Extracellular Zinc Concentration Research Articles

Comparison of Zinc Concentrations in the Broth of Commercial Automated Susceptibility Testing Devices (Vitek 2, MicroScan, BD Phoenix, and Sensititre)

ABSTRACTUp to 4-fold differences in zinc concentrations have been observed in commercial broth routinely utilized for susceptibility testing via manual broth microdilution. Herein, we report the concentration of zinc in the broth of common automated susceptibility testing (AST) platforms (Vitek, MicroScan, BD Phoenix, and Sensititre). For AST platforms with lyophilized broth contents (Vitek and MicroScan), wells were rehydrated with appropriate diluent, and contents were aliquoted out for zinc assay. Aliquots from the manufacturer-specific broth (premade cation-adjusted Mueller-Hinton broth [caMHB]) for BD Phoenix and Sensititre were also assayed by inductively coupled plasma mass spectrometry. Up to a 10-fold difference in zinc concentrations was observed across the 4 platforms (MicroScan: 0.46 mg/L; BD Phoenix: 1.16 mg/L; Vitek: 1.22 mg/L; Sensititre: 4.49 mg/L). Attention should be given to the supraphysiologic and variable zinc concentrations observed in broth used in automated platforms and the subsequent implications for susceptibility testing of metallo-β-lactamase (MBL)-harboring isolates. This variability also hampers efforts to develop a standardized method to uniformly reduce zinc concentrations in broth and mimic physiologic zinc conditions.IMPORTANCE Growing data on the impact of extracellular zinc concentration on metallo-β-lactamase-mediated resistance has shed light on the importance of susceptibility testing media. However, there are no studies documenting the amount of zinc in commonly utilized automated susceptibility testing (AST) platforms. This study reveals supraphysiologic zinc concentrations as well as large zinc variability among AST platforms and highlights the challenges this raises in the development of zinc-limited media.

Influence of physical training on intracellular and extracellular zinc concentrations.

ABSTRACTBackgroundPhysical exercise affects zinc (Zn) homeostasis. This study aimed to analyze the influence of physical training on extracellular (serum, plasma, and urine) and intracellular (erythrocytes and platelets) concentrations of Zn.MethodsForty young men, divided into a training group (TG; n = 20; 18.15 ± 0.27 years; 68.59 ± 4.18 kg; 1.76 ± 0.04 m) and a control group (CG; n = 20; 19.25 ± 0.39 years; 73.45 ± 9.04 kg; 1.79 ± 0.06 m), participated in this study. The TG was formed by semiprofessional soccer players from a youth category with a regular training plan of 10 h/week. The CG was formed by healthy men who did not practice physical exercise and had not followed any specific training plan. Plasma, serum, urine, erythrocyte, and platelet samples of Zn were obtained and analyzed by inductively coupled plasma mass spectrometry.ResultsThe TG showed elevated plasma Zn concentrations (p < 0.01) despite similar intakes. However, TG showed reduced absolute (p < 0.01) and relative (p < 0.05) Zn concentrations in erythrocytes.ConclusionsAthletes who underwent regular physical training showed elevated plasma and reduced erythrocyte Zn concentrations despite similar intakes to the CG.

Evaluation of Metallo-β-Lactamase Susceptibility Testing in a Physiologic Medium.

ABSTRACTResearch in identifying alternative growth media that better mimic host conditions is gaining ground. Relative to nutrient-rich Mueller-Hinton broth (MHB), data on the influence of physiologic or host-mimicking media on metallo-β-lactamase (MBL) resistance are lacking. The objective was to evaluate meropenem susceptibility against clinical and engineered MBL-harboring Enterobacterales strains in a physiologic medium (urine). Antimicrobial susceptibility testing (AST) by broth microdilution was conducted with a wild-type Klebsiella pneumoniae strain and two engineered isogenic variants harboring K. pneumoniae carbapenemase 2 (KPC-2) or New Delhi MBL 1 (NDM-1), as well as two clinical K. pneumoniae isolates (harboring NDM-1 and VIM-1). MICs were determined in conventional cation-adjusted MHB (caMHB) and sterile-filtered urine samples (18 patients). All KPC- and MBL-harboring isolates were meropenem resistant (MICs of ≥16 mg/liter) in caMHB. AST of the KPC isolate in urine resulted in 50% (9/18 urine samples) essential agreement (i.e., within ±1 dilution, relative to the caMHB MIC), highlighting challenges with the use of urine as a medium capable of supporting AST. In the 9 AST-viable urine samples, meropenem MICs were 2- to 9-fold lower than that in caMHB (MIC of 32 mg/liter) among MBL-harboring isolates. Zinc concentrations determined by inductively coupled plasma mass spectrometry averaged 1.25 mg/liter and ranged from 0.12 to 1.14 mg/liter in caMHB and 18 urine samples, respectively. The full extent of MBL-mediated resistance among K. pneumoniae isolates appears to be attenuated in urine. Factors influencing free bioactive zinc levels warrant further investigation.IMPORTANCE Studies assessing antibiotic susceptibility profiles in nonconventional media are lacking. MBL-mediated resistance has come under scrutiny due to the dependence on extracellular zinc concentrations, which makes the choice of testing medium influential for β-lactam MICs. This study explores human urine as a physiologically relevant matrix with which susceptibility profiles of MBL-harboring isolates can be assessed, relative to conventional broth.

General Aspects of Metal Ions as Signaling Agents in Health and Disease.

This review focuses on the current knowledge on the involvement of metal ions in signaling processes within the cell, in both physiological and pathological conditions. The first section is devoted to the recent discoveries on magnesium and calcium-dependent signal transduction—the most recognized signaling agents among metals. The following sections then describe signaling pathways where zinc, copper, and iron play a key role. There are many systems in which changes in intra- and extra-cellular zinc and copper concentrations have been linked to important downstream events, especially in nervous signal transduction. Iron signaling is mostly related with its homeostasis. However, it is also involved in a recently discovered type of programmed cell death, ferroptosis. The important differences in metal ion signaling, and its disease-leading alterations, are also discussed.

The S100B Alarmin Is a Dual-Function Chaperone Suppressing Amyloid-β Oligomerization through Combined Zinc Chelation and Inhibition of Protein Aggregation.

Amyloid beta (Aβ) aggregation and imbalance of metal ions are major hallmarks of Alzheimer's disease (AD). Indeed, amyloid plaques of AD patients are enriched in zinc and Aβ42, and AD related-cognitive decline is dependent on extracellular zinc concentration. In vitro, zinc induces the formation of polymorphic Aβ42 oligomers that delay the formation of amyloid fibers at the expense of increased cellular toxicity. S100B is an inflammatory alarmin and one of the most abundant proteins in the brain and is upregulated in AD and associated with amyloid plaques, where it exerts extracellular functions. Recent findings have uncovered novel neuroprotective functions for S100B as a suppressor of Aβ aggregation and toxicity and in the regulation of zinc homeostasis in neurons. Here we combine biophysical and kinetic approaches to demonstrate that such S100B protective functions converge, making the protein a dual-function chaperone capable of suppressing the formation of toxic Aβ oligomers through both chelation of zinc and inhibition of protein aggregation. From detailed kinetic analysis of Aβ42 aggregation monitoring ThT fluorescence, we show that substoichiometric S100B prevents the formation of toxic off-pathway oligomers that are formed by monomeric Aβ42 in the presence of zinc. Indeed, S100B is effective when added during the lag and transition phases of Aβ42 aggregation, and its action under these circumstances results from its ability to buffer zinc, as it perfectly mimics the effect obtained with the chelating agent EDTA. Further, bioimaging analysis combining transmission electron microscopy and atomic force microscopy confirms that catalytic amounts of S100B partly revert the formation of toxic oligomers. Taken together these results indicate a new role for S100B as a dual chaperone whose distinct functions are interrelated and depend on the relative levels of zinc, S100B, and Aβ, which dynamically evolve during AD.

Cytoplasmic labile zinc regulates IL-1β production in human monocytes/macrophages via mTORC1-induced glycolysis

Abstract Zinc is an essential micronutrient that plays critical roles in multiple facets of biological processes, including growth, development, proliferation, and differentiation. Since dysregulated zinc homeostasis impairs overall immune function and resultantly, increases susceptibility to infection, much attention has been recently paid to immunomodulatory roles of zinc in immune cells. Rapid and efficient production of proinflammatory cytokines, such as IL-1β and TNF-α, is a primary effect function of monocytes/macrophages for host defense. However, the molecular mechanism involved with zinc-mediated modulation of cytokine production in monocytes/macrophages needs to be better understood. In present study, we provide evidence that cytoplasmic labile zinc is essential for modulation of IL-1β production in human monocytes/macrophages via mTORC1-induced glycolysis. We found that cytoplasmic level of labile zinc in human monocytes/macrophages was largely influenced by extracellular zinc concentrations, possibly via zinc importers such as ZIP1- and ZIP8-mediated influx. The phosphorylation of S6 kinase, a substrate of mTORC1, was significantly enhanced by zinc-mediated inhibition of PP2A, a phosphatase contracting S6K phosphorylation and consequently, IL-1β production was increased by activated mTORC1-induced glycolysis. These results provide new insight how labile zinc modulate a production of cytokines by metabolic reprograming in human monocytes/macrophages.

Novel CBWD Gene Products Augment Mammalian Zinc Homeostasis

The CBWD genes (coding for COBW domain‐containing proteins) was previously identified through an in silico search of the human genome for the presence of a zinc‐responsive element known as Zinc Transcriptional Regulatory Element (ZTRE)1. The human CBWD gene family consist of six isoforms with different chromosomal and cytogenetic locations and exhibit a remarkable nucleotide and amino acid sequence conservation. The prokaryotic homologues of CBWD proteins belonging to the GOG0523 family of proteins have been reported to have roles in zinc homeostasis2, however, no known role has yet been documented for the mammalian types. The discovery that the zinc‐sensitive element (ZTRE) occurred in multiple copies of the CBWD genes was intriguing and triggered further investigations into a likely role for this gene family in mammalian cellular zinc handling and/or trafficking. Moreover, we reported a protein that acts at the ZTRE site to mediate regulation of zinc homeostatic genes and found CBWD genes to be a prominent feature3. Using Caco‐2 cells as in vitro model of human enterocytes, we demonstrate the ability of CBWD genes to respond to changes in zinc content at both transcriptional and post‐transcriptional levels evidenced by decreased transcript and overexpressed protein abundance in response to increased extracellular zinc concentration. We also show that increased expression of CBWD protein through a transgene extended cellular tolerance to extreme windows of zinc availability, demonstrating the potential of the human CBWD gene products to augment cellular zinc buffering. This potential buffering ability of CBWD gene products may be effected through cellular zinc redistribution evidenced by a less punctate zinc distribution in cells made to overexpress CBWD protein compared to control cells. Furthermore, overexpression of COBW protein attenuated the responsiveness of a zinc‐sensitive metallothionein promoter, indicating an action consistent with a role in zinc trafficking or buffering. These findings give plausible evidence in support of the involvement of the mammalian CBWD gene products in cellular zinc homeostasis.Support or Funding InformationThis work was supported by the Nigerian Tertiary Education Trust Fund through Benue State University, Makurdi, Benue State NigeriaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potential Role of Zinc Dyshomeostasis in Matrix Metalloproteinase-2 and -9 Activation and Photoreceptor Cell Death in Experimental Retinal Detachment.

We investigated whether zinc dyshomeostasis, a known mechanism of cell death in acute brain injury, contributes to the activation of matrix metalloproteinases (MMPs) and photoreceptor cell death in experimental retinal detachment (RD). RD was induced in mice by subretinal injection of 1:1 mixture of balanced salt solution and 1% sodium hyaluronate. On days 1 and 3 post RD, eyeballs were sectioned and examined for cell death (TUNEL staining), the degree of hypoxic insult (Hypoxyprobe staining), free zinc levels (TFL-Zn staining), and MMP-2 and -9 activity (gelatin zymography). In addition, we examined whether modulating extracellular zinc concentration or MMP activation in subretinal fluid affected photoreceptor cell death in RD. These changes were further examined in primary retinal cell and photoreceptor-derived cell (661W) cultures. Photoreceptor cell death peaked on day 3 post RD. Intracellular zinc markedly decreased on day 1 post RD, and subsequently accumulated on day 3. MMP-2 and -9 activity showed a concurrent increase in detached retinas. Detached retinas stained with Hypoxyprobe showed strongly positive cells, especially in the photoreceptor layer. Subretinal injection of a zinc-chelator (CaEDTA) or MMP inhibitor (GM6001, minocycline) at the time of RD significantly attenuated photoreceptor cell death in RD. Similar findings were confirmed in oxygen-glucose-deprived or zinc-exposed cell cultures. Upon RD, hypoxic retinal cells in deep layers underwent zinc dyshomeostasis, MMP activation, and ultimately death. These findings provide new insight into the possible mechanism of photoreceptor death in RD, and as such may prove useful in crafting protective measures for photoreceptor cells.

Characteristic of Extracellular Zn2+ Influx in the Middle-Aged Dentate Gyrus and Its Involvement in Attenuation of LTP

An increased influx of extracellular Zn2+ into neurons is a cause of cognitive decline. The influx of extracellular Zn2+ into dentate granule cells was compared between young and middle-aged rats because of vulnerability of the dentate gyrus to aging. The influx of extracellular Zn2+ into dentate granule cells was increased in middle-aged rats after injection of AMPA and high K+ into the dentate gyrus, but not in young rats. Simultaneously, high K+-induced attenuation of LTP was observed in middle-aged rats, but not in young rats. The attenuation was rescued by co-injection of CaEDTA, an extracellular Zn2+ chelator. Intracellular Zn2+ in dentate granule cells was also increased in middle-aged slices with high K+, in which the increase in extracellular Zn2+ was the same as young slices with high K+, suggesting that ability of extracellular Zn2+ influx into dentate granule cells is greater in middle-aged rats. Furthermore, extracellular zinc concentration in the hippocampus was increased age-dependently. The present study suggests that the influx of extracellular Zn2+ into dentate granule cells is more readily increased in middle-aged rats and that its increase is a cause of age-related attenuation of LTP in the dentate gyrus.

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.

Zinc Modulates Endotoxin-Induced Human Macrophage Inflammation through ZIP8 Induction and C/EBPβ Inhibition.

Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-κB pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNFα, IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBPβ, a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.

Stress hormone potentiates Zn2+-induced neurotoxicity via TRPM7 channel in dopaminergic neuron

Zinc toxicity is one of the key factors responsible for the neuronal injuries associated with various neurological conditions. Zinc accumulation in some cells is accompanied by the increase of blood stress hormone levels, which might indicate a functional connection between stress and zinc toxicity. However, the cellular mechanism for the effect of stress on zinc toxicity is not known. Recently, it was reported that the zinc permeable transient receptor potential melastatin 7 (TRPM7) channel may represent a novel target for neurological disorders where zinc toxicity plays an important role. To investigate the effect of stress hormone on zinc-induced cell death, neuroblastoma SH-SY5Y cells were pretreated with urocortin, a corticotropin releasing factor (CRF)-related peptide. Urocortin potentiated zinc-induced cell death at μM range of extracellular zinc concentrations. It significantly increased TRPM7 channel expression, and zinc influx into cytosol. Moreover, application of TRPM7 channel blockers and RNA interference of TRPM7 channel expression attenuated the zinc-induced cell death in urocortin-pretreated cells, indicating that TRPM7 channel may serve as a zinc influx pathway. These results suggest that TRPM7 channel may play a critical role for zinc toxicity associated with stress.

Alterations of Bio-elements, Oxidative, and Inflammatory Status in the Zinc Deficiency Model in Rats.

Our previous study showed that dietary zinc restriction induces depression-like behavior with concomitant up-regulation of the N-methyl-d-aspartate receptor (NMDAR). Because metal ions, oxidative stress, and inflammation are involved in depression/NMDAR function, in the present study, bio-elements (zinc, copper, iron, magnesium, and calcium), oxidative (thiobarbituric acid-reactive substances; protein carbonyl content), and inflammatory (IL-1α, IL-1β) factors were measured in serum, hippocampus (Hp), and prefrontal cortex (PFC) of male Sprague–Dawley rats subjected to a zinc-adequate (ZnA) (50 mg Zn/kg) or a zinc-deficient (ZnD) (3 mg Zn/kg) diet for 4 or 6 weeks. Both periods of dietary zinc restriction reduced serum zinc and increased serum iron levels. At 4 weeks, lowered zinc level in the PFC and Hp as well as lowered iron level in the PFC of the ZnD rats was observed. At 6 weeks, however, iron level was increased in the PFC of these rats. Although at 6 weeks zinc level in the PFC did not differ between the ZnA and ZnD rats, extracellular zinc concentration after 100 mM KCl stimulation was reduced in the PFC of the ZnD rats and was accompanied by increased extracellular iron and glutamate levels (as measured by the in vivo microdialysis). The examined oxidative and inflammatory parameters were generally enhanced in the tissue of the ZnD animals. The obtained data suggest dynamic redistribution of bio-elements and enhancement of oxidative/inflammatory parameters after dietary zinc restriction, which may have a link with depression-like behavior/NMDAR function/neurodegeneration.

Characterization of the GufA subfamily member SLC39A11/Zip11 as a zinc transporter

Cellular zinc influx and efflux are maintained by two major transporter families, the ZIP (SLC39A) and ZnT (SLC30A or CDF) molecules. The functions of one molecule in this class, ZIP11/SLC39A11, remain unclear. Bioinformatics analysis of the distribution and evolutionary relationships of different ZIP members in eukaryotes and prokaryotes indicated that Zip11, the sole member of gufA subfamily, is an ancient ZIP family member that might have originated in early eukaryotic ancestors. Murine Zip11 mRNA is abundantly expressed in testes and the digestive system including stomach, ileum and cecum. Analysis of cellular zinc content, metallothionein levels, and cell viability under high or low zinc conditions in cells transfected with a murine Zip11 expression plasmid, suggest that Zip11 is a zinc importer. Further, cellular zinc concentrations and metallothionein levels decreased when Zip11 was knocked down. In mice supplemented with zinc, both mRNA and protein levels of Zip11 were slightly up-regulated in several tissues. The metal response element sequences (MREs) upstream of the first exon of Zip11 responded to elevated extracellular zinc concentrations, as assessed by luciferase reporter assays. Mutagenic analysis showed that several of the MREs could regulate Zip11 promoter activity, and metal-responsive transcription factor-1 (MTF-1) was shown to be involved in this process. Collectively, these data suggest that Zip11 has unique protein sequence and structure features, it functions as a cellular zinc transporter, and its expression is at least partially regulated by zinc via hMTF-1 binding to MREs of the Zip11 promoter.

Effects of Exogenous Zinc on the Cellular Zinc Distribution and Cell Cycle of A549 Cells

As the second most abundant transition metal in humans, zinc plays essential roles in normal cellular biological functions, including metabolism, signalling, proliferation, gene expression and apoptosis. We use ZnSO(4) as a stressor in this study to investigate for the first time the effects of exogenous Zn(2+) on both the cellular distribution of zinc and zinc-related proteins and the cell cycle of human lung adenocarcinoma (A549) cells. The cellular distribution of zinc and soluble proteins was determined in the whole cell as well as in the cytoplasmic and nuclear fractions. Exogenous zinc in the tested exposure range (0-100 µM) resulted in an altered cellular distribution of both zinc and the soluble proteins, together with total glutathione (GSx), the ratio of glutathione (GSH) to glutathione disulfide (GSSG) and non-protein sulphydryl (NPSH). Surprisingly, a turning point was observed in the re-distribution trend at a concentration of approximately 50 µM ZnSO(4). It is concluded that there exists a regulatory system in A549 cells that maintains the cellular zinc content stable in the presence of a certain range of extracellular zinc concentration. In addition, an MTT assay and flow cytometric analysis showed that the ZnSO(4) treatment led to a bi-phasic variation in viability and a slight fluctuation in the apoptosis of A549 cells. Our results will help to further elucidate zinc-related cell biology and biochemistry.

Selective, quantitative measurement of releasable synaptic zinc in human autopsy hippocampal brain tissue from Alzheimer's disease patients

Aberrant central nervous system zinc homeostasis has been reported in Alzheimer's disease (AD). However, there are conflicting reports describing zinc concentration either increased or decreased in the brain of AD patients. Such discrepancies may be due to differences in the brain area examined, zinc detection method, and/or tissue composition. Furthermore, detection and measurement of the releasable zinc pool in autopsy tissue is difficult and usually unreliable. Obtaining an adequate assessment of this releasable zinc pool is of particular significance in AD research in that zinc can coordinate with and stabilize toxic amyloid beta oligomers, which are believed to play a key role in AD neuropathology. In addition, zinc released into the synaptic cleft can interact with the postsynaptic neurons causing altered signaling and synaptic dysfunction, which is a well established event in AD. The method presented here combines two approaches, biochemical fractionation and atomic absorption spectrophotometry, to allow, in addition to extracellular zinc concentration, the reliable and quantitative measurement of zinc specifically localized in synaptic vesicles, which contain the majority of the neuronal releasable zinc. Using this methodology, we found that synaptic vesicle zinc concentrations were increased in AD hippocampi compared to age-matched controls and that this increase in releasable zinc matched increased concentration of zinc in the extracellular space.

Regulation of T-cell receptor signaling by activation-induced zinc influx (109.21)

Abstract Zinc is a trace element that is essential for innate and adaptive immune responses. In addition to being a structural element of many proteins, zinc also functions as a neurotransmitter and an intracellular messenger. Although cytoplasmic zinc concentrations are strictly controlled, temporal or spatial changes in bioavailable zinc may influence the activity of several enzymes, including kinases and phosphatases. In this study, we provide evidence that zinc functions as an ionic signaling molecule after T-cell activation. Cytoplasmic zinc concentrations increased within one minute after TCR triggering by super antigen presented by myeloid dendritic cells. In contrast to activation-induced calcium influx, increased zinc concentrations were compartmentalized in the subsynaptic area. The zinc increase was dependent on the extracellular zinc concentrations and was inhibited by silencing the plasma membrane zinc transporter Zip6 consistent with influx from extracellular compartments. Increased zinc availability augmented ZAP70 phosphorylation, inhibited SHP-1 recruitment to the T cell signaling complex, slowed down Lck rephosphorylation at position 505 and sustained calcium influx. As a result, increased extracellular zinc bioavailability facilitated the induction of T cell proliferative responses to suboptimal stimuli. We propose that zinc calibrates TCR activation thresholds by inhibiting negative feedback mechanisms in TCR signaling.

Regulation of T cell receptor signaling by activation-induced zinc influx

Zinc is a trace element that is essential for innate and adaptive immune responses. In addition to being a structural element of many proteins, zinc also functions as a neurotransmitter and an intracellular messenger. Temporal or spatial changes in bioavailable zinc may influence the activity of several enzymes, including kinases and phosphatases. We provide evidence that zinc functions as an ionic signaling molecule after T cell activation. Cytoplasmic zinc concentrations increased within 1 min after T cell receptor (TCR) triggering, in particular in the subsynaptic compartment. The increase depended on the extracellular zinc concentrations and was inhibited by silencing zinc transporter Zip6. Increased zinc influx reduced the recruitment of SHP-1 to the TCR activation complex, augmented ZAP70 phosphorylation and sustained calcium influx. By calibrating TCR activation thresholds, increased extracellular zinc bioavailability facilitated the induction of T cell proliferative responses to suboptimal stimuli.

Imaging Intracellular and Intra-Mitochondrial Free Zinc Ion Concentrations Following Hypoxia/Hypoglycemia with an Expressible Fluorescence Biosensor

Zinc is a “trace” metal necessary for cellular function, but excess free zinc ion is toxic to most cells (1,2,9) We and others have observed increased intra- and extra-cellular free zinc concentrations following cerebral ischemia (3,4). Substantial evidence indicates that mitochondrial dysfunction plays a significant role in neuronal death following ischemia (5), and both mitochondrial dysfunction and increased intracellular zinc have been associated with increased production of reactive oxygen species (ROS) and ultimately apoptosis (6,7). Zinc, potently, inhibits mitochondrial enzymes involved in energy production and destruction of ROS, potentially promoting mitochondrial dysfunction (8). We targeted our expressible fluorescent zinc biosensor (9) to mitochondria of PC12 cells, to ratiometrically image the intra-mitochondrial zinc concentration at resting (pM) levels. We imaged cytoplasmic and mitochondrial zinc levels in cells deprived of oxygen and glucose (OGD), a widely used model of ischemia. Our data indicate that both intra-mitochondrial and cytoplasmic zinc concentrations increase substantially following OGD. Further experiments indicate that the zinc is released from intracellular sites rather than entering the cell from external sources. Supported by NIH EB03924. 1. Canzoniero, L.M., et al. (1999) Journal of Neuroscience 19:RC31, 1–6 2. Zodl, B, et al. (2003) Journal of Inorganic Biochemistry 97, 324–330 3. Tonder, N., et al. (1990) Neuroscience Letters 109, 247–252 4. Frederickson, C.J., et al (2006) Experimental Neurology 198, 285–293 5. Fiskum G., et al. (2008) Mitochondria and Oxidative Stress in Neurodegenerative Disorders: Annals of the New York Academy of Science. 1147, 129–138 6. Weiss, J.H., et al. (2000) Trends in Pharmacological Science 21, 395–4017. Jiang, D., et al. (2001) Journal of Biological Chemistry 276, 47524–47529 8. Gazaryan, I.G., et al. (2007) Journal of Biological Chemistry 282, 24373–24380 9. Bozym, R.A., et al. (2006) ACS Chemical Biology 1, 103–111.

Chronic treatment with zinc and antidepressants induces enhancement of presynaptic/extracellular zinc concentration in the rat prefrontal cortex.

Zinc exhibits antidepressant-like activity in preclinical tests/models. Moreover, zinc homeostasis is implicated in the pathophysiology of affective disorders. The aim of the present study was to examine the effect of chronic zinc, citalopram and imipramine intraperitoneal administration on the presynaptic and extracellular zinc concentration in the rat prefrontal cortex and hippocampus. We used two methods: zinc–selenium histochemistry (which images the pool of presynaptic-vesicle zinc) and anodic stripping voltammetry (ASV) for zinc determination in microdialysate (which assays the extracellular zinc concentration). We report that chronic (14×) zinc (hydroaspartate, 10 and 65 mg/kg) and citalopram (20 mg/kg) administration increased the pool of presynaptic zinc (by 34, 50 and 37%, respectively) in the rat prefrontal cortex. The 21% increase induced by imipramine (20 mg/kg) was marginally significant. Likewise, zinc (hydroaspartate, 65 mg/kg), citalopram and imipramine increased the extracellular zinc (although with a different pattern: time point, area under the curve and/or basal level) in this brain region. Furthermore, zinc induced an increase in presynaptic (by 65%) and extracellular zinc (by 90%) in the hippocampus, while both citalopram and imipramine did not. These results indicate that all of the treatments increase presynaptic/extracellular zinc concentrations in the rat prefrontal cortex, which may then contribute to their antidepressant mechanisms. Alterations induced by zinc (but not antidepressants) administration in the hippocampus may be related to specific zinc mechanisms. All the data (previous and present) on the effect of antidepressant treatments on the presynaptic/extracellular zinc concentrations suggest the involvement of this biometal presynaptic/synaptic homeostasis in the antidepressant mechanism(s).