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Background and Aims: Recent work has suggested that Zn(2+) plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn(2+) in gastric mucosa under baseline conditions and its regulation during secretory stimulation., Methods and Findings: Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn(2+) were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, (70)Zn(2+), from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of (70)Zn(2+) from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn(2+) was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn(2+) {[Zn(2+)](i)} during exposure to standard extracellular concentrations of Zn(2+) (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn(2+) increased with exposure to secretagogues (forskolin, carbachol/histamine) and under conditions associated with increased intracellular Ca(2+) {[Ca(2+)](i)}. Uptake of Zn(2+) was abolished following removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores, suggesting that demand for extracellular Zn(2+) increases and depends on influx of extracellular Ca(2+)., Conclusions: This study is the first to characterize the content and distribution of Zn(2+) in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca(2+) integrates basolateral demand for Zn(2+) with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues.

Background & Aims: Until recently, it has not been possible to evaluate factors that regulate the acidity of the microenvironment within the tubulovesicles and luminal (TV/L) spaces of the gastric gland. The goal of this study was to develop a method for monitoring the mechanisms that regulate acidity in the TV/L compartment., Methods: Isolated rabbit gastric glands (intact or permeabilized with S. aureus alpha-toxin) were loaded with a recently characterized fluorescent dye, LysoSensor Yellow-Blue DND 160 (Molecular Probes, Eugene, OR), which localizes to highly acidic compartments and can be used to monitor acidity ratiometrically., Results: In resting glands, the pH of the TV/L compartment was approximately 3.4. Moderate alkalizations ( approximately 0.5 to 1.0 pH unit alkalization) were observed during exposure to inhibitors of the apical H(+)/K(+) ATPase (omeprazole and SCH28080), thereby unmasking a stable, low-level leak of H(+) ions from the TV/L compartment. Similar changes were observed in alpha-toxin permeabilized glands following depletion of ATP in the cytoplasm. In intact and permeabilized glands, we used the cell-permeant, divalent cation chelator, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) to probe the effects of lowering divalent cation content of the TV/L compartment. Exposure to relatively low concentrations (20 micromol/L, 50 micromol/L) of TPEN reversibly promoted H(+) leakage. At these concentrations, simultaneous inhibition using SCH28080 led to marked enhancement of the rate of alkalization., Conclusions: The effects of low-dose TPEN suggests that acidity within the TV/L compartment of the gastric gland may be regulated, at least in part, by its content of divalent cations such as Zn(2+), for which TPEN has high affinity.

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]

We recently proposed that extracellular Ca2+ ions participate in a novel form of intercellular communication involving the extracellular Ca2+-sensing receptor (CaR). Here, using Ca2+-selective microelectrodes, we directly measured the profile of agonist-induced [Ca2+]ext changes in restricted domains near the basolateral or luminal membranes of polarized gastric acid-secreting cells. The Ca2+-mobilizing agonist carbachol elicited a transient, La3+-sensitive decrease in basolateral [Ca2+] (average ≈250 μM, but as large as 530 μM). Conversely, carbachol evoked an HgCl2- sensitive increase in [Ca2+] (average ≈400 μM, but as large as 520 μM) in the lumen of single gastric glands. Both responses were significantly reduced by pretreatment with sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump inhibitors or with the intracellular Ca2+ chelator BAPTA-AM. Immunofluorescence experiments demonstrated an asymmetric localization of plasma membrane Ca2+ ATPase (PMCA), which appeared to be partially co-localized with CaR and the gastric H+/K+-ATPase in the apical membrane of the acid-secreting cells. Our data indicate that agonist stimulation results in local fluctuations in [Ca2+]ext that would be sufficient to modulate the activity of the CaR on neighboring cells. [ABSTRACT FROM AUTHOR]