Volume 129, Issue 5, Pages 1675-1685 (November 2005) Hydrogen Peroxide Reduces Lower Esophageal Sphincter Tone in Human Esophagitis  Ling Cheng, Karen M. Harnett, Weibiao Cao, Fang Liu, Jose Behar, Claudio Fiocchi, Piero Biancani  Gastroenterology  Volume 129, Issue 5, Pages 1675-1685 (November 2005) DOI: 10.1053/j.gastro.2005.09.008 Copyright © 2005 American Gastroenterological Association Terms and Conditions

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. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

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. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

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. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

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-1β-treated LES samples. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 IL-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. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

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. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

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 μmol/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. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

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 μmol/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, F2α 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. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 8-isoPGF2α selectively reduces PGF2α-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-isoPGF2α alone, PGF2α alone, and PGF2α plus 8-isoPGF2α (10−5 mol/L). PGF2α caused a concentration-dependent contraction of LES circular muscle strips. In contrast, 8-isoPGF2α alone had little effect on tone of normal LES circular muscle strips, but preincubation with 8-isoPGF2α significantly reduced PGF2α-induced contraction (P < .001, ANOVA). Means ± SE are shown for 3 normal LES samples. (B) In esophagitis LES PGF2α did not cause contraction, perhaps because of the higher isoprostane concentration present, even though the esophagitis muscle contracted normally in response to ACh. Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions

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-1β (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 Gastroenterology 2005 129, 1675-1685DOI: (10.1053/j.gastro.2005.09.008) Copyright © 2005 American Gastroenterological Association Terms and Conditions