Circular Smooth Muscle Cells Research Articles

Anatomy of blood microcirculation in the pig epicardial ganglionated nerve plexus

Embolization of coronary arteries and their terminal arterioles causes ischemia of all tissues distributed within a cardiac wall including the intrinsic cardiac ganglionated nerve plexus (ICGP). The disturbed blood supply to the ICGP causes chronic sympathetic activation with succeeding atrial and ventricular arrhythmias. This study analyses the anatomy of microcirculation of epicardial nerves and ganglia using the hearts of 11 domestic pigs. Our findings demonstrate that thicker epicardial nerves are normally supplied with blood via 12 epineural arterioles penetrating the endoneurium regularly along a nerve, and forming an endoneurial capillary network, which drains the blood into the myocardial blood flow. The mean diameter of intraneural capillaries was 7.2 ± 0.2 µm, while the diameters of arterioles were 25.8 ± 0.7 μm and involved 45 endothelial cells accompanied by circular smooth muscle cells. Usually, two or three arterioles with a mean diameter of 28.9 ± 1.7 μm supplied blood to any epicardial ganglion, in which arterioles proceeded into a network of capillaries with a mean diameter of 6.9 ± 0.3 μm. Both the epicardial nerves and the ganglia distributed near the porta venarum of the heart had tiny arterioles that anastomosed blood vessels from the right and the left coronary arteries. The density of blood vessels in the epicardial nerves was significantly lesser compared with the ganglia. Our electron microscopic observations provided evidence that blood vessels of the pig epicardial nerves and ganglia may be considered as either arterioles or capillaries that have quantitative and qualitative differences comparing to the corresponding blood vessels in humans and, therefore, a pig should not be considered as an animal model of the first choice for further heart functional studies seeking to improve the treatment of cardiac arrhythmias via trans-coronary cardiac neuroablation. Structured abstractThis study details the anatomy of microcirculation of epicardial nerves and ganglia, from which intracardiac nerves and bundles of nerve fibers extend into all layers of the atrial and ventricular walls in the most popular animal model of experimental cardiology and cardiac surgery - the domestic pig. Our findings provided evidence that blood vessels of the pig epicardial nerves and ganglia may be considered as either arterioles or capillaries that have quantitative and qualitative differences comparing to the corresponding blood vessels in humans and, therefore, a pig should not be considered as an animal model of the first choice for further heart functional studies seeking to improve the treatment of cardiac arrhythmias via trans-coronary cardiac neuroablation.

Retracted: PKC-delta and PKD activate MAPK signal pathway in mechano-transcription of colonic smooth muscle cells.

Retraction: [PKC-delta and PKD activate MAPK signal pathway in mechano-transcription of colonic smooth muscle cells, Z. Yang, K. He, T. Wang, et al. Neurogastroenterology & Motility 2023; e14623 (https://onlinelibrary.wiley.com/doi/full/10.1111/nmo.14623)]. The above article, published online on June 6, 2023 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the Journal Editor in Chief, Maura Corsetti, and John Wiley & Sons Ltd. The retraction has been agreed due to unat[1]tributed overlap between this article and the abstract published in Gastroenterology: Li F, Sarna SK and Shi XP. Roles of PKCs and PKD in Mechanotranscription in Colonic Smooth Muscle Cells: Inhibition of Mechanotranscription as a Potential Treatment for Motility Dysfunction in Obstructive Disorders. In: 2012 Digestive Disease Week Abstract Supplement; May 19-22, San Diego, CA. Abstract 120 (https://www.gastrojournal.org/article/S0016-5085(12)60115-2/pdf).

Three-dimensional morphologic and molecular atlases of nasal vasculature

Understanding the function of the nasal vasculature in homeostasis and pathogenesis of common nasal diseases is important. Here we describe an extensive network of venous sinusoids (VSs) in mouse and human nasal mucosa. The endothelium of the VSs expressed Prox1 (considered to be a constitutive marker of lymphatic endothelium) and high levels of VCAM-1 and exhibited unusual cell-to-cell junctions. VSs are supported by circular smooth muscle cells (SMCs) and surrounded by immune cells. The nasal mucosa also showed a rich supply of lymphatic vessels with distinctive features, such as the absence of the lymphatic marker LYVE1 and sharp-ended capillaries. In mouse models of allergic rhinitis or acute Coronavirus Disease 2019 (COVID-19) infection, Prox1+ VSs were regressed or compromised. However, in aged mice, the VSs lost the SMC support and were expanded and enlarged. Our findings demonstrate three-dimensional morphological and molecular heterogeneities of the nasal vasculature and offer insights into their associations with nasal inflammation, infection and aging.

Role of PGE2 in colonic motility: PGE2 attenuates spontaneous contractions of circular smooth muscle via EP4 receptors in the rat colon

Colonic motor activity is important for the formation and propulsion of feces. The production of prostaglandins (PGs) in colonic tissue is considered to play a critical role in the generation and regulation of colonic motility. In this study, we investigated the inhibitory effects of PGE2 and selective agonists of four EP receptors on the spontaneous phasic contractions, called ‘giant contractions’ (GCs), of mucosa-free circular smooth muscle strips from the rat middle colon. Neural blockade with tetrodotoxin (TTX) increased the frequency and amplitude of the GCs by about twofold. However, inhibiting PG production with piroxicam reduced the GC frequency in the presence of TTX, but did not affect the GC amplitude. In the presence of both TTX and piroxicam, exogenous PGE2 and each EP receptor agonist were cumulatively added to the tissue bath. In this setting, PGE2, the EP2 agonist ONO-AE1-259, and the EP4 agonist ONO-AE1-329, but not the EP1 agonist ONO-AE-DI-004 or the EP3 agonist ONO-AE-248, concentration-dependently reduced the GC frequency and amplitude. The PGE2-induced inhibition of GC frequency and amplitude was inhibited by the EP4 antagonist ONO-AE3-208, but not by the EP1/2 antagonist AH6809. Immunohistochemistry revealed the EP2 and EP4 receptors were localized in perinuclear sites in circular smooth muscle cells. EP2 immunoreactivity was also located in GFAP-immunoreactive enteroglia, whereas EP4 immunoreactivity was also located in HU (embryonic lethal, abnormal vision [ELAV] protein; a marker of all myenteric neurons)-immunoreactive myenteric nerve cell bodies. These results suggest that the PGs produced in the colonic tissue inhibit the GC frequency and amplitude of circular muscle in the rat middle colon, and is mediated by EP4 receptors expressed in the smooth muscle cells.

A211 ANTIFIBROTIC ACTIVITY OF NOVEL TYROSINE KINASE INHIBITORS IN VITRO

Abstract Background Chronic inflammation in inflammatory bowel disease (IBD) causes structural alterations of the intestine. In Crohn’s disease, this can lead to stricture formation, arising from unclear interactions among local inflammatory cells, cytokines, and mesenchymal intestinal cells. Specifically, proliferation of smooth muscle and increased deposition of collagen-rich extracellular matrix leads to fibrosis and obstructive wall thickening. Since there are minimal treatment options, we explored the effects of two multimodal tyrosine kinase inhibitors, nintedanib and pirfenidone, which were recently approved for idiopathic pulmonary fibrosis (IPF), a chronic lung condition resembling Crohn’s disease. Aims To understand the basic pharmacology of two novel anti-fibrotic tyrosine kinase inhibitors and their effects on cell proliferation and collagen deposition. Methods In vitro model systems of adult rat colonic circular smooth muscle cells (CSMS), rat IEC-18 intestinal epithelial cells or mouse 3T3 were assessed for response to serum or the mesenchymal growth factor PDGF-BB, evaluating growth responses by proliferation assay, and type I collagen expression by immunocytochemistry and western blotting. Programmed cell death was detected by ICC and western blotting for caspase-dependent apoptotic markers. Results Both 3T3 fibroblasts and rat ISMC showed concentration-dependent proliferation in response to serum or PDGF application. Nintedanib reduced this response to baseline at EC100 of 2.3 ± 0.7 (n=5) µM, and EC50 of 0.3 ± 0.1 (n=6) µM without cytotoxicity, while pirfenidone was ineffective at levels ≤ 5mM. Nintedanib (10 µM) was ineffective against serum-induced growth of rat IEC-18 cells while blocking growth of rat CSMC or mouse 3T3 fibroblasts in parallel assays, suggesting a selective effect on mesenchymal cell types. In nintedanib-treated cultures, nuclear staining showed concentration-dependent reduction of mitotic figures with proportional appearance of nuclear fragmentation, typical of apoptosis. Western blotting for collagen I identified at 125kD band for both CSMC and 3T3 cells and suggested down-regulation with both nintedanib and pirfenidone. Conclusions Novel anti-fibrotic therapies for chronic idiopathic pulmonary disease display distinctive effects on ISMC proliferation and extracellular matrix production. These address molecular mechanisms of fibrosis that are in common with IBD, and so the translation of therapeutic approaches may be a promising treatment option. Detecting specific mechanisms of action of nintedanib, such as apoptosis, leads to better targets for therapeutic options. Funding Agencies NSERC

A132 SMOOTH MUSCLE BIOPSIES FROM POEM PROCEDURES FOR ACHALASIA SHOW LACK OF FUNCTIONAL INNERVATION

Abstract Background Idiopathic achalasia is a disease of the esophagus causing impaired peristalsis and absent lower esophageal sphincter relaxation. The etiopathogenesis of the disease is unclear but most histopathologic studies from surgical resections show an absence of nitric oxide-producing neurons in the myenteric plexus. The peroral endoscopic myotomy (POEM) procedure has evolved in the last decade to treat achalasia and has provided a unique way to sample diseased tissue from an otherwise inaccessible tissue compartment. Aims To study the effects of achalasia on innervation of smooth muscle and smooth muscle phenotype. Methods Patients with a diagnosis of achalasia based on high resolution manometry undergoing a POEM at Kingston general hospital were approached for enrollment between June 2017 to September 2018. Demographic information including age, symptom duration, previous treatments, and Eckhardt score was collected. Intraoperatively, biopsies of the circular smooth muscle (CSM) layer were taken at the proximal and distal extents of the myotomy and placed in formalin. Tissue was embedded in wax, sectioned and stained using immunocytochemistry (ICC) for neuronal, axonal and smooth muscle cell markers. This was compared to control tissue from patients undergoing gastroesophagectomy for adjacent malignancy. Results Control tissue from 3 separate esophagectomies were obtained. Samples were obtained from a total of 25 patients (13 males), with a median age of 50 (IQR 38–67). Most patients had Type 2 achalasia (19 [76%]) followed by Type 1 and 3 (2, [8%] respectively). Two cases had conflicting manometric findings. The median duration of symptoms was 3 years (IQR 1.5–10.5) and the median Eckhardt score was 6.5 (IQR 5–9). In sample tissues, no neuronal cell bodies were detected in the CSM layer. ICC for the axonal marker PGP9.5 showed that CSM of achalasia samples were almost completely devoid of axon structure, independent of the subtype of achalasia, compared to abundant axon presence in control tissues. In parallel, ICC showed that cholinergic (ChAT) or nitrergic (nNOS) axonal subtypes were absent in biopsy CSM while abundant in controls. CSM cells displayed hypertrophy with no detection of proliferation by ICC (KI67) or alterations in the phenotypic marker SM-22. Conclusions Preliminary results from advanced immunohistochemical techniques show the absence of functional innervation of the CSM layer of all patients with achalasia. This is characterized by a depleted excitatory and inhibitory axon population. Further studies are focused on defining differences in smooth muscle phenotype and the presence or absence of inflammatory cells within the CSM. Funding Agencies None

The effects of TJ-14 (Hange-shashin-to), Japanese kampo medicines, on spontaneous electrical activity of circular smooth muscle cells in the mouse proximal colon

The effects of TJ-14 (Hange-shashin-to), Japanese kampo medicines, on spontaneous electrical activity of circular smooth muscle cells in the mouse proximal colon

Immune/Inflammatory Response and Hypocontractility of Rabbit Colonic Smooth Muscle After TNBS-Induced Colitis.

The contractility of colonic smooth muscle is dysregulated due to immune/inflammatory responses in inflammatory bowel diseases. Inflammation in vitro induces up-regulation of regulator of G-protein signaling 4 (RGS4) expression in colonic smooth muscle cells. To characterize the immune/inflammatory responses and RGS4 expression pattern in colonic smooth muscle after induction of colitis. Colitis was induced in rabbits by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Innate/adaptive immune response RT-qPCR array was performed using colonic circular muscle strips. At 1-9weeks after colonic intramuscular microinjection of lentivirus, the distal and proximal colons were collected, and muscle strips and dispersed muscle cells were prepared from circular muscle layer. Expression levels of RGS4 and NFκB signaling components were determined by Western blot analysis. The biological consequences of RGS4 knockdown were assessed by measurement of muscle contraction and phospholipase C (PLC)-β activity in response to acetylcholine (ACh). Contraction in response to ACh was significantly inhibited in the inflamed colonic circular smooth muscle cells. RGS4, IL-1, IL-6, IL-8, CCL3, CD1D, and ITGB2 were significantly up-regulated, while IL-18, CXCR4, CD86, and C3 were significantly down-regulated in the inflamed muscle strips. RGS4 protein expression in the inflamed smooth muscles was dramatically increased. RGS4 stable knockdown in vivo augmented ACh-stimulated PLC-β activity and contraction in colonic smooth muscle cells. Inflamed smooth muscle exhibits up-regulation of IL-1-related signaling components, Th1 cytokines and RGS4, and inhibition of contraction. Stable knockdown of endogenous RGS4 in colonic smooth muscle increases PLC-β activity and contractile responses.

Stress increases descending inhibition in mouse and human colon.

A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues. Stress was applied using water avoidance stress (WAS) in the animal model or mimicked using stress hormones, adrenaline (5 nM), and corticosterone (1 μM). Intracellular recordings were obtained from colonic circular smooth muscle cells in isolated smooth muscle/myenteric plexus preparations and the inhibitory junction potential (IJP) was elicited by nerve stimulation or balloon distension oral to the site of recording. Water avoidance stress increased the number of fecal pellets compared to control (p < 0.05). WAS also caused a significant increase in IJP amplitude following balloon distension. Stress hormones also increased the IJP amplitude following nerve stimulation and balloon distension (p < 0.05) in control mice but had no effect in colons from stressed mice. No differences were observed with application of ATP between stress and control tissues, suggesting the actions of stress hormones were presynaptic. Stress hormones had a large effect in the nerve stimulated IJP in human colon (increased >50%). Immunohistochemical studies identified alpha and beta adrenergic receptor immunoreactivity on myenteric neurons in human colon. These studies suggest that WAS and stress hormones can signal via myenteric neurons to increase inhibitory neuromuscular transmission. This could lead to greater descending relaxation, decreased transit time, and subsequent diarrhea.

Short-Term Effects of Relamorelin on Descending Colon Motility in Chronic Constipation: A Randomized, Controlled Trial.

The pentapeptide ghrelin agonist, relamorelin, accelerates colonic transit in patients with chronic constipation (CC). In a murine model, relamorelin decreased excitability of colonic circular smooth muscle cells and colonic intraluminal pressure. To determine short-term effects of relamorelin on colonic motility measured by barostat and multilumen manometry in CC. In a placebo-controlled, single-dose, double-blind, randomized study in patients with CC, we investigated the motor effects of relamorelin, 100 μg, SQ (12 patients) compared to placebo SQ (six patients). A motility-barostat balloon assembly was used to measure colonic compliance; tone and phasic pressure activity were measured before and after a 1000-kcal milkshake meal (administered ~60 min post-medication). Overall "background" phasic pressure activity was assessed by: average amplitude and motility index (MI = ln[sum amplitudes × #contractions + 1]) over defined periods. High-amplitude propagating contractions (HAPCs) were characterized by amplitude >75 mmHg and propagating contractions >50 mmHg; both were propagated over at least 10 cm. Postprandial HAPCs were the primary end point. The study sample had 80% power to detect an increase of 3.3 HAPCs in the hour post-meal. Relamorelin, 100 μg, significantly induced more pre-meal propagated contractions [PCs of either >50 or >75 mmHg] compared to placebo (p < 0.05). Relamorelin also induced more post-meal PCs >50 or >75 mmHg than placebo. Relamorelin did not significantly alter colonic compliance, fasting or postprandial phasic pressure activity (20 min pre-meal fasting MI) or tone, and 60 min postprandial phasic pressure amplitude or MI, or tone. Relamorelin stimulates propagated colonic contractions without alteration of background irregular contractions in CC. ClinicalTrial.Gov registration number: NCT 01781104.

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

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

Lymphaticosclerosis: a new way of thinking about lymphatic vessel obstruction.

ORIGINAL ARTICLE, p 1286 Lymphoedema is a progressive condition that often manifests following cancer surgeries that involve removal or irradiation of lymph nodes. The symptoms may range from mild to severe and debilitating, and in the latter case can significantly increase susceptibility to infection and restrict quality of life.1 2 It is clear that removal of lymph nodes or severing of lymphatic vessels can cause an immediate interruption of the normal flow of lymph from the upstream vessel network into the downstream collecting vessels.3 However, the pathological mechanisms that progressively lead to severe secondary (acquired) lymphoedema are unclear, as are the reasons why different individuals receiving the same procedure may exhibit wide‐ranging severities of lymphatic dysfunction. In this issue of the BJD, Ogata et al.4 have investigated the change in phenotype of lymphatic smooth muscle cells during lymphoedema. The authors studied lymphatic vessels from 29 randomly chosen patients who had developed secondary leg lymphoedema after surgical removal of tumours, were refractory to congenital conservative therapy and received a lymphaticovenous anastomosis procedure. Typically, the collecting lymphatics that are responsible for pumping of lymph in the limbs have a thin layer of circular smooth muscle cells, which intrinsically contracts. The phasic contractions of these cells, in concert with intraluminal one‐way valves composed of endothelial cells and connective tissue, allow forward propulsion of lymph through the system.5 Ogata et al.4 observed a striking change in the collecting lymphatic wall in patients with chronic lymphoedema, with a markedly thickened smooth muscle layer, increased collagen deposition and apparent invasion of smooth muscle cells into the intimal layer. In cases of more severe lymphoedema, stenosis or ‘lymphaticosclerosis’ of the lymphatic lumen due to overgrowth of lymphatic smooth muscle cells was apparent. In addition, labelling of the myosin heavy chain isoforms SM1 and SM2 was reduced in lymphatic smooth muscle, and transmission electron microscopy revealed an increase in synthetic organelles, such as sarcoplasmic reticulum and Golgi complex, while contractile myofilaments were reduced.4 Collectively, these findings suggest that the progression of surgery‐induced lymphatic injury into lymphoedema involves re‐entry of lymphatic smooth muscle cells into a proliferative stage to respond to the injury, followed by their failure to mature and remodel appropriately. This change in the phenotype of lymphatic smooth muscle is akin to a common problem with angioplasty: restenosis of the lumen after plaque removal due to the synthetic vascular smooth muscle phenotype.6

Bisphenol A inhibits duodenal movement ex vivo of rat through nitric oxide-mediated soluble guanylyl cyclase and α-adrenergic signaling pathways.

The gastrointestinal tract is directly exposed to bisphenol A (BPA)-tainted foods and beverages stored in polycarbonate plastic containers. The effect of BPA on the movement of small intestine has not been reported until now. We report here the effect of BPA on the movement of the duodenum ex vivo in a rat model. We found significant inhibition of duodenal movement by BPA (10-320 µM). We suggest that BPA-induced inhibition of duodenal movement might be due to the suppression of stimulatory and/or activation of inhibitory motor neurons in enteric plexuses innervating the longitudinal and circular visceral smooth muscle cells in the duodenal wall. We observed a significant reversal of BPA-induced depression of duodenal movement by methylene blue, a soluble guanylyl cyclase blocker and N-ω-nitro-L-arginine methyl ester, a nitric oxide (NO) synthase inhibitor; but significant potentiation of the movement by sodium nitroprusside, a NO donor. From the results, we may suggest that BPA-induced inhibition of the movement might be partially due to activation of inhibitory motor neurons that secrete NO, a relaxant, on to smooth muscle cells. Furthermore, we found significant reversal of BPA-induced depression of the movement in phentolamine, an α-adrenergic receptor blocker, pretreated preparation. This result proves that norepinephrine secreting motor neurons may also be involved in BPA-induced inhibition of the movement. From the results, we conclude that BPA inhibits the movement of the duodenum through NO-mediated soluble guanylyl cyclase and α-adrenergic signaling pathways in visceral smooth muscle cells.

High‐fat Diet Causes Loss of Nitric Oxide Motor Neurons and Impairs Inhibitory Neuromuscular Communication in the Mouse Distal Colon

High‐fat (HF) diet increases the risks of gastrointestinal (GI) motility complications, however, the mechanisms that underlie these complications are still not entirely understood. We tested the hypothesis that HF diet causes oxidative stress, loss of nitric oxide (NO) motor neurons in the myenteric plexus, disrupted inhibitory neuromuscular communication and impaired GI motility.MethodsDistal colon segments from mice fed a control (10 Kcal%) or HF diet (60 Kcal%) were used in vitro. Alexa fluor 546 and 680 conjugated maleimides were used to assess the oxidized/reduced glutathione ratio in myenteric NO neurons. NO neuronal density was assessed using nNOS immunohistochemistry. Basal NO availability was measured using NO induced fluorescence of DAF‐FM diacetate. Electrical field stimulation was applied across varying voltages (60‐120V) and train (200‐1000ms) durations to evoke inhibitory junction potentials (IJPs) in colonic circular smooth muscle cells. Amplitude and area of IJP were examined to assess fast and slow IJPs respectively.ResultsWe observed an increase (17%, p&lt;0.01) in oxidized/reduced glutathione fluorescence ratio in nNOS neurons of HF mice. HF mice had lower (28%, p&lt;0.01) nNOS neurons per ganglionic area than controls. Fluorescence intensity of DAF‐FM diacetate was also lower (20%, p&lt;0.01) in HF mice. HF mice showed a significant loss (52%, p&lt;0.05) in IJP area, when stimulated at higher train duration (800‐1000ms).ConclusionWe conclude that HF diet causes oxidative stress, leading to loss of inhibitory NO neurons, lower NO availability, and disrupted neuromuscular transmission in the mouse distal colon. Supported by NIH (DK094932).

Intestinal smooth muscle phenotype determines enteric neuronal survival via GDNF expression

Intestinal inflammation causes initial axonal degeneration and neuronal death, as well as the proliferation of intestinal smooth muscle cells (ISMC), but subsequent axonal outgrowth leads to re-innervation. We recently showed that expression of glial cell-derived neurotrophic factor (GDNF), the critical neurotrophin for the post-natal enteric nervous system (ENS) is upregulated in ISMC by inflammatory cytokines, leading us to explore the relationship between ISMC growth and GDNF expression. In co-cultures of myenteric neurons and ISMC, GDNF or fetal calf serum (FCS) was equally effective in supporting neuronal survival, with neurons forming extensive axonal networks among the ISMC. However, only GDNF was effective in low-density cultures where neurons lacked contact with ISMC. In early-passage cultures of colonic circular smooth muscle cells (CSMC), polymerase chain reaction (PCR) and western blotting showed that proliferation was associated with expression of GDNF, and the successful survival of neonatal neurons co-cultured on CSMC was blocked by vandetanib or siGDNF. In tri-nitrobenzene sulfonic acid (TNBS)-induced colitis, immunocytochemistry showed the selective expression of GDNF in proliferating CSMC, suggesting that smooth muscle proliferation supports the ENS in vivo as well as in vitro. However, high-passage CSMC expressed significantly less GDNF and failed to support neuronal survival, while expressing reduced amounts of smooth muscle marker proteins. We conclude that in the inflamed intestine, smooth muscle proliferation supports the ENS, and thus its own re-innervation, by expression of GDNF. In chronic inflammation, a compromised smooth muscle phenotype may lead to progressive neural damage. Intestinal stricture formation in human disease, such as inflammatory bowel disease (IBD), may be an endpoint of failure of this homeostatic mechanism.

Spontaneous transient hyperpolarizations in the rabbit small intestine.

Four types of electrical activity were recorded and related to cell structure by intracellular recording and dye injection into impaled cells in muscles of rabbit small intestine. The specific cell types from which recordings were made were longitudinal smooth muscle cells (LSMCs), circular smooth muscle cells (CSMCs), interstitial cells of Cajal distributed in the myenteric region (ICC-MY) and fibroblast-like cells (FLCs). Slow waves (slow wavesSMC) were recorded from LSMCs and CSMCs. Slow waves (slow wavesICC) were of greatest amplitude (>50mV) and highest maximum rate of rise (>10Vs(-1)) in ICC-MY. The dominant activity in FLCs was spontaneous transient hyperpolarizations (STHs), with maximum amplitudes above 30mV. STHs were often superimposed upon small amplitude slow waves (slow wavesFLC). STHs displayed a cyclical pattern of discharge irrespective of background slow wave activity. STHs were inhibited by MRS2500 (3μm), a P2Y1 antagonist, and abolished by apamin (0.3μm), a blocker of small conductance Ca(2+)-activated K(+) channels. Small amplitude STHs (<15mV) were detected in smooth muscle layers, whereas STHs were not resolved in cells identified as ICC-MY. Electrical field stimulation evoked purinergic inhibitory junction potentials (IJPs) in CSMCs. Purinergic IJPs were not recorded from ICC-MY. These results suggest that FLCs may regulate smooth muscle excitability in the rabbit small intestine via generation of rhythmic apamin-sensitive STHs. Stimulation of P2Y1 receptors modulates the amplitudes of STHs. Our results also suggest that purinergic inhibitory motor neurons regulate the motility of the rabbit small intestine by causing IJPs in FLCs that conduct to CSMCs.

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.

Mechanical stress is a pro-inflammatory stimulus in the gut: in vitro, in vivo and ex vivo evidence.

AimsInflammatory infiltrates and pro-inflammatory mediators are found increased in obstructive and functional bowel disorders, in which lumen distention is present. However, what caused the low level inflammation is not well known. We tested the hypothesis that lumen distention- associated mechanical stress may induce expression of specific inflammatory mediators in gut smooth muscle.MethodsStatic mechanical stretch (18% elongation) was applied in vitro in primary culture of rat colonic circular smooth muscle cells (RCCSMCs) with a Flexercell FX-4000 Tension Plus System. Mechanical distention in vivo was induced in rats with an obstruction band placed in the distal colon.ResultsIn the primary culture of RCCSMCs, we found that static stretch significantly induced mRNA expression of iNOS, IL-6, and MCP-1 in 3 hours by 6.0(±1.4), 2.5(±0.5), and 2.2(±0.5) fold (n = 6∼8, p<0.05), respectively. However, gene expression of TNF-α, IL-1β, and IL-8 was not significantly affected by mechanical stretch. In the in vivo model of colon obstruction, we found that gene expression of iNOS, IL-6, and MCP-1 is also significantly increased in a time-dependent manner in the mechanically distended proximal segment, but not in the sham controls or distal segments. The conditioned medium from the muscle strips of the stretched proximal segment, but not the distal segment or control, significantly induced translocation and phosphorylation of NF-κB p65. This treatment further increased mRNA expression of inflammatory mediators in the naïve cells. However, treatment of the conditioned medium from the proximal segment with neutralizing antibody against rat IL-6 significantly attenuated the activation of NF-κB and gene expression of inflammatory mediators.ConclusionsOur studies demonstrate that mechanical stress induces gene expression of inflammatory mediators i.e. iNOS, IL-6, and MCP-1 in colonic SMC. Further ex vivo study showed that mechanical stress functions as a pro-inflammatory stimulus in the gut.

Damage from dissection is associated with reduced neuro-musclar transmission and gap junction coupling between circular muscle cells of guinea pig ileum, in vitro.

Excitatory and inhibitory junction potentials of circular smooth muscle cells in guinea pig ileum and colon are suppressed 30–90 min after setting up in vitro preparations. We have previously shown this “unresponsive” period is associated with a transient loss of dye coupling between smooth muscle cells, which subsequently recovers over the ensuing 30–90 min; junction potentials recover in parallel with dye coupling (Carbone et al., 2012). Here, we investigated which components of dissection trigger the initial loss of coupling. Intracellular recordings were made from circular muscle cells of guinea pig ileum with micropipettes containing 5% carboxyfluorescein. After allowing 90–120 min for junction potentials to reach full amplitude, we re-cut all 4 edges of the preparation more than 1 mm from the recording sites. This caused a reduction in the amplitude of IJPs from 17.2 ± 0.7 mV to 9.5 ± 1.5 mV (P < 0.001, n = 12) and a significant reduction in dye coupling. Both recovered within 60 min. We repeated this experiment (n = 4), recording both 1 and 4 mm from the cut edge: both sites were equally affected by re-cutting the sides of the preparation. Equilibrated preparations were stretched to 150% of their original length, this had no significant effect on junction potentials or dye coupling. Setting up preparations in low calcium solution did not prevent the initial suppression of IJPs and dye coupling. Application of 3 μM indomethacin (n = 3), 10 μM ketotifen (n = 4) or 10 μM forskolin during dissection did not prevent the suppression of IJPs and dye coupling. If dissection damage was reduced, by leaving the mucosa and submucosa attached to the circular muscle, IJPs showed less initial suppression than in preparations where the layers were dissected off. We conclude that physical damage to the gut wall triggers loss of gap junction coupling and neuromuscular transmission, this is not due to stretch, influx of calcium ions, release of prostaglandins or mast cell degranulation. The mechanisms underlying this potent effect remain to be determined.

Sa2046 Effect of RM-131 on Circular Smooth Muscle Cells in Human and Mouse Colon and on Colonic Intraluminal Pressure in Conscious Mice

Background and Aims: RM-131 is a synthetic ghrelin agonist that has been found to significantly accelerate gastric emptying, intestinal transit and colonic filling. The mechanism(s) through which smooth muscle cells of gastrointestinal tract respond to RM-131 is not known. We studied the effect of RM-131 on circular smooth muscle cells of human and mouse colon in vitro and on colonic intraluminal pressure in conscious mice. Methods: SJL/J mice and human colonic tissue were used in this study. Normal human colon was obtained from surgical excess tissue. The use of mice was approved by Mayo Institutional Animal Care and Use Committee and the use of human biopsy specimens was approved by Institutional Review Board. Muscle strips without the mucosa from mouse colon and human colon strips were placed in a recording chamber for intracellular recording. The recording chamber was superfused with oxygenized normal Krebs solution at 37°C. A bipolar stimulating electrode was placed on both sides of the muscle strip for electric field stimulation to elicit inhibitory junction potentials (IJPs). To test the effect of RM-131 on colonic intraluminal pressure in conscious mice, a miniaturized pressure transducer catheter was introduced into the colon such that the middle of the pressure sensor was 2.5 cm proximal to the anus. Results: RM-131 (0.25 μM) significantly hyperpolarized the resting membrane potential for human colonic circular smooth muscle cells by 2.8±0.7 mV (P<0.01, n=9) and in mouse preparations by 3.4±0.4 mV (P<0.01, n=6). RM-131 had no significant effect on either the fast IJP or the slow IJP. Ghrelin (1 μM) also hyperpolarized the resting membrane potential of circular smooth muscle cells in all cells recorded from human preparations by 3.5±1.4 mV (P=0.06, n=3) and in mouse preparations by 3.8±0.8 mV (P<0.01, n=7). Ghrelin also had no significant effect on either the fast IJP or slow IJP. For intraluminal pressure recordings, RM-131 or control saline was applied via intraperitoneal injection. Intraluminal pressure changes with RM-131 or saline were normalized with their pre-control. In the RM-131 treated group, intraluminal pressure was 0.57±0.05 (n=8) which was significantly less compared to the control group (0.82±0.08, n=8), suggesting that RM131 significantly reduced colonic tone. Conclusions: Our results suggest that RM-131 decreases colonic tone by hyperpolarizing the resting membrane potential of circular smooth muscle cells and decreases the excitability of colonic circular smooth muscle cells. These effects of RM-131 are likely the basis for the decrease in colonic intraluminal pressure. The previously reported increase in upper GI contractility together with a decrease in colonic intraluminal pressure position RM-131 for the treatment of functional gastrointestinal disorders.