Porcine Lower Esophageal Sphincter Research Articles

Investigating the Mechanisms Underlying U46619-Induced Contraction on Porcine Lower Esophageal Sphincter.

Gastroesophageal reflux disease (GERD) is associated with an incompetent lower esophageal sphincter (LES), resulting in the reflux of gastric contents into the esophagus. U46619, a thromboxane A2 (TXA2) receptor agonist, induces contractions in various smooth muscles. Therefore, this study aimed to investigate the effects and mechanisms of action of U46619 on the porcine LES. To achieve this, contractions of the clasp and sling strips of the porcine LES, induced by U46619, were measured using isometric transducers. Furthermore, the contractile mechanism of U46619 in the porcine LES was investigated by pretreating the strips with atropine (a muscarinic receptor antagonist), tetrodotoxin (a neuronal sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution. Additionally, reverse transcription polymerase chain reaction (PCR) and immunohistochemistry (IHC) were performed to determine the presence of the TXA2 receptor in porcine LES. The results of this study demonstrated that U46619 caused marked concentration-dependent contractions in both porcine sling and clasp strips. The mechanism of U46619-induced contraction of the porcine LES was found to be related to calcium channels. Furthermore, the reverse transcription PCR analysis and IHC revealed that the TXA2 receptor was expressed in the clasp and sling fibers of porcine LES. Consequently, this study suggests that U46619 mediates the contraction of porcine LES through calcium channels and has potential as a therapeutic approach for treating GERD. SIGNIFICANCE STATEMENT: This study establishes that U46619 induces concentration-dependent contractions in porcine LES, primarily mediated by calcium channels. The presence of the TXA2 receptor in LES clasp and sling fibers is confirmed. These findings highlight U46619's potential as a GERD therapeutic by targeting calcium channels for LES contraction modulation.

Arecae pericarpium extract induces porcine lower-esophageal-sphincter contraction via muscarinic receptors

BackgroundGastroesophageal reflux disease (GERD) is associated with lower esophageal sphincter (LES) incompetence. In some patients, GERD is refractory to acid reduction therapy which is the main treatment for GERD. So far, medications that can increase LES tone are few. Arecae pericarpium (A. pericarpium) is a medication in Traditional Chinese Medicine known to promote intestinal motility.MethodsWe investigated the effect of A. pericarpium extracts on porcine LES motility. In addition, we used tetrodotoxin (TTX) and atropine to study the underlying mechanism of A. pericarpium extracts-induced contractions of LES.ResultsThe results of this study showed that A. pericarpium extracts and their main active ingredient, arecoline, can induce the contractions of porcine LES sling and clasp muscles in a dose-response manner. TTX did not have an inhibitory effect on the contractions induced by A. pericarpium extracts and arecoline in LES. However, atropine significantly inhibited A. pericarpium extracts- and arecoline-induced contractions of LES.ConclusionA. pericarpium extracts can induce the contractions of porcine LES in a dose dependent manner, possibly through muscarinic receptors, and hence, may be worth developing as an alternative therapy for GERD.

Involvement of interstitial cells of Cajal in nicotinic acetylcholine receptor-induced relaxation of the porcine lower esophageal sphincter

The interstitial cells of Cajal (ICCs) play an important role in coordinated gastrointestinal motility. The present study aimed to elucidate whether or how ICCs are involved in the lower esophageal sphincter (LES) relaxation induced by stimulation of the nicotinic acetylcholine receptor. The application of 1,1-dimethyl-4-phenyl-piperazinium (DMPP; a nicotinic acetylcholine receptor agonist) induced a transient relaxation in the circular smooth muscle of the porcine LES. DMPP-induced relaxation was abolished by not only 1 μM tetrodotoxin but also the inhibition of ICC activity by pretreatment with 100 μM carbenoxolone (a gap junction inhibitor), pretreatment with 100 μM CaCCinh-A01 (an anoctamin-1 blocker acting as a calcium-activated chloride channel inhibitor), and pretreatment with Cl−-free solution. However, pretreatment with 100 μM Nω-nitro-L-arginine methyl ester had little effect on DMPP-induced relaxation. Furthermore, DMPP-induced relaxation was inhibited by pretreatment with 1 mM suramin, a purinergic P2 receptor antagonist, but not by 1 μM VIP (6–28), a vasoactive intestinal peptide (VIP) receptor antagonist. Stimulation of the purinergic P2 receptor with adenosine triphosphate (ATP) induced relaxation, which was abolished by the inhibition of ICC activity by pretreatment with CaCCinh-A01. In conclusion, membrane hyperpolarization of the ICCs via the activation of anoctamin-1 plays a central role in DMPP-induced relaxation. ATP may be a neurotransmitter for inhibitory enteric neurons, which stimulate the ICCs. The ICCs act as the interface of neurotransmission of nicotinic acetylcholine receptor in order to induce LES relaxation.

Long-Chain Fatty Acid Receptors Mediate Relaxation of the Porcine Lower Esophageal Sphincter.

Long-chain fatty acids activate the free fatty acid receptor 1 (FFA1) and FFA4. In the gastrointestinal system, FFA1 and FFA4 have been found in the pancreas and intestine. Fatty food and decreased lower esophageal sphincter (LES) motility are associated with gastroesophageal reflux disease. The effect of long-chain fatty acids on the esophageal motility is unknown. The purpose of this study is to investigate the effects of long-chain fatty acids on the porcine LES motility ex vivo using isometric transducers. In endothelin 1-precontracted porcine LES strips, the FFA1 selective agonists, fasiglifam, TUG424, and GW9508, caused marked relaxations in a concentration-dependent manner. The relative efficacies to elicit relaxation were GW9508 > TUG424 > fasiglifam in both clasp and sling strips. In contrast, the FFA4 specific agonists, TUG891 and GSK137647, produced mild relaxations. In addition, the endogenous FFA1 agonist DHA caused a mild relaxation whereas GW1100, an FFA1 antagonist, inhibited GW9508 induced relaxation of the porcine LES clasp and sling muscle. Both real-time PCR and immunohistochemistry revealed that FFA1 and FFA4 were expressed in the porcine LES. Real-time PCR analysis showed that the FFA4 expression was much lower than FFA1. Taken together, long-chain fatty acid receptor agonists elicit relaxation of the porcine LES. FFA1 might influence LES motility.

Su1314 – Interstitial Cells of Cajal Play a Crucial Role in the Neurotransmission of Nicotinic Acetylcholine Receptor in Porcine Lower Esophageal Sphincter

Su1314 – Interstitial Cells of Cajal Play a Crucial Role in the Neurotransmission of Nicotinic Acetylcholine Receptor in Porcine Lower Esophageal Sphincter

Estradiol mediates relaxation of porcine lower esophageal sphincter

Most pregnant women have symptoms of gastroesophageal reflux disease (GERD) during pregnancy. Postmenopausal hormone replacement therapy is associated with GERD. The effects of estradiol on lower esophageal sphincter (LES) motility and GERD are not clearly known. The purpose of this study is to investigate the effects of estradiol on the motility of the porcine LES. Relaxations of clasp and sling strips of porcine LES caused by estradiol were measured using isometric transducers. We investigated the mechanism of estradiol-induced relaxation of the porcine LES using tetraethylammonium, apamine, iberiotoxin, glibenclamide, KT5720, KT5823, NG-nitro-l-arginine, tetrodotoxin, and ω-conotoxin GVIA. Reverse transcription polymerase chain reaction (PCR) analysis and immunohistochemistry (IHC) were performed to determine the existence of the G protein-coupled estrogen receptor (GPER) in the porcine LES. In endothelin-1-precontracted porcine LES strips, estradiol caused marked relaxations in a concentration-dependent manner. The mechanism of estradiol-induced relaxation on the porcine LES was associated with the potassium channel. Reverse transcription PCR analysis and IHC revealed that GPER was expressed in the sling and clasp fibers of the porcine LES. This finding suggests that GPER mediates the relaxation of the porcine LES. Estradiol may play a role in LES motility.

Tu1601 - Nicotine Caused the Dual Regulation of Tone in the Porcine Lower Esophageal Sphincter Through Excitatory and Inhibitory Enteric Innervation

Tu1601 - Nicotine Caused the Dual Regulation of Tone in the Porcine Lower Esophageal Sphincter Through Excitatory and Inhibitory Enteric Innervation

Sa1997 - Myoarchitecture of the Human and Porcine Lower Esophageal Sphincter Studied with Micro CT Imaging Reveals Circular Smooth Muscle Fibers Cross Midline at the Angle of His and Continue as the Sling Fibers of the Stomach

Sa1997 - Myoarchitecture of the Human and Porcine Lower Esophageal Sphincter Studied with Micro CT Imaging Reveals Circular Smooth Muscle Fibers Cross Midline at the Angle of His and Continue as the Sling Fibers of the Stomach

Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na+/Ca2+ Exchanger.

Background and AimsHydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question.MethodsIsometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function.ResultsPorcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-β-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp–induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice.ConclusionsEndogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.

Mo1484 - Rho Kinase-Associated Myogenic Contraction Compensates for Reduced Force Induced by Release After Stretching in the Porcine Lower Esophageal Sphincter

Mo1484 - Rho Kinase-Associated Myogenic Contraction Compensates for Reduced Force Induced by Release After Stretching in the Porcine Lower Esophageal Sphincter

Mechanism of bombesin-induced tonic contraction of the porcine lower esophageal sphincter.

Gastroesophageal reflux disease (GERD) is a disorder that is related to an incompetent lower esophageal sphincter (LES). Previous studies showed that bombesin could increase LES pressure in humans and opossums. The aim of the present study was to characterize the effects of bombesin on porcine LES contraction. We used the selective agonists, neuromedin B (NMB), gastrin-releasing peptide (GRP), and [D-Tyr6,Apa-4Cl11,Phe13,Nle14]bombesin-(6-14) (DTACPN-BN), as well as receptor antagonists of bombesin receptor subtype 2 (BB2), and 3 (BB3) for ex vivo contraction studies. Atropine, nifedipine, tetrodotoxin, and ω-conotoxin GVIA were used to explore the agonist-induced LES contraction mechanism. Reverse transcription polymerase chain reaction and immunohistochemistry were applied to detect bombesin receptor expression. Our results indicate that GRP and DTACPN-BN, but not NMB, induced tonic contractions of the porcine LES in a dose-dependent manner, and the contractions were inhibited with selective BB2 and BB3 antagonists. The GRP-induced contraction is mainly caused by L-type Ca2+ channel-mediated Ca2+ influx. However, DTACPN-BN-induced contractions are associated with neuronal conduction. RT-PCR and immunohistochemistry revealed that BB2 and BB3 were expressed in the porcine LES. Bombesin-induced tonic contraction of the LES is mediated through BB2 and BB3. Bombesin, BB2, and BB3 agonists might have the potential to treat GERD.

Trypsin-induced biphasic regulation of tone in the porcine lower esophageal sphincter

The lower esophageal sphincter (LES) plays an important role in coordinated esophageal motility. The present study aimed to elucidate how trypsin affects LES contractility. Porcine LES circular smooth muscle strips were prepared. Contractile responses to trypsin were assessed. Trypsin (300nM) induced a transient contraction. At concentrations of 1μM or higher, trypsin induced biphasic responses, consisting of a transient contraction followed by a transient relaxation. Pretreatment with either 1μM tetrodotoxin or carbenoxolone had no effect on these responses. In contrast, trypsin-induced responses were completely blocked by pretreatment with the serine protease inhibitor. Pretreatment with 10μM FSLLRY-NH2, a PAR2 antagonist, significantly inhibited trypsin-induced biphasic responses. Trypsin (1μM)-induced contractions were partially inhibited by pretreatment with 10μM Y-27632. In addition, trypsin (10μM)-induced relaxation was partially inhibited by pretreatment with 10μM Y-27632, 10μM PD98059 or 10μM SB203580. Trypsin-induced relaxation was abolished by increasing the extracellular K+ concentration to 40mM, but not by pretreatment with l-arginine methyl ester. Furthermore, trypsin-induced relaxation was partially inhibited by pretreatment with 10μM glibenclamide or 1μM 4-aminopyridine. Trypsin causes biphasic regulation of LES tone by directly acting on smooth muscle. Rho-associated protein kinase (ROK) is involved in trypsin-induced contraction, whereas ROK, ERK1/2, p38MAPK, and membrane hyperpolarization are involved in relaxation. The regulation of LES tone by trypsin may play a role in esophageal motility.

Tu1829 Trypsin Induced BiPhasic Contraction and Relaxation in the Porcine Lower Esophageal Sphincter

Tu1829 Trypsin Induced BiPhasic Contraction and Relaxation in the Porcine Lower Esophageal Sphincter

Selective stimulation of excitatory and inhibitory motor neurons in porcine lower esophageal sphincter

Lecea, B.1 3; Farré, R.3; Martínez, E.2 3; Aulí, M.1 3; Suñol, X.1; Clavé, P.1 3 Author Information

Heat shock proteins and lower esophageal sphincter tone

BACKGROUND: Abnormal lower esophageal sphincter (LES) tone results in various clinical disorders, such as achalasia and gastroesophageal reflux. Investigation into the cellular mechanisms regulating smooth muscle contraction have revealed that increased phosphorylation of the heat shock-related protein 20 (HSP20) results in relaxation. HSP27 has also been implicated in the contraction of smooth muscle. We hypothesized that these proteins would mediate LES tone. METHODS: Strips of porcine LES were suspended in a muscle bath.

Pharmacologic Characterization of Intrinsic Mechanisms Controlling Tone and Relaxation of Porcine Lower Esophageal Sphincter

The neurotransmitters mediating relaxation of lower esophageal sphincter (LES) were studied using circular LES strips from adult pigs in organ baths. LES relaxation by sodium nitroprusside (1 nM-3 microM), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP; 1 nM-1 microM), ATP (10 microM-30 mM), and tricarbonyldichlororuthenum dimer (1 microM-1 mM) was unaffected by tetrodotoxin (1 microM) or l-N(G)-nitroarginine methyl ester (l-NAME; 100 microM). Calcitonin gene-related peptide (CGRP; 1 nM-1 microM) did not affect LES tone. ATP relaxation was blocked by 1 microM apamin and the P2Y(1) antagonist MRS 2179 (N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate; 10 microM). Apamin inhibited PACAP relaxation. VIP and PACAP relaxation was blocked by 10 U/ml alpha-chymotrypsin. L-NAME (-62.52 +/- 13.13%) and 1H-[1,2,4]oxadiazole-[4,3-alpha]quinoxalin-1-one (ODQ; 10 microM, -67.67 +/- 6.80%) similarly inhibited electrical LES relaxation, and apamin blocked non-nitrergic relaxation. Nicotine relaxation (100 microM) was inhibited by L-NAME (-60.37 +/- 10.8%) and ODQ (-41.90 +/- 7.89%), and apamin also blocked non-nitrergic relaxation. Non-nitrergic and apamin-sensitive LES relaxation by electrical stimulation or nicotine was strongly inhibited by MRS 2179, slightly inhibited by alpha-chymotrypsin and the P2X(1,2,3) receptor antagonist NF 279 (8,8 cent-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt; 10 microM), and unaffected by tin protoporphyrin IX (100 microM). Porcine LES relaxation after stimulation of intrinsic inhibitory motor neurons is mediated by two main neuromuscular pathways: nitric oxide through guanylate cyclase signaling and apamin-insensitive mechanisms and by non-nitrergic apamin-sensitive neurotransmission mainly mediated by ATP, ADP, or a related purine acting on P2Y1 receptors and a minor contribution of purinergic P2X1,2,3 receptors and PACAP. Nitrergic and purinergic co-transmitters show parallel effects of similar magnitude without major interplay. Our study shows no role for CGRP and only a minor one for VIP and carbon monoxide in porcine LES relaxation.

A Porcine Model of Gastroesophageal Reflux

The exact mechanism by which the lower esophageal sphincter (LES) prevents gastroesophageal reflux disease (GERD) remains controversial. The two main reasons for this controversy are the lack of a good animal model and the only recent technologic advances in this field. To date, no animal models exist that use 24-hour pH studies, manometry, and endoscopy in evaluating GERD. The purpose of this study is to create a good model of GERD that uses this technology and to delineate the true nature of the LES more clearly. Recently, three-dimensional vector volume studies were performed and through detailed, cadaveric, anatomic studies, the vector volume profiles were correlated to distinct groups of muscle fibers at the gastroesophageal junction in humans. In this study, an anatomic analysis of the porcine gastroesophageal junction revealed an arrangement of muscle fibers similar to that in humans. A myectomy of the porcine oblique fibers was performed in nine piglets weighing 10 to 12 kg. The authors were able to demonstrate manometric attenuation of the porcine LES, gastroesophageal reflux by ambulatory 24-hour pH monitoring (P< 0.0025), and the creation of GERD by documenting esophagitis grossly and histologically. In conclusion, this model of GERD works and lends further support to the notion that the LES may be a discrete anatomic entity.

Occurrence, Distribution and Motor Effects of Galanin in the Porcine Lower Esophageal Sphincter

Specimens from the lower esophageal sphincter (LES) region of the pig were analyzed for galanin-like immunoreactivity (GAL-LI) using radioimmunoassay and immunohistochemistry. The mean concentration of GAL-LI was 3.4 pmol/g tissue. GAL-LI nerve fibers were observed surrounding muscle bundles in the smooth circular muscle layer, and in ganglion cells of the myenteric plexus. Bolus injection of 40-200 pmol/kg of galanin directly into the arterial supply of the resting LES increased the LES pressure (LESP) dose-dependently, whereas slow infusion of 2-100 pmol galanin/kg/min had no effect on resting LESP. The vagally increased LESP was not modulated by simultaneous galanin infusion, and the concentration of galanin in the venous effluent of the LES declined insignificantly during vagal stimulation. It is concluded that galanin may be an important neuropeptide for the modulation of resting LES tone.