 23-year-old nulliparous heathy woman, to examine the effect of contracting one or more individual muscle layers on the urethral closure pressure (UCP). The lofted urethra turned out to be both curved and asymmetric. The model results led us to reject the current hypothesis that the striated and smooth muscles contribute equally to UCP. While a simulated contraction of the outer (circular) striated muscle increased closure pressure, a similar contraction of the large inner longitudinal smooth muscle both reduced closure pressure and shortened urethral length, suggesting a role in initiating micturition. When age-related atrophy of the posterior striated muscle was simulated, a reduced and asymmetric UCP distribution developed in the transverse plane. Lastly, a simple 2D axisymmetric model of the vascular plexus and lumen suggests arteriovenous pressure plays and important role in helping to maintain luminal closure in the proximal urethra and thereby functional urethral length. More work is needed to examine interindividual differences and validate such models in vivo.

Dysregulation of epithelial ion transport and neurochemical changes in the colon of a parkinsonian primate

The pathological changes underlying gastrointestinal (GI) dysfunction in Parkinson’s disease (PD) are poorly understood and the symptoms remain inadequately treated. In this study we compared the functional and neurochemical changes in the enteric nervous system in the colon of adult, L-DOPA-responsive, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmoset, with naïve controls. Measurement of mucosal vectorial ion transport, spontaneous longitudinal smooth muscle activity and immunohistochemical assessment of intrinsic innervation were each performed in discrete colonic regions of naïve and MPTP-treated marmosets. The basal short circuit current (Isc) was lower in MPTP-treated colonic mucosa while mucosal resistance was unchanged. There was no difference in basal cholinergic tone, however, there was an increased excitatory cholinergic response in MPTP-treated tissues when NOS was blocked with L-Nω-nitroarginine. The amplitude and frequency of spontaneous contractions in longitudinal smooth muscle as well as carbachol-evoked post-junctional contractile responses were unaltered, despite a decrease in choline acetyltransferase and an increase in the vasoactive intestinal polypeptide neuron numbers per ganglion in the proximal colon. There was a low-level inflammation in the proximal but not the distal colon accompanied by a change in α-synuclein immunoreactivity. This study suggests that MPTP treatment produces long-term alterations in colonic mucosal function associated with amplified muscarinic mucosal activity but decreased cholinergic innervation in myenteric plexi and increased nitrergic enteric neurotransmission. This suggests that long-term changes in either central or peripheral dopaminergic neurotransmission may lead to adaptive changes in colonic function resulting in alterations in ion transport across mucosal epithelia that may result in GI dysfunction in PD.

499 SUSTAINED ESOPHAGEAL CONTRACTIONS (SEC) BY FLIP PROVIDES ADDITIONAL CHARACTERISTICS ON ESOPHAGEAL DYSMOTILITY IN PATIENTS WITH ESOPHAGEAL SYMPTOMS

Abstract Sustained esophageal contractions (SEC) have previously been described on high frequency esophageal ultrasound, high resolution manometry (HRM), and more recently on real time impedance planimetry with functional lumen imaging probe (FLIP). SEC may demonstrate a discoordination between circular and longitudinal esophageal smooth muscle contractions. The aim of this study was to determine the clinical characterization of SEC as detected on FLIP compared to HRM. Methods We performed a retrospective chart review of patients who underwent both FLIP and HRM at our Center for Esophageal Diseases between May 2017 and November 2019. FLIP endoscopic reports were reviewed for the presence of SECs which were defined as vigorous lumen closure ≥5 seconds at the LES or esophageal body as noted by the endoscopist during the real time FLIP examination of a sedated endoscopy. Primary variables recorded included EGJ distensibility index (EGJ-DI), diameter, and the presence of SECs at the 40 mL and 60 mL fill volumes. HRM diagnosis, clinical symptoms and outcomes were also reviewed. Results Of 165 patients who underwent HRM and FLIP imaging, 64 patients (mean age 53.7 years, 20 male) with SECs were included in the study. Among these patients, HRM demonstrated a wide variety of motility disorders (Figure 1). 40% of those with EGJOO, Type II or Type III achalasia, and all patients with jackhammer esophagus had SECs on FLIP imaging. Of 56 patients with normal motility or IEM, 22 had SECs, and 18 patients underwent an endoscopic intervention. Among those who had an intervention, 11 (61%) reported improvement in symptoms with improvement in Eckardt score from 4.9 to 3.9. Conclusion SECs are frequently present in hypercontractile esophageal disorders. Interestingly, 39% of patients with normal motility or IEM have SECs noted on FLIP, and 82% of these patients had an intervention performed based on FLIP findings with 61% reporting symptom improvement. Overall, 19% of patients with normal motility or IEM improved due to interventions performed based on SECs found on FLIP. Detection of SEC during FLIP imaging can guide treatment aimed at relaxation of esophageal muscles.

Shugan Decoction Alleviates Colonic Dysmotility in Female SERT-Knockout Rats by Decreasing M3 Receptor Expression.

BackgroundIrritable bowel syndrome (IBS) is a functional gut disease characterized by visceral hypersensitivity and gut motor dysfunction. Serotonin (5-hydroxytryptamine, 5-HT) is an important enteric neurotransmitter. High levels of 5-HT aggravate IBS symptoms. The serotonin reuptake transporter (SERT) is a membrane-embedded transporter involved in IBS pathogenesis that plays an important role in regulating 5-HT signaling.AimWe investigated whether gut motor function was altered in SERT-knockout (SERT-KO) rats. Additionally, we sought to determine whether Shugan decoction (SGD), a clinically experienced prescription for the treatment of IBS, exerts regulatory effects on intestinal motility in SERT-KO rats, and attempted to identify the mechanisms involved.MethodSERT-KO rats were produced by transcription activator-like effector nuclease (TALEN) technology. Fecal pellet output was measured for ten consecutive days to estimate distal colonic motility. Small intestinal motility was measured by charcoal-meal experiments. The colonic and small intestinal muscle contractile activities were measured by organ bath study. Western blot was used to analyze the muscarinic receptor expression in colon tissue.ResultCompared with that in wild-type (WT) rats, the defecation amount, amplitude of spontaneous contraction, and the tension of ACh-induced contraction of colonic longitudinal smooth muscle in SERT-KO rats were significantly increased. The expression of muscarinic receptor subtype-3 (M3R) in the colons of SERT-KO rats was also elevated. SGD can decrease defecation of SERT-KO rats. Moreover, SGD reduced the amplitude of spontaneous contraction, the frequency and tension of ACh-induced contraction of colonic longitudinal smooth muscle, and the expression of M3R in the colon in SERT-KO rats.ConclusionsSERT-KO rats showed increased defecation accompanied by enhanced colonic motility and M3R expression. The findings suggest that SGD modifies colonic dysmotility and reduces defecation in SERT-KO rats by down-regulating M3R expression in the colon.

Involvement of different receptor subtypes in prostaglandin E2-induced contraction and relaxation in the lower esophageal sphincter and esophageal body

Prostaglandin E2 (PGE2) plays a role in the pathogenesis of gastro-esophageal reflux disease (GERD). There are 4 subtypes of PGE2, PGE2 receptor 1, 2, 3 and 4 (EP 1–4). In GERD patents, PGE2, EP2 and EP4 are upregulated. However, the effects of PGE2 on esophageal motility remain elusive.We examined how PGE2 regulates motility in the porcine circular smooth muscle of the lower esophageal sphincter (LES), and the circular and longitudinal smooth muscle of the esophagus body in organ bath.PGE2 induced tonic relaxation in the LES and circular smooth muscle, but transient contraction in longitudinal smooth muscle. The relaxation of the LES and circular smooth muscle was similar in pattern and mechanism, but was much larger in the LES. The relaxation was completely blocked by a voltage-gated K+ channel blocker or 40 mM K+ depolarization, indicating the involvement of K+ channel. Longitudinal smooth muscle contraction was completely blocked by an L-type Ca2+ channel blocker, showing the contribution of Ca2+ movement. The involvement of the EP receptor in motility was examined with selective receptor agonists and antagonists. Activation of EP2 and EP4 caused relaxation in the LES and circular smooth muscle. Compatible with PGE2, EP2 and EP4 agonists caused more significant relaxation in the LES than in circular smooth muscle. EP1 contributed to the longitudinal smooth muscle contraction.The different effects of PGE2 in the LES, circular and longitudinal smooth muscle contributes to esophageal motility, their impairment might increase the amount and frequency of esophageal reflux.

Embryogenesis of the peristaltic reflex.

Neurogenic gut movements start after longitudinal smooth muscle differentiation in three species (mouse, zebrafish, chicken), and at E16 in the chicken embryo. The first activity of the chicken enteric nervous system is dominated by inhibitory neurons. The embryonic enteric nervous system electromechanically couples circular and longitudinal spontaneous myogenic contractions, thereby producing a new, rostro-caudally directed bolus transport pattern: the migrating motor complex. The response of the embryonic gut to mechanical stimulation evolves from a symmetric, myogenic response at E12, to a neurally mediated, polarized, descending inhibitory, 'law of the intestine'-like response at E16. High resolution, whole-mount 3D reconstructions are presented of the enteric nervous system of the chicken embryo at the neural-control stage E16 with the iDISCO+ tissue clarification technique. Gut motility is a complex transport phenomenon involving smooth muscle, enteric neurons, glia and interstitial cells of Cajal. Because these different cells differentiate and become active at different times during embryo development, studying the ontogenesis of motility offers a unique opportunity to 'time-reverse-engineer' the peristaltic reflex. Working on chicken embryo intestinal explants in vitro, we found by spatio-temporal mapping and signal processing of diameter and position changes that motility follows a characteristic sequence of increasing complexity: (1) myogenic circular smooth muscle contractions from E6 to E12 that propagate as waves along the intestine, (2) overlapping and independent, myogenic, low-frequency, bulk longitudinal smooth muscle contractions around E14, and (3) tetrodotoxin-sensitive coupling of longitudinal and circular contractions by the enteric nervous system as from E16. Inhibition of nitric oxide synthase neurons shows that the coupling consists in nitric oxide-mediated relaxation of circular smooth muscle when the longitudinal muscle layer is contracted. This mechanosensitive coupling gives rise to a directional, cyclical, propagating bolus transport pattern: the migrating motor complex. We further reveal a transition to a polarized, descending, inhibitory reflex response to mechanical stimulation after neuronal activity sets in at E16. This asymmetric response is the elementary mechanism responsible for peristaltic transport. We finally present unique high-resolution 3D reconstructions of the chicken enteric nervous system at the neural-control stage based on confocal imaging of iDISCO+ clarified tissues. Our study shows that the enteric nervous system gives rise to new peristaltic transport patterns during development by coupling spontaneous circular and longitudinal smooth muscle contraction waves.

Inhibitory effects of patchouli alcohol on stress-induced diarrhea-predominant irritable bowel syndrome.

AIMTo elucidate the mechanism of patchouli alcohol (PA) in treatment of rat models of diarrhea-predominant irritable bowel syndrome (IBS-D).METHODSWe studied the effects of PA on colonic spontaneous motility using its cumulative log concentration (3 × 10−7 mol/L to 1 × 10−4 mol/L). We then determined the responses of the proximal and distal colon segments of rats to the following stimuli: (1) carbachol (1 × 10−9 mol/L to 1 × 10−5 mol/L); (2) neurotransmitter antagonists including Nω-nitro-L-arginine methyl ester hydrochloride (10 μmol/L) and (1R*, 2S*)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (1 μmol/L); (3) agonist α,β-methyleneadenosine 5′-triphosphate trisodium salt (100 μmol/L); and (4) single KCl doses (120 mmol/L). The effects of blockers against antagonist responses were also assessed by pretreatment with PA (100 μmol/L) for 1 min. Electrical-field stimulation (40 V, 2-30 Hz, 0.5 ms pulse duration, and 10 s) was performed to observe nonadrenergic, noncholinergic neurotransmitter release in IBS-D rat colon. The ATP level of Kreb’s solution was also determined.RESULTSPA exerted a concentration-dependent inhibitory effect on the spontaneous contraction of the colonic longitudinal smooth muscle, and the half maximal effective concentration (EC50) was 41.9 μmol/L. In comparison with the KCl-treated IBS-D group, the contractile response (mg contractions) in the PA + KCl-treated IBS-D group (11.87 ± 3.34) was significantly decreased in the peak tension (P < 0.01). Compared with CCh-treated IBS-D rat colon, the cholinergic contractile response of IBS-D rat colonic smooth muscle (EC50 = 0.94 μmol/L) was significantly decreased by PA (EC50 = 37.43 μmol/L) (P < 0.05). Lack of nitrergic neurotransmitter release in stress-induced IBS-D rats showed contraction effects on colonic smooth muscle. Pretreatment with PA resulted in inhibitory effect on L-NAME-induced (10 μmol/L) contraction (P < 0.05). ATP might not be the main neurotransmitter involved in inhibitory effects of PA in the colonic relaxation of stress-induced IBS-D rats.CONCLUSIONPA application may serve as a new therapeutic approach for IBS-D.

Nitrergic signaling via interstitial cells of Cajal and smooth muscle cells influences circular smooth muscle contractility in murine colon.

Regulation of gastrointestinal motility involves excitatory and inhibitory neurotransmission. Nitric oxide (NO), the major inhibitory neurotransmitter, acts via its receptor NO-sensitive guanylyl cyclase (NO-GC). In the GI tract, NO-GC is expressed in several cell types such as smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Using cell-specific knockout mice, we have previously shown that NO-GC modulates spontaneous contractions in colonic longitudinal smooth muscle. However, its detailed role in the colonic circular smooth muscle is still unclear. Myography was performed to evaluate spontaneous contractions in rings of proximal colon (2.5mm) from global (GCKO) and cell-specific knockout mice for NO-GC. Immunohistochemistry and in situ hybridization were used to specify NO-GC expression. Colonic circular smooth muscle showed three different contraction patterns: high-frequency ripples, slow phasic contractions, and large contractions. Ripples formed independently of NO-GC. Slow phasic contractions occurred intermittently in WT, SMC-GCKO, and ICC-GCKO tissue, whereas they were more prominent and prolonged in GCKO and SMC/ICC-GCKO tissue. Tetrodotoxin and the NO-GC inhibitor ODQ transformed slow phasic contractions of WT and single cell-specific knockout into GCKO-like contractions. ODQ increased the frequency of large contractions in WT and ICC-GCKO colon but not in GCKO, SMC-GCKO, and SMC/ICC-GCKO preparations. Tetrodotoxin and hexamethonium abolished large contractions. We conclude that short rings of murine colon can be effectively used to record spontaneous contractions. Although NO-GC in SMC determines smooth muscle tone, concerted action of NO-GC in both SMC and ICC modulates slow phasic contractions and large contractions.

Impact of prenatal and postnatal exposure to the pesticide chlorpyrifos on the contraction of rat ileal muscle strips: involvement of an inducible nitric oxide synthase-dependent pathway.

Prenatal/postnatal exposure to insecticides has been linked to developmental disorders in adulthood. Chlorpyrifos (CPF) is a widely used organophosphorus acetylcholinesterase (AChE)-inhibiting insecticide. The present study established whether prenatal and postnatal exposure to CPF is associated with intestinal motor dysfunction in adult rats. Three groups of pregnant rats were exposed to either CPF (1 or 5mg/kg/day; the CPF1 and CPF5 groups) or vehicle (the control group) by gavage from gestational day 1 until weaning. At weaning, the pups were separated from their dams and individually gavaged (with the same dose) until postnatal day 60. We then measured in vivo intestinal transit and the in vitro contractile responses of ileal smooth muscle strips to electrical field stimulation. Expression of inducible nitric oxide synthase (iNOS) in the ileum was determined using qRT-PCR and immunoblots. Morphometry and AChE assays were also performed. At adulthood, the mean body mass was lower in the CPF1 and CPF5 groups than in controls. CPF5 exposure was associated with weaker in vitro contraction of ileal muscle strips, which was reversed by adding the NOS inhibitor (L-NAME). There was no significant intergroup difference in the mean in vivo transit time. Exposure to CPF was associated with greater iNOS expression, lower AChE activity and reduced circular and longitudinal smooth muscle thickness. Prenatal and postnatal exposure to CPF in the rat is associated with weaker contraction of ileal longitudinal smooth muscle via a nitrergic mechanism with increased iNOS expression.

The effect of alfalfa saponins on the contractility of bovine isolated abomasum and duodenum preparations

Saponins, a vast group of secondary plant metabolites, occur naturally in plants that are commonly present in ruminants’ diet or plants that are used as phytogenic feed additives. They have gained particular attention among ruminant nutritionists due to numerous health benefits resulting in improved animal performance and feed conversion efficiency. Despite the broad knowledge of saponins’ effects of on cattle, little is known about their impact on gut motility. Therefore, the effect of two structurally divergent alfalfa saponins on the contractility of abomasum and duodenum smooth muscle was examined.The study was conducted on tissues obtained from routinely slaughtered, healthy cows. The experiments were carried out on longitudinal and circular smooth muscle preparations under isometric conditions. The effect of hederagenin and medicagenic acid in a concentration range of 0.001–100µM was verified in a non-cumulative manner on acetylcholine-precontracted smooth muscle specimens. The results are expressed as percentage of the control contraction induced by acetylcholine.Generally, both triterpenoid saponins caused significant enhancement of acetylcholine-induced contraction of abomasum and duodenum preparations. Hederagenin generated dose-dependently significant increase of acetylcholine-evoked contraction of abomasum strips. The strongest contraction caused by hederagenin in a concentration of 100µM was observed in abomasal circular smooth muscle and amounted to 184.1±15.5% of the control treatment. Similarly, in case of duodenum hederagenin increased smooth muscle contractility dose-dependently. Hederagenin applied in the highest tested concentration doubled the force of acetylcholine-induced contraction in duodenal longitudinal smooth muscle. Medicagenic acid caused a remarkable increase of abomasum but not duodenum smooth muscle contractility. The contractile effect of medicagenic acid was observed in a concentration range of 0.001–10µM. The highest contractions of abomasal longitudinal and circular smooth muscle generated by medicagenic acid exceeded 150% of the control treatment with acetylcholine. All reactions caused by hederagenin and medicagenic acid were reversible and did not abolish the spontaneous motoric activity of specimens.Taking into consideration the physicochemical properties of saponins it is postulated that the contractile effect of hederagenin and medicagenic acid results from their impact on the fluidity or plasticity of the membrane and subsequently the ion flow through cell membrane. The increased ability of cells to depolarize can explain the enhancement of the response to acetylcholine. The reversibility of induced contractions, preserved spontaneous contractility and remained reactivity of smooth muscle after saponin treatment indicate that the effect caused by alfalfa saponin does not result from cell membrane damage.

Characterization of Esophageal Physiology Using Mechanical State Analysis.

The esophagus functions to transport swallowed fluids and food from the pharynx to the stomach. The esophageal muscles governing bolus transport comprise circular striated muscle of the proximal esophagus and circular smooth muscle of the distal esophagus. Longitudinal smooth muscle contraction provides a mechanical advantage to bolus transit during circular smooth muscle contraction. Esophageal striated muscle is directly controlled by neural circuits originating in the central nervous system, resulting in coordinated contractions. In contrast, the esophageal smooth muscle is controlled by enteric circuits modulated by extrinsic central neural connections resulting in neural relaxation and contraction. The esophageal muscles are modulated by sensory information arising from within the lumen. Contraction or relaxation, which changes the diameter of the lumen, alters the intraluminal pressure and ultimately inhibits or promotes flow of content. This relationship that exists between the changes in diameter and concurrent changes in intraluminal pressure has been used previously to identify the “mechanical states” of the circular muscle; that is when the muscles are passively or actively, relaxing or contracting. Detecting these changes in the mechanical state of the muscle has been difficult and as the current interpretation of esophageal motility is based largely upon pressure measurement (manometry), subtle changes in the muscle function during peristalsis can be missed. We hypothesized that quantification of mechanical states of the esophageal circular muscles and the pressure-diameter properties that define them, would allow objective characterization of the mechanisms that govern esophageal peristalsis. To achieve this we analyzed barium swallows captured by simultaneous videofluoroscopy and pressure with impedance recording. From these data we demonstrated that intraluminal impedance measurements could be used to determine changes in the internal diameter of the lumen comparable with measurements from videofluoroscopy. Our data indicated that identification of mechanical state of esophageal muscle was simple to apply and revealed patterns consistent with the known neural inputs activating the different muscles during swallowing.

A simple, inexpensive model to demonstrate how contraction of GI longitudinal smooth muscle promotes propulsion.

peristalsis is a propulsive activity that involves both circular and longitudinal muscle layers of the esophagus, distal stomach, and small and large intestines. During peristalsis, the circular smooth muscle contracts behind (on the orad side) the bolus and relaxes in front (on the aborad side) of

Effect of gingerol on colonic motilityviainhibition of calcium channel currents in rats

To investigate the effect of gingerol on colonic motility and the action of L-type calcium channel currents in this process. The distal colon was cut along the mesenteric border and cleaned with Ca(2+)-free physiological saline solution. Muscle strips were removed and placed in Ca(2+)-free physiological saline solution, which was oxygenated continuously. Longitudinal smooth muscle samples were prepared by cutting along the muscle strips and were then placed in a chamber. Mechanical contractile activities of isolated colonic segments in rats were recorded by a 4-channel physiograph. Colon smooth muscle cells were dissociated by enzymatic digestion. L-type calcium currents were recorded using the conventional whole-cell patch-clamp technique. Gingerol inhibited the spontaneous contraction of colonic longitudinal smooth muscle in a dose-dependent manner with inhibition percentages of 13.3% ± 4.1%, 43.4% ± 3.9%, 78.2% ± 3.6% and 80.5% ± 4.5% at 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L, respectively (P < 0.01). Nifedipine, an L-type calcium channel blocker, diminished the inhibition of colonic motility by gingerol. Gingerol inhibited L-type calcium channel currents in colonic longitudinal myocytes of rats. At a 75 μmol/L concentration of gingerol, the percentage of gingerol-induced inhibition was diminished by nifedipine from 77.1% ± 4.2% to 42.6% ± 3.6% (P < 0.01). Gingerol suppressed IBa in a dose-dependent manner, and the inhibition rates were 22.7% ± 2.38%, 35.77% ± 3.14%, 49.78% ± 3.48% and 53.78% ± 4.16% of control at 0 mV, respectively, at concentrations of 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L (P < 0.01). The steady-state activation curve was shifted to the right by treatment with gingerol. The value of half activation was -14.23 ± 1.12 mV in the control group and -10.56 ± 1.04 mV in the 75 μmol/L group (P < 0.05) with slope factors, Ks, of 7.16 ± 0.84 and 7.02 ± 0.93 (P < 0.05) in the control and 75 μmol/L groups, respectively. However, the steady-state inactivation curve was not changed, with a half-inactivation voltage, 0.5 V, of -27.43 ± 1.26 mV in the control group and -26.56 ± 1.53 mV in the 75 μmol/L gingerol group (P > 0.05), and a slope factor, K, of 13.24 ± 1.62 in the control group and 13.45 ± 1.68 (P > 0.05) in the 75 μmol/L gingerol group. Gingerol inhibits colonic motility by preventing Ca(2+) influx through L-type calcium channels.

Involvement of the P2X7 purinergic receptor in colonic motor dysfunction associated with bowel inflammation in rats.

Background and PurposeRecent evidence indicates an involvement of P2X7 purinergic receptor (P2X7R) in the fine tuning of immune functions, as well as in driving enteric neuron apoptosis under intestinal inflammation. However, the participation of this receptor in the regulation of enteric neuromuscular functions remains undetermined. This study was aimed at investigating the role of P2X7Rs in the control of colonic motility in experimental colitis.Experimental ApproachColitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. P2X7R distribution was examined by immunofluorescence analysis. The effects of A804598 (selective P2X7R antagonist) and BzATP (P2X7R agonist) were tested on contractions of longitudinal smooth muscle evoked by electrical stimulation or by carbachol in the presence of tetrodotoxin.Key ResultsP2X7Rs were predominantly located in myenteric neurons, but, in the presence of colitis, their expression increased in the neuromuscular layer. In normal preparations, A804598 elicited a negligible increase in electrically induced contractions, while a significant enhancement was recorded in inflamed tissues. In the presence of Nω-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor) the A804598 effects were lost. P2X7R stimulation with BzATP did not significantly affect electrical-induced contractions in normal colon, while a marked reduction was recorded under inflammation. The inhibitory effect of BzATP was antagonized by A804598, and it was also markedly blunted by NPA. Both P2X7R ligands did not affect carbachol-induced contractions.Conclusions and ImplicationsThe purinergic system contributes to functional neuromuscular changes associated with bowel inflammation via P2X7Rs, which modulate the activity of excitatory cholinergic nerves through a facilitatory control on inhibitory nitrergic pathways.

Molecular and functional diversity of GABA-A receptors in the enteric nervous system of the mouse colon.

The enteric nervous system (ENS) provides the intrinsic neural control of the gastrointestinal tract (GIT) and regulates virtually all GI functions. Altered neuronal activity within the ENS underlies various GI disorders with stress being a key contributing factor. Thus, elucidating the expression and function of the neurotransmitter systems, which determine neuronal excitability within the ENS, such as the GABA-GABAA receptor (GABAAR) system, could reveal novel therapeutic targets for such GI disorders. Molecular and functionally diverse GABAARs modulate rapid GABAergic-mediated regulation of neuronal excitability throughout the nervous system. However, the cellular and subcellular GABAAR subunit expression patterns within neurochemically defined cellular circuits of the mouse ENS, together with the functional contribution of GABAAR subtypes to GI contractility remains to be determined. Immunohistochemical analyses revealed that immunoreactivity for the GABAAR gamma (γ) 2 and alphas (α) 1, 2, 3 subunits was located on somatodendritic surfaces of neurochemically distinct myenteric plexus neurons, while being on axonal compartments of submucosal plexus neurons. In contrast, immunoreactivity for the α4-5 subunits was only detected in myenteric plexus neurons. Furthermore, α-γ2 subunit immunoreactivity was located on non-neuronal interstitial cells of Cajal. In organ bath studies, GABAAR subtype-specific ligands had contrasting effects on the force and frequency of spontaneous colonic longitudinal smooth muscle contractions. Finally, enhancement of γ2-GABAAR function with alprazolam reversed the stress-induced increase in the force of spontaneous colonic contractions. The study demonstrates the molecular and functional diversity of the GABAAR system within the mouse colon providing a framework for developing GABAAR-based therapeutics in GI disorders.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

The signaling pathways mediating sustained contraction of mouse colonic longitudinal smooth muscle and the mechanisms involved in hypercontractility of this muscle layer in response to cytokines and TNBS-induced colitis have not been fully explored. In control longitudinal smooth muscle cells, ACh acting via m3 receptors activated sequentially Gα12, RhoGEF (LARG), and the RhoA/Rho kinase pathway. There was abundant expression of MYPT1, minimal expression of CPI-17, and a notable absence of a PKC/CPI-17 pathway. LARG expression was increased in longitudinal muscle cells isolated from muscle strips cultured for 24 h with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice. The increase in LARG expression was accompanied by a significant increase in ACh-stimulated Rho kinase and ZIP kinase activities, and sustained muscle contraction. The increase in LARG expression, Rho kinase and ZIP kinase activities, and sustained muscle contraction was abolished in cells pretreated with the Jun kinase inhibitor, SP600125. Expression of the MLCP activator, telokin, and MLCP activity were also decreased in longitudinal muscle cells from TNBS-treated mice or from strips treated with IL-1β or TNF-α. In contrast, previous studies had shown that sustained contraction in circular smooth muscle is mediated by sequential activation of Gα13, p115RhoGEF, and dual RhoA-dependent pathways involving phosphorylation of MYPT1 and CPI-17. In colonic circular smooth muscle cells isolated from TNBS-treated mice or from strips treated with IL-1β or TNF-α, CPI-17 expression and sustained muscle contraction were decreased. The disparate changes in the two muscle layers contribute to intestinal dysmotility during inflammation.

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.

Role of PGE2 in the colonic motility: PGE2 generates and enhances spontaneous contractions of longitudinal smooth muscle in the rat colon

The aim of this study was to determine which PGE2 receptors (EP1-4 receptors) influence colonic motility. Mucosa-free longitudinal smooth muscle strips of the rat middle colon spontaneously induced frequent phasic contractions (giant contractions, GCs) in vitro, and the GCs were almost completely abolished by a cyclooxygenase inhibitor, piroxicam, and by an EP3 receptor antagonist, ONO-AE3-240, but enhanced by tetrodotoxin (TTX). In the presence of piroxicam, exogenous PGE2, both ONO-AE-248 (EP3 agonist), and ONO-DI-004 (EP1 agonist) induced GC-like contractions, and increased the frequency and amplitude. These effects of EP receptor agonists were insensitive to TTX and ω-conotoxins. In immunohistochemistry, the EP1 and EP3 receptors were expressed in the longitudinal smooth muscle cells. These results suggest that the endogenous PGE2 spontaneously generates and enhances the frequent phasic contractions directly activating the EP1 and EP3 receptors expressed on longitudinal smooth muscle cells in the rat middle colon.

Uridine adenosine tetraphosphate induces contraction of circular and longitudinal gastric smooth muscle by distinct signaling pathways

Extracellular nucleotides uridine-5'-triphosphate (UTP) and adenosine-5'-triphosphate (ATP) induce contraction of gastric smooth muscle (SM). The dinucleotide uridine adenosine tetraphosphate (Up4 A), an endothelium-derived contraction factor, induces vascular SM contraction. Its effect on gastric SM contractions, however, is unknown. We addressed the hypothesis that Up4 A induces gastric SM contraction via a mechanism that may differ between circular and longitudinal muscle (CM and LM, respectively). CM and LM were isolated from rat gastric fundus for the measurement of isometric tension. Up4 A induced transient contractile responses in both CM and LM, which were similar to those induced by ATP and UTP. Up4 A failed to induce contraction of either LM or CM in the absence of extracellular Ca(2+) or in the presence of nimodipine, an inhibitor of voltage-gated Ca(2+) channels. P2X1, 2, 4, 5 and 7 and P2Y1, 2, 4 and 6 receptor expression was detected in gastric SM by reverse transcription-polymerase chain reaction. IP5 I (a P2X receptor antagonist) and α,β-methylene-ATP (a P2X receptor agonist) had no effect on Up4 A-induced contractions of either LM or CM, and α,β-methylene-ATP alone failed to induce a contractile response in either tissue. Suramin (a P2Y receptor antagonist), on the other hand, significantly inhibited Up4 A-induced contraction of CM, but not LM. Up4 A-induced contraction of CM, but not LM, was also inhibited by pretreatment with Y-27632, an inhibitor of Rho-associated kinase. We conclude that Up4 A induces extracellular Ca(2+) -dependent contractions of rat gastric LM and CM, and Up4 A-induced contraction of CM is mediated by suramin-sensitive P2Y receptors and subsequent activation of the Rho-associated kinase pathway.