FluoZin-3 Fluorescence Research Articles

Dietary Zinc Supplementation in Steers Modulates Labile Zinc Concentration and Zinc Transporter Gene Expression in Circulating Immune Cells.

Zinc (Zn) is critical for immune function, and marginal Zn deficiency in calves can lead to suboptimal growth and increased disease susceptibility. However, in contrast to other trace minerals such as copper, tissue concentrations of Zn do not change readily in conditions of supplementation or marginal deficiency. Therefore, the evaluation of Zn status remains challenging. Zinc transporters are essential for maintaining intracellular Zn homeostasis, and their expression may indicate changes in Zn status in the animal. Here, we investigated the effects of dietary Zn supplementation on labile Zn concentration and Zn transporter gene expression in circulating immune cells isolated from feedlot steers. Eighteen Angus crossbred steers (261 ± 14 kg) were blocked by body weight and randomly assigned to two dietary treatments: a control diet (58 mg Zn/kg DM, no supplemental Zn) or control plus 150 mg Zn/kg DM (HiZn; 207 mg Zn/kg DM total). After 33 days, Zn supplementation increased labile Zn concentrations (as FluoZin-3 fluorescence) in monocytes, granulocytes, and CD4 T cells (P < 0.05) but had the opposite effect on CD8 and γδ T cells (P < 0.05). Zn transporter gene expression was analyzed on purified immune cell populations collected on days 27 or 28. ZIP11 and ZnT1 gene expression was lower (P < 0.05) in CD4 T cells from HiZn compared to controls. Expression of ZIP6 in CD8 T cells (P = 0.02) and ZnT7 in B cells (P = 0.01) was upregulated in HiZn, while ZnT9 tended (P = 0.06) to increase in B cells from HiZn. These results suggest dietary Zn concentration affects both circulating immune cell Zn concentrations and Zn transporter gene expression in healthy steers.

Chloroquine disrupts zinc storage granules in primary Malpighian tubule cells of Drosophila melanogaster.

Contrasting reports exist in the literature regarding the effect of chloroquine treatment on cellular zinc uptake or secretion. Here, we tested the effect of chloroquine administration in the Drosophila model organism. We show that larvae grown on a diet supplemented with 2.5mg/ml chloroquine lose up to 50% of their stored zinc and around 10% of their total potassium content. This defect in chloroquine-treated animals correlates with the appearance of abnormal autophagolysosomes in the principal cells of the Malpighian tubules, where zinc storage granules reside. We further show that the reported increase of Fluozin-3 fluorescence following treatment of cells with 300 μM chloroquine for 1 h may not reflect increased zinc accumulation, since a similar treatment in Madin-Darby canine kidney cells results in a 36% decrease in their total zinc content. Thus, chloroquine should not be considered a zinc ionophore. Zinc supplementation plus chloroquine treatment restored zinc content both in vivo and in vitro, without correcting autophagic or other ionic alterations, notably in potassium, associated with the chloroquine treatment. We suggest that chloroquine or hydroxychloroquine administration to patients could reduce intracellular zinc storage pools and be part of the drug's mechanism of action.

The ZIP3 Zinc Transporter Is Localized to Mossy Fiber Terminals and Is Required for Kainate-Induced Degeneration of CA3 Neurons

Tight regulation of neuronal Zn2+ is critical for physiological function. Multiple Zn2+ transporters are expressed in the brain, yet their spatial distribution and distinct roles are largely unknown. Here, we show developmental regulation of the expression of Zn2+ transporters ZIP1 and ZIP3 in mouse hippocampal neurons, corresponding to previously described increase in neuronal vesicular Zn2+ during the first postnatal month. Rates of Zn2+ uptake in cultured mouse hippocampal neurons, monitored using FluoZin-3 fluorescence, were higher in mature neurons, which express higher levels of ZIP1 and ZIP3. Zn2+ uptake was attenuated by ∼50% following silencing of either ZIP1 or ZIP3. Expression of both ZIP1 and ZIP3 was ubiquitous on somas and most neuronal processes in the cultured neurons. In contrast, we observed distinct localization of the transporters in adult mouse hippocampal brain, with ZIP1 predominantly expressed in the CA3 stratum pyramidale, and ZIP3 primarily localized to the stratum lucidum. Consistent with their localization, silencing of ZIP1 expression in vivo reduced Zn2+ uptake in CA3 neurons while ZIP3 silencing reduced Zn2+ influx into dentate gyrus (DG) granule cells in acute hippocampal slices. Strikingly, in vivo silencing of ZIP3, but not ZIP1, protected CA3 neurons from neurodegeneration following kainate-induced seizures. Our results indicate that distinct Zn2+ transporters control Zn2+ accumulation and toxicity in different neuronal populations in the hippocampus and suggest that selective regulation of Zn2+ transporters can prevent seizure induced brain damage.SIGNIFICANCE STATEMENT Zinc plays a major role in neuronal function and its dysregulation is associated with neurodegeneration. Multiple zinc transporters are expressed in neurons, yet little is known on their distinct roles. Here, we show that the plasma membrane ZIP1 and ZIP3 zinc transporters are expressed on distinct neuronal populations in the CA3 region of the hippocampus. We show that ZIP1 mediates zinc influx into postsynaptic cells, while ZIP3 is responsible for zinc re-uptake from this synapse into dentate granule cells. We further show that silencing of ZIP3, but not ZIP1, can rescue the postsynaptic cells from kainate-induced neurodegeneration. This suggests that neuronal zinc toxicity and degeneration can be modulated by regulation of specific zinc transporters function.

Zinc ionophores isolated from Terminalia bellirica fruit rind extract protect against cardiomyocyte hypoxia/reoxygenation injury

The study aimed to isolate and characterize zinc ionophores from Terminalia bellirica fruit using a liposome assay and test its utility in H9c2 rat cardiomyoblasts cells subjected to hypoxia/reoxygenation. Ethyl acetate extract that exhibited zinc ionophore activity was resolved to yield three polyphenols that were characterized as epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and epigallocatechin (EGC) by nuclear magnetic resonance and electrospray ionization-mass spectra. The polyphenols enhanced the uptake of zinc into the liposomes and increased FluoZin-3 fluorescence. These polyphenols in the presence of 10 μM ZnCl2 enhanced the zinc import into H9c2 cells, whose intracellular zinc levels were otherwise lowered upon hypoxia/reoxygenation. EGCG proved to be more potent than ECG, which indeed was more effective than EGC in improving cellular zinc levels and in attenuating the apoptosis of H9c2 cells after hypoxia/reoxygenation injury. The polyphenols required zinc for anti-apoptotic effect. The cardioprotective effect is indeed due to enhanced zinc uptake mediated by these polyphenols.

Thiopental Elevates Steady-State Levels of Intracellular Ca2+ and Zn2+ in Rat Thymic Lymphocytes

Thiopental is an ultra-short-acting barbiturate and has been used commonly in the induction phase of general anesthesia. However, the toxic effect of thiopental is not completely clear. The effect of thiopental on intracellular Ca2+ ([Ca2+]i) levels was investigated in non-excitable cells. Experiments were carried out using a flow-cytometric technique, rat thymic lymphocytes (as non-excitable cells), and appropriate fluorescent probes. Treatment of cells with 300 µM thiopental increased Fluo-3 fluorescence intensity, indicating elevation of [Ca2+]i. This increase was partially attenuated by a chelator of intracellular Zn2+. Thus, thiopental elevated both [Ca2+]i and intracellular Zn2+ ([Zn2+]i) levels. Under intracellular Zn2+-free conditions, 100–300 µM thiopental was still able to induce a statistically significant increase in [Ca2+]i, whereas removal of extracellular Ca2+ greatly reduced the increase in [Ca2+]i induced by this dose of thiopental. Therefore, the thiopental-induced increase in [Ca2+]i was mainly due to an increased influx of Ca2+. Treatment of cells with 300 µM thiopental increased FluoZin-3 fluorescence intensity, indicating the presence of [Zn2+]i, both in the presence and absence of extracellular Zn2+. The thiopental-induced elevation of [Zn2+]i was due to an increase in both influx of Zn2+ and intracellular Zn2+ release. Concanavalin A (10 µg/mL) augmented Fluo-3 fluorescence in the presence of an intracellular Zn2+ chelator. The combination of concanavalin A and 100–300 µM thiopental synergistically increased [Ca2+]i. Results suggest that thiopental increases [Ca2+]i in both quiescent and activated lymphocytes, possibly resulting in modulation of immune system function.&#x0D;

Agonist-Evoked Increases in Intra-Platelet Zinc Couple to Functional Responses.

Background Zinc (Zn 2+ ) is an essential trace element that regulates intracellular processes in multiple cell types. While the role of Zn 2+ as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated. Objectives This article determines whether cytosolic Zn 2+ concentrations ([Zn 2+ ] i ) change in platelets in response to agonist stimulation, in a manner consistent with a secondary messenger, and correlates the effects of [Zn 2+ ] i changes on activation markers. Methods Changes in [Zn 2+ ] i were quantified in Fluozin-3 (Fz-3)-loaded washed, human platelets using fluorometry. Increases in [Zn 2+ ] i were modelled using Zn 2+ -specific chelators and ionophores. The influence of [Zn 2+ ] i on platelet function was assessed using platelet aggregometry, flow cytometry and Western blotting. Results Increases of intra-platelet Fluozin-3 (Fz-3) fluorescence occurred in response to stimulation by cross-linked collagen-related peptide (CRP-XL) or U46619, consistent with a rise of [Zn 2+ ] i . Fluoresence increases were blocked by Zn 2+ chelators and modulators of the platelet redox state, and were distinct from agonist-evoked [Ca 2+ ] i signals. Stimulation of platelets with the Zn 2+ ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn 2+ ] i , resulting in myosin light chain phosphorylation, and cytoskeletal re-arrangements which were sensitive to cytochalasin-D treatment. Cq stimulation resulted in integrin α IIb β 3 activation and release of dense, but not α, granules. Furthermore, Zn 2+ -ionophores induced externalization of phosphatidylserine. Conclusion These data suggest that agonist-evoked fluctuations in intra-platelet Zn 2+ couple to functional responses, in a manner that is consistent with a role as a secondary messenger. Increased intra-platelet Zn 2+ regulates signalling processes, including shape change, α IIb β 3 up-regulation and dense granule release, in a redox-sensitive manner.

Ternary Zn(II) Complexes of FluoZin-3 and the Low Molecular Weight Component of the Exchangeable Cellular Zinc Pool.

Knowledge of the nature of exchangeable (labile) intracellular Zn(II) is increasingly important for biomedical research. The detection and quantitative determination of Zn(II) ions is usually performed by using Zn(II)-specific fluorescent sensors, among which 2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) has been used most widely. Selectivity of this sensor for Zn(II) over other divalent cations was demonstrated, but possible interference in its performance by other compounds has not been investigated. Many potential low molecular weight ligands for Zn(II) ions (LMWLs) are abundant in the cell. In this study we demonstrate that FluoZin-3 is susceptible to competition for Zn(II) from LMWLs and also forms strong ternary complexes with some of them. We determined the set of conditional stability constants C Ktern for ternary Zn(FluoZin-3)(LMWL) complexes using fluorescence titrations and applied it to model the response of exchangeable zinc to FluoZin-3. We found that competition and formation of ternary complexes with LMWLs together strongly affect (net reduce) the Zn(FluoZin-3) fluorescence. This effect may cause a significant underestimation of exchangeable Zn(II). We also demonstrated a strong pH dependence of this effect. Reduced glutathione (GSH) emerged as the most important Zn(II) partner among the LMWLs, characterized with Ktern = 2.8 ± 0.2 × 106 M-1. Our experiments and calculations suggest that Zn(LMWL) complexes contribute to the exchangeable cellular zinc pool.

Zinc Dyshomeostasis in Cardiomyocytes after Acute Hypoxia/Reoxygenation.

Zinc dyshomeostasis may play a role in the pathogenesis of myocardial ischemia/reperfusion injury. The objective of this study was to investigate the expression profile of zinc regulated transporter like- and iron-regulated transporter-like proteins (ZIPs) and zinc transporter proteins (ZnTs) in cardiomyocytes and their modulation in response to hypoxia and reoxygenation. Adult rat ventricular myocytes (ARVMs) were subjected to 6h of hypoxia, followed by 18h of reoxygenation. Intracellular and extracellular zinc concentrations were determined using Fluozin-3 and Newport Green fluorescence, respectively. Expression of ZnTs 1, 2, 5, and 9 along with ZIPs 1, 2, 3, 6, 7, 9, 10, 11, 13, and 14 was detectable in the cardiomyocytes by real-time reverse transcriptase polymerase chain reaction. Hypoxia elicited accumulation of intracellular free zinc, but subsequent reoxygenation resulted in striking loss of intracellular free zinc and decreased the cardiomyocyte viability. Concomitantly, extracellular zinc levels dropped rapidly during hypoxia, but increased after reoxygenation. Immunoblotting analysis revealed that hypoxia increased the expression of ZnT1, but reoxygenation significantly increased the expression of ZnTs 2 and 5. Neither hypoxia nor reoxygenation altered the levels of ZnT9. Increased intracellular zinc at the end of hypoxia is related to enhanced expression of ZIPs, whereas decreased intracellular zinc during reoxygenation appears to be due to lowered expression of all ZIPs, in addition to elevated levels of ZnTs 2 and 5. These results thus suggest that there is impaired accumulation of intracellular zinc during reoxygenation, due to overexpression of specific ZnTs and downregulation of ZIP expression.

Glucose deprivation stimulates Cu(2+) toxicity in cultured cerebellar granule neurons and Cu(2+)-dependent zinc release.

Copper chloride (0.01mM, 2h) did not have significant influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution. However, CuCl2 caused severe neuronal damage by glucose deprivation (GD). The glutamate NMDA-receptors blocker MK-801 partially and antioxidant N-acetyl-l-cysteine (NAC) or Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) almost entirely protected CGNs from this toxic effect. Measurements of intracellular calcium ions using Fluo-4 AM, or zinc ions with FluoZin-3 AM demonstrated that 1 h-exposure to GD induced intensive increase of Fluo-4 but not FluoZin-3 fluorescence in neurons. The supplementation of solution with CuCl2 caused an increase of FluoZin-3, Fluo-4 and CellROX Green (reactive oxygen species probe) fluorescence by GD. The stimulation of Fluo-4 but not FluoZin-3 fluorescence by copper could be prevented partially by MK-801 and as well as CellROX Green fluorescence by NAC at GD. This data imply that during GD copper ions induce intense displacement zinc ions from intracellular stores, in addition free radical production, glutamate release and Ca(2+) overload of CGNs, that causes death of neurons as a result.

Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells.

BackgroundSeveral chemicals have been widely used to evaluate the involvement of free Ca2+ in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca2+-sensitive reagents in diverse cultured cells.Methodology/Principal FindingsIn rat astrocytic C6 glioma cells loaded with the fluorescent Ca2+ dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca2+ chelator EGTA irrespective of added CaCl2. The addition of the Ca2+ ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn2+ ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters.Conclusions/SignificanceTaken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca2+ in diverse cells enriched of Zn2+.

Copper-uptake is critical for the down regulation of synapsin and dynamin induced by neocuproine: modulation of synaptic activity in hippocampal neurons.

Extracellular and intracellular copper and zinc regulate synaptic activity and plasticity, which may impact brain functionality and human behavior. We have found that a metal coordinating molecule, Neocuproine, transiently increases free intracellular copper and zinc levels (i.e., min) in hippocampal neurons as monitored by Phen Green and FluoZin-3 fluorescence, respectively. The changes in free intracellular zinc induced by Neocuproine were abolished by the presence of a non-permeant copper chelator, Bathocuproine (BC), indicating that copper influx is needed for the action of Neocuproine on intracellular Zn levels. Moreover, Neocuproine decreased the mRNA levels of Synapsin and Dynamin, and did not affect the expression of Bassoon, tubulin or superoxide dismutase (SOD). Western blot analysis showed that protein levels of synapsin and dynamin were also down regulated in the presence of Neocuproine and that these changes were accompanied by a decrease in calcium transients and neuronal activity. Furthermore, Neocuproine decreased the number of active neurons, effect that was blocked by the presence of BC, indicating that copper influx is needed for the action of Neocuproine. We finally show that Neocuproine blocks the epileptiform-like activity induced by bicuculline in hippocampal neurons. Collectively, our data indicates that presynaptic protein configuration and function of primary hippocampal neurons is sensitive to transient changes in transition metal homeostasis. Therefore, small molecules able to coordinate transition metals and penetrate the blood-brain barrier might modify neurotransmission at the Central Nervous System (CNS). This might be useful to establish therapeutic approaches to control the neuronal hyperexcitabiltity observed in brain conditions that are associated to copper dyshomeotasis such as Alzheimer’s and Menkes diseases. Our work also opens a new avenue to find novel and effective antiepilepsy drugs based in metal coordinating molecules.

Changes in cellular thiol content and intracellular Zn2+ level by 1,4-naphthoquinone in rat thymocytes

1,4-Naphthoquinone is an active metabolite of naphthalene and it is also found in diesel exhaust particles. It is known to cause oxidative stress. In this study, we characterized 1,4-naphthoquinone-mediated cytotoxicity and its effects on the levels of non-protein thiols and intracellular Zn2+ in rat thymocytes (thymic lymphocytes) by using 5-chloromethylfluorescein (5-CMF) fluorescence and FluoZin-3 fluorescence, respectively. Low concentrations of 1,4-naphthoquinone (0.3μM) increased the intensity of 5-CMF fluorescence, which is used to measure non-protein thiols. In contrast, 5-CMF intensity decreased at higher concentrations (1–3μM) of 1,4-naphthoquinone. Removal of intracellular Zn2+ attenuated the 1,4-naphthoquinone-induced augmentation of 5-CMF fluorescence. Additionally, 1,4-naphthoquinone (0.3–3μM) increased FluoZin-3 fluorescence, which is used to assess intracellular Zn2+, in a concentration-dependent manner. The augmentation of FluoZin-3 fluorescence by 1,4-naphthoquinone was due to the release of intracellular Zn2+, because the removal of extracellular Zn2+ did not affect the augmentation of FluoZin-3 fluorescence. These results suggest that sublethal concentrations of 1,4-naphthoquinone (0.3–1μM) affect the cellular levels of non-protein thiols and intracellular Zn2+. The difference in the observed decrease in cellular thiol content due to 1,4-naphthoquinone treatment and increase due to Zn2+ release following 1,4-naphthoquinone treatment likely confers the change in cellular thiol content. Further, the increase in intracellular Zn2+ concentration after 1,4-naphthoquinone exposure may change the activity of thymocytes because thymulin, a thymus-specific hormone, requires Zn2+ for its biological activity.

Clioquinol-induced increase and decrease in the intracellular Zn2 + level in rat thymocytes

AimsClioquinol is emerging as a potential therapy for some diseases, such as Alzheimer disease and cancer. This agent is a lipophilic chelator of Zn2+. In this study, the effect of clioquinol on the intracellular Zn2+ level was examined in order to gain insights into the toxicological profile of clioquinol. Main methodsThe effect of clioquinol was estimated using a flow cytometer and FluoZin-3, a fluorescent indicator for Zn2+, in rat thymocytes. Key findingsClioquinol, at concentrations ranging from 10 to 300nM, augmented FluoZin-3 fluorescence in a concentration-dependent manner. However, the effect induced by 1μM clioquinol was less than that by 300nM clioquinol. Removal of extracellular Zn2+, using the membrane impermeable Zn2+-chelator diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA), abolished the clioquinol-induced augmentation of FluoZin-3 fluorescence. Clioquinol did not augment Fluo-3 fluorescence, an indicator of intracellular Ca2+, in the presence of DTPA. The results suggested that clioquinol caused an extracellular Zn2+-dependent increase in the intracellular Zn2+ concentration. However, in the presence of DTPA, clioquinol at micromolar concentrations (1–10μM) attenuated FluoZin-3 fluorescence in a concentration-dependent manner. Clioquinol even at 10μM did not affect FluoZin-3 fluorescence under cell-free condition. The concentration–response relationship for the clioquinol induced change in Zn2+ level appeared to be bell-shaped. These results indicate that micromolar concentrations of clioquinol, without chelated Zn2+, decrease intracellular Zn2+ concentration. SignificanceThe effect of clioquinol on the intracellular Zn2+ level varies, depending on the extracellular Zn2+ concentration and the clioquinol concentration. Clioquinol may therefore exert various types of Zn2+-dependent cytotoxicity.

Yttrium decreases the intracellular Zn2+ concentration in rat thymocytes by attenuating a temperature-sensitive Zn2+ influx

Yttrium is used in the production of various electronic devices because the alloy it contains enhances or modifies the properties of other elements. In order to study the cytotoxic action of yttrium, the effect of yttrium chloride (YCl3) on the intracellular Zn2+ level was examined in rat thymocytes using a flow cytometer with FluoZin-3-AM and propidium iodide. The application of YCl3 significantly decreased the intensity of the FluoZin-3 fluorescence, suggesting a decrease in the intracellular Zn2+ level or quenching of the FluoZin-3 fluorescence by Y3+. However, since Y3+ did not attenuate the FluoZin-3 fluorescence under cell-free conditions, the latter suggestion was ruled out. Rat thymocytes possess a temperature-sensitive membrane pathway that carries Zn2+ into the cells. The application of YCl3 attenuated the FluoZin-3 fluorescence augmented by externally applied ZnCl2 in a concentration-dependent manner. This suggested that Y3+ inhibited the Zn2+ influx, resulting in the decrease in the intracellular Zn2+ level. Yttrium may induce dyshomeostasis of intracellular Zn2+, leading to some cytotoxic actions.

ZnT4 provides zinc to zinc-dependent proteins in the trans-Golgi network critical for cell function and Zn export in mammary epithelial cells

Zinc (Zn) transporter 4 (ZnT4) plays a key role in mammary gland Zn metabolism. A mutation in ZnT4 (SLC30A4) that targets the protein for degradation is responsible for the "lethal milk" (lm/lm) mouse phenotype. ZnT4 protein is only detected in the secreting mammary gland, and lm/lm mice have ∼35% less Zn in milk, decreased mammary gland size, and decreased milk secretion. However, the precise contribution of ZnT4 is unknown. We used cultured mouse mammary epithelial cells (HC11) and determined that ZnT4 was localized to the trans-Golgi network (TGN) and cell membrane and transported Zn from the cytoplasm. ZnT4-mediated Zn import into the TGN directly contributed to labile Zn accumulation as ZnT4 overexpression increased FluoZin3 fluorescence. Moreover, ZnT4 provided Zn for metallation of galactosyltransferase, a Zn-dependent protein localized within the TGN that is critical for milk secretion, and carbonic anhydrase VI, a Zn-dependent protein secreted from the TGN into milk. We further noted that ZnT4 relocalized to the cell membrane in response to Zn. Together these studies demonstrated that ZnT4 transports Zn into the TGN, which is critical for key secretory functions of the mammary cell.

Glucose starvation stimulates Zn2+ toxicity in cultures of cerebellar granule neurons

Zinc chloride (0.02 mM, 3h) did not have any influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution (BSS). However, in the absence of glucose ZnCl(2) caused severe neuronal damage, decreasing cell survival to 12±2%. Either the blockade of ionotropic glutamate NMDA-receptors with MK-801 or APV or supplementation the medium with ruthenium red (mitochondrial Ca(2+) uniporter blocker) almost entirely protected CGNs from the toxic effect of ZnCl(2) during glucose deprivation (GD). However, NBQX (AMPA/kainate glutamate receptor blocker) did not show protective effect. Measurements of intracellular calcium ions concentration using fluorescent probe (Fluo-4 AM) and zinc ions (FluoZin-3AM) demonstrated that 1.5h-exposure to GD induced intensive increase of Fluo-4 fluorescence and small increase of FluoZin-3 fluorescence in neurons. The supplementation of medium with ZnCl(2) caused equal increase of FluoZin-3 fluorescence at both GD and normoglycemia, whereas the potentiation of Fluo-4 fluorescence by zinc was observed only under GD and could be prevented by MK-801. However, neither MK-801 nor NBQX could influence [Zn(2+)](i) increase caused by zinc addition under GD, while ruthenium red did cause significant increase of [Zn(2+)](i). This data implies that zinc ions during GD induce an additional overload of CGNs with calcium ions that get transported through activated NMDA-channel. Zinc and calcium ions accumulate in mitochondria and amplify individual destructive action on these organelles leading to neuronal death.

Triclosan, an antibacterial agent, increases intracellular Zn2+ concentration in rat thymocytes: Its relation to oxidative stress

Triclosan is used as an antibacterial agent in household items and personal care products. Since this compound is found in maternal milk of humans and bodies of wild animals, there is growing concern among some consumer groups and scientific community that triclosan is adverse for humans and wild animals. In order to estimate adverse actions of triclosan, the effects of triclosan on intracellular Zn2+ concentration and cellular thiol content were studied in rat thymocytes by the use of flow cytometer with appropriate fluorescent probes. Triclosan at 1–3μM (sublethal concentrations) increased the intensity of FluoZin-3 fluorescence (intracellular Zn2+ concentration) and decreased the intensity of 5-chloromethylfluorescein (5-CMF) fluorescence (cellular thiol content). Negative correlation (r=−0.985) between triclosan-induced changes in FluoZin-3 and 5-CMF fluorescences was found. Removal of external Zn2+ did not significantly affect the triclosan-induced augmentation of FluoZin-3 fluorescence, suggesting an intracellular Zn2+ release by triclosan. These actions of triclosan were similar to those of H2O2 and triclosan significantly potentiated the cytotoxicity of H2O2. Therefore, the results may suggest that triclosan at sublethal concentrations induces oxidative stress that decreases cellular thiol content, resulting in an increase in intracellular Zn2+ concentration by Zn2+ release from intracellular store(s). Since recent studies show many physiological roles of intracellular Zn2+ in cellular functions, the triclosan-induced disturbance of cellular Zn2+ homeostasis may induce adverse actions on the cells.

Putative role of intracellular Zn2+ release during oxidative stress: a trigger to restore cellular thiol content that is decreased by oxidative stress

Although the ability of zinc to retard the oxidative process has been recognized for many years, zinc itself has been reported to induce oxidative stress. In order to give some insights into elucidating the role of intracellular Zn(2+) in cells suffering from oxidative stress, the effects of N-ethylmaleimide (NEM) and ZnCl(2) on cellular thiol content and intracellular Zn(2+) concentration were studied by use of 5-chloromethylfluorescein diacetate (5-CMF-DA) and FluoZin-3 pentaacetoxymethyl ester (FluoZin-3-AM) in rat thymocytes. The treatment of cells with NEM attenuated 5-CMF fluorescence and augmented FluoZin-3 fluorescence in a dose-dependent manner. These NEM-induced phenomena were observed under external Zn(2+)-free conditions. Results suggest that NEM decreases cellular thiol content and induces intracellular Zn(2+) release. Micromolar ZnCl(2) dose-dependently augmented both FluoZin-3 and 5-CMF fluorescences, suggesting that the elevation of intracellular Zn(2+) concentration increases cellular thiol content. Taken together, it is hypothesized that intracellular Zn(2+) release during oxidative stress is a trigger to restore cellular thiol content that is decreased by oxidative stress.

Central role for metallothionein (MT) in Zn dependent nitric oxide (NO) mediated resistance to LPS‐induced apoptosis in sheep pulmonary artery endothelial cells (SPAEC)

We have previously shown that: a) iNOS or chemically derived (S-nitroso-N-acetylpenicillamine (SNAP)) NO is associated with resistant phenotype to LPS induced apoptosis in a zinc dependent fashion; and b) NO mediated elevations in intracellular zinc ([Zn]i) are critically dependent upon MT in cultured pulmonary mouse fibroblasts and endothelial cells. In the current study, we used RNA interference (siRNA) to couple these observations and reveal a central role for NO→MT→ [Zn]i↑ in affecting sensitivity of SPAEC to LPS-induced apoptosis. We exposed SPAEC to a combination of siRNA to sheep MT-Ia, b, c and MT-II and noted a lack of change in [Zn]i (as detected microspectrofluorometrically with the zinc indicator, FluoZin-3) compared to a huge increase (6X increase in FluoZin-3 fluorescence) after SNAP in wildtype SPAEC after a brief exposure to large concentrations of SNAP. Pretreatment of SPAEC with SNAP (250μM, 6h) made them resistant to LPS (1 ug/ml) -induced apoptosis. Importantly, the anti-apoptotic effect of SNAP was abrogated in SPAEC treated with non-toxic (1 uM) doses of zinc chelator, TPEN. Collectively, these data confirm that: a) increases in labile [Zn]i are an important component of NO mediated resistance to LPS induced aopotosis; and b) s-nitrosation of MT (with subsequent zinc release) may be a critical source of such NO mediated increases in labile zinc.

Clioquinol induces autophagy in cultured astrocytes and neurons by acting as a zinc ionophore

Recent studies have demonstrated that clioquinol, an antibiotic with an anti-amyloid effect, acts as a zinc ionophore under physiological conditions. Because increases in labile zinc may induce autophagy, we examined whether clioquinol induces autophagy in cultured astrocytes in a zinc-dependent manner.Within 1h of exposure to 0.1–10μM clioquinol, the levels of microtubule-associated protein 1 light chain 3 (LC3)-II, a marker of autophagy, began to increase in astrocytes. Confocal live-cell imaging of GFP-LC3-transfected astrocytes showed the formation of LC3(+) autophagic vacuoles (AVs), providing a further indication that clioquinol induced autophagy. Addition of 3-methyladenine or small-interfering RNA against autophagy-related gene 6 (ATG6/Beclin-1) blocked clioquinol-induced increases in LC3-II. FluoZin-3 fluorescence microscopy showed that, like the zinc ionophore pyrithione, clioquinol increased intracellular zinc levels in the cytosol and AVs in an extracellular zinc-dependent manner. Zinc chelation with N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) reduced, and addition of zinc increased the levels of LC3-II and LC3(+) puncta, indicating that zinc influx plays a key role therein. Moreover, astrocytes and SH-SY5Y cells expressing mutant huntingtin (mHttQ74) accumulated less aggregates when treated with clioquinol, and this effect was reversed by TPEN. These results indicate that clioquinol-induced autophagy is likely to be physiologically functional.The present study demonstrates that clioquinol induces autophagy in a zinc-dependent manner and contributes to clearance of aggregated proteins in astrocytes and neurons. Hence, in addition to its metal-chelating effect in and around amyloid beta (Aβ) plaques, clioquinol may contribute to the reduction of Aβ loads by activating autophagy by increasing or normalizing intracellular zinc levels in brain cells.