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METHODS IN CELL PHYSIOLOGY

Thiol-oxidant monochloramine mobilizes intracellular Ca²⁺ in parietal cells of rabbit gastric glands

Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 15 April 2006 ; accepted in final form 27 January 2007

In Helicobacter pylori-induced gastritis, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the gastric mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH₂Cl), to serve as agonists of Ca²⁺ accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for Ca²⁺ in the cytoplasm (fura-2 AM) or intracellular stores (mag-fura-2 AM). Conditions were adjusted to screen out contributions from metal cations such as Zn²⁺, for which these reporters have affinity. Exposure to NH₂Cl (up to 200 µM) led to dose-dependent increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i), in the range of 200–400 nM above baseline levels. These alterations were prevented by pretreatment with the oxidant scavenger vitamin C or a thiol-reducing agent, dithiothreitol (DTT), which shields intracellular thiol groups from oxidation by chlorinated oxidants. Introduction of vitamin C during ongoing exposure to NH₂Cl arrested but did not reverse accumulation of Ca²⁺ in the cytoplasm. In contrast, introduction of DTT or N-acetylcysteine permitted arrest and partial reversal of the effects of NH₂Cl. Accumulation of Ca²⁺ in the cytoplasm induced by NH₂Cl is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. Ca²⁺-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca²⁺]_i. Under certain conditions, NH₂Cl may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-NH₂Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents.

calcium; Helicobacter pylori; oxidative stress