Human Lower Esophageal Sphincter Research Articles

Expression and function of vasoactive intestinal peptide receptors in human lower esophageal sphincter.

Vasoactive intestinal peptide (VIP) is an important neurotransmitter involved in the modulation of gastrointestinal function through the stimulation of VIP receptors. However, the expression of VPAC1R, VPAC2R and PAC1R in the human Lower esophageal sphincter (LES) has not been fully clarified. Therefore, the purpose of this study is to explore the expression of these receptors in the human Lower esophageal sphincter, the responses of the Lower esophageal sphincter to Vasoactive intestinal peptide, and the role of Vasoactive intestinal peptide receptors in the responses. Sling and clasp fiber samples of LES were acquired from patients undergoing subtotal esophagectomy, while circular muscle bundles from the esophagus and gastric fundus were used as control groups. Western blotting and RT-PCR technology were performed to determine the expression of the three VIP receptor subtypes. The isometric tension responses of the muscle sample strips to Ro25-1553 and PG99-465, and the effect of electrical field stimulation (EFS) on the sling and clasp fibers were studied. We found that VPAC2R messenger RNA (mRNA) and protein were expressed in the sling and clasp fibers of human LES. However, no VPAC1R or PAC1R mRNA and protein expressions were found in the LES samples. The sling and clasp fibers of the LES produced significant concentration-dependent relaxation following exposure to Ro25-1553 and EFS could induce them to produce frequency-dependent relaxation. Furthermore, the relaxation responses of the LES were inhibited by PG99-465 and induced by EFS and Ro25-1553. VPAC2R, but not VPAC1R or PAC1R, is expressed by the human LES. The relaxation responses of the LES generated by the VIP receptor agonist Ro25-1553 and EFS could be inhibited by the selective VPAC2 receptor antagonist PG99-465. VPAC2R may be important for the generation of relaxation and functional regulation of the LES.

Axial Movements and Length Changes of the Human Lower Esophageal Sphincter During Respiration and Distension-induced Secondary Peristalsis Using Functional Luminal Imaging Probe.

Background/AimsEfficient transport through the esophago-gastric junction (EGJ) requires synchronized circular and longitudinal muscle contraction of the esophagus including relaxation of the lower esophageal sphincter (LES). However, there is a scarcity of technology for measuring esophagus movements in the longitudinal (axial) direction. The aim of this study is to develop new analytical tools for dynamic evaluation of the length change and axial movement of the human LES based on the functional luminal imaging probe (FLIP) technology and to present normal signatures for the selected parameters.MethodsSix healthy volunteers without hiatal hernia were included. Data were analyzed from stepwise LES distensions at 20, 30, and 40 mL bag volumes. The bag pressure and the diameter change were used for motion analysis in the LES. The cyclic bag pressure frequency was used to distinguish dynamic changes of the LES induced by respiration and secondary peristalsis.ResultsCyclic fluctuations of the LES were evoked by respiration and isovolumetric distension, with phasic changes of bag pressure, diameter, length, and axial movement of the LES narrow zone. Compared to the respiration-induced LES fluctuations, peristaltic contractions increased the contraction pressure amplitude (P < 0.001), shortening (P < 0.001), axial movement (P < 0.001), and diameter change (P < 0.01) of the narrow zone. The length of the narrow zone shortened as function of the pressure increase.ConclusionsFLIP can be used for evaluation of dynamic length changes and axial movement of the human LES. The method may shed light on abnormal longitudinal muscle activity in esophageal disorders.

Three-Dimensional Myoarchitecture of the Lower Esophageal Sphincter and Esophageal Hiatus Using Optical Sectioning Microscopy

Studies to date have failed to reveal the anatomical counterpart of the lower esophageal sphincter (LES). We assessed the LES and esophageal hiatus morphology using a block containing the human LES and crural diaphragm, serially sectioned at 50 μm intervals and imaged at 8.2 μm/pixel resolution. A 3D reconstruction of the tissue block was reconstructed in which each of the 652 cross sectional images were also segmented to identify the boundaries of longitudinal (LM) and circular muscle (CM) layers. The CM fascicles on the ventral surface of LES are arranged in a helical/spiral fashion. On the other hand, the CM fascicles from the two sides cross midline on dorsal surface and continue as sling/oblique muscle on the stomach. Some of the LM fascicles of the esophagus leave the esophagus to enter into the crural diaphragm and the remainder terminate into the sling fibers of the stomach. The muscle fascicles of the right crus of diaphragm which form the esophageal hiatus are arranged like a “noose” around the esophagus. We propose that circumferential squeeze of the LES and crural diaphragm is generated by a unique myo-architectural design, each of which forms a “noose” around the esophagus.

Expression of serotonin receptors in human lower esophageal sphincter.

Serotonin (5-HT) is a neurotransmitter and vasoactive amine that is involved in the regulation of a large number of physiological functions. The wide variety of 5-HT-mediated functions is due to the existence of different classes of serotonergic receptors in the mammalian gastrointestinal tract and nervous system. The aim of this study was to explore the expression of multiple types of 5-HT receptor (5-HT1AR, 5-HT2AR, 5-HT3AR, 5-HT4R, 5-HT5AR, 5-HT6R and 5-HT7R) in sling and clasp fibers from the human lower esophageal sphincter (LES). Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing esophagogastrectomy, and circular muscle strips from the esophagus and stomach were used as controls. Reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR and western blotting were used to investigate the expression of the various 5-HT receptor types. Messenger RNA for all seven 5-HT receptor types was identified in the sling and clasp fibers of the LES. At the mRNA level, the expression levels were highest for 5-HT3AR and 5-HT4R, and lowest for 5-HT5AR, 5-HT6R and 5-HT7R. At the protein level, the expression levels were highest for 5-HT3AR and 5-HT4R, followed by 5-HT1AR and 5-HT2AR; 5-HT7R was also detected at a low level. The expression of 5-HT5AR and 5-HT6R proteins was not confirmed. The results indicate that a variety of 5-HT receptor types can be detected in the human LES and probably contribute to LES function.

Expression of lysophosphatidic acid receptors in the human lower esophageal sphincter

Lysophosphatidic acid (LPA) is a bioactive lipid that is involved in a variety of physiological and pathological processes occurring in the gastrointestinal tract. It acts via six distinct types of receptors, LPA1, LPA2, LPA3, LPA4, LPA5 and LPA6, which belong to the family of G protein-coupled receptors. The aim of the present study was to detect the expression of the LPA receptors in the human lower esophageal sphincter (LES). Quantitative polymerase chain reaction and western blotting were used to analyze the expression of LPA1-6 receptors in sling and clasp fibers from the human LES. The results showed that the protein and mRNA expression levels of various LPA receptors were significantly different. Specifically, the mRNA and protein expression levels of the LPA1 receptor were higher compared with those of the other receptors. The prevalence of the LPA1 receptor mRNA and protein indicates that the LPA1 receptor is likely to be involved in the regulation of human LES functions.

Expression of dopamine receptors in human lower esophageal sphincter

Dopamine (DA) is considered to be an important modulator of enteric function. Recent experiments have suggested that DA receptors are widely expressed in animal gastrointestinal tract. The aim of this study was to explore the expression of DA receptors (D(1) R, D(2) R, D(3) R, D(4) R, D(5) R) in sling fibers and clasp fibers from the human lower esophageal sphincter (LES). Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing esophago-gastrectomy, and circular muscle strips from the esophagus and stomach were used as controls. Reverse transcription-polymerase chain reaction and western blotting were used to determine the expression of the five subtypes of DA receptors. Messenger RNA and protein for three of five DA receptors were identified in the sling and clasp fibers of the LES. Expression was highest for D(1) R, then D(5) R and D(2) R in decreasing levels. D(3) R and D(4) R mRNA and protein were not identified in the muscle strips. D(1) R, D(2) R, D(5) R can be detected in the human LES, and probably contribute to LES function. D(3) R and D(4) R are not expressed, and probably do not contribute to LES function in humans.

Effects of acetylcholine on sling and clasp fibers of the human lower esophageal sphincter

Manometric studies on the human lower esophageal sphincter (LES) have shown radial asymmetry of the high-pressure zone (HPZ). The aim of this study was to compare the functional properties of human LES clasp and sling muscles, and to look at their relationship with the expression of muscarinic receptors and intracellular Ca(2+) concentration. Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing subtotal esophagectomy. Isometric tension responses of the strips to acetylcholine were studied. Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression of five subtypes of muscarinic receptors. Intracellular Ca(2+) ([Ca(2+) ]i) was measured using laser scanning confocal microscopy. Acetylcholine caused a concentration-dependent increase in the tension of sling and clasp strips, the sling strip being stronger than clasp (P=0.00). Messenger RNA and protein for the five muscarinic acetylcholine receptor (mAChRs) expressed in the sling and clasp muscles were highest for M2, and then in decreasing levels: M(3)>M(1)>M(4)>M(5) . Acetylcholine caused significant elevation of [Ca(2+) ]i in sling and clasp muscle cells in the presence of extracellular Ca2+ (1.5mmol/L), and Ach-induced [Ca(2+) ]i elevation was 1.6 times greater in sling cells than in clasp cells. Variation of intracellular concentrations of Ca(2+) may be the reason for differential responses to acetylcholine for sling versus clasp fibers. However, these differences are not associated with the distribution and the level of expression of the five mAChRs between the two muscle types. Further study should focus on the ligand affinity and signal transduction pathway.

The expression of motilin receptor in lower esophageal sphincter and its contraction responses to motilin

Objective The aim of the study was to investigate the expression of motilin receptor in sling and clasp fibers of the human lower esophageal sphincter and different contraction. responses of the two muscles to motilin. Methods Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect MTLR in the human sling fibers and clasp fibers. Inverted microscope was used to measure length changes of the sling and clasp fibers which were induced by motilin. Results MTLR were expressed both in the sling fibers and clasp fibers. Expression of MTLR in sling fibers was higher than in clasp fibers. The contraction in the sling fibers was stronger than in clasp fibers. Conclusion The contraction of the sling and clasp fibers in human esophageal sphincter is induced by motilin through MTLR. The contraction in the sling fibers is stronger than in clasp fibers. Key words: MTLR; Lower esophageal sphincter; Contraction; RT-PCR; Western blot

Presence of GABAB Receptors Forming Heterodimers With GABAB1 and GABAB2 Subunits in Human Lower Esophageal Sphincter

Baclofen, a GABAB-receptor (GABABR) agonist has been proposed to be useful as therapeutic agent for the management of gastro-esophageal reflux disease, but whether the compound acts directly at the lower esophageal sphincter (LES) remains to be elucidated. We performed the present study to assess the presence of GABABR in human LES. Western blot analysis showed that both proteins of GABAB1(a)/GABAB1(b) and GABAB2 subunits were present in the muscle layer of LES. Immunohistochemical findings showed that both GABAB1- and GABAB2-subunit proteins were located on the neurons within the myenteric plexus, and furthermore, both proteins were observed in the same neurons. Reverse transcriptase-polymerase chain reaction analysis also revealed the presence of mRNAs for both subunits of GABABR and also mRNAs for 6 isoforms of GABAB1 subunits, from GABAB1(a) to GABAB1(g), except GABAB1(d), in human LES. Thus, the functional GABABR-forming heterodimers with subunits of GABAB1 and GABAB2 are located on the myenteric neurons in human LES, suggesting that GABABR agonists and antagonists act at least, at the level of the peripheral nervous system.

Calcium Sensitization in Human Esophageal Muscle: Role for RhoA Kinase in Maintenance of Lower Esophageal Sphincter Tone

A rise in intracellular-free calcium ([Ca(2+)](i)) concentration is important for initiating contraction of smooth muscles, and Ca(2+) sensitization involving RhoA kinase can sustain tension. We previously found that [Ca(2+)](i) was comparable in cells from the esophageal body (EB) and lower esophageal sphincter (LES) muscles, despite the fact that the LES maintains resting tone. We hypothesized that Ca(2+) sensitization contributes to contraction in human esophageal muscle. Tension and [Ca(2+)](i) were measured simultaneously in intact human EB and LES muscles using the ratiometric Ca(2+)-sensitive dye fura-2. Spontaneous oscillations in EB muscle tension were associated with transient elevations of [Ca(2+)](i). Carbachol caused a large increase in tension, compared with spontaneous oscillations, although the rise of [Ca(2+)](i) was similar, suggesting Ca(2+) sensitization. The RhoA-kinase blockers (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and 1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride (HA-1077) reduced carbachol- and nerve-evoked contraction of the EB, accompanied by smaller reduction in the rise of [Ca(2+)](i). Protein kinase C inhibitors reduced force to a lesser extent. RhoA-kinase blockers caused concentration-dependent reduction of tension in spontaneously contracted LES muscles. Moreover, RhoA-kinase blockers reduced intrinsic nerve-evoked and carbachol-evoked contraction. However, there was no effect on nerve- or nitric oxide-mediated relaxation of LES. Ca(2+) sensitization mediated by the RhoA-kinase pathway has an important role in contraction of human EB muscles and LES tonic contraction, a feature not previously recognized.

Inhibitory purinergic neurotransmission in human lower esophageal sphincter

Inhibitory purinergic neurotransmission in human lower esophageal sphincter

Hydrogen Peroxide Reduces Lower Esophageal Sphincter Tone in Human Esophagitis

We have previously used the normal lower esophageal sphincter (N-LES) of human organ donors to examine the physiologic signal transduction of lower esophageal sphincter (LES) circular muscle. Now, for the first time, we have obtained a human LES specimen with esophagitis (E-LES) and characterized its pathophysiologic mechanical and inflammatory profiles. E-LES was examined histologically, and its in vitro circular muscle contraction and production of inflammatory mediators were compared with those of N-LES. E-LES exhibited scattered erosions and displayed inflammatory cells in the epithelial layer, basal zone hyperplasia, and elongation of lamina propria papillae, characteristic of chronic reflux esophagitis. E-LES muscle strips developed lower in vitro tone (0.78 g) than N-LES (3.3 +/- 0.2 g). E-LES tone was essentially restored to normal by the H2O2 scavenger catalase, suggesting that H2O2 was responsible for reduction of tone. NOX5 cDNA was higher and H2O2 levels were 4 times higher in E-LES circular muscle (0.85 nmol/mg protein) than in N-LES (0.19 +/- 0.05 nmol/mg protein). When N-LES smooth muscle was incubated in H2O2 (70 micromol/L, 2 hours), platelet activating factor (PAF), prostaglandin E2 (PGE2), and F2-isoprostane increased 2.5, 5.2, and 36 times, respectively. In E-LES, levels of PAF, PGE2, and F2-isoprostane were 4, 6, and 40 times, respectively, higher than in N-LES. PAF, PGE2, and F2 isoprostane produced dose-dependent reductions in tone of N-LES muscle strips. We conclude that an excessive production of H2O2 triggers an increased production of PAF, PGE2, and F2-isoprostane, which are responsible for reducing LES tone in human esophagitis.

AGA technical review on the clinical use of esophageal manometry

AGA technical review on the clinical use of esophageal manometry

Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle.

Transient receptor potential channel (TRPC) genes encode Ca(2+)-permeable channels mediating capacitative Ca(2+) entry (CCE), which maintains intracellular Ca(2+) stores. We compared TRPC gene expression and CCE in human esophageal body (EB) and lower esophageal sphincter (LES), because these smooth muscles have distinct contractile functions that are likely associated with different Ca(2+) regulatory mechanisms. Circular layer smooth muscle cells were grown in primary culture. Transcriptional expression of TRPC genes was compared by semiquantitative RT-PCR. CCE was measured by fura 2 Ca(2+) fluorescence after blockade of sarcoplasmic reticulum Ca(2+)-ATPase with thapsigargin. mRNA for TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 was identified in EB and LES. TRPC3 and TRPC4 were more abundant in LES than EB. Basal concentration of free intracellular Ca(2+) ([Ca(2+)](i)) was similar in cells from LES (138 +/- 8 nmol/l) and EB (110 +/- 6 nmol/l) and increased with ACh (10 micromol/l; 650 +/- 28 and 590 +/- 21 nmol/l, respectively). With zero Ca(2+) in bath, thapsigargin (2 micromol/l) increased [Ca(2+)](i) more in LES (550 +/- 22 nmol/l) than EB (250 +/- 15 nmol/l, P < 0.001). Subsequent external application of 1 mmol/l Ca(2+) increased [Ca(2+)](i) more in LES (585 +/- 35 nmol/l) than EB (295 +/- 21 nmol/l, P < 0.001), indicating enhanced CCE in LES. This demonstrates CCE and TRPC transcriptional expression in human esophageal smooth muscle. In LES cells, enhanced CCE and expression of TRPC3 and TRPC4 may contribute to the physiological characteristics that distinguish LES from EB.

Pharmacological and molecular characterization of muscular cholecystokinin receptors in the human lower oesophageal sphincter.

In vitro cholecystokinin (CCK) contracts the human lower oesophageal sphincter by stimulating muscular receptors. The aim of this study was to characterize the muscular CCK receptor subtypes in the human lower oesophageal sphincter. Twenty-five circular strips from six patients were studied. RNA was extracted, reverse transcribed, and cDNAs were amplified with primers for human CCK-A and B receptors. The potency of the contraction induced by CCK-8, desulphated CCK-8, and gastrin-I, and the effect of the CCK-A (loxiglumide and SR 27897) and the CCK-B (YM022 and L-365 260) specific receptor antagonists were compared. Both CCK-A and CCK-B receptor mRNAs were found in functional lower oesophageal sphincter strips. The potency of the CCK-8 concentration-dependent contraction was two and three orders of magnitude higher than that of desulphated CCK-8 and gastrin-I, respectively. The CCK-8-induced contraction was blocked by the CCK-A receptor antagonists loxiglumide (IC50 11 micromol L-1) and SR 27897 (IC50 74 nmol L-1) but not by CCK-B receptor antagonists (1 micromol L-1). Our data suggest that, although the human lower oesophageal sphincter expresses both CCK-A and CCK-B receptors, the contractile effect of CCK-8 on the circular muscle is mainly due to the activation of CCK-A receptors.

Group I secreted PLA2 in the maintenance of human lower esophageal sphincter tone

In cat spontaneous lower esophageal sphincter (LES), tone is maintained by the activity of group I secreted phospholipase A2 (sPLA2-I) that produces arachidonic acid. Arachidonic acid metabolites activate G proteins linked to phospholipases, producing second messengers and activation of a protein kinase C-dependent pathway to maintain tone. We examined the role of sPLA2-I in the maintenance of tone in human LES samples obtained from organ donors. In vitro LES tone and sPLA2-I-induced contraction of enzymatically isolated LES smooth muscle cells were measured in the absence or presence of inhibitors. Cell permeabilization by saponin allowed use of G-protein antibodies. In vitro LES tone was reduced by inhibitors of sPLA2-I, by indomethacin, by the phosphatidylcholine-specific phospholipase C inhibitor D609, and by the protein kinase C inhibitor chelerythrine. sPLA2-I-induced contraction of isolated LES smooth muscle cells was reduced by indomethacin, pertussis toxin, Gi3 antibodies, D609, and by chelerythrine. Human LES tone is maintained by the activity of sPLA2-I that produces arachidonic acid and metabolites and activation of Gi3-linked receptors and of phosphatidylcholine-specific phospholipase C, resulting in production of diacylglycerol, activation of PKC, and maintenance of tone through a protein kinase C-dependent contractile pathway.

Anatomic dilatation of the cardia and competence of the lower esophageal sphincter: a clinical and experimental study

Anatomic and clinical data suggest that the gastroesophageal junction on cardia in patients with gastroesophageal reflux disease (GERD) may be dilated. We hypothesized that anatomic dilatation of the cardia induces a lower esophageal sphincter dysfunction that may be corrected by narrowing the gastroesophageal junction (i.e., calibration of the cardia). We measured the perimeter of the cardia during surgery in control subjects and patients with GERD and Barrett's esophagus. We then tested our hypothesis in a mechanical model. The model was based on a pig gastroesophageal specimen with perpendicularly placed elastic bands around the cardia simulating the action of the “sling” and “clasp” fibers. “Dilatation” of the cardia was induced by displacing the sling band laterally and decreasing its tension. “Calibration” of the cardia was performed by reapproximation of the sling band toward the esophagus but maintaining the same tension as the dilated model. In the “basal,” “dilated,” and “calibrated” states, the perimeter of the cardia was noted and rapid mechanized pullback manometry with a water-perfused catheter was performed. The opening pressure was determined, and three-dimensional sphincter pressure images were analyzed. The average cardia perimeter was 6.3 cm in control subjects, 8.9 cm in GERD patients, and 13.8 cm in patients with Barrett's esophagus. The arrangement of the bands in the experimental model generated a manometric high-pressure zone similar to that in the human lower esophageal sphincter. Dilatation of the cardia resulted in a decrease in the resting pressure, length, and vector volume of the high-pressure zone, and reduced the opening pressure. Calibration restored the resting and opening pressure, and normalized the three-dimensional pressure image. In patients with GERD and Barrett's esophagus, the cardia is dilated. Our model supports the hypothesis that lower esophageal sphincter function is compromised by anatomic dilatation of the cardia and can be restored by approximation of the “sling” fibers toward the lesser curvature (“clasp” fibers). This provides evidence for a correlation between gastroesophageal sphincter dysfunction in reflux disease and its correction by antireflux surgery.

Calcium regulation in the human lower esophageal sphincter

Calcium regulation in the human lower esophageal sphincter

Effects of acid perfusion on the human lower esophageal sphincter (LES) and esophageal contractions

Effects of acid perfusion on the human lower esophageal sphincter (LES) and esophageal contractions

The role of motilin and cisapride in the enteric nervous system of the lower esophageal sphincter in humans.

To assess the pharmacophysiological significance of the enteric nervous system and the responses of the human lower esophageal sphincter (LES) to motilin and cisapride, the mechanical responses of esophageal tissues from six patients with esophageal cancer and seven patients with gastric cancer were investigated. Circular muscle reactions were recorded to evaluate the in vitro esophageal responses to electrical field stimulation (EFS), motilin, and cisapride, evoking the adrenergic and cholinergic nerves before and after treatment with various autonomic nerve blockers. The findings of this study revealed that: cholinergic nerves are mainly involved in the regulation of enteric nerves in the steady state, while non-adrenergic non-cholinergic (NANC) inhibitory nerves also exist; motilin may act both via nerves and also directly on the LES smooth muscle; and cisapride releases acetylcholine from the end of the postganglionic fiber of the cholinergic nerve in human LES thereby inducing contraction of the LES. These results suggest that cholinergic and NANC inhibitory nerves play an important role in human LES, and that motilin and cisapride is clinically useful for improving the impaired LES of patients with gastroesophageal reflux.