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Monochloramine (N[H.sub.2]Cl) 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 [Ca.sup.2+] homeostasis and disrupt mucosal integrity. In this report, we show that monochloramine also leads to disturbances in intracellular free zinc concentration ([[[Zn.sup.2+]].sub.i]) in the gastric gland of the rabbit and that the increased [Zn.sup.2+] within the cell causes an independent decrease in cell viability. Changes in [[[Zn.sup.2+]].sub.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 [[[Zn.sup.2+]].sub.i] is increased in gastric glands exposed to N[H.sub.2]Cl and that elevated [[[Zn.sup.2+]].sub.i] decreases cell viability. Chelation of [Zn.sup.2+] with tetrakis-(2-pyridylmethyl) ethylenediamine decreases the toxicity of N[H.sub.2]Cl, 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 [[[Zn.sup.2+]].sub.i] early in the process and that zinc chelation can protect, but not rescue, gastric glands exposed to toxic doses of N[H.sub.2]Cl. gastritis; thiol oxidation; zinc doi: 10.1152/ajpgi.00355.2009.

Secretory compartments of neurons, endocrine cells, and exocrine glands are acidic and contain high levels of labile [Zn.sup.2+]. Previously, we reported evidence that acidity is regulated, in part, by the content of [Zn.sup.2+] in the secretory [i.e., tubulovesicle (TV)] compartment of the acid-secreting gastric parietal cell. Here we report studies focusing on the mechanisms of [Zn.sup.2+] transport by the TV compartment in the mammalian (rabbit) gastric parietal cell. Uptake of [Zn.sup.2+] by isolated TV structures was monitored with a novel application of the fluorescent [Zn.sup.2+] reporter N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ). Uptake was suppressed by removal of external ATP or blockade of [H.sup.+]-[K.sup.+]-ATPase that mediates luminal acid secretion. Uptake was diminished with dissipation of the proton gradient across the TV membrane, suggesting [Zn.sup.2+]/[H.sup.+] antiport as the connection between [Zn.sup.2+] uptake and acidity in the TV lumen. In isolated gastric glands loaded with the reporter fluozin-3, inhibition of [H.sup.+]-[K.sup.+]-ATPase arrested the flow of [Zn.sup.2+] from the cytoplasm to the TV compartment and secretory stimulation with forskolin enhanced vectorial movement of cytoplasmic [Zn.sup.2+] into the tubulovesicle/lumen (TV/L) compartment. Our findings suggest that [Zn.sup.2+] accumulation in the TV/L compartment is physiologically coupled to secretion of acid. These findings offer novel insight into mechanisms regulating [Zn.sup.2+] homeostasis in the gastric parietal cell and potentially other cells in which acidic subcellular compartments serve signature functional roles. gastric gland; tubulovesicles; zinc; acidity regulation doi: 10.1152/ajpcell.00577.2008

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 (N[H.sub.2]C1), to serve as agonists of [Ca.sup.2+] accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for [Ca.sup.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.sup.2+], for which these reporters have affinity. Exposure to N[H.sub.2]Cl (up to 200 [micro]M) led to dose-dependent increases in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.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 N[H.sub.2]Cl arrested but did not reverse accumulation of [Ca.sup.2+] in the cytoplasm. In contrast, introduction of DTT or N-acetylcysteine permitted arrest and partial reversal of the effects of N[H.sub.2]Cl. Accumulation of [Ca.sup.2+] in the cytoplasm induced by N[H.sub.2]Cl is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. [Ca.sup.2+]-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [[[Ca.sup.2+]].sub.i]. Under certain conditions, N[H.sub.2]Cl may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-N[H.sub.2]Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents. calcium; Helicobacter pylori; oxidative stress

During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (N[H.sub.2]Cl), to serve as agonists of [Ca.sup.2+] and [Zn.sup.2+] accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to [Ca.sup.2+] and [Zn.sup.2+] in the cytoplasm. Responses to different concentrations of N[H.sub.2]Cl (50, 100, and 200 [micro]M) were monitored. Subsequent studies were designed to identify the sources and mechanisms of N[H.sub.2]Cl-induced increases in [Ca.sup.2+] and [Zn.sup.2+] in the cytoplasm. Exposure to N[H.sub.2]Cl led to dose-dependent increases in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]]i) in the range of 200-400 nM above baseline levels. Further studies indicated that N[H.sub.2]Cl-induced accumulation of [Ca.sup.2+] in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular [Ca.sup.2+] through store-operated channels. In addition, exposure to N[H.sub.2]Cl resulted in dose-dependent and sustained increases in intracellular [Zn.sup.2+] concentration ([[Zn.sup.2+]]i) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that [Ca.sup.2+]- and [Zn.sup.2+]-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [[Ca.sup.2+]]i and [[Zn.sup.2+]]i. Under certain conditions, N[H.sub.2]Cl may act not as a toxin but as an agent that activates intracellular signaling pathways. key words

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and post lactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance. [ABSTRACT FROM AUTHOR]