Christian J. Stork

Intracellular Zinc Elevation Measured with a “Calcium-Specific” Indicator during Ischemia and Reperfusion in Rat Hippocampus: A Question on Calcium Overload

Much of our current evidence concerning of the role of calcium (Ca2+) as a second messenger comes from its interaction with fluorescent probes; however, many Ca2+ probes also have a higher affinity for another divalent cation: zinc (Zn2+). In this study, using a selective Zn2+ probe (Newport Green), we investigated the accumulation of intracellular Zn2+ transients in acute rat hippocampal slices during ischemia, simulated by oxygen and glucose deprivation (OGD). Subsequent reperfusion with glucose-containing oxygenated medium resulted in an additional increase in intracellular Zn2+. Such observations compelled us to investigate the contribution of Zn2+ to the alleged intracellular Ca2+ overload occurring in ischemia and reperfusion. Using confocal fluorescent microscopy of Calcium Green-1, a widely used Ca2+ indicator, we detected increases in fluorescence intensity during OGD and reperfusion. However, application of a Zn2+ chelator, at the peak of the fluorescence elevation (interpreted as Ca2+ overload), resulted in a significant drop in intensity, suggesting that rising Zn2+ is the primary source of the increasing Calcium Green-1 fluorescence. Finally, staining with the cell viability indicator propidium iodide revealed that Zn2+ is responsible for the ischemic neuronal cell death, because Zn2+ chelation prevented cells from sustaining ischemic damage. Current cellular models of ischemic injury center on Ca2+-mediated excitotoxicity. Our results indicate that Zn2+ elevation contributes to conventionally recognized Ca2+ overload and also suggest that the role of Ca2+ in neurotoxicity described previously using Ca2+ probes may need to be re-examined to determine whether effect previously attributed to Ca2+ could, in part, be attributable to Zn2+.

Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons

Background Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP3-gated thapsigargin sensitive intracellular calcium (Ca2+) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn2+) that has been reported to play a role in cell signaling. Results In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn2+ indicators, we showed that intracellular regions of Zn2+ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn2+ chelator TPEN, indicating that the local Zn2+ fluorescence represented free Zn2+ localized to the ER in the basal condition. Blockade of the ER Ca2+ pump by thapsigargin produced a steady increase of intracellular Zn2+. Furthermore, we determined that the thapsigargin-induced Zn2+ increase was not dependent on extracellular Ca2+ or extracellular Zn2+, suggesting that it was of intracellular origin. The applications of caged IP3 or IP3-3Kinase inhibitor (to increase available IP3) produced a significant increase in intracellular Zn2+. Conclusions Taken together, these results suggest that Zn2+ is sequestered into thapsigargin/IP3-sensitive stores and is released upon agonist stimulation.

Measuring cell viability with membrane impermeable zinc fluorescent indicator

Recent findings suggest that the accumulation of cytoplasmic zinc [Zn2+]i is a ubiquitous component in the cell death cascade. Zn2+ can be liberated from intracellular stores following oxidative stress and contribute to cell death processes. Here we show that the membrane/cell impermeable Zn2+ fluorescent indicator Newport Green (NG), which is non-toxic and impermeable to the membranes of healthy cells, can label unhealthy cells in tissue slices in a manner comparable to the traditional viability indicator propidium iodide (PI). Using confocal microscopy, we detected PI labeled nuclei colocalized with NG fluorescence. Our results indicate that cells which absorbed PI into their nuclei also allowed cell-impermeable Zn2+ dye to penetrate their plasma membranes, subsequently exhibiting cytosolic and nuclear fluorescence. As in PI staining, we observed marked increases in NG fluorescence in damaged/dead cells of tissue slices. Two other cell impermeable fluorescent Zn2+ dyes, Fluozin-3 and Zinpyr-4, also stained cytosolic Zn2+ in PI labeled cells. Our data indicates that the application of a Zn2+ fluorescent indicator is a fast, simple, non-toxic and reliable method for visualizing cell viability within in vitro tissue preparations. Accordingly, we demonstrate that intracellular accumulation of Zn2+ correlates with neuronal death.

UV irradiation‐induced zinc dissociation from commercial zinc oxide sunscreen and its action in human epidermal keratinocytes

Zinc oxide (ZnO) is an active ingredient in sunscreen owing to its properties of broadly filtering the ultraviolet (UV) light spectrum and it is used to protect against the carcinogenic and photodamaging effects of solar radiation on the skin. This study investigated the dissociation of zinc (Zn2+) from ZnO in commercial sunscreens under ultraviolet type B light (UVB) irradiation and assessed the cytotoxicity of Zn2+ accumulation in human epidermal keratinocytes (HEK). Using Zn2+ fluorescent microscopy, we observed a significant increase in Zn2+ when ZnO sunscreens were irradiated by UVB light. The amount of Zn2+ increase was dependent on both the irradiation intensity as well as on the ZnO concentration. A reduction in cell viability as a function of ZnO concentration was observed with cytotoxic assays. In a real‐time cytotoxicity assay using propidium iodide, the treatment of UVB‐irradiated ZnO sunscreen caused a late‐ or delayed‐type cytotoxicity in HEK. The addition of a Zn2+ chelator provided a protective effect against cellular death in all assays. Furthermore, Zn2+ was found to induce the production of reactive oxygen species (ROS) in HEK. Our data suggest that UVB irradiation produces an increase in Zn2+ dissociation in ZnO sunscreen and, consequently, the accumulation of free or labile Zn2+ from sunscreen causes cytotoxicity and oxidative stress.

Exploring the Zinc Metallome of Cultured Cortical Neurons Using Synchroton Radiation X-Ray Fluorescence Microscopy

Zinc is abundant in the brain, and dysregulation of zinc ion homeostasis has been implicated in Alzheimer’s disease pathology and neuronal death after brain ischemia. Many studies have established that cytosolic free zinc ion concentrations are much lower than measured total average cellular concentrations, suggesting a large capacity for intracellular zinc buffering. It is generally believed that protein binding constitutes a major source of intracellular zinc ion buffering. In this study, we explored the subcellular distribution of zinc-binding sites in neurons using synchrotron radiation X-ray fluorescence microscopy. We observed that nearly all the intracellular zinc in resting neurons is bound and that zinc-binding sites were uniformly distributed in neurons resulting in similar average zinc concentrations (≈300 μM) throughout the cell body including the nucleus, peri-nuclear region, and processes. However, sites of elevated zinc concentration were observed in resting neurons, and were prevalent throughout the neuron (including the cell body and processes) when neurons were loaded with zinc. These sites of elevated zinc could represent either zinc bound to proteins or zinc sequestered in subcellular organelles. Additional studies are needed to completely describe the neuronal zinc metallome and the function of zinc-binding proteins in buffering zinc loads.