Lower Esophageal Sphincter Circular Muscle Research Articles

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.

Pressure morphology of the relaxed lower esophageal sphincter: the formation and collapse of the phrenic ampulla

This study aimed to apply novel high-resolution manometry with eight-sector radial pressure resolution (3D-HRM technology) to resolve the deglutitive pressure morphology at the esophagogastric junction (EGJ) before, during, and after bolus transit. A hybrid HRM assembly, including a 9-cm-long 3D-HRM array, was used to record EGJ pressure morphology in 15 normal subjects. Concurrent videofluoroscopy was used to relate bolus movement to pressure morphology and EGJ anatomy, aided by an endoclip marking the squamocolumnar junction (SCJ). The contractile deceleration point (CDP) marked the time at which luminal clearance slowed to 1.1 cm/s and the location (4 cm proximal to the elevated SCJ) at which peristalsis terminated. The phrenic ampulla spanned from the CDP to the SCJ. The subsequent radial and axial collapse of the ampulla coincided with the reconstitution of the effaced and elongated lower esophageal sphincter (LES). Following ampullary emptying, the stretched LES (maximum length 4.0 cm) progressively collapsed to its baseline length of 1.9 cm (P < 0.001). The phrenic ampulla is a transient structure comprised of the stretched, effaced, and axially displaced LES that serves as a "yield zone" to facilitate bolus transfer to the stomach. During ampullary emptying, the LES circular muscle contracts, and longitudinal muscle shortens while that of the adjacent esophagus reelongates. The likely LES elongation with the formation of the ampulla and shortening to its native length after ampullary emptying suggest that reduction in the resting tone of the longitudinal muscle within the LES segment is a previously unrecognized component of LES relaxation.

Viewpoints on Acid-Induced Inflammatory Mediators in Esophageal Mucosa

We have focused on understanding the onset of gastroesophageal reflux disease by examining the mucosal response to the presence of acid in the esophageal lumen. Upon exposure to HCl, inflammation of the esophagus begins with activation of the transient receptor potential channel vanilloid subfamily member-1 (TRPV1) in the mucosa, and production of IL-8, substance P (SP), calcitonin gene related peptide (CGRP) and platelet activating factor (PAF). Production of SP and CGRP, but not PAF, is abolished by the neural blocker tetrodotoxin suggesting that SP and CGRP are neurally released and that PAF arises from non neural pathways. Epithelial cells contain TRPV1 receptor mRNA and protein and respond to HCl and to the TRPV1 agonist capsaicin with production of PAF. PAF, SP and IL-8 act as chemokines, inducing migration of peripheral blood leukocytes. PAF and SP activate peripheral blood leukocytes inducing the production of H2O2. In circular muscle, PAF causes production of IL-6, and IL-6 causes production of additional H2O2, through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Among these, NADPH oxidase 5 cDNA is significantly up-regulated by exposure to PAF; H2O2 content of esophageal and lower esophageal sphincter circular muscle is elevated in human esophagitis, causing dysfunction of esophageal circular muscle contraction and reduction in esophageal sphincter tone. Thus esophageal keratinocytes, that constitute the first barrier to the refluxate, may also serve as the initiating cell type in esophageal inflammation, secreting inflammatory mediators and pro-inflammatory cytokines and affecting leukocyte recruitment and activity.

Neurotransmission in lower esophageal sphincter ofW/Wvmutant mice

To address the controversy surrounding the role of interstitial cells of Cajal (ICC) in nitrergic neurotransmission to gastrointestinal smooth muscle, circular smooth muscle from the lower esophageal sphincter (LES) of W/W(v) wild-type and mutant (ICC-deficient) mice were studied by using intracellular and tension recordings in vitro. Resting membrane potential was more negative, and the spontaneous unitary potentials diminished in mutant mice. In wild-type mice, nerve stimulation induced a biphasic inhibitory junction potential (IJP) consisting of a fast initial IJP followed by a long-lasting slow IJP (LSIJP). The IJP was markedly impaired in a significant proportion of mutant mice, whereas in others it was normal. Pharmacological studies in the mice with markedly impaired IJPs revealed that cholinergic and purinergic components of the nerve-mediated responses appeared intact. In wild-type mice, caffeine hyperpolarized smooth muscle cells, inhibited the initial fast IJP, and completely abolished the LSIJP. In mutant mice, caffeine depolarized smooth muscle cells and abolished the impaired LSIJP but did not affect the initial fast IJP. Immunohistochemical staining for c-Kit confirmed deficiency of ICC in mutant mice with a normal nitrergic IJP. Rings of LES circular smooth muscle from W/W(v) mutant mice generated significantly less spontaneous tone than controls. When tone was restored with carbachol, normal nitrergic LES relaxation was recorded. These data suggest that 1) there is significant variability in the generation of nitrergic neurotransmission in the LES of W/W(v) mutant mice, whereas purinergic and cholinergic neurotransmission are intact; 2) the altered nitrergic responses appear to be associated with abnormal Ca2+-dependent signaling initiated by spontaneous Ca2+ release from sarcoplasmic reticulum in smooth muscle cells; and 3) c-Kit-positive ICC are not essential for nitrergic neurotransmission in mouse LES smooth muscle.

Functional evidence for Na+-activated K+ channels in circular smooth muscle of the opossum lower esophageal sphincter

Na(+) reduction induces contraction of opossum lower esophageal sphincter (LES) circular smooth muscle strips in vitro; however, the mechanism(s) by which this occurs is unknown. The purpose of the present study was to investigate the electrophysiological effects of low Na(+) on opossum LES circular smooth muscle. In the presence of atropine, quanethidine, nifedipine, and substance P, conventional intracellular electrodes recorded a resting membrane potential (RMP) of -37.5 +/- 0.9 mV (n = 4). Decreasing [Na(+)] from 144.1 to 26.1 mM by substitution of equimolar NaCl with choline Cl depolarized the RMP by 7.1 +/- 1.1 mV. Whole cell patch-clamp recordings revealed outward K(+) currents that began to activate at -60 mV using 400-ms stepped test pulses (-120 to +100 mV) with increments of 20 mV from holding potential of -80 mV. Reduction of [Na(+)] in the bath solution inhibited K(+) currents in a concentration-dependent manner. Single channels with conductance of 49-60 pS were recorded using cell-attached patch-clamp configurations. The channel open probability was significantly decreased by substitution of bath Na(+) with equimolar choline. A 10-fold increase of [K(+)] in the pipette shifted the reversal potential of the single channels to the positive by -50 mV. These data suggest that Na(+)-activated K(+) channels exist in the circular smooth muscle of the opossum LES.

IL-1β signaling in cat lower esophageal sphincter circular muscle

In a cat model of acute experimental esophagitis, resting in vivo lower esophageal sphincter (LES) pressure and in vitro tone are lower than in normal LES, and the LES circular smooth muscle layer contains elevated levels of IL-1beta that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1beta-induced reduction in LES tone. IL-1beta significantly reduced acetylcholine-induced Ca(2+) release in Ca(2+)-free medium, and this effect was partially reversed by catalase, demonstrating a role of H(2)O(2) in these changes. IL-1beta significantly increased the production of H(2)O(2), and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA(2). IL-1beta-induced cPLA(2) phosphorylation was blocked by SB-203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating cPLA(2). The IL-1beta-induced reduction in LES tone was partially reversed by AACOCF3 and by the Ca(2+)-insensitive PLA(2) inhibitor bromoenol lactone (BEL). IL-1beta significantly increased cyclooxygenase (COX)-2 and PGE(2) levels. The increase in PGE(2) was blocked by SB-203580, AACOCF3, BEL, and the COX-2 inhibitor NS-398 but not by PD-98059 or the COX-1 inhibitor valeryl salicylate. The data suggested that IL-1beta reduces LES tone by producing H(2)O(2), which may affect Ca(2+)-release mechanisms and increase the synthesis of COX-2 and PGE(2). Both H(2)O(2) and PGE(2) production depend on sequential activation of p38 MAPK and cPLA(2). cPLA(2) activates NADPH oxidases, producing H(2)O(2), and may produce arachidonic acid, converted to PGE(2) via COX-2.

Signal-Transduction Pathways that Regulate Smooth Muscle Function I. Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles

Contraction of esophageal (Eso) and lower esophageal sphincter (LES) circular muscle depends on distinct signal-transduction pathways. ACh-induced contraction of Eso muscle is linked to phosphatidylcholine metabolism, production of diacylglycerol and arachidonic acid (AA), and activation of the Ca(2+)-insensitive PKCepsilon. Although PKCepsilon does not require Ca(2+) for activation, either influx of extracellular Ca(2+) or release of Ca(2+) from stores is needed to activate the phospholipases responsible for hydrolysis of membrane phospholipids and production of second messengers, which activate PKCepsilon. In contrast, the LES uses two distinct intracellular pathways: 1) a PKC-dependent pathway activated by low doses of agonists or during maintenance of spontaneous tone, and 2) a Ca(2+)-calmodulin-myosin light chain kinase (MLCK)-dependent pathway activated in response to maximally effective doses of agonists during the initial phase of contraction. The Ca(2+) levels, released by agonist-induced activity of phospholipase C, determine which contractile pathway is activated in the LES. The Ca(2+)-calmodulin-MLCK-dependent contractile pathway has been well characterized in a variety of smooth muscles. The steps linking activation of PKC to myosin light chain (MLC20) phosphorylation and contraction, however, have not been clearly defined for LES, Eso, or other smooth muscles. In addition, in LES circular muscle, a low-molecular weight pancreatic-like phospholipase A2 (group I PLA2) causes production of AA, which is metabolized to prostaglandins and thromboxanes. These AA metabolites act on receptors linked to heterotrimeric G proteins to induce activation of phospholipases and production of second messengers to maintain contraction of LES circular muscle. We have examined the signal-transduction pathways activated by PGF(2alpha) and by thromboxane analogs during the initial contractile phase and found that these pathways are the same as those activated by other agonists. In response to low doses of agonists or during maintenance of tone, presumably due to low levels of calcium release, a PKC-dependent pathway is activated, whereas at high doses of PGF(2alpha) and thromboxane analogs, in the initial phase of contraction, calmodulin is activated, PKC activity is reduced, and contraction is mediated, in part, through a Ca(2+)-calmodulin-MLCK-dependent pathway. The PKC-dependent signaling pathways activated by PGF(2alpha) and by thromboxanes during sustained LES contraction, however, remain to be examined, but preliminary data indicate that a distinct PKC-dependent pathway may be activated during maintenance of tonic contraction, which is different from the one activated during the initial contractile response. The initial contractile response to low levels of agonists depends on activation of G(q). Sustained contraction in response to PGF(2alpha) may involve activation of the monomeric G protein RhoA, because the contraction is inhibited by the RhoA-kinase antagonist Y27632. This shift in signal-transduction pathways between initial and sustained contraction has been recently reported in intestinal smooth muscle.

H2O2: a mediator of esophagitis-induced damage to calcium-release mechanisms in cat lower esophageal sphincter

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca(2+) stores. We therefore measured cytosolic Ca(2+) in response to a maximally effective dose of ACh in fura 2-AM-loaded lower esophageal sphincter (LES) circular muscle cells and examined the contribution of H(2)O(2) to the reduction in Ca(2+) signal. In normal cells, the ACh-induced Ca(2+) increase was the same in normal-Ca(2+) and Ca(2+)-free medium and was abolished by the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C inhibitor U-73122, confirming that the initial ACh-induced contraction depends on Ca(2+) release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced Ca(2+) increase in normal-Ca(2+) medium was significantly lower in esophagitis than in normal cells and was further reduced ( approximately 70%) when the cells were incubated in Ca(2+)-free medium. This reduction was partially reversed by the H(2)O(2) scavenger catalase. H(2)O(2) measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with H(2)O(2), the ACh-induced Ca(2+) increase was significantly reduced in normal-Ca(2+) and Ca(2+)-free medium and was similar to that observed in animals with esophagitis. The initial ACh-induced contraction was also reduced in normal cells incubated with H(2)O(2). H(2)O(2), when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca(2+) signal in normal cells. H(2)O(2)-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, H(2)O(2) reduced TG-induced contraction, suggesting that TG and H(2)O(2) may operate through similar mechanisms. Ca(2+)-ATPase activity measurement indicates that H(2)O(2) and TG reduced Ca(2+)-ATPase activity, confirming similarity of mechanism of action. We conclude that H(2)O(2) may be at least partly responsible for impairment of Ca(2+) release in acute experimental esophagitis by inhibiting Ca(2+) uptake and refilling Ca(2+) stores.

Endothelin receptors in lower esophageal sphincter circular smooth muscle

To characterize endothelin (ET) receptors in the lower esophageal sphincter, we measured contraction of transverse strips from the guinea-pig lower esophageal sphincter induced by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the lower esophageal sphincter circular muscle. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. ET-1 or ET-2 alone did not cause contraction or relaxation in resting strips. However, in carbachol precontracted lower esophageal sphincter strips, ET-1 and ET-2 caused marked, tetrodotoxin-insensitive relaxation. The ET-1-induced relaxation was abolished by BQ-123, an ET A receptor selective antagonist, but not inhibited by BQ-788, an ET B receptor selective antagonist. ET-3 and sarafotoxin S6c, a selective ET B receptor agonist, did not cause relaxation in the carbachol precontracted muscle strips. These clearly indicate that ET A receptors mediate relaxation. On the other hand, ET-3 and sarafotoxin S6c caused tetrodotoxin and atropine-insensitive contraction in the resting strips. The sarafotoxin S6c-induced contraction was inhibited by BQ-788, but not by BQ-123. Furthermore, ET-1 and ET-2 caused contraction of the resting muscle strips after pretreatment with BQ-123. This ET-1-induced contraction was also inhibited by BQ-788. Taken together, these indicate that ET B receptors mediate contraction. Autoradiography localized 125I-ET-1 binding to the lower esophageal sphincter circular muscle as well as longitudinal muscle of the esophagus. Binding of 125I-ET-1 to cell membranes prepared from the circular smooth muscle was saturable and specific. Analysis of dose–inhibition curves indicated the presence of two classes of receptors, ET A and ET B receptors. These results demonstrate that the guinea-pig lower esophageal sphincter possesses ET A receptors mediating relaxation and ET B receptors mediating contraction.

Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle

Myogenic lower esophageal sphincter (LES) tone is maintained by arachidonic acid metabolites, such as PGF(2alpha) and thromboxane A(2)/B(2). Experimental esophagitis in cat reduces LES in vivo pressure and in vitro tone. Because IL-1beta may mediate esophagitis-associated reduction in ACh release in esophagus, we examined whether IL-1beta may also play a role in esophagitis-induced reduction of LES tone. A cat model of experimental esophagitis was obtained by repeated esophageal perfusion with HCl (Biancani P, Barwick K, Selling J, and McCallum R. Gastreonterology 87: 8-16, 1984 and Sohn UD, Harnett KM, Cao W, Rich H, Kim N, Behar J, and Biancani P. J Pharmacol Exp Ther 283: 1293-1304, 1997.). LES circular muscle strips were examined in muscle chambers as previously described (Biancani P, Billett G, Hillemeier C, Nissenshon M, Rhim BY, Sweczack S, and Behar J. Gastroenterology 103: 1199-1206, 1992). Levels of inflammatory mediators were measured. IL-1beta levels were higher in esophagitis than in normal LES. IL-1beta reduced normal LES tone, and the reduction was reversed by catalase, suggesting a role of H(2)O(2). This was confirmed by IL-1beta-induced production of H(2)O(2) in normal LES and elevated H(2)O(2) levels in esophagitis. H(2)O(2) by itself is sufficient to explain the changes that occur in the muscle, reducing its ability to contract. H(2)O(2) increased PGE(2) in normal LES, and PGE(2) levels were elevated in esophagitis LES, whereas PGF(2alpha) levels were unchanged. H(2)O(2) also increased levels of 8-isoprostanes, stable prostaglandin-like compounds formed by free radical-induced peroxidation of arachidonic acid, and 8-isoprostane levels were elevated in esophagitis. The PGF(2alpha) analog 8-iso-PGF(2alpha) caused little contraction of LES strips but reduced PGF(2alpha) binding and contraction of normal LES. In esophagitis, PGF(2alpha) binding and contraction were reduced in LES, suggesting that isoprostanes may contribute to reduction in tone in esophagitis. The data suggest that, in esophagitis, IL-1beta causes production of H(2)O(2). H(2)O(2) increases PGE(2), which relaxes the LES, and 8-iso-F(2alpha), which blocks PGF(2alpha)-mediated contraction.

Regional differences in L-type Ca2+ channel expression in feline lower esophageal sphincter.

In humans and cats, muscle from the lower esophageal sphincter (LES) circular region exhibits greater spontaneous tone than LES sling muscle, whereas the sling muscle is much more responsive to cholinergic stimulation. Despite physiological and pharmacological evidence for the presence of L-type Ca2+ channel current (ICa,L) activity in LES circular muscle, the identity of this channel has not been demonstrated biochemically or electrophysiologically fingerprinted. Furthermore, there is no information on the channel's presence and role in the sling region of the LES. We hypothesized that regional differences in the expression of ICa,L between LES circular and sling muscles, if present, could contribute to the functional asymmetry observed within the LES. ICa,L expression was compared between circular and sling regions of the LES by Western blot analysis. The patch-clamp technique was used to study ICa,L. Muscle strip studies assessed ICa,L contribution to contractile activity. We found both protein expression of ICa,L and ICa,L density to be greater in LES circular muscle than sling muscle. ICa,L voltage- and time-dependent activation and inactivation curves were similar in cells from both regions. ICa,L blockade with nifedipine inhibited spontaneous tone and ACh-induced contractions only in circular muscle but was able to abolish depolarization (KCl)-induced contractions in both sling and circular muscles. In contrast, La3+ inhibited tone and ACh-induced contractions in muscles from both regions. Therefore, regional myogenic differences in ICa,L expression within the LES circular and sling muscle exist and provide one explanation for the differential contribution of sling and circular muscle to LES contractility.

Diversity of K&lt;sup&gt;+&lt;/sup&gt; channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.

Opposing roles of K(+) and Cl(-) channels in maintenance of opossum lower esophageal sphincter tone.

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 microM) reduced basal tone to 27.7 +/- 3.8% of control. The K(+) channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl(-) channel blockers DIDS (500 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 microM), as well as niflumic acid (0.1-300 microM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K(+) and Ca(2+)-activated Cl(-) channels have important opposing roles in the genesis of LES tone.

Group I secreted PLA2 and arachidonic acid metabolites in the maintenance of cat LES tone.

Spontaneous tone of in vitro lower esophageal sphincter (LES) circular muscle is associated with elevated levels of arachidonic acid (AA), PGF(2alpha), and increased [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Gq-, Gi3-, and G(i1/i2)-like G proteins. Tone and AA levels were reduced by inhibitors of a pancreatic-like (group I) secreted phospholipase A2 (sPLA2), by the cyclooxygenase inhibitor indomethacin, and by the thromboxane A2 antagonist SQ-29548. In addition, pertussis toxin (PTX) reduced LES tone, confirming a role of PTX-sensitive G proteins in maintenance of LES tone. PGF(2alpha) contracted LES smooth muscle (strips and cells) and increased [35S]GTPgammaS binding to Gq and Gi3 in solubilized LES circular muscle membranes. PGF(2alpha)-induced contraction of LES permeable muscle cells was inhibited by Gq and Gi3 but not by G(i1/i2) and Go antibodies. The thromboxane A2 analog U-46619 contracted LES smooth muscle and increased Gq binding. U-46619-induced contraction was inhibited by Gq but not by Gi3, G(i1/i2), and Go antibodies. LES tone and [(35)S]GTPgammaS binding were significantly reduced by indomethacin. We conclude that group I sPLA2 may mediate "spontaneous" LES tone by producing AA, which is metabolized to PGF(2alpha) and thromboxane A2. These AA metabolites activate receptors linked to Gi3 and Gq to maintain LES contraction.

Signal Transduction Pathways in Esophageal and Lower Esophageal Sphincter Circular Muscle

Esophageal reflux is a common condition that affects children and 1 in 10 adults, and if untreated may result in chronic esophagitis, aspiration pneumonia, esophageal strictures, and Barrett’s esophagus, a premalignant condition. Although esophagitis is a multifactorial disease that may depend on transient lower esophageal sphincter (LES) relaxation, speed of esophageal clearance, mucosal resistance, and other factors, impairment of LES pressure is a common finding in patients complaining of chronic heartburn. Our data suggest that esophageal and LES circular muscle utilize distinct Ca 2+ sources, phospholipid pools, and signal transduction pathways to contract in response to acetylcholine (ACh): (1) In esophageal muscle ACh-induced contraction requires influx of extracellular Ca 2+ and may be linked to phosphatidylcholine metabolism, production of diacylglycerol (DAG) and arachidonic acid, and activation of a protein kinase C (PKC)-dependent pathway. (2) In LES muscle ACh-induced contraction utilizes intracellular Ca 2+ release arising from metabolism of phosphatidylinositol (PI), and a calmodulin-myosin light chain kinase-dependent pathway. Resting LES tone, on the other hand, may be due to relatively low basal PI hydrolysis resulting in submaximal levels of inositol triphosphate (IP 3)-induced calcium release and interaction with DAG to activate PKC. (3) After induction of experimental esophagitis, basal levels of PI hydrolysis and intracellular calcium stores are substantially reduced, resulting in a reduction of resting tone. In addition the signal transduction pathway responsible for LES contraction in response to ACh changes from one that depends on IP 3 production, calcium release, and calmodulin activation to one that relies on influx of extracellular calcium and activation of PKC.

Asymmetry of lower esophageal sphincter pressure: is it related to the muscle thickness or its shape?

Lower esophageal sphincter (LES) pressure shows axial and circumferential asymmetry, the reasons for which are not clear. Our aim was to determine whether the muscle thickness and shape of the LES were the reasons for the axial and circumferential asymmetry in the LES pressure. High-frequency, catheter-based intraluminal ultrasonography was performed to obtain images of the human LES and esophagus. Station pull-through manometry was performed to record the axial and circumferential asymmetry of LES pressure. Circular and longitudinal muscle were thicker in the LES compared with in the esophagus. There was a close correlation between the axial asymmetry in LES pressure and circular muscle thickness. The thickness of LES muscle increased and decreased with an increase and decrease in the LES pressure, respectively. The circumferential asymmetry in resting LES pressure was related to the noncircular shape of the LES. A swallow-induced LES relaxation was followed by its contraction. During the contraction phase, the LES and esophagus showed relative symmetry in pressure and shape. We conclude that the axial asymmetry of LES pressure is explained by its muscle thickness. On the other hand, circumferential asymmetry is related to the noncircular shape of the LES.

The role of nitric oxide in ethanol-induced gastrointestinal dysfunction.

We recently showed that acute ethanol inhibits contractility of the lower esophageal sphincter (LES) and the lower esophageal body (LEB) both in vivo and in vitro. To evaluate the mechanism of this inhibitory effect of ethanol, we investigated the role of nitric oxide (NO) on contractility of isolated LES and LEB circular muscle strips using inhibitors of NO synthase (NOS), NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine. Ethanol significantly decreased LES basal tone. This effect was not mediated by NO, because inhibition was not prevented by inhibitors of NOS. Electrical field stimulation caused an On-response relaxation from LES strips, and an Off-response contraction from both LES and LEB strips. Inhibitors of NOS prevented the On-response relaxation of LES, but had no significant effect on LES Off-response contraction. Ethanol potentiated the On-response relaxation of the LES Off-response contraction. Ethanol potentiated the On-response relaxation of LES, but had no significant effect on Off-response contraction. Ethanol's potentiating effect of the On-response relaxation is NO-mediated, because it was abolished by NOS inhibitors. Ethanol also inhibited carbachol-induced LES contractility. This inhibitory affect was NO-mediated, because NOS inhibitors abolished it. Ethanol inhibited both the Off-response contraction and carbachol-induced contraction of LEB strips. These effects were not NO-mediated, because they were not affected by NOS inhibitor. These data suggest that NO is not a mediator for the inhibitory effect of ethanol on LEB contractility, and that NO seems to be a mediator of ethanol inhibition of some aspects of LES motor functions.

Nitric oxide pathway in cat esophagus: localization of nitric oxide synthase and functional effects.

In the cat lower esophageal sphincter (LES) and esophageal body, nitric oxide synthase (NOS) immunoreactive nerves were abundant in the circular smooth muscle layer, especially in the LES region. NADPH diaphorase staining showed an identical pattern. The ability to form L-citrulline from L-arginine corresponded roughly to the distribution of NOS. Confocal microscopic analysis indicated colocalization within neurons of vasoactive intestinal peptide (VIP) in 65% of NOS-positive nerves. In LES circular smooth muscle preparations, electrically induced relaxations (single train stimuli) were generally abolished by NG-nitro-L-arginine (L-NNA). Continuous electrical stimulation for 2 min evoked a relaxation in the presence of L-NNA. This relaxation was inhibited by VIP antiserum and followed by a decrease in guanosine 3',5'-cyclic monophosphate, but not by any consistent change in adenosine 3',5'-cyclic monophosphate levels. K+ (124 mM) induced a biphasic relaxation, with L-NNA inhibiting the first phase but not the second. We conclude that nitric oxide (NO) has a major role as the mediator responsible for relaxation in the cat esophagus. NO seems also to initiate the release and enhance the effect of another transmitter.

Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh.

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (Emax) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-([(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122). Basal Ins(1,4,5)P3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by Emax of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P3 concentrations activated a PKC-dependent pathway, whereas contraction induced by Emax of Ins(1,4,5)P3 was calmodulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute experimental esophagitis impairs signal transduction in cat lower esophageal sphincter circular muscle

It has been previously shown that induction of experimental esophagitis in the cat by esophageal perfusion for 30 minutes with 0.1N HCl for 4 consecutive days results in a significant reduction of in vivo lower esophageal sphincter (LES) resting pressure and in vitro spontaneous tone without affecting esophageal response to KCl. It has also been shown that basal LES tone and LES contraction in response to acetylcholine depend on the release of calcium from intercellular stores, whereas esophageal contraction is mediated by extracellular calcium. The present report shows that esophageal acid perfusion impairs the transduction pathway mediating lower esophageal sphincter contraction in response to acetylcholine through release of intracellular calcium because LES strips and single cells no longer contract in response to acetylcholine if calcium is removed from the physiologic salt solution. This suggests that either the intracellular calcium stores or the release mechanisms that mediate maintenance of tone and contraction in response to acetylcholine may be damaged. However, the acid perfusion has no effect on the acetylcholine response in the esophagus, which is mediated by the influx of extracellular calcium. In the LES circular muscle, the injury results in reduced levels of inositol phosphates without affecting resting levels of 5′-cyclic adenosine monophosphate or 5′-cyclic guanosine monophosphate. The reduced levels of 1,4,5-inositol trisphosphate are consistent with impairment in the mechanisms responsible for release of intracellular calcium, although concurrent damage to calcium stores may also occur.