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 (NH 2 Cl), to serve as agonists of Ca 2+ accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for Ca 2+ 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 Zn 2+ , for which these reporters have affinity. Exposure to NH 2 Cl (up to 200 µM) led to dose-dependent increases in intracellular Ca 2+ concentration ([Ca 2+ ] 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 NH 2 Cl arrested but did not reverse accumulation of Ca 2+ in the cytoplasm. In contrast, introduction of DTT or N -acetylcysteine permitted arrest and partial reversal of the effects of NH 2 Cl. Accumulation of Ca 2+ in the cytoplasm induced by NH 2 Cl is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. Ca 2+ -handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca 2+ ] i . Under certain conditions, NH 2 Cl may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-NH 2 Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents. calcium; Helicobacter pylori ; oxidative stress Address for reprint requests and other correspondence: D. I. Soybel, Dept. of Surgery, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (e-mail: dsoybel{at}partners.org )