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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]

Adipose tissue is an important energy depot and endocrine organ, and the degree of adiposity impacts the host response to infection. However, little is known regarding the mechanisms by which white adipose tissue (WAT) is lost acutely and then restored after the resolution of sepsis. Therefore, the signaling pathways governing protein synthesis, autophagy, apoptosis, and the ubiquitin-proteasome were investigated to identify potential mechanisms mediating the acute (24 h) loss of WAT after cecal ligation and puncture as well as the failure to replenish WAT during recovery (day 10). While whole body fat mass was decreased equally in pair-fed control and septic mice at 5 days after cecal ligation and puncture, fat mass remained 35% lower in septic mice at day 10. During sepsis-recovery, protein synthesis in epididymal WAT was increased compared with control values, and this increase was associated with an elevation in eukaryotic translation initiation factor (eIF)2Bε but no change in mammalian target of rapamycin complex 1 activity (eIF4E-binding protein-1 or S6 kinase 1 phosphorylation). Protein breakdown was increased during sepsis-recovery, as evidenced by the elevation in ubiquitin-proteasome activity. Moreover, indexes of autophagy (light chain 3B-II, autophagy-related protein 5/12, and beclin) were increased during sepsis-recovery and associated with increased AMP-activated kinase-dependent Ser555-phosphorylated Unc-51-like autophagy activating kinase-1. Apoptosis was increased, as suggested by the increased cleavage of caspase-3 and poly(ADP-ribose) polymerase. These changes were associated with increased inflammasome activity (increased NLR family, pyrin domain containing 3; TMS1; and caspase-1 cleavage) and the endoplasmic reticulum stress response (increased eIF2α and activating transcription factor-4) and browning (uncoupling protein-1) in epididymal WAT. Our data suggest that WAT stores remain depleted during recovery from sepsis due to sustained inflammation and elevations in protein and cellular degradation, despite the increase in protein synthesis. [ABSTRACT FROM AUTHOR]

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]

Secretory compartments of neurons, endocrine cells, and exocrine glands are acidic and contain high levels of labile Zn2t Previously, we reported evidence that acidity is regulated, in part, by the content of Zn2+ in the secretory [i.e., tubulovesicle (TV)] compartment of the acid-secreting gastric parietal cell. Here we report studies focusing on the mechanisms of Zn2+ transport by the TV compartment in the mammalian (rabbit) gastric parietal cell. Uptake of Zn2+ by isolated TV structures was monitored with a novel application of the fluorescent Zn2+ reporter N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ). Uptake was suppressed by removal of external AlP or blockade of H+-K+-ATPase that mediates luminal acid secretion. Uptake was diminished with dissipation of the proton gradient across the TV membrane, suggesting Zn2+/H+ antiport as the connection between Zn2+ uptake and acidity in the TV lumen. In isolated gastric glands loaded with the reporter fluozin-3, inhibition of H+-K+-ATPase arrested the flow of Zn2+ from the cytoplasm to the TV compartment and secretory stimulation with forskolin enhanced vectorial movement of cytoplasmic Zn2+ into the tubulovesicle/lumen (TV/L) cornpartment. Our findings suggest that Zn2+ accumulation in the TV/L compartment is physiologically coupled to secretion of acid. These findings offer novel insight into mechanisms regulating Zn2+ homeostasis in the gastric parietal cell and potentially other cells in which acidic subcellular compartments serve signature functional roles. [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]

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 (NH2CI), to serve as agonists of Ca2+ and Zn2+ 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 Ca2+ and Zn2+ in the cytoplasm. Responses to different concentrations of NH2Cl (50, 100, and 200 µM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH2Cl-induced increases in Ca2+ and Zn2+ in the cytoplasm. Exposure to NH2CI led to dose-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) in the range of 200-400 nM above baseline levels. Further studies indicated that NH2CI-induced accumulation of Ca2+ in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular Ca2+ through store-operated channels. In addition, exposure to NH2CI resulted in dose-dependent and sustained increases in intracellular Zn2+ concentration ([Zn2+]i) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that Ca2+- and Zn2+-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [Ca2+]i and [Zn2+]i. Under certain conditions, NH2CI may act not as a toxin but as an agent that activates intracellular signaling pathways. [ABSTRACT FROM AUTHOR]

Presents a study using reverse transcriptase-polymerase chain reaction to generate a 1,200-bp fragment specific to a basolateral isoform of the Na+-K+-Cl- cotransporter in the gastric fundus of Necturus maculosus. Findings suggesting that this basolateral transporter has a dominant and previously unsuspected role in secretion of HCL across the apical membrane.

Examines the genetic properties and localization of extracellular Ca2+-sensing receptor (CaR) in small and large intestine of rats. Distribution of CaR mRNA in small and large intestine by RT-PCR; Distribution of CaR mRNA in various intestinal segments by Northern blot analysis; Immunocytochemistry of CaR in small and large intestine.