Hydrogen Peroxide Reduces Lower Esophageal Sphincter Tone in Human Esophagitis

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GASTROENTEROLOGY 2005;129:1675–1685

CASE REPORT Hydrogen Peroxide Reduces Lower Esophageal Sphincter Tone in Human Esophagitis LING CHENG,* KAREN M. HARNETT,* WEIBIAO CAO,* FANG LIU,* JOSE BEHAR,* CLAUDIO FIOCCHI,‡ and PIERO BIANCANI* *Division of Gastroenterology, Rhode Island Hospital and Brown University, Providence, Rhode Island; and ‡Division of Gastroenterology, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, Ohio

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 ⴞ 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 ⴞ 0.05 nmol/mg protein). When N-LES smooth muscle was incubated in H2O2 (70 ␮mol/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.

reflux of gastric contents may occur as a result of transient relaxation of the lower esophageal sphincter (TLESR)1 unrelated to swallowing or secondary peristalsis2– 4 and that repeated episodes of reflux over time lead to impairment of lower esophageal sphincter (LES) tone and esophageal peristaltic contractions,4,5 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.6 Many substances considered critical to reflux esophagitis are classical inflammatory products, such as prostanoids and reactive oxygen species (ROS).7–9 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.10 –12 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.

astroesophageal 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,

Abbreviations used in this paper: LES, lower esophageal sphincter; E-LES, human LES specimen with esophagitis; N-LES, normal lower esophageal sphincter; PAF, platelet activating factor; PGE2, prostaglandin E2; PGF2␣, prostaglandin F2␣; TLESR, transient relaxation of the lower esophageal sphincter. © 2005 by the American Gastroenterological Association 0016-5085/05/$30.00 doi:10.1053/j.gastro.2005.09.008

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In normal LES circular muscle, spontaneous tone is associated with elevated levels of arachidonic acid (AA), which is metabolized to PGF2␣ 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 F2␣, which inhibit prostaglandin (PGF2␣)-mediated contraction.13 In this model, however, LES function returns to normal within 3 weeks after suspension of acid perfusion,14 whereas, in humans, even after healing of the mucosa by endoscopic and histologic criteria, LES and esophageal function do not return to normal.5,15–19 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.20 In this model, onset of chronic esophagitis may occur after more than 6 months post-LES myotomy.20 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,

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Figure 1. An esophageal specimen obtained from an organ donor exhibited hyperemia of the mucosa with scattered hemorrhagic erosions in the lower esophagus, extending across the cardia.

and we have carefully characterized its pathophysiologic mechanical and inflammatory profiles.

Materials and Methods Tissues Specimens 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.

Measurement of In Vitro LES Tone The LES was isolated, and circular muscle strips were excised as previously described.21 LES strips (2 mm) were

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mounted in separate 1-mL muscle chambers and equilibrated with continuous perfusion of oxygenated physiologic salt solution (PSS), as previously described in detail.21,22 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.

Effect of Agonist and Antagonists on LES Tone 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.21 After tone had stabilized, cumulative dose responses were obtained for PGF2␣. PGF2␣ has been shown to participate in maintenance of resting LES tone.23 To examine the effect of isoprostanes on LES response to PGF2␣, cumulative dose response relationships were obtained before and after incubation in isoprostane F2␣ (10⫺5 mol/L) for 1.5 hours.

Preparation of Circular Smooth Muscle Tissue 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 PGF2␣ isoprostane.

RT-PCR 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

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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.

Real-Time RT-PCR 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 ␮L 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.

Measurement of H2O2 in Smooth Muscle Tissue 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.13

Measurement of PAF 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

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Figure 2. (A) Erosions, basal zone hyperplasia, and elongation of lamina propria papillae with congestion. The enlarged area (B) shows inflammatory cells present in the epithelial layer. These findings are characteristic of reflux esophagitis.

factor [3H] (PAF) scintillation proximity assay system (TRK990, Amersham Pharmacia Biotech Inc Piscataway, NJ).

Measurement of PGE2 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 ␮g/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.13

F2-Isoprostane Measurement 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 ␮mol/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 ␮L supernatant. F2-Isoprostane was isolated using an affinity column and quantified using an 8-Isoprostane Enzyme Immunoassay kit (Cayman Chemical Co) as previously described.13

Protein Determination The amount of protein present was determined by colorimetric analysis (Bio-Rad, Melville, NY) according to the method of Bradford.24

Materials The 8-isoprostane affinity column, 8-isoprostane enzyme immunoassay (EIA) kit, prostaglandin E2 affinity col-

umn, prostaglandin E2 EIA kit-monoclonal, and 8-iso prostanglandin F2␣ 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 F2␣ were purchased from Biomol (Plymouth Meeting, PA).

Statistical Analysis 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.

Results 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)

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Figure 3. LES. Circular muscle strips were mounted in muscle chambers, stretched to 2.5 g to bring them near conditions of optimum force development, and equilibrated for 4 – 6 hours while perfused continuously with oxygenated physiologic salt solution (PSS) at 37°C. During this time, the tension in LES strips increased, attaining a steady level at 3– 4 hours. An esophagitis specimen was obtained from an organ donor. In LES circular muscle strips from the esophagitis specimen, tone was lower than in normal strips and increased almost to a normal level with catalase. These data suggest that H2O2 may be responsible for the reduced tone in esophagitis. Means ⫾ SE are shown for 3 normal LES samples.

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 upregulated 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

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Figure 4. LES circular muscle was examined by RT-PCR for the presence of NADPH oxidases. (A) We used published primers for NOX1, NOX2, NOX3, NOX4, and NOX5 and found that these 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. (B) Increased NOX5 cDNA in the esophagitis specimen was confirmed by real-time RT-PCR when compared with normal human esophageal circular muscle. Means ⫾ SE are shown for 3 normal (control) and for 3 IL-1␤-treated LES samples.

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-1␤ did not increase NOX5 cDNA, even though IL-1␤ 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 5. IL-1␤ 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-1␤ (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-1␤-treated LES samples.

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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-1␤13 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)-1␤ (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.13 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 ␮mol/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 esoph-

Figure 6. Esophagitis and H2O2 increase PAF production in LES. The PAF concentration was quantified by using a platelet activating factor [3H] (PAF) scintillation proximity assay system (Amersham Pharmacia Biotech Inc). In esophagitis circular muscle, PAF levels were 4 times higher than in normal LES. In normal LES muscle, PAF levels were significantly increased (P ⬍ .01, unpaired t test) by H2O2 (70 mmol/L, 2 hours). PAF concentration-dependently decreased tone of normal LES muscle strips (P ⬍ .001, ANOVA). Means ⫾ SE are shown for 6 normal LES and for 3 H2O2-treated LES samples.

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Figure 7. Esophagitis and H2O2 increase PGE2 production in LES. PGE2 levels were measured in untreated normal LES circular smooth muscle, in normal muscle treated with H2O2 (70 ␮mol/L for 2 hours), and in LES from the esophagitis specimen. PGE2 was purified using a PGE2 purification column and then measured by enzyme immunoassay. PGE2 production was higher in H2O2-treated (P ⬍ .01, unpaired t-test) LES and eophagitis LES compared with normal LES. PGE2 concentration-dependently decreased tone of normal LES muscle strips (P ⬍ .001, ANOVA). Means ⫾ SE are shown for 3 normal LES and for 3 H2O2-treated LES samples.

agitis sample exceeded those of the normal sample ⫹ 2 standard deviations. Incubation of N-LES circular muscle in H2O2 (70 ␮mol/L, 2 hours) significantly increased PGE2 levels (P ⬍ .01, unpaired t test; Figure 7). PGE2 decreases LES tone,25–27 and this was confirmed in human LES muscle by demonstrating that its tone was concentration-dependently reduced (P ⬍ .001) by PGE2. Lipid peroxidation of membrane phospholipids by H2O2 may increase the production of isoprostanes,28 which are stable prostaglandin analogs formed in situ on the cell membrane by free radical-induced peroxidation of arachidonic acid. F2 isoprostane levels were 288 pg/mg protein in E-LES circular muscle and 7.2 ⫾ 1 pg/mg protein in N-LES (Figure 8). The F2 isoprostane levels in the esophagitis sample exceeded those of the normal sample ⫹ 2 standard deviations. In addition, incubation of N-LES circular muscle with H2O2 (70 ␮mol/L) for 2 hours caused a significant increase in F2 isoprostane levels (P ⬍ .001) to levels comparable with the F2 isoprostane levels found in E-LES. To test whether an interaction between PGF2␣ and F2 isoprostane would affect the LES tone, we examined N-LES circular muscle strips incubated with indomethacin (10⫺5 mol/L) to eliminate any contribution of endogenous prostaglandins and thromboxanes. Figure 9 (panel A) shows that PGF2␣ caused a concentrationdependent contraction of indomethacin-treated normal human LES circular muscle strips. In contrast, 8-iso PGF2␣ alone had little effect on tone of normal LES circular muscle, although preincubation with 8-iso PGF2␣ (10⫺5 mol/L) essentially abolished PGF2␣-induced contraction (Figure 9, panel A; P ⬍ .001). These

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results suggest that 8-iso PGF2␣ may selectively inhibit binding of PGF2␣ to its receptors because of structural similarities of these two AA-derived autacoids. To confirm a functional role of isoprostanes in reducing PGF2␣mediated tone in esophagitis, we examined PGF2␣-induced contraction of LES muscle from the esophagitis specimen. PGF2␣-induced contraction was abolished in E-LES (P ⬍ .001), but ACh-induced contraction was unchanged (Figure 8, panel B) and similar to AChinduced contraction of N-LES, demonstrating the selective inhibition of PGF2␣ response in the esophagitis specimen.

Discussion GERD is an inflammatory disorder that affects esophageal and LES smooth muscle function. In a cat model of acute esophagitis, inflammation induced by acid reduces LES pressure in vivo and LES tone in vitro and causes the production of classical inflammatory products, such as prostanoids, ROS,7,8 and proinflammatory cytokines such as IL-1␤, which are thought to derive from inflammatory cells infiltrating acid-damaged tissue.29 Preliminary evidence in the cat suggests that proinflammatory cytokines (such as IL-1␤ or IL-6) are produced in the mucosa in response to inflammation and may diffuse to the circular muscle layer30 causing overproduction of H2O2. H2O2 is known to cause lipid

Figure 8. Esophagitis and H2O2 increase PAF production in LES. F2 isoprostane levels were measured in untreated normal LES circular smooth muscle in muscle treated with H2O2 (70 ␮mol/L for 2 hours) and in LES muscle from the esophagitis specimen. F2 isoprostane was purified using an 8-isoprostane affinity column and then measured by enzyme immunoassay. In esophagitis circular muscle, F2␣ isoprostane levels were 40 times higher than in normal LES. Incubation of normal LES circular muscle with H2O2 caused a significant increase in F2 isoprostane levels (P ⬍ .001, unpaired t test). Means ⫾ SE are shown for 3 normal LES and for 3 H2O2-treated LES samples.

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Figure 9. 8-isoPGF2␣ selectively reduces PGF2␣-induced contraction of LES. (A) After LES strips had equilibrated and tension had reached a steady level, they were incubated in indomethacin (10⫺4 mol/L, 30 minutes) to eliminate production of endogenous prostaglandin. After tone had stabilized at this lower level, cumulative concentration responses were obtained for 8-isoPGF2␣ alone, PGF2␣ alone, and PGF2␣ plus 8-isoPGF2␣ (10⫺5 mol/L). PGF2␣ caused a concentration-dependent contraction of LES circular muscle strips. In contrast, 8-isoPGF2␣ alone had little effect on tone of normal LES circular muscle strips, but preincubation with 8-isoPGF2␣ significantly reduced PGF2␣-induced contraction (P ⬍ .001, ANOVA). Means ⫾ SE are shown for 3 normal LES samples. (B) In esophagitis LES PGF2␣ did not cause contraction, perhaps because of the higher isoprostane concentration present, even though the esophagitis muscle contracted normally in response to ACh.

peroxidation and release of calcium from intracellular stores and, in addition, may diffuse to the nucleus altering protein expression in the cell. Pharmacologic identification of the effect of H2O2 may be achieved by examining the modulatory effects of specific scavengers, such as catalase. H2O2 diffuses across biologic membranes31 and may be neutralized by extracellular scavengers, such as catalase, that do not cross the cell membrane. Our results suggest that overproduction of oxygen radicals may be a central feature of chronic inflammation as it occurs in esophagitis13 or ulcerative colitis32,33 and may be responsible for delayed recovery of muscle function. The finding that catalase almost restores tone of E-LES supports a central role of H2O2 in esophagitisassociated reduction of LES tone. To investigate a possible mechanism for overproduction of H2O2, we examined whether nonphagocytic NADPH oxidases may be present in the circular muscle layer. Phagocytic NADPH oxidase34 consists of 6 subunits that are partitioned between different subcellular locations in the resting state. Two of these subunits, p22phox and gp91phox, are integral membrane proteins and form a heterodimeric flavocytochrome, also known as cytochrome b558, which constitutes the catalytic core of the enzyme. The remaining oxidase components reside in the cytosol and include the small GTPase Rac as well as a complex of p40phox, p47phox, and p67phox. Activation of NADPH oxidase is initiated by phosphorylation of phox proteins, which are believed to induce conformational

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changes that lead to rearrangements, affecting interactions within the cytosolic p40-p47-p67phox complex. These events culminate in the translocation of this complex to the membrane and association with both RacGTP and cytochrome b558 to form the active enzyme.34 Superoxide-generating homologues of phagocytic gp91phox (NOX1-NOX5, DUOX1, DUOX2) and homologues of other subunits, such as p41 or NOXO1 (homologue of p47phox), p51 or NOXA1 (homologue of p67phox), have been found in several cell types.34 –36 The presence of these homologues in nonphagocytic cells suggests that ROS generated in these cells may have a nonmitochondrial origin and have distinctive cellular functions related to immunity, signal transduction, and modification of the extracellular matrix.34 Human LES smooth muscle contains NOX1–NOX5, but NOX5 cDNA is the only one that is significantly increased by induction of esophagitis. Up-regulation of NADPH oxidases and overproduction of free radicals may cause further up-regulation of transcription factors, ultimately resulting in alterations of cell phenotype. H2O2 may be at least partly responsible for impairment of Ca2⫹ release in a cat model of acute experimental esophagitis by acting as a Ca2⫹ ATPase inhibitor and inhibiting Ca2⫹uptake and refilling of Ca2⫹ stores37 and thus contributing to decreased LES tone, which depends on release of Ca2⫹ from intracellular stores.38 It is notable that, whereas esophagitis up-regulates NOX5, IL-1␤ increases production of H2O2 without having any effect on NOX5 cDNA levels. This is not entirely surprising because NADPH oxidases can be regulated by multiple mechanisms34 but suggests that inflammatory mediators other than IL-1␤ may be present in esophagitis and may be responsible for up-regulation of NOX5. We have reported that cat and human LES tone depends on the activity of a group I PLA221,23 that produces AA and AA metabolites. The AA metabolites PGF2␣ and TX A2/B2 bind to their respective receptors activating Gq and Gi3, which are coupled to phosphatidylcholinespecific phospholipase C and to phosphatidylinositolspecific phospholipase C. Activation of these phospholipases results in the generation of second messengers that activate PKC and maintain a sustained PKC-dependent contraction of LES circular muscle.38 Because LES tone is reduced and H2O2 levels were elevated in the human sample of reflux esophagitis, we examined whether H2O2 reduced tone by altering AA metabolism. A mechanism for H2O2-induced reduction in tone is through increased production of PGE2, which is known to relax LES smooth muscle.25 H2O2 increases production of PGE2 in LES circular smooth muscle, and PGE2 levels are significantly higher in acute cat experimental esoph-

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agitis than in normal LES. Similarly, in the present study, PGE2 levels were elevated in the human esophagitis specimen, and H2O2 treatment of normal human LES caused a significant increase in PGE2, which reduces LES tone25 as confirmed in this study with human LES. In contrast to PGE2, the levels of PGF2␣, which contribute to the maintenance of LES tone, were unchanged after induction of experimental esophagitis in the cat. However, the effectiveness of PGF2␣ was reduced by the presence of elevated levels of isoprostanes found in cat esophagitis LES circular smooth muscle. Isoprostanes are stable prostaglandin-like compounds that are formed enzymatically39 and nonenzymatically in vivo. F2-isoprostanes may bind with varying affinity to PGF2␣ receptors,40 acting as weak agonists,41 and may bind to specific isoprostane receptors.42 Because isoprostanes are not metabolized, prolonged exposure to isoprostanes may eventually cause desensitization or sequestration of PGF2␣ receptors.40,41 In the present study, we show that N-LES muscle strips do not contract in response to 8-iso PGF2␣ and that 8-iso PGF2␣ inhibits PGF2␣-induced contraction of NLES strips. LES muscle strips from the esophagitis specimen, which contained elevated levels of isoprostanes, did not contract in response to PGF2␣ but contracted normally in response to ACh. These data suggest that the elevated levels of F2 isoprostanes in E-LES reduce contraction by binding to PGF2␣ receptors and blocking the effect of endogenous PGF2␣, perhaps by causing receptor internalization and reducing the number of receptors available for activation by PGF2␣. Thus, a second mechanism responsible for the reduced tone in esophagitis may be H2O2-induced production of 8-iso PGF2␣ that may prevent PGF2␣-mediated tone development, possibly by causing PGF2␣ receptor internalization. In addition to the production of isoprostanes, oxidative attack of phosphatidylcholines by ROS yields a series of potent PAF mimetics43– 45 termed “PAF-like agents.” The structure of these bioactive lipid products differs slightly from PAF whose sn-2 residue is exclusively derived from acetyl-CoA. PAF-like agents, created by oxidation of phospholipids,46 activate PAF receptors in a variety of experimental preparations,43– 46 and their biologic activity is completely blocked by specific PAF receptor antagonists.46 In the present study, we show that PAF concentration-dependently decreases normal human LES tone, that H2O2 treatment can increase PAF levels in normal LES muscle, and that PAF levels are elevated in the human esophagitis specimen. In the cat model of experimental esophagitis, IL-1␤ levels are elevated in the circular smooth muscle layer and lead to an increased production of H2O2. IL-1␤

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Figure 10. The illustration summarizes data obtained in this and other investigations. H2O2 increases in LES (or esophageal) circular muscle in response to esophagitis-associated increases in IL-1␤ (as shown in the present investigation), or of IL-653 or in response to other inflammatory mediators. H2O2, in turn, increases production of PAF, PGE2, and isoprostanes, all contributing to decreased LES tone. In addition, H2O2 inhibits Ca2⫹-ATPase activity, preventing calcium uptake in intracellular stores and resulting in depletion of releasable calcium,37,54 which may also decrease LES tone.38

activates IL-1␤ receptors, a subfamily of Toll-like receptors,47 which can result in the activation of NADPH oxidase, a cytosolic enzyme similar to neutrophil NADPH oxidase, which produces H2O2 independently of NADH and of the mitochondrial respiratory process.48 –51 We have demonstrated that the inflammatory cytokine IL-1␤ increases production of H2O2 in normal human LES muscle. It is possible that, in chronic reflux esophagitis, elevated levels of cytokines such as IL-1␤ may cause up-regulation of NADPH oxidase, which would maintain elevated levels of H2O2 in the circular smooth muscle layer. The restoration of tone of the esophagitis specimen by catalase supports this hypothesis. The most common approach to treatment of GERD is acid suppression therapy. By decreasing the acidity of gastric contents, the refluxed material is rendered less irritating, and the symptoms and mucosal injury improve.52 Although mucosal inflammation is decreased, motor dysfunction of the body of the esophagus and LES continues, consistent with an increased and persistent production of H2O2, as demonstrated in this study. Thus, E-LES has little basal tone and high levels of H2O2 in the circular muscle layer. When muscle strips from this esophagitis specimen were treated with the H2O2 scavenger catalase, tone was restored to levels comparable with those of N-LES muscle. These findings suggest that persistent inflammationinduced production of H2O2 plays a major role in the motor dysfunction in LES muscle. The overproduction of H2O2 could be secondary to up-regulation of H2O2producing enzymes such as NADPH oxidase, as demonstrated in this investigation.

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Our conclusions are illustrated in Figure 10, which includes other data from our laboratory: H2O2 increases in LES (or in esophageal) circular muscle in response to esophagitis-associated increases in IL-1␤ (as shown in the present investigation) or of IL-653 or in response to other inflammatory mediators. H2O2, in turn, increases production of PAF, PGE2, and isoprostanes, all contributing to decreased LES tone. In addition, H2O2 inhibits Ca2⫹ATPase activity, preventing calcium uptake in intracellular stores and resulting in depletion of releasable calcium,37,54 which may also decrease LES tone.38 In summary, we report that H2O2 plays a central role in mediating the decrease in LES tone characteristically found in GERD. We demonstrate that scavenging H2O2 reverses this decrease, pointing to inhibition of ROS as a novel therapeutic approach focused on restoring LES function.

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Received January 12, 2005. Accepted March 16, 2005. Address requests for reprints to: Piero Biancani, M.D., RI Hospital, Gastrointestinal Motor Function Research Laboratory, 55 Claverick St, Room 333, Providence, Rhode Island 02903. e-mail: piero_biancani@ brown.edu; fax: (401) 444-5890. Supported by NIH grant RO1-0K-57030.

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