Small Intestinal Smooth Muscle Research Articles

Divergent Effect of Dezocine, Morphine and Sufentanil on Intestinal Motor Function in Rats

Background: Opioid induced bowel dysfunction is the most common side effect of preoperatively administrated morphine, fentanyl and its derivative. However, the influence of dezocine on intestinal mobility is rarely reported. This study was designed to investigate the effects of dezocine, morphine and sufentanil on both intestinal smooth muscle contraction and propulsion in rats.Methods: Contractile tension and frequency of isolated rat small intestine smooth muscle were measured using tension transducer after incubation with different concentrations of dezocine, morphine and sufentanil. The propulsive rate of methylene blue in rat intestinal tract was measured 30 minutes after intraperitoneal injection of morphine, sufentanil and dezocine. Percent of change in contractile tension and contraction frequency compared to baseline level were calculated to evaluate muscle contraction. Propulsive rate of methylene blue was calculated as the percentage of methylene blue moving distance in intestinal tract compared to the length of the small intestine.Results: Morphine and sufentanil significantly increased the contractile tension of isolated small intestine smooth muscle at high doses. The contraction frequency did not change significantly among the 3 tested doses. Increasing the dose of dezocine from 1.7 mg.L-1 to 10.2 mg.L-1 did not change either the contractile tension or the contraction frequency. The propulsive rate of methylene blue in intestinal tract was significantly decreased after the treatment with morphine, sufentanil and dezocine (45.6%, 43.7%, and 42.1% respectively) compared to control group(57.1%), while the difference among the 3 drug groups were not significant.Conclusion: Morphine and sufentanil may dose dependently increase the contractile tension and contraction ability of isolated rat small intestine smooth muscle, while dezocine has no significant effect on intestine smooth muscle contraction. However, all these opioids might impair small intestinal propulsion.

Ranolazine Blocks NaV1.5 Peak Current and Shear Sensitivity in Human Colonic Circular Smooth Muscle

Introduction:SCN5A-encoded NaV1.5 is a voltage-gated mechanosensitive sodium channel expressed in circular layer smooth muscle cells (SMCs) and interstitial cells of Cajal of human small intestine. In human small intestinal smooth muscle, NaV1.5 contributes to resting membrane potential and slow waves. SCN5A mutations are found in 2-3% of patients with irritable bowel syndrome (IBS). The majority of these SCN5A mutations are associated with constipation predominant IBS. A Na+ current was previously described in colonic circular SMCs, and we recently identified SCN5A mRNA in human colonic circular muscle suggesting NaV1.5 is also expressed in the colon. Ranolazine is a clinically available NaV1.5 antagonist used in cardiac disease that also blocks NaV1.5 mechanosensitivity. Constipation is a common adverse effect of ranolazine. The aim of this study was to examine effect of ranolazine on Na+ current and shear sensitivity in human colonic circular SMCs. Methods: Whole cell inward currents were recorded from freshly dissociated human colonic circular SMCs with and without 50 μmol/L ranolazine in bath. Shear stress was induced by 10 mL/min flow of bath solution containing 0 or 50 μmol/L ranolazine. The distinct time and voltage dependent characteristics of fast activating Na+ current and slower activating Ca2+ current were used to separate the components of inward current. The voltage dependence of activation was fitted by a Boltzmann equation. Comparisons were made by paired t-test of means ±SEM. Results: In colonic circular SMCs, ranolazine (50 μmol/L) inhibited NaV1.5 peak currents by 43±9% (-104±33 to -58±20 pA/pF; n=6; P<0.05). Ranolazine shifted the half-point of the voltage dependence of activation (V1/2) from -37±1.7 to -43±1.4 mV (n=6; P<0.05). Mechanical stimulation by flow (shear stress) increased colonic SMC NaV1.5 peak current by 18±2% (−104±33 to −122±39 pA/pF; n=6; P<0.05). V1/2 was hyperpolarized with shear stress from -37±1.7 to -40±2.2 mV (n=6; P<0.05). Shear stress accelerated time to peak activation at -30 mV from 5.4±0.6 to 2.9±0.4 ms (n=5; P<0.05). Sensitivity to shear was blocked by ranolazine (-58±20 to -60±28 pA/pF; n=6; P=0.2). Ranolazine also blocked change in V1/2 and activation caused by shear (-43±1.4 mv to -43±1.1; n=6; P=0.7 for V1/2 and 2.9±0.4 to 4.3±1.4 ms, n=3; P<0.05 for activation time with ranolozine). Conclusion: Ranolazine blocked human colonic circular SMC Na+ currents and also blocked Na+ current shear sensitivity. These effects of ranolazine may help explain the gastrointestinal side effects of the drug and may be of clinical interest for the treatment of disorders such as diarrhea-predominant IBS. Supported by NIH DK 57266 and DK 57061.

Ano1, a Ca2+-activated Cl- channel, coordinates contractility in mouse intestine by Ca2+ transient coordination between interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca(2+)-activated Cl(-) channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca(2+) transients between myenteric ICC (ICC-MY) of small intestine. First, we found spontaneous Ca(2+) transients in ICC-MY in both Ano1 WT and knockout (KO) mice. However, Ca(2+) transients within the ICC-MY network in Ano1 KO mice were uncoordinated, while ICC-MY Ca(2+) transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca(2+) transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA-mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca(2+) transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca(2+) transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non-rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca(2+) transients in ICC-MY.

Su2024 IL-17A and IL-1β Exert IκBζ-Mediated, but Oppositely-Directed, Effects on Contractility and RGS4 Translocation in Small Intestinal Smooth Muscle Cells, Whose Directions Are Determined by the Balance Between P38mapk and JNK Activities

Su2024 IL-17A and IL-1β Exert IκBζ-Mediated, but Oppositely-Directed, Effects on Contractility and RGS4 Translocation in Small Intestinal Smooth Muscle Cells, Whose Directions Are Determined by the Balance Between P38mapk and JNK Activities

Voltage Dependent Potassium Channel Remodeling in Murine Intestinal Smooth Muscle Hypertrophy Induced by Partial Obstruction

Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

Small intestinal smooth muscle cell apoptosis in rats with severe acute pancreatitis

Small intestinal smooth muscle cell apoptosis in rats with severe acute pancreatitis

In vitro evaluation of bi-layer silk fibroin scaffolds for gastrointestinal tissue engineering

Silk fibroin scaffolds were investigated for their ability to support attachment, proliferation, and differentiation of human gastrointestinal epithelial and smooth muscle cell lines in order to ascertain their potential for tissue engineering. A bi-layer silk fibroin matrix composed of a porous silk fibroin foam annealed to a homogeneous silk fibroin film was evaluated in parallel with small intestinal submucosa scaffolds. AlamarBlue analysis revealed that silk fibroin scaffolds supported significantly higher levels of small intestinal smooth muscle cell, colon smooth muscle cell, and esophageal smooth muscle cell attachment in comparison to small intestinal submucosa. Following 7 days of culture, relative numbers of each smooth muscle cell population maintained on both scaffold groups were significantly elevated over respective 1-day levels—indicative of cell proliferation. Real-time reverse transcription polymerase chain reaction and immunohistochemical analyses demonstrated that both silk fibroin and small intestinal submucosa scaffolds were permissive for contractile differentiation of small intestinal smooth muscle cell, colon smooth muscle cell, esophageal smooth muscle cell as determined by significant upregulation of α-smooth muscle actin and SM22α messenger RNA and protein expression levels following transforming growth factor-β1 stimulation. AlamarBlue analysis demonstrated that both matrix groups supported similar degrees of attachment and proliferation of gastrointestinal epithelial cell lines including colonic T84 cells and esophageal epithelial cells. Following 14 days of culture on both matrices, spontaneous differentiation of T84 cells toward an enterocyte lineage was confirmed by expression of brush border enzymes, lactase, and maltase, as determined by real-time reverse transcription polymerase chain reaction and immunohistochemical analyses. In contrast to small intestinal submucosa scaffolds, silk fibroin scaffolds supported spontaneous differentiation of esophageal epithelial cells toward a suprabasal cell lineage as indicated by significant upregulation of cytokeratin 4 and cytokeratin 13 messenger RNA transcript levels. In addition, esophageal epithelial cells maintained on silk fibroin scaffolds also produced significantly higher involucrin messenger RNA transcript levels in comparison to small intestinal submucosa counterparts, indicating an increased propensity for superficial, squamous cell specification. Collectively, these data provide evidence for the potential of silk fibroin scaffolds for gastrointestinal tissue engineering applications.

Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element

The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.

Sympathetic axonopathies and hyperinnervation in the small intestine smooth muscle of aged Fischer 344 rats

It is well documented that the intrinsic enteric nervous system of the gastrointestinal (GI) tract sustains neuronal losses and reorganizes as it ages. In contrast, age-related remodeling of the extrinsic sympathetic projections to the wall of the gut is poorly characterized. The present experiment, therefore, surveyed the sympathetic projections to the aged small intestine for axonopathies. Furthermore, the experiment evaluated the specific prediction that catecholaminergic inputs undergo hyperplastic changes. Jejunal tissue was collected from 3-, 8-, 16-, and 24-month-old male Fischer 344 rats, prepared as whole mounts consisting of the muscularis, and processed immunohistochemically for tyrosine hydroxylase, the enzymatic marker for norepinephrine, and either the protein CD163 or the protein MHCII, both phenotypical markers for macrophages. Four distinctive sympathetic axonopathy profiles occurred in the small intestine of the aged rat: (1) swollen and dystrophic terminals, (2) tangled axons, (3) discrete hyperinnervated loci in the smooth muscle wall, including at the bases of Peyer's patches, and (4) ectopic hyperplastic or hyperinnervating axons in the serosa/subserosal layers. In many cases, the axonopathies occurred at localized and limited foci, involving only a few axon terminals, in a pattern consistent with incidences of focal ischemic, vascular, or traumatic insult. The present observations underscore the complexity of the processes of aging on the neural circuitry of the gut, with age-related GI functional impairments likely reflecting a constellation of adjustments that range from selective neuronal losses, through accumulation of cellular debris, to hyperplasias and hyperinnervation of sympathetic inputs.

Growth hormone secretagogue participates in two-way regulation of the motility of small intestinal smooth muscle in rats

To investigate the effect of growth hormone secretagogue(ghrelin) on the contraction and relaxation of small intestinal smooth muscle in rats and its mechanism. Twenty-four vagotomized rats were injected intraperitoneally with different concentrations of ghrelin (0, 20, 40, 80 μg/kg). The small intestinal transit were observed. The effect of ghrelin(0.01, 0.1, 0.5, 1.0 μmol/L) on the contraction and relaxation of rat small intestinal smooth muscle strips was observed in vitro in the presence of carbachol(50 nmol/L), the locations of ghrelin receptors(GHS-R1a) on different cells in small intestinal muscle layers were detected by immunofluorescence. With the increase of concentrations, ghrelin elevated the percentage of small intestinal transit[(25.4±1.0)%, (33.7±1.9)%, (39.3±2.4)%, (44.7±2.1)%] in a dose-dependent manner, and the differences were statistically significant among groups(P<0.05). Ghrelin could also enhance the contraction [(67.0±2.4)%,(149.5±3.3)%, (187.1±4.7)%, (213.5±3.4)%] and relaxation[(35.3±1.1)%, (62.9±3.8)%, (79.6±2.7)%, (94.6±2.2)%] of smooth muscle strips mediated by Cch in a dose-dependent manner, and the differences were statistically significant among groups(P<0.05). Immunofluorescence revealed that ghrelin receptors mainly located on membrane of the nerve cells in the muscle layers, while no receptors were observed on membrane of the smooth muscle cells. Ghrelin may enhance the effect of the contraction and relaxation of the rat small intestinal smooth muscle mediated by cholinergic neurotransmitters by activating the nerve cells in the enteric plexus.

Inhibition of cell proliferation by a selective inhibitor of the Ca2+-activated Cl− channel, Ano1

BackgroundIon channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca2+-activated Cl− channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16Ainh-A01 as a specific inhibitor of Ano1. AimTo investigate the effect of the T16Ainh-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. MethodsInhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16Ainh-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. ResultsT16Ainh-A01 inhibited Ca2+-activated Cl− currents by 60% at 10μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16Ainh-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16Ainh-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC. ConclusionsThe selective Ano1 inhibitor T16Ainh-A01 inhibited Ca2+-activated Cl− currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.

Chemical constituents from rauvolfia verticillata and bioactivities research

A new acridone alkaloid, named 9-hydroxynoracronycine (1), together with four known compounds, including coumarins, lignan, and indole alkaloid, was isolated and identified from the roots and rhizomes of Rauvolfia verticillata. The structure of the new compound was determined by spectroscopic means (UV, IR, MS, and NMR). The acridone alkaloid and coumarins were identified as new constituents of Rauvolfia genus. The cytotoxic activities of the new compound were tested against human breast cancer cell line MCF-7 and human promyelocytic leukemia HL-60. Results showed that compound 1 decreased MCF-7 cell proliferation in a statistically IC50 significant manner at 102.8 μmol/L. In addition, compound 1 exhibited moderate small intestine smooth muscle relaxation.

Effect of the ginsenoside Rb1 on the spontaneous contraction of intestinal smooth muscle in mice

To investigate the effect and the possible mechanism of ginsenoside Rb1 on small intestinal smooth muscle motility in mice. Intestinal smooth muscle strips were isolated from male ICR mice (5 wk old), and the effect of ginsenoside Rb1 on spontaneous contraction was recorded with an electrophysiolograph. The effect of ginsenoside Rb1 on ion channel currents, including the voltage-gated K⁺ channel current (IK(V)), calcium-activated potassium channel currents (IK(Ca)), spontaneous transient outward currents and ATP-sensitive potassium channel current (IK(ATP)), was recorded on freshly isolated single cells using the whole-cell patch clamp technique. Ginsenoside Rb1 dose-dependently inhibited the spontaneous contraction of intestinal smooth muscle by 21.15% ± 3.31%, 42.03% ± 8.23% and 67.23% ± 5.63% at concentrations of 25 μmol/L, 50 μmol/L and 100 μmol/L, respectively (n = 5, P < 0.05). The inhibitory effect of ginsenoside Rb1 on spontaneous contraction was significantly but incompletely blocked by 10 mmol/L tetraethylammonium or 0.5 mmol/L 4-aminopyridine, respectively (n = 5, P < 0.05). However, the inhibitory effect of ginsenoside Rb1 on spontaneous contraction was not affected by 10 μmol/L glibenclamide or 0.4 μmol/L tetrodotoxin. At the cell level, ginsenoside Rb1 increased outward potassium currents, and IK(V) was enhanced from 1137.71 ± 171.62 pA to 1449.73 ± 162.39 pA by 50 μmol/L Rb1 at +60 mV (n = 6, P < 0.05). Ginsenoside Rb1 increased IK(Ca) and enhanced the amplitudes of spontaneous transient outward currents from 582.77 ± 179.09 mV to 788.12 ± 278.34 mV (n = 5, P < 0.05). However, ginsenoside Rb1 (50 μmol/L) had no significant effect on IK(ATP) (n = 3, P < 0.05). These results suggest that ginsenoside Rb1 has an inhibitory effect on the spontaneous contraction of mouse intestinal smooth muscle mediated by the activation of IK(V) and IK(Ca), but the K(ATP) channel was not involved in this effect.

Effect of insulin on small intestinal smooth muscle in diabetic mice

Objective To investigate the effects of regular insulin (RI)on duodenal smooth muscle in diabetic mice. Methods Diabetes mellitus (DM) model was established by intraperitoneal injection of 150 mg/kg streptozotocin (STZ) in male BALB/c mice. The model mice were divided into DM group and DM treated with RI group with 6 each. Meanwhile, 6 normal mice were served as controls. The mice in treatment group were intraperitoneally injected with 40 U/kg of RI daily.Whereas the mice in DM and control groups were intraperitoneally injected with phosphate buffer solution (pH = 7. 40). After 6 weeks, the small intestinal transit rate of mice was determined by lavage of Indian ink. Interstitial cells of cajal (ICC) in duodenal myenteric plexus were counted using immunohistochemical staining. Slow waves of duodenal smooth muscle cells were recorded with intracellular recordings. Data were analysed by SPSS 17.0 software, and comparisons among three groups were done using LSD test. Results After intervention for 6 months, the clinical presentations,such as more water and food intake and polyuria, were improved in treatment group. The body weight was increased in treatment group [(23.33±3.13) g] compared with DM group [(15.42±1.40) g,P＜0.01] ,but dereased compared with control group [(26.78 ± 2.09) g, P＜0.05]. The level of blood glucose in DM group was significantly higher than that in control and treatment groups(P＜0.01). Small intestine transmission rate was significantly reduced in DM group than that in control and treatment groups (P＜0.01), but it was slower in treatment group than that in control group (P＜ 0. 01 ). Immunohistochemical study showed that the number of c-kit positive cells reduced obviously in DM group than that in control group and treatment group (P＜0.05), whereas it was lower in treatment group than that in control group (P ＜ 0.05). The slow wave frequency and amplitude of duodenal smooth muscle cells in DM group were reduced when compared with control and treatment groups (P＜0.01) and both were lower in treatment group than that in control group (P＜0. 01 ). Conclusion The findings indicate that DM mice have gastrointestinal dysmotility and exogenous insulin may improve small intestinal dysmotility in DM mice. Key words: Diabetes mellitus; Insulin; Duodenum; Myocytes, smooth muscle

Morphological changes in the small intestinal smooth muscle layers of horses suffering from small intestinal strangulation. Is there a basis for predisposition for reduced contractility?

Intestinal strangulation often leads to enterectomy after which ileus can develop. This has prompted research to look into possible pathophysiological processes triggering equine ileus. However, morphological changes of the small intestinal smooth muscle in relation to equine colic have not yet been studied. The presence of some smooth muscle proteins was morphologically assessed and quantified in control and colic horses. In addition, the up- or down-regulation of heat shock proteins (HSP20 and HSP27) influencing the contractility of smooth muscles was studied. Cranial resection margins of 18 strangulated small intestinal samples were collected. Small intestinal control samples were collected from 11 horses subjected to euthanasia for other than gastrointestinal-related reasons. Formaldehyde-fixed tissue was paraffin-embedded and processed for conventional staining and immunohistochemistry. Snap-frozen full-thickness biopsies were collected for western blot analyses. Evaluating the muscle layer microscopically, colic samples showed significantly more signs of degradation than controls (P = 0.026) of which vacuolar degeneration was most prominent (P = 0.009). In colic samples, myosin protein levels were decreased (P = 0.022) whereas desmin (P = 0.049) and HSP20 protein levels (P = 0.005) were elevated. In colic samples, microscopic lesions at the level of the muscle layer indicate a stress response. In addition, modified amounts of structural proteins such as myosin and desmin together with increased HSP20 levels could perhaps provide a basis for explaining the malfunctioning of the intestinal muscle layer. Post operative ileus, following small intestinal strangulation and resection, could be related in part to a dysfunctional muscle layer. In addition to microscopic signs of degeneration, myosin and HSP20 were affected. Pharmacological interventions might alter HSP20 expressions and thus serve a protective effect.

Diffuse Abnormal Layering of Small Intestinal Smooth Muscle is Present in Patients With FLNA Mutations and X-linked Intestinal Pseudo-obstruction

X-linked intestinal pseudo-obstruction, a rare disorder caused by mutations in FLNA, the gene encoding the cytoskeletal protein filamin A, has been regarded as a hereditary enteric neuropathy largely on the basis of sparse and incomplete pathologic studies. Diffuse abnormal layering of small intestinal smooth muscle (DAL) is a rare malformation, which has only been described in 4 patients (all male, 3 in the same family) with intestinal pseudo-obstruction. We report DAL in 5 male patients (2 families) with intestinal pseudo-obstruction and mutations in FLNA. Light microscopic, ultrastructural, and immunohistochemical studies showed abnormal lamination of the small intestinal muscularis propria with associated absent or severely reduced FLNA immunoreactivity. Intestinal samples from the oldest patient in the series, a teenager, showed multinucleate myocytes in small and large intestine, along the submucosal surface of the muscularis propria. As neither DAL nor the pattern of myocyte multinucleation observed in our patients have been described outside the context of X-linked intestinal pseudo-obstruction, these histopathologic features may be specific for this hereditary disorder and suggest an underlying myopathic basis for dysmotility in affected patients.

The transwall gradient across the mouse colonic circular muscle layer is carbon monoxide dependent

Gastric and small intestinal circular smooth muscle layers have a transwall resting membrane potential (RMP) gradient that is dependent on release of carbon monoxide (CO) from interstitial cells of Cajal (ICCs). Our aim was to determine whether a RMP gradient exists in the mouse colon and whether the gradient is CO dependent. Microelectrodes were used to record RMPs from muscle cells at different depths of the circular muscle layer from wild-type and heme oxygenase-2-knockout (HO-2-KO) mice. A transwall RMP gradient was present in wild-type mice. The CO scavenger oxyhemoglobin (20 μM) and the heme oxygenase inhibitor chromium mesoporphyrin IX (CrMP, 5 μM) abolished the transwall gradient. The gradient was absent in HO-2-KO mice. Tetrodotoxin (1 μM) caused a significant depolarization in circular smooth muscle cells throughout the circular muscle layer and abolished the transwall gradient. Removal of the submucosal neurons abolished the gradient. The majority of submucosal neurons contained HO-2 immunoreactivity (HO-2-IR), while ICCs did not. These data show for the first time that a transwall gradient exists across the circular smooth muscle layer of the mouse colon, that the gradient is due to CO, and that the source of CO is the submucosal neurons.

Damage of the interstitial cells of Cajal and myenteric neurons causing ileus in acute necrotizing pancreatitis rats

Small intestinal motility is impaired in acute necrotizing pancreatitis (ANP). The present study was designed to detect the impairment in small intestinal motility and to assess the role of interstitial cells of Cajal (ICC), myenteric neurons and the associated mechanism in the pathogenesis of ileus during experimentally induced acute pancreatitis. ANP was induced by intraperitoneal injections of 30% L-ornithine at a dose of 3 g/kg at hourly intervals. The alterations of small intestine electrical activity--migrating myoelectric complexes (MMCs), and slow waves--were measured 24 hr after ANP induction. The spontaneous mechanical activity and the contractile response to ACh, KCl, tetrodotoxin (TTX) and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) were evaluated by organ bath technique, and the morphologic alterations of the network of ICC, myenteric neurons and neuronal nitric oxide synthase (nNOS) immunoreactive cells were evaluated using the markers of c-Kit, PGP9.5, and nNOS, respectively. To demonstrate the deficiencies in enteric neuronal origin, we also measured nNOS expression in the muscular layer of ileum. L-ornithine-induced necrotizing pancreatitis manifests with multiple symptoms, including decreased amplitude of spontaneous contractions in small intestinal smooth muscle, declined contractile response to ACh, TTX, and L-NNA in vitro, disrupted MMC cycle length, decreased dominant frequency and dominant power of slow waves in vivo. Furthermore, the morphologic studies demonstrated the damage of ICC (ANP group versus control; P = .000), myenteric neurons (ANP group versus control; P = .001) and nNOS immunoreactive neurons (ANP group versus control; P = .000). We also observed a substantial loss in the expression of nNOS protein in muscular layer of the small intestine (ANP group versus control; P = .032). Our results suggest that the pathogenesis of the small intestinal paralysis in ANP may be related to the deficiencies in ICC and nNOS neurons.

Dopamine induces contraction in the proximal, but relaxation in the distal rat isolated small intestine

In the gut, dopamine is released by enteric neurons and modulates motility of small intestine smooth muscle cells. Here, we systematically analyzed the dopamine-induced effects on the longitudinal smooth muscle of different sections of the rat isolated small intestine. We found that exogenous dopamine had biphasic effects and could lead to both an early contraction and a late relaxation, depending on the region of small intestine. Thus, dopamine-induced early contractions were commonly observed in the duodenum, but less frequently in the jejunum, and rarely in the ileum. The amplitudes of these early contractions showed a striking regional dependence (duodenum > jejunum > ileum) and were significantly blocked by SCH23390 and raclopride. Conversely, dopamine-induced late relaxations were regularly obtained in the ileum and in the jejunum, but less frequently in the duodenum. Interestingly, the amplitudes of these relaxations showed an inverse regional dependence (ileum > jejunum > duodenum), and were insensitive to dopamine receptor antagonists. Rather, they were significantly inhibited by propranolol and prazosin. We conclude that dopamine exerts differential effects on smooth muscle motility in different regions within the rat small intestine. In proximal parts, dopamine predominantly causes D 1 and D 2 dopamine receptor-dependent contraction, whereas it leads to α and β adrenoceptor-dependent relaxation in more distal parts.

Smooth muscle NOS, colocalized with caveolin‐1, modulates contraction in mouse small intestine

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca2+] through L-type Ca2+ channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 μM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-β cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-ω-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 μM) blocked the responses to KCl and LNNA confirming the role of L-type Ca2+ channels. ODQ (1 μM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism.