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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]

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]