Work reported previously from our laboratory indicates that the transport of Zn2+
by the parietal cell is a critical feature of its ability to regulate acidity within its secretory compartment, which includes the tubulovesicles of the parietal cell and the lumen of the gland. Chelation of Zn2+
within these compartments led to marked alkalization, suggesting that the presence of Zn2+
insulates these compartments against leakage of H+
. Recently we have reported evidence that the secretory compartments serve as a reservoir for Zn2+
, accumulating it in response to proton gradients and failing to accumulate it when secretion is blocked by proton pump inhibition 
The studies reported here provide three sets of observations with respect to utilization of zinc within the gastric mucosa: first, as demonstrated by μXRF, that zinc accumulates differently in distinct micro-anatomic regions of the mucosa; second, as observed following in vivo intravenous injection, the non-radioactive isotope 70Zn2+ accumulates readily within both gastric mucosa and in the acid-secreting gastric glands; and third, as suggested by in vitro studies of isolated gastric glands, that exposure to well-recognized secretory agonists accelerates the uptake of Zn2+ from the nutrient side of the epithelium and requires intact intracellular stores of Ca2+. The studies reported here refine and extend the concept that demand for Zn2+ from the circulation and nutrient compartment may be regulated acutely by alterations in H+ secretion to the lumen.
Heterogeneous distribution of Zn2+ within the gastric mucosa
To our knowledge, this is the first report of the use of μXRF to evaluate the distribution of Zn2+
and other cations in the mucosa of a specific region of the gastrointestinal tract. The basis of the method is fluorescence produced by synchrotron-derived x-rays, which can be focused to localize elemental content within isolated cells 
or tissues 
. In vitro
, quantification within individual cells and resolution of individual isotopes of different metals is feasible 
. However, interpretation of differences in distribution within tissues depends on unique structural features 
or co-localizing markers 
for cellular or sub-cellular areas of interest.
The gastric mucosa is divided into two spatially and functionally distinct regions 
: the gastric glands, which secrete acid and pepsinogen; and the surface epithelium, which secretes mucus and bicarbonate and is thought to provide the protective barrier against back-diffusion of luminal H+
ions. Within the gastric gland, the region toward the surface is populated with mucus neck cells; the middle region is dominated by the acid-secreting parietal cells, and the deepest regions harbor pepsinogen-secreting chief cells 
. The precise localization and quantification of metal signals in the stomach awaits development of co-localizing markers suitable for use in tissues prepared for μXRF. However, the distinct histologic structure of the mucosa permits general conclusions regarding distribution of zinc in the regions of the gastric glands and the surface epithelium. Our μXRF studies thus demonstrate a consistent signal for zinc within the glandular regions. At the same time they do not suggest major variation within different regions of the gastric gland.
Curiously, we find higher levels of Zn2+ present at the interface of the lumen and the surface epithelium ( and ), more consistently than other metals such as Cu2+ and Fe2+, and not in conjunction with other intracellular cations (K+, Ca2+) that are more likely to be free and labile than bound. This observation offers the possibility that Zn2+ is secreted with the mucus by the surface epithelium or, possibly, is selectively trapped within the mucus layer after discharge into the gastric lumen with secretion. It is tempting to speculate that associations of Zn2+ or other metals with the mucin layer might be important in its structure or ability to resist back-diffusion of H+ ions. The development of a method for evaluating distribution of metal ion species in a complex and highly hydrated mucosal surface provides opportunities to evaluate alterations in content and distribution of metal ion species under pathologically relevant conditions, for example, in response to systemic stress or during infestation with pathogenic organisms such as Helicobacter pylori.
Demonstrating and monitoring the demand for Zn2+: in vivo and in vitro studies
The concept of “demand” for any nutrient is based on the need of a cell, tissue or organism to maintain homeostasis. In studies utilizing sector field ICP-MS, following injection of a non-radioactive and naturally scarce isotope, 70Zn2+, uptake is demonstrated within the mucosa as a whole and in individual glands. Such movements can be tracked within hours of injection into the circulation and provide evidence that demands of the tissues are readily replenished by movement of Zn2+ from the circulation into the mucosa.
To more precisely characterize the avidity of individual glands, we developed an in vitro assay to monitor uptake of 70Zn2+ by isolated glands, observing that it can be detected within minutes when extracellular concentrations are in the 10 nanomolar range and very consistently when concentrations are 100 nM. Moreover, rates of uptake are accelerated during stimulation with a powerful secretory agonist, forskolin. The use of rare, non-radioactive isotopes such as 70Zn2+ permits investigation of Zn2+ distribution into whole tissues and cell preparations, without the restrictions required when radioactive isotopes (i.e., 65Zn2+) would be used. The specificity of these methods for individual metal species not only permits quantification of rates of entry into cells and tissues, but also increases the confidence in designing studies utilizing potentially less specific reporters of Zn2+ movements, such as the fluorescent reporter fluozin-3.
Connection between intracellular Ca2+ and uptake of Zn2+ across the basolateral membrane of the gastric parietal cell
Based on studies with 70Zn2+, we were able to design experiments to study conditions of uptake under baseline conditions and during exposure to well-recognized secretory agonists, using utilizing relatively inexpensive and rapidly responsive fluorescence imaging and fluorometric methods. Utilizing these methods, we were able to confirm enhanced uptake of Zn2+ across the basolateral membrane during secretory stimulation. In addition, we found that manipulation of [Ca2+]i homeostasis leads to alterations in the ability of the parietal cell to take up and preserve [Zn2+]i. Thus, intracellular accumulation of Zn2+ is nearly abolished when intracellular stores of Ca2+ are depleted by exposure to thapsigargin and Ca2+-depleted Ringer's. Following exposure to thapsigargin, uptake of Zn2+ was observed—albeit at a reduced level—if Ca2+ is present in the extracellular solution. This uptake was arrested when the chelator DTPA (10 µM) was added. These observations indicate that the increases in [Zn2+]i are indeed due to influx from extracellular sources and not release from intracellular stores. They argue that, under baseline conditions, uptake of Zn2+ across the basolateral membrane depends on adequate stores of intracellular Ca2+. With stimulation by powerful agonists such as forskolin and carbachol, demand for extracellular Zn2+ increases and depends on influx of extracellular Ca2+. In addition, these studies illustrate physiologically relevant approaches for using both real-time imaging of individual glands, which is time-intensive, and 96-well platforms, which would be amenable to rapid through-put approaches for small molecule screens.
Additional considerations: technical
The fluorescent reporter utilized here was fluozin-3, which has been utilized in its free acid form for monitoring extracellular secretion of Zn2+
from insulin-secreting beta cells 
. When conjugated to an acetoxymethyl ester group, it has proven useful for monitoring intracellular concentrations of free Zn2+
] in different cell types 
, including epithelial cells of the gastrointestinal tract 
. Importantly, this reporter is unresponsive to Ca2+
under a number of experimentally relevant conditions 
. However, it is possible for release or sequestration of other divalent cations to influence responses of the reporter 
. It is for this reason that caution must be used in applying relationships that have been used to calculate free concentration of Zn2+
from the reported Kd
and from maximum and minimum fluorescence responses, as was originally proposed for Ca2+
. Thus, we have refrained from providing direct estimates of [Zn2+
, based on fluozin-3 measurements.
Additional considerations: connection between intracellular Ca2+ and movements of Zn2+ across the basolateral membrane of the gastric parietal cell
In the current set of studies, we find that Ca2+
facilitates optimal uptake of Zn2+
across the cell membrane, implying that it is either a counter-ion in exchange or it is acting as a regulatory second-messenger. Membrane proteins that facilitate Zn2+
transport constitute the SLC30A (ZnT) and SLC39A (Zip) gene families 
. To date, fourteen proteins that facilitate import of Zn2+
to the cytoplasm (either from extracellular sources or possibly intracellular compartments) have been identified in mammals 
. Current information on the mechanisms of Zn2+
transport does not implicate Ca2+
in the former role and emerging information for ZIP, ZnT and related transporters in yeast suggest that counter exchanging ions are likely to be protons 
. Based on the well-recognized role of Ca2+
as a second messenger in responses to secretagogues 
, our findings offer the novel conclusion that Ca2+
is a second messenger that can match the basolateral demand for Zn2+
with the secretory response to physiologic stimulation. It seems likely that similar connections will be found in other secretory cells and tissues—neural, endocrine and exocrine.
Additional considerations: pathophysiological implications of Ca2+-regulated Zn2+ uptake
Compared to levels of free Ca2+
, the amount of free Zn2+
within the cytoplasm is even more tightly controlled. Our studies have suggested that, in different epithelial cells, the baseline concentration of [Zn2+
is in the nanomolar range 
. This consideration alone suggests tight regulation of the activities of transporters that modulate demand for Zn2+
and its disposal from the cytoplasm. Observations in other cell types such as neurons suggest that exposure to elevated levels of Zn2+
can be cytotoxic, within minutes 
. Preliminary studies in epithelial cells of the gastrointestinal tract confirm that oxidant stress induces increases in [Zn2+
that can influence pathways of cell death and the balance between necrosis and apoptosis 
. Dysregulation of Ca2+
homeostasis is also a consequence of such oxidant-related stress 
. The findings of the current study suggest that oxidative dysregulation of intracellular Zn2+
could be amplified by dysregulation of intracellular Ca2+
homeostasis. Further studies will help to clarify this potential relationship, not only in gastric mucosa but in other secretory cell types or tissues.