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

Weibiao Cao, Karen M. Harnett, Ling Cheng, Michael T. Kirber, Jose Behar, Piero Biancani

Abstract

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca2+ stores. We therefore measured cytosolic Ca2+ 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 H2O2 to the reduction in Ca2+ signal. In normal cells, the ACh-induced Ca2+ increase was the same in normal-Ca2+ and Ca2+-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 Ca2+ release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced Ca2+ increase in normal-Ca2+ medium was significantly lower in esophagitis than in normal cells and was further reduced (∼70%) when the cells were incubated in Ca2+-free medium. This reduction was partially reversed by the H2O2 scavenger catalase. H2O2 measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with H2O2, the ACh-induced Ca2+ increase was significantly reduced in normal-Ca2+ and Ca2+-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 H2O2. H2O2, when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca2+ signal in normal cells. H2O2-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, H2O2 reduced TG-induced contraction, suggesting that TG and H2O2 may operate through similar mechanisms. Ca2+-ATPase activity measurement indicates that H2O2 and TG reduced Ca2+-ATPase activity, confirming similarity of mechanism of action. We conclude that H2O2 may be at least partly responsible for impairment of Ca2+ release in acute experimental esophagitis by inhibiting Ca2+ uptake and refilling Ca2+ stores.

  • acetylcholine
  • smooth muscle
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