Longitudinal Smooth Muscle Research Articles

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.

Topographic Anatomy of the Anal Sphincter Complex and Levator Ani Muscle as It Relates to Intersphincteric Resection for Very Low Rectal Disease.

Intersphincteric resection has become a widely used treatment for patients with rectal cancer. However, the detailed anatomy of the anal canal related to this procedure has remained unclear. The purpose of this study was to clarify the detailed anatomy of the anal canal. This is a descriptive study. Histologic evaluations of paraffin-embedded tissue specimens were conducted at a tertiary referral hospital. Tissue specimens were obtained from cadavers of 5 adults and from 13 patients who underwent abdominoperineal resection for rectal cancer. Sagittal sections from 9 circumferential portions of the cadaveric anal canal (histologic staining) and 3 circumferential portions from patients were studied (immunohistochemistry for smooth and skeletal muscle fibers). Longitudinal fibers between the internal and external anal sphincters consisted primarily of smooth muscle fibers that continued from the longitudinal muscle of the rectum. The levator ani muscle attached directly to the lateral surface of the longitudinal smooth muscle of the rectum. The length of the attachment was longer in the anterolateral portion and shorter in the posterior portion of the anal canal. In the lateral and posterior portions, the levator ani muscle partially overlapped the external anal sphincter; however, there was less overlap in the anterolateral portion. In the posterior portion, thick smooth muscle was present on the surface of the levator ani muscle and it continued to the longitudinal muscle of the rectum. We observed only limited portions in some surgical specimens because of obstruction by tumors. The levator ani muscle attaches directly to the longitudinal muscle of the rectum. The spatial relationship between the smooth and skeletal muscles differed in different portions of the anal canal. For intersphincteric resection, dissection must be performed between the longitudinal muscle of the rectum and the levator ani muscle/external anal sphincter, and the appropriate surgical lines must be selected based on the specific structural characteristics of each portion.

Multi-phenotypic Role of Serum Response Factor in the Gastrointestinal System.

Serum response factor (SRF) is a master transcription factor of the actin cytoskeleton that binds to highly conserved CArG boxes located within the majority of smooth muscle cell (SMC)-restricted promoters/enhancers. Although most studies of SRF focus on skeletal muscle, cardiac muscle, and vascular SMCs, SRF research has recently expanded into the gastrointestinal (GI) system. Genome scale analyses of GI SMC transcriptome and CArG boxes (CArGome) have identified new SRF target genes. In addition to circular and longitudinal smooth muscle layers, SRF is also expressed in GI mucosa and cancers. In the GI tract, SRF is the central regulator of genes involved in apoptosis, dedifferentiation, proliferation, and migration of cells. Since SRF is the cell phenotypic modulator, it may play an essential role in the development of myopathy, hypertrophy, ulcers, gastric and colon cancers within the GI tract. Given the multi-functional role displayed by SRF in the digestive system, SRF has received more attention emerging as a potential therapeutic target. This review summarizes the findings in SRF research pertaining to the GI tract and provides valuable insight into future directions.

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.

Morphometric Analyses of Longitudinal and Circular Smooth Muscle and Lacteals of the Small Intestine of Zucker Lean and Obese Rats

Lymph lacteals are the entry point of absorbed fats and cholesterol in the villi of the small intestine. In a recent study, lacteals and the leakiness of the submucosal lympahtic network was implicated as a source for adult‐onset obesity (Harvey et al. 2005). The purpose of this study was to determine what structural differences may exist in the lacteals, submucosa, and other layers of the ileum between lean and obese Zucker rats using histochemistry (H&E) and immunofluorescence microscopy. We hypothesized that due to increased food intake and levels of digested fats in the obese Zucker rat, the lacteals and the smooth muscle layers of the ileum will modify from their normal appearance to accommodate the increased intake of fats and volume of food respectively. Ileum from lean and obese Zucker rats was harvested, fixed in buffered formalin overnight, processed and embedded in paraffin. Cross sections (2–3 μm) of the ileum were mounted on slides and stained with H&E to measure longitudinal and circular smooth muscle layer thickness. Similar sections were incubated with a LYVE‐1 antibody to identify lymphatic endothelial cells of the lacteals and a smooth muscle actin (SMA) antibody to label smooth muscle cells extending into the villi. Layers of longitudinal and circular smooth muscle, identified in the ileum with H&E staining, were significantly thicker in obese Zucker rats compared to lean rats. Preliminary immunolabeling with LYVE‐1 identified positive endothelial cells in crypts of the sub mucosae and SMA‐positive smooth muscle cells that extended into the villi (lamina propria). SMA was also detected in the circular and longitudinal muscle layers and the muscularis muscosae as predicted. Histological data obtained from the ileum support the hypothesis that increased food intake increases intestinal smooth muscle layer thickness in obese Zucker rats.Support or Funding InformationThis work was supported by an APS/NHLBI STRIDE Undergraduate Summer Research Fellowship (NIH R25HL115473) and NIH grant L40HL097863.

Acute Aerobic Swimming Exercise Induces Distinct Effects in the Contractile Reactivity of Rat Ileum to KCl and Carbachol.

Aerobic exercise promotes short-term physiological changes in the intestinal smooth muscle associated to the ischemia-reperfusion process; however, few studies have demonstrated its effect on the intestinal contractile function. Thus, this work describes our observations regarding the influence of acute aerobic swimming exercise in the contractile reactivity, oxidative stress, and morphology of rat ileum. Wistar rats were divided into sedentary (SED) and acutely exercised (EX-AC) groups. Animals were acclimated by 10, 10, and 30 min of swimming exercise in intercalated days 1 week before exercise. Then they were submitted to forced swimming for 1 h with a metal of 3% of their body weight attached to their body. Animals were euthanized immediately after the exercise section and the ileum was suspended in organ baths for monitoring isotonic contractions. The analysis of lipid peroxidation was performed in order to determinate the malondialdehyde (MDA) levels as a marker of oxidative stress, and intestinal smooth muscle morphology by histological staining. Cumulative concentration-response curves to KCl were altered in the EX-AC with an increase in both its efficacy and potency (Emax = 153.2 ± 2.8%, EC50 = 1.3 ± 0.1 × 10−2 M) compared to the SED group (Emax = 100%, EC50 = 1.8 ± 0.1 × 10−2 M). Interestingly, carbachol had its efficacy and potency reduced in the EX-AC (Emax = 67.1 ± 1.4%, EC50 = 9.8 ± 1.4 × 10−7 M) compared to the SED group (Emax = 100%, EC50 = 2.0 ± 0.2 × 10−7 M). The exercise did not alter the MDA levels in the ileum (5.4 ± 0.6 μ mol/mL) in the EX-AC compared to the SED group (8.4 ± 1.7 μ mol/mL). Moreover, neither the circular nor the longitudinal smooth muscle layers thickness were modified by the exercise (66.2 ± 6.0 and 40.2 ± 2.6 μm, respectively), compared to the SED group (61.6 ± 6.4 and 34.8 ± 3.7 μm, respectively). Therefore, the ileum sensitivity to contractile agents is differentially altered by the acute aerobic swimming exercise, without affecting the oxidative stress and the morphology of ileum smooth muscle.

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.

Immunolocalization of AMPA receptor subunits within the enteric nervous system of the mouse colon and the effect of their activation on spontaneous colonic contractions.

The appropriate expression of specific neurotransmitter receptors within the cellular networks that compose the enteric nervous system (ENS) is central to the regulation of gastrointestinal (GI) functions. While the ENS expression patterns of the neurotransmitter glutamate have been well documented, the localization of its receptors on ENS neurons remains to be fully characterized. We investigated the expression patterns of glutamate receptor AMPA subunits within ENS neurons of the mouse colon and the consequences of their pharmacological activation on spontaneous colonic contractility. RT-PCR was used to detect individual AMPA receptor (GluR 1-4) subunit expression at the mRNA level in mouse colon tissue. Immunohistochemistry and confocal microscopy was used to localize the expression of the GluR1 and 4 subunits in colon tissue. Brain tissue was used as a positive control. Organ bath preparations were used to determine the effect of AMPA receptors activation on the force and frequency of colonic longitudinal smooth muscle spontaneous contractions. GluR1, 3, 4 mRNA was detected in the mouse colon. Immunoreactivity for GluR1 and 4 subunits was detected on the somatic and dendritic surfaces of subpopulations of neurochemically defined ENS neurons. The pharmacological activation of AMPA receptors increased the force but not frequency of spontaneous colonic contractions. Molecularly distinct AMPA receptor subtypes are differentially expressed within the neural networks of the mouse colon and have a direct role in motility. These data provide the rationale for the development of AMPA-selective ligands for the therapeutic delivery to the GIT in motility disorders.

Modification of abomasum contractility by flavonoids present in ruminants diet: in vitro study

Flavonoid supplementation is likely to be beneficial in improving rumen fermentation and in reducing the incidence of rumen acidosis and bloat. Flavonoids are also said to increase the metabolic performance during the peripartum period. Ruminants are constantly exposed to flavonoids present in feed. However, it is not clear if these phytochemicals can affect the activity of the gut smooth muscle. Therefore, the aim of the study was to verify the effect of three flavonoids on bovine isolated abomasum smooth muscle. The study was carried out on bovine isolated circular and longitudinal abomasal smooth muscle specimens. All experiments were conducted under isometric conditions. The effect of apigenin, luteolin and quercetin (0.001 to 100 µM) was evaluated on acetylcholine-precontracted preparations. The effect of multiple, but not cumulative, treatment and single treatment with each flavonoid on abomasum strips was compared. Apigenin (0.1 to 100 µM) dose-dependently showed myorelaxation effects. Luteolin and quercetin applied in low doses increased the force of the ACh-evoked reaction. However, if used in high doses in experiments testing a wide range of concentrations, their contractile effect either declined (luteolin) or was replaced by an antispasmodic effect (quercetin). Surprisingly, the reaction induced by flavonoids after repeated exposure to the same phytochemical was not reproducible in experiments testing only single exposure of abomasum strips to the same flavonoid used in a high concentration. Taking into account the physicochemical properties of flavonoids, this data suggests the ability of flavonoids to interfere with cell membranes and, subsequently, to modify their responsiveness. Assuming ruminant supplementation with luteolin or quercetin or their presence in daily pasture, a reduction of the likelihood of abomasum dysmotility should be expected.

Morphological features of the distal ileum and ceca of the common pigeon (Columba livia)

Background to the study: Common pigeon ( Columba livia ) is widely distributed worldwide and feeds mainly on grains or seeds and insects and small invertebrates. Some studies have demonstrated variations in the morphology and function of avian alimentary tract. Methods: Ten apparently healthy adult common pigeons were examined in this study. Following euthanasia, gross and histological techniques were used to investigate the morphology of the distal ileum and ceca of common pigeon. Results: At the ileo-ceco-rectal junction, the tunica muscularis of the distal ileum exhibited a thickened layer of oblique smooth muscle fibers positioned between the middle circular and outer longitudinal smooth muscle layers. The ceca of the common pigeon were paired, small (approximately 0.33 cm), blind-ended sacs situated at acute angles with the distal ileum. Histologically, each cecum exhibited a lamina propria-submucosa that contained aggregates of lymphocytes and few glandular acini throughout the length of the organ. Conclusion: It was suggested that the ceca of the common pigeon may be important in immunological response of the bird to invasion by antigens while playing some roles in the digestion of food materials. Moreover, the thickened oblique layer of smooth muscle fibers in the wall of the distal ileum may serve to regulate the passage of materials through the ileo-ceco-rectal orifice. Keywords: Cecum, common pigeon, distal ileum, lamina propria‑submucosa, sphincter

Effect of Aurantii Fructus Immaturus Flavonoid on the Contraction of Isolated Gastric Smooth Muscle Strips in Rats.

This study was designed to investigate the effect of Aurantii fructus immaturus flavonoid (AFIF) on the contraction of isolated gastric smooth muscle in rats and explore its underlying mechanisms. Isolated antral longitudinal smooth muscle strip (ALSMS) and pyloric circular smooth muscle strip (PCSMS) of rats were suspended in tissue chambers. The responses of ALSMS and PCSMS to administration of AFIF were observed. Cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) levels of PCSMS were measured by ELISA kits. In this study, AFIF showed no significant effect on ALSMS contraction, but it dose-dependently reduced the mean contraction amplitude of PCSMS. When the concentration of AFIF reached 3000 μg/mL, 6000 μg/mL, and 10000 μg/mL, its inhibitory effect on PCSMS contraction was significant. This effect of AFIF was weakened in Ca2+-rich environment. And Nω-nitro-L-arginine methyl (L-NAME), the inhibitor of nitric oxide synthase (NOS), significantly inhibited AFIF's action in comparison with control (P < 0.05). After incubation with AFIF for 30 min, levels of cGMP and PKG in PCSMS were significantly increased compared with control (P < 0.05). Our results suggest that AFIF has a dose-dependent diastolic effect on PCSMS in rats, which may be related to the regulatory pathway of NO/cGMP/PKG/Ca2+.

Effects of ghrelin and motilin on smooth muscle contractility of the isolated gastrointestinal tract from the bullfrog and Japanese fire belly newt

Ghrelin has been identified in some amphibians and is known to stimulate growth hormone release and food intake as seen in mammals. Ghrelin regulates gastrointestinal motility in mammals and birds. The aim of this study was to determine whether ghrelin affects gastrointestinal smooth muscle contractility in bullfrogs (anuran) and Japanese fire belly newts (urodelian) in vitro. Neither bullfrog ghrelin nor rat ghrelin affected longitudinal smooth muscle contractility of gastrointestinal strips from the bullfrog. Expression of growth hormone secretagogue receptor 1a (GHS-R1a) mRNA was confirmed in the bullfrog gastrointestinal tract, and the expression level in the gastric mucosa was lower than that in the intestinal mucosa. In contrast, some gastrointestinal peptides, including substance P, neurotensin and motilin, and the muscarinic receptor agonist carbachol showed marked contraction, indicating normality of the smooth muscle preparations. Similar results were obtained in another amphibian, the Japanese fire belly newt. Newt ghrelin and rat ghrelin did not cause any contraction in gastrointestinal longitudinal muscle, whereas substance P and carbachol were effective causing contraction. In conclusion, ghrelin does not affect contractility of the gastrointestinal smooth muscle in anuran and urodelian amphibians, similar to results for rainbow trout and goldfish (fish) but different from results for rats and chickens. The results suggest diversity of ghrelin actions on the gastrointestinal tract across animals. This study also showed for the first time that motilin induces gastrointestinal contraction in amphibians.

Oxidative Stress Contributes to Overactive Bladder in the Transgenic Sickle Cell Mouse

Introduction: Lower urinary tract symptoms (LUTS) such as nocturia, urinary frequency, and urgency have been clinically reported in children and adults with sickle cell disease (SCD; Portocarrero et al J Urol 2012; Anele et al Neurourol Urodyn 2015), but little is known about the pathophysiology of micturition dysfunction in SCD patients. Transgenic sickle cell mice have been employed to better understand SCD pathophysiology. Recently, we reported that Berkeley SCD mice display underactive bladder and reduced detrusor smooth muscle contractions (Claudino et al Plos One, 2015). The homozygous Townes transgenic sickle cell mouse is another model for SCD, but urinary bladder dysfunction has never been studied in this model. Considering that SCD is associated with elevated ROS production, we hypothesized that increased ROS levels lead to alterations in the urinary bladder function of Townes SCD mice. Thus, the aim of this study was to evaluate the micturition and detrusor smooth muscle contractions in Townes transgenic sickle cell mice.Methods: Townes transgenic sickle cell mice and C57BL/6 mice (control) aged 3 to 4 months old were used. Cystometry was performed to evaluate the urinary function in vivo. In separate protocols, the urinary bladder was removed and placed in Krebs solution. Two longitudinal detrusor smooth muscle strips with intact urothelium were obtained from each bladder. The strips were mounted in 4-ml organ baths containing Krebs-Henseleit solution at 37°C continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4). Cumulative concentration-response curves to muscarinic receptor agonist carbachol (CCh; 0.01-100 μM), chloride potassium (KCl; 1-300mM) and frequency-response curves for electrical field stimulation (EFS; 1-32Hz) were obtained in strips of detrusor smooth muscle from both control and SCD mice.mRNA expression for gp91phox and SOD-1 in bladder were also evaluated.Results: Micturition in SCD mice was irregular, and characterized by significant (P < 0.05) increases in the frequency of voiding contractions and nonvoiding contractions (n=5). Voiding pressure, basal pressure, capacity and threshold pressure were not changed in SCD mice. Carbachol (0.01 - 100 µM) induced concentration-dependent detrusor smooth muscle contractions in both control and SCD mice, but maximal contractile responses were significantly lower (P < 0.05) in SCD compared to control mice (1.40 ± 0.4 and 2.9 ± 0.2 mN/mg, respectively; n=5). Likewise, EFS-induced neurogenic detrusor contractions in SCD mice were 29% lower (P < 0.05) compared to the control (n=10). Contractile responses induced by KCl (1 - 300 mM) did not differ between control and SCD group (n=5). The mRNA expression for the NADPH subunit gp91phox in bladder was about 2-fold higher (P<0.05) in SCD mice compared to the control group, whereas no changes in the mRNA expression for SOD-1 were observed among groups (n=6). Infiltration by inflammatory cells (mononuclear cells) was seen in the submucosa and muscular area of bladder from SCD mice (n=4).Conclusion: Our study shows that the overactive bladder seen in SCD mouse is associated with upregulation of NADPH oxidase subunit gp91phox. Interestingly, in the contrast, the Berkeley SCD mouse exhibits an atonic detrusor smooth muscle and underactive bladder (Claudino et al Plos One 2015). One may speculate therefore that the overactive bladder in Townes SCD may progress to underactive bladder at latter stages. To confirm this hypothesis, we will evaluate the urinary bladder function in older SCD mice.Financial Support: FAPESP DisclosuresNo relevant conflicts of interest to declare.

Differences in time to peak carbachol-induced contractions between circular and longitudinal smooth muscles of mouse ileum.

The muscular layer in the GI tract consists of an inner circular muscular layer and an outer longitudinal muscular layer. Acetylcholine (ACh) is the representative neurotransmitter that causes contractions in the gastrointestinal tracts of most animal species. There are many reports of muscarinic receptor-mediated contraction of longitudinal muscles, but few studies discuss circular muscles. The present study detailed the contractile response in the circular smooth muscles of the mouse ileum. We used small muscle strips (0.2 mm × 1 mm) and large muscle strips (4 × 4 mm) isolated from the circular and longitudinal muscle layers of the mouse ileum to compare contraction responses in circular and longitudinal smooth muscles. The time to peak contractile responses to carbamylcholine (CCh) were later in the small muscle strips (0.2 × 1 mm) of circular muscle (5.7 min) than longitudinal muscles (0.4 min). The time to peak contractile responses to CCh in the large muscle strips (4 × 4 mm) were also later in the circular muscle (3.1 min) than the longitudinal muscle (1.4 min). Furthermore, a muscarinic M2 receptor antagonist and gap junction inhibitor significantly delayed the time to peak contraction of the large muscle strips (4 × 4 mm) from the circular muscular layer. Our findings indicate that muscarinic M2 receptors in the circular muscular layer of mouse ileum exert a previously undocumented function in gut motility via the regulation of gap junctions.

Histology and ultrastructure of the uterus of African giant rat (Cricetomys Gambianus, Waterhouse) during oestrous cycle.

This study describes histology and ultrastructure of uterus in the African giant rat during oestrous cycle. Endometrial histology displayed glandular mucosa consisting of lamina epithelialis and lamina propria mucosae. Its epithelium varied between simple and pseudostratified columnar. The myometrium consisted of inner circular and outer longitudinal smooth muscles with medium sized arteries and veins in-between. The perimetrium contained simple squamous epithelium. Endometrial ultrastructures were variable during oestrous cycle. At mid oestrus, hemidesmosomes anchored undulating basement membrane of the mucosal epithelium. Preponderance spherical mitochondria, lipofuscin granules concentration, flocculent homogenous materials and indented nuclei were displayed. At mid metoestrus, late metoestrus/early dioestrus and mid dioestrus, the base of the mucosal columnar epithelium lain on relatively straight basement membrane and their cytoplasmic ultrastructure displayed variation to mid oestrus. Epithelial apex showed intermediate filament, microvilli and junctional complexes. The uterine glands occurred in variable numbers and sizes during oestrous cycle and shared similar ultrastructure. Mid dioestrus showed cell ultrastructure of uterine glands having apical accumulation of secretory vesicles. Some actively secreting uterine glands were lined by simple ciliated columnar epithelium mingled with pseudostratified epithelium. The findings of the study indicate that giant rat endometrial ultrastructure varies during oestrous cycle and glandular secretion is merocrine.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

Selective 5-HT4 receptor agonists such as prucalopride are used as human prokinetics, since activation of 5-HT4 receptors on intestinal cholinergic neurons facilitates acetylcholine release. 5-HT4 receptors, linked to adenylyl cyclase, act via generation of cAMP. None of the 4 in vitro studies on 5-HT in horses provided evidence for neuronal 5-HT4 receptors, but none used the protocol as described in human studies [1-4]. To investigate whether functional 5-HT4 receptors are present in the equine small intestine. In vitro organ bath set up, applying electrical field stimulation (EFS) in longitudinal and circular smooth muscle strips. Results were similar in both muscle layers. In the presence of 0.3 mmol/l NG-Nitro-L-arginine methyl ester and 0.3 μmol/l apamine, excluding effects of the inhibitory transmitters NO and ATP, EFS induced voltage-dependent on-contractions; these were neurogenic as they were abolished by 3 μmol/l tetrodotoxin. At a voltage inducing 50% of the maximal amplitude, the submaximal EFS-induced contractions were cholinergic as atropine (1 μmol/l) abolished them. Prucalopride (0.3 μmol/l) did not increase the amplitude of these submaximal EFS-induced contractions. Even in the presence of the nonselective phosphodiesterase inhibitor IBMX, previously shown to enhance the effect of neuronal 5-HT4 receptors by inhibiting breakdown of their 2nd messenger cAMP [5], prucalopride (3 μmol/l) had no influence. Also 5-HT (10 μmol/l), a full agonist at 5-HT4 receptors, tested in the presence of methysergide and granisetron to exclude interaction with other 5-HT receptor subtypes, did not enhance EFS-induced submaximal contractions. There is no evidence for presence of 5-HT4 receptors on the cholinergic neurons of the equine small intestine. These results question the application of 5-HT4 prokinetic drugs in horses. Ethical animal research: Research ethics committee oversight not currently required by this conference: the study was performed on material collected at an abattoir. Sources of funding: None. Competing interests: None declared.

3D Topography of the Young Adult Anal Sphincter Complex Reconstructed from Undeformed Serial Anatomical Sections.

BackgroundPelvic-floor anatomy is usually studied by artifact-prone dissection or imaging, which requires prior anatomical knowledge. We used the serial-section approach to settle contentious issues and an interactive 3D-pdf to make the results widely accessible.Method3D reconstructions of undeformed thin serial anatomical sections of 4 females and 2 males (21–35y) of the Chinese Visible Human database.FindingsBased on tendinous septa and muscle-fiber orientation as segmentation guides, the anal-sphincter complex (ASC) comprised the subcutaneous external anal sphincter (EAS) and the U-shaped puborectal muscle, a part of the levator ani muscle (LAM). The anococcygeal ligament fixed the EAS to the coccygeal bone. The puborectal-muscle loops, which define the levator hiatus, passed around the anorectal junction and inserted anteriorly on the perineal body and pubic bone. The LAM had a common anterior attachment to the pubic bone, but separated posteriorly into puborectal and “pubovisceral” muscles. This pubovisceral muscle was bilayered: its internal layer attached to the conjoint longitudinal muscle of the rectum and the rectococcygeal fascia, while its outer, patchy layer reinforced the inner layer. ASC contraction makes the ano-rectal bend more acute and lifts the pelvic floor. Extensions of the rectal longitudinal smooth muscle to the coccygeal bone (rectococcygeal muscle), perineal body (rectoperineal muscle), and endopelvic fascia (conjoint longitudinal and pubovisceral muscles) formed a “diaphragm” at the inferior boundary of the mesorectum that suspended the anorectal junction. Its contraction should straighten the anorectal bend.ConclusionThe serial-section approach settled contentious topographic issues of the pelvic floor. We propose that the ASC is involved in continence and the rectal diaphragm in defecation.

Nitrergic signalling via interstitial cells of Cajal regulates motor activity in murine colon.

In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO-sensitive guanylyl cyclase (NO-GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide-sensitive guanylyl cyclase (NO-GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild-type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle-specific guanylyl cyclase knockout (SMC-GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC-specific guanylyl cyclase knockout (ICC-GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (L-NAME, ODQ) did not significantly affect contractions of GCKO and ICC-GCKO colon whereas the arrhythmic contractile patterns of WT and SMC-GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field-stimulated neuronal NO release was similar in SMC-GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon.

Characterization of the peristaltic reflex in murine distal colon.

Ascending and descending neuromuscular reflexes play an important role in gastrointestinal motility. However, the underlying mechanisms in colon are incompletely understood. Nerve stimulation (NS)- and balloon distention (BD)-mediated reflexes in distal colonic circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of mice were investigated using conventional intracellular recordings. In the CSM, NS evoked ascending purinergic inhibitory junction potentials (IJPs), whereas BD induced atropine-sensitive ascending depolarization with superimposed action potentials (APs). The ascending depolarization reached a peak ∼4-7 s after the onset of distention and gradually returned to baseline after termination of the distention. In the LSM, NS produced an ascending biphasic IJP followed by a train of atropine-sensitive APs. Both stimuli produced similar descending IJPs in CSM and LSM, which were blocked by MRS-2500 and MRS-2179, putative purinergic receptor blockers. These data indicate that in the murine distal colon, descending purinergic inhibition in both CSM and LSM occurs. Ascending responses are more complex, with NS producing both inhibition and excitation to CSM and LSM, and BD evoking only cholinergic excitation.

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