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Monochloramine (NH2Cl) is a potent, thiol-directed oxidant capable of oxidizing thiol (S-H) residues in a wide variety of proteins. Generated in the stomach by the interaction of bacterial and host products, monochloramine has been shown to dysregulate Ca2+ homeostasis and disrupt mucosal integrity. In this report, we show that monochloramine also leads to disturbances in intracellular free zinc concentration ([Zn2+]i) in the gastric gland of the rabbit and that the increased Zn2+ within the cell causes an independent decrease in cell viability. Changes in [Zn2+]i were measured by using the fluorescent reporter FluoZin-3, whereas cell viability was assessed by measuring the conversion of calcein-AM to fluorescent calcein, an assay that is not affected by intracellular oxidation state. Cell death was confirmed using propidium iodide and YO-PRO-1 dye uptake measurements. Our experiments demonstrate that [Zn2+]i is increased in gastric glands exposed to NH2Cl and that elevated [Zn2+]i decreases cell viability. Chelation of Zn2+ with tetrakis-(2-pyridylmethyl) ethylenediamine decreases the toxicity of NH2Cl, but only when administered concurrently. These findings suggest that the toxic effect of thiol oxidants present during chronic gastritis is partially due to dysregulation of [Zn2+]i early in the process and that zinc chelation can protect, but not rescue, gastric glands exposed to toxic doses of NH2Cl. [ABSTRACT FROM AUTHOR]

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 NH2CI (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 NH2CI 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 NH2CI. Accumulation of Ca2+ in the cytoplasm induced by NH2CI 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, NH2CI 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. [ABSTRACT FROM AUTHOR]