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Hydrogen Peroxide Reduces Lower Esophageal Sphincter Tone in Human Esophagitis

2005; Elsevier BV; Volume: 129; Issue: 5 Linguagem: Inglês

10.1053/j.gastro.2005.09.008

1528-0012

Ling Cheng, Karen M. Harnett, Weibiao Cao, Fang Liu, José Behar, Claudio Fiocchi, Piero Biancani,

Dysphagia Assessment and Management

Background & Aims: 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. Methods: E-LES was examined histologically, and its in vitro circular muscle contraction and production of inflammatory mediators were compared with those of N-LES. Results: 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 μmol/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. Conclusions: 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. Background & Aims: 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. Methods: E-LES was examined histologically, and its in vitro circular muscle contraction and production of inflammatory mediators were compared with those of N-LES. Results: 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 μmol/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. Conclusions: 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. Gastroesophageal reflux disease (GERD) is a common esophageal disorder that may lead to the development of serious complications, including ulcers, strictures, bleeding, intestinal metaplasia (Barrett’s esophagus), and, eventually, adenocarcinoma of the esophagus. It is currently thought that, in the early stages of GERD, reflux of gastric contents may occur as a result of transient relaxation of the lower esophageal sphincter (TLESR)1Mittal R.K. Holloway R.H. Penagini R. Blackshaw L.A. Dent J. Transient lower esophageal sphincter relaxation.Gastroenterology. 1995; 109: 601-610Abstract Full Text PDF PubMed Scopus (678) Google Scholar unrelated to swallowing or secondary peristalsis2Mittal R.K. McCallum R.W. Characteristics and frequency of transient relaxations of the lower esophageal sphincter in patients with reflux esophagitis.Gastroenterology. 1988; 95: 593-599Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 3Dent J. Dodds W.J. Friedman R.H. Sekiguchi T. Hogan W.J. Arndorfer R.C. Petrie D.J. Mechanism of gastroesophageal reflux in recumbent asymptomatic human subjects.J Clin Invest. 1980; 65: 256-267Crossref PubMed Scopus (800) Google Scholar, 4Dent J. Holloway R.H. Touli J. Dodds W.J. Mechanism of lower esophageal sphincter incompetence in patients with symptomatic gastroesophageal reflux.Gut. 1988; 29: 1020-1028Crossref PubMed Scopus (558) Google Scholar and that repeated episodes of reflux over time lead to impairment of lower esophageal sphincter (LES) tone and esophageal peristaltic contractions,4Dent J. Holloway R.H. Touli J. Dodds W.J. Mechanism of lower esophageal sphincter incompetence in patients with symptomatic gastroesophageal reflux.Gut. 1988; 29: 1020-1028Crossref PubMed Scopus (558) Google Scholar, 5Kahrilas P.J. Dodds W.J. Hogan W.J. Kern M. Arndorfer R.C. Reece A. Esophageal peristaltic dysfunction in peptic esophagitis.Gastronterology. 1986; 91: 897-904PubMed Google Scholar prolonging the esophageal exposure to acid and resulting in erosive esophagitis. Some attention has been paid to the role of injurious agents in gastric reflux, such as hydrochloric acid and pepsin, and the mechanisms of esophageal epithelium’s failure to resist chemical aggression.6Rai A.M. Orlando R.C. Gastroesophageal reflux disease.Curr Opin Gastroenterol. 1998; 14: 329-333Crossref Scopus (36) Google Scholar Many substances considered critical to reflux esophagitis are classical inflammatory products, such as prostanoids and reactive oxygen species (ROS).7Ottington Y. Alber D. Moussard C. Esophageal mucosal prostaglandin E2 levels in health and gastroesopaheal reflux disease.Prost Leuk Med. 1987; 29: 141-151Abstract Full Text PDF Scopus (20) Google Scholar, 8Wetscher G.J. Hinder R.A. Klingler P. Gadenstatter M. Perdikis G. Hinder P.R. Reflux esophagitis in humans is a free radical event.Dis Esophagus. 1997; 10: 29-32Crossref PubMed Scopus (90) Google Scholar, 9Wetscher G.J. Hinder R.A. Bagchi D. Hinder P.R. Bagchi M. Perdikis G. McGinn T. Reflux esophagitis in humans is mediated by oxygen-derived free radicals.Am J Surg. 1995; 170: 552-556Abstract Full Text PDF PubMed Scopus (126) Google Scholar These products are thought to derive from inflammatory cells infiltrating acid-damaged tissue. It is important to examine the relationship between inflammatory mechanisms and mechanisms responsible for LES tone to unravel the sequence of pathophysiologic events contributing to GERD. We have extensively used the cat, and occasional human esophageal specimens, to study esophageal and lower esophageal sphincter function. The LES tone is regulated mostly through myogenic mechanisms.10Christensen J. Freeman B.Q. Miller J.K. Some physiological characteristics of the esophagogastric junction in the opossum.Gastroenterology. 1973; 64: 1119Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 11Christensen J. Conklin J.L. Freeman B.W. Physiologic specialization at the esophagogastric junction in three species.Am J Physiol. 1973; 225: 1265Crossref PubMed Scopus (68) Google Scholar, 12Goyal R.K. Rattan S. Genesis of basal sphincter pressure effect of tetrodotoxin on lower esophageal sphincter pressure in opossum in vivo.Gastroenterology. 1976; 71: 62-67Abstract Full Text PDF PubMed Scopus (138) Google Scholar We have characterized how inflammation induced by acid affects the signal transduction mechanisms mediating contraction of LES circular smooth muscle and begun to identify the inflammatory mediators responsible for these changes. In normal LES circular muscle, spontaneous tone is associated with elevated levels of arachidonic acid (AA), which is metabolized to PGF2α and thromboxane A2 to maintain LES contraction. An animal model of acute esophagitis has been used for some time in our laboratory, obtained by perfusing the cat esophagus with 0.1 N HCl for 45 minutes during 3 successive days and with experiments carried out on the fourth day. In this model of acute esophagitis, we have shown that in vivo LES resting pressure and in vitro LES tone are significantly reduced because of release of inflammatory mediators in response to acid-induced inflammation and cell damage. In animals with esophagitis, LES circular muscle contains high levels of H2O2, and addition of H2O2 to normal muscle produces a concentration-dependent decrease of in vitro LES tone. H2O2 increases production of lipid mediators, such as prostaglandin E2 (PGE2) and platelet activating factor (PAF), which relax LES circular muscle, and of products of lipid peroxidation such as isoprostanes 8-iso F2α, which inhibit prostaglandin (PGF2α)-mediated contraction.13Cheng L. Cao W. Behar J. Biancani P. Harnett K.M. Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G787-G797Crossref PubMed Scopus (26) Google Scholar In this model, however, LES function returns to normal within 3 weeks after suspension of acid perfusion,14Eastwood G.L. Castell D.O. Higgs R.H. Experimental esophagitis in cats impairs lower esophageal sphincter pressure.Gastroenterology. 1976; 69: 146-153Google Scholar whereas, in humans, even after healing of the mucosa by endoscopic and histologic criteria, LES and esophageal function do not return to normal.5Kahrilas P.J. Dodds W.J. Hogan W.J. Kern M. Arndorfer R.C. Reece A. Esophageal peristaltic dysfunction in peptic esophagitis.Gastronterology. 1986; 91: 897-904PubMed Google Scholar, 15Howard S. Chan-Yeung M. Martin L. Phaneuf S. Salari H. Polyphosphoinositide hydrolysis and protein kinase C activation in guinea pig tracheal smooth muscle cells in culture by leukotriene D4 involve a pertussis toxin sensitive G-protein.Eur J Pharmacol. 1992; 227: 123-129Crossref PubMed Scopus (24) Google Scholar, 16Russell C. Pope C. Gannan R. Allen F. Velasco N. Hill L. Does surgery correct esophageal motor dysfunction in gastroesophageal reflux?.Ann Surg. 1981; 194: 290-296Crossref PubMed Scopus (52) Google Scholar, 17Timmer R. Breumelhof R. Nadorp J.H. Smout A.J. Oesophageal motility and gastro-oesophageal reflux before and after healing of reflux oesophagitis. A study using 24 hour ambulatory pH and pressure monitoring.Gut. 1994; 35: 1519-1522Crossref PubMed Scopus (83) Google Scholar, 18Volker E. Does healing of esophagitis improve esophageal motor function?.Dig Dis Sci. 1988; 33: 161Crossref PubMed Scopus (84) Google Scholar, 19Williams D. Thompson D. Heggie L. O’Hanrahan T. Bancewicz J. Esophageal clearance function following treatment of esophagitis.Gastroenterology. 1994; 106: 108-116PubMed Google Scholar It is likely that inflammatory mechanisms may produce time-dependent damage and irreversible alterations in motor function. Using an animal model of myotomy-induced experimental esophagitis, we demonstrated that ranitidine added at 6 months after sphincterotomy restored LES muscle function, suggesting that LES muscle function may be reversible in the early stages before permanent damage takes place.20Poorkhalkali N. Rich H.G. Jacobson I. Amaral J. Migliori S. Chrostek C. Biancani P. Cabero J.L. Helander H.F. Chronic oesophagitis in the cat.Scand J Gastroenterol. 2001; 36: 904-909Crossref PubMed Scopus (12) Google Scholar In this model, onset of chronic esophagitis may occur after more than 6 months post-LES myotomy.20Poorkhalkali N. Rich H.G. Jacobson I. Amaral J. Migliori S. Chrostek C. Biancani P. Cabero J.L. Helander H.F. Chronic oesophagitis in the cat.Scand J Gastroenterol. 2001; 36: 904-909Crossref PubMed Scopus (12) Google Scholar Over the course of several years, we have obtained some human esophageal specimens, and we have extended results initially obtained in the cat to the human LES. We found that, similar to the cat, AA contributes to maintenance of human LES tone by producing prostaglandins and thromboxanes. A role of AA metabolites in the maintenance of human LES tone is consistent with the reported association of esophagitis with aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), which are known to inhibit prostaglandin and thromboxane production. Human esophageal specimens from organ donors with reflux esophagitis are exceedingly infrequent and rarely available for study. For the first time, we now report that we have obtained a human LES specimen with histologically proven erosive esophagitis from an organ donor, and we have carefully characterized its pathophysiologic mechanical and inflammatory profiles. Human esophagi from organ donors, including the LES and a stomach cuff, were provided by National Disease Research Interchange (Philadelphia, PA). The experimental protocols were approved by the Human Research Institutional Review Committee at Rhode Island Hospital. The specimens were pinned on a wax block and opened along the lesser curvature. Upon gross examination, one of the specimens exhibited obvious hyperemia of the mucosa with scattered hemorrhagic erosions in the lower esophagus, extending across the cardia as shown in Figure 1. The mucosa was removed by sharp dissection and sent to pathology for histologic diagnosis. The LES was isolated, and circular muscle strips were excised as previously described.21Cao W. Harnett K.M. Behar J. Biancani P. Group I secreted PLA2 in the maintenance of human lower esophageal sphincter tone.Gastroenterology. 2000; 119: 1243-1252Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar LES strips (2 mm) were mounted in separate 1-mL muscle chambers and equilibrated with continuous perfusion of oxygenated physiologic salt solution (PSS), as previously described in detail.21Cao W. Harnett K.M. Behar J. Biancani P. Group I secreted PLA2 in the maintenance of human lower esophageal sphincter tone.Gastroenterology. 2000; 119: 1243-1252Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 22Biancani P. Hillemeier C. Bitar K.N. Makhlou G.M. Contraction mediated by Ca++ influx in the esophagus and by Ca++ release in the LES.Am. J. Physiol. 1987; 253: G760-G766PubMed Google Scholar During this time, the tension in LES strips increased, attaining a steady level at 4–6 hours. The PSS contained the following (in mmol/L): NaC1, 116.6; NaHCO3, 21.9; NaH2PO4, 1.2; KC1, 3.4; CaC12 2.5; glucose, 5.4; and MgC12, 1.2. The solution was equilibrated with a gas mixture containing 95% O2 and 5% CO2 at pH 7.4 and 37°C. Muscle strips were stimulated with square wave pulses of supramaximal voltage, 0.2-millisecond, I-Hz, and 10-second trains, delivered by a stimulator (model S48; Grass Instruments, Quincy, MA) through platinum wire electrodes, placed longitudinally on either side of the strip. Electrical stimulation was used to document further the nature of the strips. LES strips relaxed during the stimulus train. Smooth muscle tension was recorded on a chart recorder (Grass Instruments). Passive force was obtained at the end of the experiment by completely relaxing the strips with excess EDTA until no further decrease in resting force was observed. Basal LES tone is the difference between resting and passive force. A single dose of catalase (800 U/mL) was used after tone had reached a steady state. PGE2 and PAF cumulative dose–response relationships were obtained. Each dose was maintained until tone had stabilized before recording the response and/or adding the next dose. To examine the effect of exogenous prostaglandins, LES strips were incubated in indomethacin (10−4 mol/L, 30 minutes) to eliminate production of endogenous prostaglandin and thromboxanes. We have previously shown that indomethacin causes a 50% reduction in human LES tone.21Cao W. Harnett K.M. Behar J. Biancani P. Group I secreted PLA2 in the maintenance of human lower esophageal sphincter tone.Gastroenterology. 2000; 119: 1243-1252Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar After tone had stabilized, cumulative dose responses were obtained for PGF2α. PGF2α has been shown to participate in maintenance of resting LES tone.23Cao W.B. Harnett K.M. Chen Q. Jain M.K. Behar J. Biancani P. Group I secreted PLA2 (sPLA2) and arachidonic acid metabolites in the maintenance of cat LES tone.Am J Physiol. 1999; 277: G585-G598PubMed Google Scholar To examine the effect of isoprostanes on LES response to PGF2α, cumulative dose response relationships were obtained before and after incubation in isoprostane F2α (10−5 mol/L) for 1.5 hours. The LES was excised, circular muscle layer was cut into 0.5-mm-thick slices with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA), and tissue squares were made by cutting twice with a 2-mm blade block, the second cut at right angles to the first. This circular smooth muscle tissue was used for reverse-transcription polymerase chain reaction (RT-PCR) detection of NOX1–NOX5 and for measurements of H2O2, PAF, PGE2 and 8-iso PGF2α isoprostane. Total RNA is purified by Trizol reagent (Invitrogen) from LES circular muscle. Two micrograms of total RNA were reverse transcribed and then subjected to PCR by using a kit SUPERSCRIPT First-Strand Synthesis System for RT-PCR (Invitrogen). Primers for NOX5 are 5′-AAGACTCCATCACGGGGCTGCA-3′ (sense) and 5′-CCCTTCAGCACCTTGGCCAGAG-3′ (antisense). Primers for GAPDH are 5′-ATGACCACAGTCCATGCCATCAC-3′ (sense) and 5′-GAGGTCCACCACCCTGTTGCTGTA-3′ (antisense). PCR products were electrophoresed in 1.5% agarose gels and visualized by ethidium bromide staining. Total RNA is purified by Trizol reagent (Invitrogen) for cultured cells and tissues. Total RNA are reverse transcribed by using a kit SUPERSCRIPT First-Strand Synthesis System for RT-PCR (Invitrogen). All real-time PCR reactions are performed in triplicate in 25 μL total volume containing Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA), sense and antisense primers, cDNA, and reference dye. Reactions are carried out in a Stratagene Mx4000 multiplex quantitative PCR system (Stratagene). Fluorescence values of SYBR Green I dye, representing the amount of product amplified at that point in the reaction, are recorded in real-time at the annealing and extension step of each cycle. The Ct, defined as the point at which the fluorescence signal is statistically significant above background, is calculated by using Stratagene Mx4000 software. This value is then used to determine the relative amount of amplification in each sample. Transcript level of each specific gene is normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification. LES and esophageal circular smooth muscle (100 mg) were homogenized in PBS buffer, and H2O2 content was measured by a Bioxytech H2O2 kit (Oxis International, Inc, Portland, OR) as previously described.13Cheng L. Cao W. Behar J. Biancani P. Harnett K.M. Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G787-G797Crossref PubMed Scopus (26) Google Scholar Smooth muscle tissue (100 mg) was homogenized in 1.5 mL cold methanol on ice with 10 bursts of 20-sec duration with a Tissue Tearer (Biospec, Racine, WI); 0.1 mL of the homogenate was used for protein measurement. Chloroform and water were added to the remaining homogenate to obtain a chloroform/methanol/H2O ratio of 0.5:1:0.4 by volume. The mixture was vortexed and left at room temperature for 1 hour then centrifuged (5000g, 5 minutes), and the supernatant was removed. The pellet was resuspended with 1.9 mL chloroform/methanol/H2O solution for another 1 hour then centrifuged, and the supernatants from the 2 extractions were combined. Chloroform (2 mL) and 1 mol/L NaCl (2 mL) were added to the supernatant then centrifuged (5000g, 5 minutes). The upper phase was aspirated and discarded, and the lower phase was washed once with 4 mL 1 mol/L NaCl/methanol (9:1 by volume), dried under nitrogen, and stored at −20°C. Measurement of PAF were performed within 72 hours. The PAF concentration was quantified by using a platelet activating factor [3H] (PAF) scintillation proximity assay system (TRK990, Amersham Pharmacia Biotech Inc Piscataway, NJ). LES circular smooth muscle tissue (100 mg) was homogenized in PGE2 homogenization buffer (0.1 mol/L phosphate buffer, pH 7.4), containing 1 mmol/L EDTA, 20 μg/mL indomethacin at 4°C, purified by an affinity column (Cayman Chemical Co, Ann Arbor, MI), and quantified using a PGE2 Competitive Enzyme Immunoassay kit (Cayman Chemical Co) as previously described.13Cheng L. Cao W. Behar J. Biancani P. Harnett K.M. Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G787-G797Crossref PubMed Scopus (26) Google Scholar LES smooth muscle squares (150 mg) were homogenized in 2.5 mL buffer solution on ice with 3, 10–20-second bursts with a Tissue Tearer (Biospec), followed by 50 strokes with a Dounce homogenizer (Wheaton, Melville, NJ). The homogenized buffer solution was 0.1 mol/L phosphate buffer, pH 7.4, containing 1 mmol/L EDTA, 10 μmol/L indomethacin (Cayman catalog No. 70270), 0.005% BHT (Butylated hydroxytcluene, from Sigma). The samples were centrifuged at 1500g for 15 minutes at 40°C. Protein content was measured in 100 μL supernatant. F2-Isoprostane was isolated using an affinity column and quantified using an 8-Isoprostane Enzyme Immunoassay kit (Cayman Chemical Co) as previously described.13Cheng L. Cao W. Behar J. Biancani P. Harnett K.M. Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G787-G797Crossref PubMed Scopus (26) Google Scholar The amount of protein present was determined by colorimetric analysis (Bio-Rad, Melville, NY) according to the method of Bradford.24Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214409) Google Scholar The 8-isoprostane affinity column, 8-isoprostane enzyme immunoassay (EIA) kit, prostaglandin E2 affinity column, prostaglandin E2 EIA kit-monoclonal, and 8-iso prostanglandin F2α were purchased from Cayman Chemical Co; PAF [3H] scintillation proximity assay (SPA) system (TRK990) was purchased from Amersham Pharmacia Biotech Inc (Piscataway, NJ); Bioxytech H2O2-560 quantitative hydrogen peroxide assay was purchased from Oxis International, Inc (Portland, OR); prostaglandin E2 and prostaglandin F2α were purchased from Biomol (Plymouth Meeting, PA). Data are expressed as mean ± SEM. Statistical differences between means were determined by Student t test. Differences between multiple groups were tested using analysis of the variance (ANOVA) for repeated measures and checked for significance using Scheffè F test. For comparison of the esophagitis sample (n = 1) with normal samples, we compared the normal values ± 2 standard deviations (SD) of the mean to the value for the esophagitis sample. Although rigorous statistics cannot be performed because of the small sample size, values lying outside the mean ± 2SD may be considered to be outside the norm. Histologic examination of the specimen displaying hyperemia of the mucosa and hemorrhagic erosions in the lower esophagus extending across the cardia showed erosions, basal zone hyperplasia, and elongation of lamina propria papillae with inflammatory cells present in the epithelial layer (Figure 2). All these findings are characteristic of reflux esophagitis. When mounted in a warm (37°C) oxygenated muscle chamber, N-LES circular muscle strips gradually develop tone and reach a steady state tone (3.25 ± 0.2 grams) after 3–4 hours. In contrast, circular muscle strips from E-LES developed little tone (0.7 grams), a value falling outside of the normal tone minus 2 standard deviations. When E-LES circular muscle strips were exposed to catalase (800 U/mL), tone increased within 15–30 minutes and reached a value of 2.7 g, similar to that of N-LES strips (Figure 3). These findings suggest that H2O2 may be present in E-LES circular muscle and responsible for reduced tone when inflammation is present. Data for catalase-treated N-LES are not shown because, in the normal LES, catalase caused a significant reduction in resting tone. It is possible that low levels of H2O2 may contribute to maintenance of tone, perhaps by facilitating release of calcium from intracellular stores. Higher levels of H2O2, such as those found in esophagitis, however, reduce LES tone because neutralizing H2O2 returns tone almost to normal levels. Up-regulation of NADPH oxidases is a possible mechanism for overproduction of H2O2. We therefore examined by RT-PCR whether any NADPH oxidases are present in human esophageal circular muscle and up-regulated by esophagitis. We designed primers for NOX1, NOX2, NOX3, NOX4, and NOX5 and found that these membrane-bound catalytic components of the NADPH oxidase enzyme are all present in esophageal circular muscle, but only NOX5 mRNA was increased in the human specimen with esophagitis, as shown in Figure 4A. Increased NOX5 cDNA in the esophagitis specimen was confirmed by real-time RT-PCR when compared with normal human esophageal circular muscle, as shown in Figure 4B. The Figure shows increased levels of NOX5 cDNA in the esophagitis specimen when compared with normal specimens. Three-hour exposure to IL-1β did not increase NOX5 cDNA, even though IL-1β increases H2O2 levels in normal circular muscle as shown in Figure 5. This is not surprising because NADPH oxidase-mediated production of H2O2 may be regulated at multiple levels, even when its expression is not up-regulated.Figure 5IL-1β and esophagitis increase H2O2 levels in LES circular smooth muscle. H2O2 levels of LES circular smooth muscle were measured by colorimetric assay. An esophagitis specimen was obtained from an organ donor. Treatment of normal LES circular smooth muscle with IL-1β (200 U/mL, 2 hours) increased H2O2 levels compared with untreated muscle. H2O2 levels were 4-fold higher in LES muscle from the esophagitis specimen compared with normal muscle. Means ± SE are shown for 3 normal and for 3 IL-1β-treated LES samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Figure 5 shows H2O2 levels in normal and esophagitis circular muscle tissue. Basal H2O2 levels of N-LES circular muscle were 0.2 ± 0.04 nmol/mg protein and 0.82 nmol/mg protein in E-LES circular muscle, a value exceeding that of normal + 2 standard deviations (Figure 5). Because we have previously shown that, in cat LES, IL-1β13Cheng L. Cao W. Behar J. Biancani P. Harnett K.M. Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G787-G797Crossref PubMed Scopus (26) Google Scholar and other cytokines, presumably released by inflammatory cells or the damaged esophageal epithelium, may contribute to production of H2O2, this possibility was tested in N-LES circular muscle. Exposure to interleukin (IL)-1β (200 U/mL, 2 hours) increased production of H2O2 to 0.57 ± 0.13 nmol/mg protein, a level approaching that of esophagitis muscle (Figure 5). In our cat model of acute experimental esophagitis, elevated levels of H2O2 in the LES smooth muscle layer enhance the production of lipid inflammatory mediators, such as PAF, PGE2, and F2-isoprostane, which reduces LES tone.13Cheng L. Cao W. Behar J. Biancani P. Harnett K.M. Inflammation induced changes in arachidonic acid metabolism in cat LES circular muscle.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G787-G797Crossref PubMed Scopus (26) Google Scholar We therefore tested whether these mediators are elevated in the human esophagitis specimen and whether H2O2 enhances their production in N-LES muscle. Figure 6 shows that PAF levels were 1.9 ng/mg protein in the human esophagitis sample and 0.5 ± 0.07 ng/mg protein in N-LES muscle. The PAF levels in the esophagitis sample exceeded those of the normal sample + 2 standard deviations. Incubation of N-LES circular muscle in H2O2 (70 μmol/L, 2 hours) significantly increased PAF levels (P < .01). The right panel in Figure 5 shows that LES tone is concentration-dependently reduced by PAF (P < .001). PGE2 levels (Figure 7) were 1009 pg/mg protein in the human esophagitis sample and 161 ± 32 pg/mg protein in N-LES muscle. The PGE2 levels in the esophagitis sample exceeded those of the normal sample + 2 standard deviations. Incubation of N-LES circular muscle in H2O2 (70 μmol/L, 2 hours) significantly increased PGE2 levels (P < .01, unpaired t test; Figure 7). PGE2 decreases LES tone,25Daniel E.E. Sarna S. Waterfall W. Crankshaw J. Role of endogenous prostaglandins in regulating the tone of opossum lower esophageal sphincter in vivo.Prostaglandins. 1979; 17: 641-648Crossref PubMed Scopus (8) Google Scholar, 26Goyal R.K. Rattan S. Mechanism of the lower esophageal sphincter relaxation. Action of prostaglandin E1 and theophilline.J Clin Invest. 1973; 55: 337Crossref Scopus (92) Google Scholar, 27Goyal R.K. Rattan S. Hersh T. Comparison of the effects of prostaglandins E1, E2, and A2, and of hypovolumic hypotension on the lower esophageal sphincter.Gastroenterology. 1973; 65: 608-612PubMed Google Scholar and this was confirmed in human LES muscle by demonstrating that its tone was