Cytosolic Zn Research Articles

Activation of β3-Adrenoceptors Induces Increase in Intracellular Free Zinc Ion via No Signaling Pathway in Hyperglycemic Cardiomyocytes

Hyperglycemia is a major etiological factor causing cardiovascular dysfunction. Changes in intracellular ionic homeostasis via a series of biochemical changes initiated by hyperglycemia directly affect cellular function resulting abnormal cardiac remodeling and development of diabetic complications. Since hyperglycemia regulates free cytosolic Zn2+ ([Zn2+]i) and attenuates protein S-nitrosylation via NO-cGMP-PKG, which induces depressed cardiac contractility, in part contribution of activated β3-adrenergic receptor (β3-AR). We postulated a possible cross-talk between hyperglycemia, activation of β3-AR and NO availability, [Zn2+]i increase and contractile dysfunction. [Zn2+]i levels were determined by imaging freshly isolated adult rat cardiomyocytes from either normoglycemic or hyperglycemic rat loaded with FluoZin-3. We investigated the distribution of β-ARs subtypes by measuring the expression/protein levels of β1-, β2-, β3-AR as well as their left ventricular agonist responsiveness using isolated perfused hearts from diabetic rats. We found that hyperglycemia influenced both function and the mRNA/protein levels of selective β1- and β3-AR although the total β-AR did not change. In vitro experiments showed that β3-AR agonist (BRL-37,344) induced significant increase in [Zn2+]i while this effect was prevented with L-NAME exposure in normoglycemic cardiomyocytes. Our data demonstrated that either chronic or acute hyperglycemia initiates a series of interconnected biochemical changes in cardiomyocytes including a cross-talk between activation of β3-AR and NO signaling pathway including increased [Zn2+]i and then this cross-talk meadiates abnormal cardiac remodeling and the development of diabetic cardiomyopathy. Therefore, it can be proposed a pathway that a dynamic association of intracellular free Zn2+ with sulfur in protein cysteine clusters under hyperglycemia, from which the metal is released by NO and other thiol oxidant species is one of the mechanisms which plays important role in the Zn2+ contribution to maintaining cellular redox balance. (Supported by TUBITAK-SBAG-109S267 & 111S042)

Zinc-binding and structural properties of the histidine-rich loop of Arabidopsis thaliana vacuolar membrane zinc transporter MTP1

The vacuolar Zn2+/H+ antiporter of Arabidopsis thaliana, AtMTP1, has a cytosolic histidine-rich loop (His-loop). We characterized the structures and Zn2+-binding properties of the His-loop and other domains. Circular dichroism analyses revealed that the His-loop partly consists of a polyproline type II structure and that its conformational change is induced by Zn2+ as well as the C-terminal domain. Isothermal titration calorimetry of the His-loop revealed a binding number of four Zn2+ per molecule. Numbers of Ni and Co associated with the His-loop were approximately one ion per molecule and the thermodynamic parameters of the association with these ions were different from that of Zn2+. These results suggest the involvement of the His-loop in sensing cytosolic Zn2+ and in the regulation of zinc transport activity through Zn2+-induced structural change.

Zn2+‐induced ERK activation mediates PARP‐1‐dependent ischemic‐reoxygenation damage to oligodendrocytes

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca(2+)](i), loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia-like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia-induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O(2) (reperfusion-like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high-affinity Zn(2+) chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high-affinity Zn(2+) probe, we showed that ischemia induced an intracellular Zn(2+) rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia-induced oligodendrocyte death. Moreover, UO126 blocked the ischemia-induced increase in poly-[ADP]-ribosylation of proteins, and the poly[ADP]-ribose polymerase 1 (PARP-1) inhibitor DPQ significantly inhibited ischemia-induced oligodendroglial cell death-demonstrating that PARP-1 was required downstream in the Zn(2+)-ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn(2+), blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia-reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult.

New Alternately Colored FRET Sensors for Simultaneous Monitoring of Zn2+ in Multiple Cellular Locations

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn2+based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn2+ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn2+ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn2+. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn2+ uptake simultaneously in two compartments, revealing that nuclear Zn2+ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn2+ more slowly and with a delay of 600–700 sec. Lastly, these studies provide the first glimpse of nuclear Zn2+ and reveal that nuclear Zn2+ is buffered at a higher level than cytosolic Zn2+.

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn 2+ over Cd 2+ selectivity

Zinc and cadmium are similar metal ions, but though Zn(2+) is an essential nutrient, Cd(2+) is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn(2+) vs. Cd(2+) suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn(2+) transport, but reject Cd(2+), thus constituting the first mammalian metal transporter with a refined selectivity against Cd(2+). Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn(2+) and Cd(2+). A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn(2+) transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd(2+) by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn(2+) and Cd(2+), and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd(2+) binding.

Demonstration of an Olfactory Bulb–Brain Translocation Pathway for ZnO Nanoparticles in Rodent Cells In Vitro and In Vivo

ZnO nanoparticles (ZnO-NPs) are widely used in the engineering and cosmetic industries, and inhaled airborne particles pose a known hazard to human health; their translocation into humans is a recognized public health concern. The pulmonary-blood pathway for ZnO-NP toxicity is well documented, but whether translocation of these particles can also occur via an olfactory bulb-brain route remains unclear. The potential toxicity of ZnO-NPs for the human central nervous system (CNS) is predicated on the possibility of their translocation. Our study investigated translocation of ZnO-NPs both in vitro using the neuronal cell line PC12 and in vivo in a Sprague-Dawley rat model. Our findings indicate that the zinc-binding dye, Newport-Green DCF, binds ZnO stoichiometrically and that ZnO-NP concentration can therefore be measured by the fluorescence intensity of the bound dye in confocal fluorescence microscopy. Confocal data obtained using Newport-Green DCF-2K(+)-conjugated ZnO-NPs along with the membrane probe FM1-43 demonstrated endocytosis of ZnO-NPs by PC12 cells. In addition, Fluozin-3 measurement showed elevation of cytosolic Zn(2+) concentration in these cells. Following in vivo nasal exposure of rats to airborne ZnO-NPs, olfactory bulbs and brains that were examined by Newport-Green fluorescence and TEM particle measurement clearly showed the presence of ZnO-NPs in brain. We conclude that an olfactory bulb-brain translocation pathway for airborne ZnO-NPs exists in rats, and that endocytosis is required for interneuron translocation of these particles.

Transient Increase in Zn2+ in Hippocampal CA1 Pyramidal Neurons Causes Reversible Memory Deficit

The translocation of synaptic Zn2+ to the cytosolic compartment has been studied to understand Zn2+ neurotoxicity in neurological diseases. However, it is unknown whether the moderate increase in Zn2+ in the cytosolic compartment affects memory processing in the hippocampus. In the present study, the moderate increase in cytosolic Zn2+ in the hippocampus was induced with clioquinol (CQ), a zinc ionophore. Zn2+ delivery by Zn-CQ transiently attenuated CA1 long-term potentiation (LTP) in hippocampal slices prepared 2 h after i.p. injection of Zn-CQ into rats, when intracellular Zn2+ levels was transiently increased in the CA1 pyramidal cell layer, followed by object recognition memory deficit. Object recognition memory was transiently impaired 30 min after injection of ZnCl2 into the CA1, but not after injection into the dentate gyrus that did not significantly increase intracellular Zn2+ in the granule cell layer of the dentate gyrus. Object recognition memory deficit may be linked to the preferential increase in Zn2+ and/or the preferential vulnerability to Zn2+ in CA1 pyramidal neurons. In the case of the cytosolic increase in endogenous Zn2+ in the CA1 induced by 100 mM KCl, furthermore, object recognition memory was also transiently impaired, while ameliorated by co-injection of CaEDTA to block the increase in cytosolic Zn2+. The present study indicates that the transient increase in cytosolic Zn2+ in CA1 pyramidal neurons reversibly impairs object recognition memory.

Zinc Oxide Nanoparticles Interfere With Zinc Ion Homeostasis to Cause Cytotoxicity

The toxicological effects of zinc oxide nanoparticles (ZnO-NPs) are attracting increasing concern as the field of nanotechnology progresses. Although the literature suggests that toxicity of ZnO-NPs may be related to their dissolution, the mechanism for ZnO-NP perturbation of cytosolic zinc concentration ([Zn(2+)](c)) homeostasis remains obscure. Using FluoZin-3 and RhodZin-3, this study investigated changes in both [Zn(2+)](c) and mitochondrial free Zn(2+) concentration ([Zn(2+)](m)) under conditions of ZnO-NP treatment in vivo and in vitro. In human leukemia Jurkat cells and human lung carcinoma H1355 cells, ZnO-NP treatment resulted in an elevation of both [Zn(2+)](c) and [Zn(2+)](m). In H1355 cells, ZnO-NP treatment induced depolarization of mitochondrial membrane potential, as well as caspase-3 activation and lactic dehydrogenase (LDH) release. In our in vivo experiments, when rats were exposed to ZnO-NPs, higher [Zn(2+)](c) and [Zn(2+)](m) were recorded in both broncho-alveolar lavage (BAL) cells and white blood cells isolated from ZnO-NP-exposed rats, compared with high efficiency particulate air-filter-protected controls LDH levels were also elevated in the BAL of ZnO-NP-exposed rats compared with controls. A mechanical toxicological pathway for ZnO-NP toxicity is suggested by these results: an elevation in [Zn(2+)](c) resulting from ZnO-NP dissolution in the intracellular endosome; cytosolic Zn(2+) sequestration by mitochondria; and elevated [Zn(2+)](m) leading to mitochondrial dysfunction, caspase activation, and cell apoptosis. We conclude that exposure to ZnO-NPs interferes with the homeostasis of [Zn(2+)](c,) and that elevated [Zn(2+)](c) results in cell apoptosis.

Glucose Regulates Free Cytosolic Zn2+ Concentration, Slc39 (ZiP), and Metallothionein Gene Expression in Primary Pancreatic Islet β-Cells

Zn2+ is an important cofactor for insulin biosynthesis and storage in pancreatic β-cells. Correspondingly, polymorphisms in the SLC30A8 gene, encoding the secretory granule Zn2+ transporter ZnT8, are associated with type 2 diabetes risk. Using a genetically engineered (FRET)-based sensor (eCALWY-4), we show here that elevated glucose time-dependently increases free cytosolic Zn2+ ([Zn2+]cyt) in mouse pancreatic β-cells. These changes become highly significant (853 ± 96 pm versus 452 ± 42 pm, p < 0.001) after 24 h and are associated with increased expression of the Zn2+ importer family members Slc39a6, Slc39a7, and Slc39a8, and decreased expression of metallothionein 1 and 2. Arguing that altered expression of the above genes is not due to altered [Zn2+]cyt, elevation of extracellular (and intracellular) [Zn2+] failed to mimic the effects of high glucose. By contrast, increases in intracellular cAMP prompted by 3-isobutyl-1-methylxanthine and forskolin partially mimicked the effects of glucose on metallothionein, although not ZiP, gene expression. Modulation of intracellular Ca2+ and insulin secretion with pharmacological agents (tolbutamide and diazoxide) suggested a possible role for changes in these parameters in the regulation of Slc39a6 and Slc39a7 but not Slc39a8, nor metallothionein expression. In summary, 1) glucose induces increases in [Zn2+]cyt, which are then likely to facilitate the processing and/or the storage of insulin and its cocrystallization with Zn2+, and 2) these increases are associated with elevated expression of zinc importers. Conversely, a chronic increase in [Zn2+]cyt following sustained hyperglycemia may contribute to β-cell dysfunction and death in some forms of diabetes.

Intracellular zinc release-activated ERK-dependent GSK-3β–p53 and Noxa–Mcl-1 signaling are both involved in cardiac ischemic-reperfusion injury

Oxidative stress and nitrosative stress are both suggested to be involved in cardiac ischemia-reperfusion (I/R) injury. Using time-lapse confocal microscopy of cardiomyocytes and high-affinity O(2)(-•) and Zn(2+) probes, this study is the first to show that I/R, reactive oxygen species (ROS), and reactive nitrogen species (RNS) all cause a marked increase in the [O(2)(-•)](i), resulting in cytosolic and mitochondrial Zn(2+) release. Exposure to a cell-penetrating, high-affinity Zn(2+)(i) chelator, TPEN, largely abolished the Zn(2+)(i) release and markedly protected myocytes from I/R-, ROS-, RNS-, or Zn(2+)/K(+) (Zn(2+)(i) supplementation)-induced myocyte apoptosis for at least 24 h after TPEN removal. Flavonoids and U0126 (a MEK1/2 inhibitor) largely inhibited the myocyte apoptosis and the TPEN-sensitive I/R- or Zn(2+)(i) supplement-induced persistent extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, dephosphorylation of p-Ser9 on glycogen synthase kinase 3β (GSK-3β), and the translocation into and accumulation of p-Tyr216 GSK-3β and p53 in, the nucleus. Silencing of GSK-3β or p53 expression was cardioprotective, indicating that activation of the ERK-GSK-3β-p53 signaling pathway is involved in Zn(2+)-sensitive myocyte death. Moreover, the ERK-dependent Noxa-myeloid cell leukemia-1 (Mcl-1) pathway is also involved, as silencing of Noxa expression was cardioprotective and U0126 abolished both the increase in Noxa expression and in Mcl-1 degradation. Thus, acute upstream Zn(2+)(i) chelation at the start of reperfusion and the use of natural products, that is, flavonoids, may be beneficial in the treatment of cardiac I/R injury.

Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn 2+ with genetically encoded sensors

Zn(2+) plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn(2+) levels. Although much is known about total Zn(2+) in cells, very little is known about its subcellular distribution. Yet defining the location of Zn(2+) and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn(2+) levels as well as flux of Zn(2+) into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn(2+) that is 100 times lower than the cytosol. Both organelles take up Zn(2+) when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn(2+) and thus have the potential to play a role in influencing Zn(2+) toxicity. ER Zn(2+) homeostasis is perturbed by small molecule antagonists of Ca(2+) homeostasis and ER Zn(2+) is released upon elevation of cytosolic Ca(2+) pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca(2+) signaling can influence Zn(2+) homeostasis and vice versa, that Zn(2+) dynamics may modulate Ca(2+) signaling.

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate – the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.

Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn(2+) binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn(2+) was buffered at 0.4 nM in pancreatic beta cells, and we found substantially higher Zn(2+) concentrations in insulin-containing secretory vesicles.

Inducibility of metallothionein biosynthesis in the whole soft tissue of zebra mussels Dreissena polymorpha exposed to cadmium, copper, and pentachlorophenol

Freshwater mussels Dreissena polymorpha (Pallas, 1771) were exposed to the elevated concentrations of Cd (10, 50, 100, and 500 microg/L), Cu (10, 30, 50, and 80 microg/L), and an organochlorinated pesticide, pentachlorophenol (PCP) (1, 10, and 100 microg/L). Induced synthesis of biomarker metallothionein (MT) and changes in concentrations of cytosolic Cd, Cu, and Zn in the whole soft tissue of mussels were monitored after a 7-day laboratory exposure to the contaminants. A clear dose-dependent elevation in the MT concentration was observed after exposure to Cd at doses of 10-100 microg/L, and this increase of MT content was accompanied with a linear increase of cytosolic Cd. Cd concentration of 500 microg/L caused no additional increase of MT and Cd in mussel cytosol, suggesting possible toxic effects due to exceeding cellular inducible/defense capacity. Cu exposure resulted with variable changes in MT concentrations, with no clear linear relationship between MT and Cu concentrations in water, although a progressive dose-dependent accumulation of Cu in the soluble fraction of mussel tissues was recorded. A decrease of cytosolic Zn was evident at higher exposure concentrations of both metals used. PCP in concentrations applied was unable to induce MT synthesis, but the higher concentrations of PCP influenced the cytosolic metal concentrations. In conclusion, the results obtained confirm the specificity of MT induction in D. polymorpha as an biological response on metal stimulation, especially by cadmium, being more closely correlated to MT than copper within the ecologically relevant concentration range. The strong induction potential of cadmium as well as an absence of MT induction following exposure to PCP as an organic chemical contaminant are supporting evidences for usage of zebra mussel MT as a specific biomarker of Cd exposure in biomonitoring programs.

Intracellular Zn2+Accumulation Contributes to Synaptic Failure, Mitochondrial Depolarization, and Cell Death in an Acute Slice Oxygen–Glucose Deprivation Model of Ischemia

Despite considerable evidence for contributions of both Zn(2+) and Ca(2+) in ischemic brain damage, the relative importance of each cation to very early events in injury cascades is not well known. We examined Ca(2+) and Zn(2+) dynamics in hippocampal slices subjected to oxygen-glucose deprivation (OGD). When single CA1 pyramidal neurons were loaded via a patch pipette with a Ca(2+)-sensitive indicator (fura-6F) and an ion-insensitive indicator (AlexaFluor-488), small dendritic fura-6F signals were noted after several (approximately 6-8) minutes of OGD, followed shortly by sharp somatic signals, which were attributed to Ca(2+) ("Ca(2+) deregulation"). At close to the time of Ca(2+) deregulation, neurons underwent a terminal increase in plasma membrane permeability, indicated by loss of AlexaFluor-488 fluorescence. In neurons coloaded with fura-6F and a Zn(2+)-selective indicator (FluoZin-3), progressive rises in cytosolic Zn(2+) levels were detected before Ca(2+) deregulation. Addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) significantly delayed both Ca(2+) deregulation and the plasma membrane permeability increases, indicating that Zn(2+) contributes to the degenerative signaling. Present observations further indicate that Zn(2+) is rapidly taken up into mitochondria, contributing to their early depolarization. Also, TPEN facilitated recovery of the mitochondrial membrane potential and of field EPSPs after transient OGD, and combined removal of Ca(2+) and Zn(2+) markedly extended the duration of OGD tolerated. These data provide new clues that Zn(2+) accumulates rapidly in neurons during slice OGD, is taken up by mitochondria, and contributes to consequent mitochondrial dysfunction, cessation of synaptic transmission, Ca(2+) deregulation, and cell death.

Intracellular Zn2+ increases contribute to the progression of excitotoxic Ca2+ increases in apical dendrites of CA1 pyramidal neurons

Sustained intracellular Ca2+ elevation is a well-established contributor to neuronal injury following excessive activation of N-methyl-d-aspartic acid (NMDA)–type glutamate receptors. Zn2+ can also be involved in excitotoxic degeneration, but the relative contributions of these two cations to the initiation and progression of excitotoxic injury is not yet known. We previously concluded that extended NMDA exposure led to sustained Ca2+ increases that originated in apical dendrites of CA1 neurons and then propagated slowly throughout neurons and caused rapid necrotic injury. However the fluorescent indicator used in those studies (Fura-6F) may also respond to Zn2+, and in the present work we examine possible contributions of Zn2+ to indicator signals and to the progression of degenerative signaling along murine CA1 dendrites. Selective chelation of Zn2+ with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) significantly delayed, but did not prevent the development and progression of sustained high-level Fura-6F signals from dendrites to somata. Rapid indicator loss during the Ca2+ overload response, which corresponds to rapid neuronal injury, was also not prevented by TPEN. The relationship between cytosolic Zn2+ and Ca2+ levels was assessed in single CA1 neurons co-loaded with Fura-6F and the Zn2+-selective indicator FluoZin-3. NMDA exposure resulted in significant initial increases in FluoZin-3 increases that were prevented by TPEN, but not by extracellular Zn2+ chelation with Ca-EDTA. Consistent with this result, Ca-EDTA did not delay the progression of Fura-6F signals during NMDA. Removal of extracellular Ca2+ reduced, but did not prevent FluoZin-3 increases. These results suggest that sustained Ca2+ increases indeed underlie Fura-6F signals that slowly propagate throughout neurons, and that Ca2+ (rather than Zn2+) increases are ultimately responsible for neuronal injury during NMDA. However, mobilization of Zn2+ from endogenous sources leads to significant neuronal Zn2+ increases, that in turn contribute to mechanisms of initiation and progression of progressive Ca2+ deregulation.

Zn2+Influx Is Critical for Some Forms of Spreading Depression in Brain Slices

Spreading depression (SD) is wave of profound depolarization that propagates throughout brain tissue and can contribute to the spread of injury after stroke or traumatic insults. The contribution of Ca(2+) influx to SD differs depending on the stimulus, and we show here that Zn(2+) can play a critical complementary role in murine hippocampal slices. In initial studies, we used the Na(+)/K(+) ATPase inhibitor ouabain and found conditions in which SD was always prevented by L-type Ca(2+) channel blockers; however, Ca(2+) influx was not responsible for L-type effects. Cytosolic Ca(2+) increases were not detectable in CA1 neurons before SD, and removal of extracellular Ca(2+) did not prevent ouabain-SD. In contrast, cytosolic Zn(2+) increases were observed in CA1 neurons before ouabain-SD, and L-type channel block prevented the intracellular Zn(2+) rises. A slow mitochondrial depolarization observed before ouabain-SD was abolished by L-type channel block, and Zn(2+) accumulation contributed substantially to initial mitochondrial depolarizations. Selective chelation of Zn(2+) with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) abolished SD, implying that Zn(2+) entry can play a critical role in the generation of ouabain-SD. TPEN was most effective when synaptic activity was reduced by adenosine A(1) receptor activation, and a combination of Ca(2+) and Zn(2+) removal was required to prevent ouabain-SD when A(1) receptors were blocked. Similarly, Zn(2+) chelation could prevent SD triggered by oxygen/glucose deprivation but Zn(2+) accumulation did not contribute to SD triggered by localized high K(+) exposures. These results identify Zn(2+) as a new target for the block of spreading depolarizations after brain injury.

Metallothioneins and cytosolic metals in Neomysis integer exposed to cadmium at different salinities

In the present study the induction of metallothioneins (MTs) and its relation to cytosolic metal concentrations (Zn, Cu and Cd) in the euryhaline crustacean Neomysis integer exposed to Cd at different salinities was studied. N. integer was exposed to the same free cadmium ion activity of 5.74 x 10(-9) mol l(-1) (i.e. 1/5 of the 96 h LC(50) value expressed as cadmium activity) in hypo-osmotic (5 psu), isosmotic (16 psu) and hyper-osmotic media (25 psu) for 7 days. In this way, the effect of salinity on cadmium speciation was eliminated and therefore the physiological effect of salinity on Cd accumulation and MT induction could be studied. The accumulation of cytosolic Cd in N. integer changed with salinity from 1.11+/-0.05 micromol l(-1) at 5 psu up to 1.43+/-0.17 micromol l(-1) at 25 psu. This could indicate that the physiological response of euryhaline estuarine invertebrates like N. integer to salinity changes can influence the rate of trace metal uptake from solution. While the salinity changes did not cause significant differences in cytosolic Zn concentrations (mean value of all tested salinities: 34.4+/-2.8 micromol l(-1)), an inverse relationship between salinity and cytosolic Cu concentration was observed. The highest concentration of 15.7+/-2.3 micromol Cul(-1) was determined at 5 psu and the lowest 10.9+/-1.4 micromol Cul(-1) at 25 psu. This could point to a possible relationship between the copper concentration and the hemocyanin metabolism in N. integer. This is the first time that differential pulse voltammetry method was applied to MT assays with N. integer. Although the exposure to Cd resulted in a higher Cd cytosolic concentration, no subsequent MT increase was detected. The significant positive correlation between MT levels and cytosolic Cu concentrations (Spearman correlation coefficient r(s)=0.356, p=0.009) implies a strong relationship between MT and Cu in N. integer.

Insights into Zn2+ homeostasis in neurons from experimental and modeling studies

To understand the mechanisms of neuronal Zn2+ homeostasis better, experimental data obtained from cultured cortical neurons were used to inform a series of increasingly complex computational models. Total metals (inductively coupled plasma-mass spectrometry), resting metallothionein, (65)Zn2+ uptake and release, and intracellular free Zn2+ levels using ZnAF-2F were determined before and after neurons were exposed to increased Zn2+, either with or without the addition of a Zn2+ ionophore (pyrithione) or metal chelators [EDTA, clioquinol (CQ), and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine]. Three models were tested for the ability to match intracellular free Zn2+ transients and total Zn2+ content observed under these conditions. Only a model that incorporated a muffler with high affinity for Zn2+, trafficking Zn2+ to intracellular storage sites, was able to reproduce the experimental results, both qualitatively and quantitatively. This "muffler model" estimated the resting intracellular free Zn2+ concentration to be 1.07 nM. If metallothionein were to function as the exclusive cytosolic Zn2+ muffler, the muffler model predicts that the cellular concentration required to match experimental data is greater than the measured resting concentration of metallothionein. Thus Zn2+ buffering in resting cultured neurons requires additional high-affinity cytosolic metal binding moieties. Added CQ, as low as 1 microM, was shown to selectively increase Zn2+ influx. Simulations reproduced these data by modeling CQ as an ionophore. We conclude that maintenance of neuronal Zn2+ homeostasis, when challenged with Zn2+ loads, relies heavily on the function of a high-affinity muffler, the characteristics of which can be effectively studied with computational models.