Zinc chloride-induced TRPA1 activation does not contribute to toxicity in vitro (original) (raw)
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Clioquinol and pyrithione activate TRPA1 by increasing intracellular Zn2+
Proceedings of the National Academy of Sciences, 2009
The antifungal and amoebicidal drug clioquinol (CQ) was withdrawn from the market when it was linked to an epidemic of subacute myelo-optico-neuropathy (SMON). Clioquinol exerts its anti-parasitic actions by acting as a Cu/Zn chelator and ionophore. Here we show that local injections of CQ produce mechanical hyperalgesia and cold hypersensitivity through a mechanism involving TRPA1 in mice. We also show that CQ activates TRPA1 in a Zn 2؉ -dependent manner. Using a different Zn 2؉ -ionophore, zinc pyrithione (ZnPy), we demonstrate that low, nanomolar concentrations of intracellular Zn 2؉ ([Zn 2؉ ]i) stimulate TRPA1. Direct application of Zn 2؉ to the intracellular face of excised, inside-out patches activates TRPA1 with an EC50 value of 7.5 ؎ 1 nM. TRPA1 is expressed in a subpopulation of nociceptive dorsal root ganglion (DRG) neurons, where it acts as a sensory receptor for environmental irritants and oxidants. Using cultured DRG neurons from wild-type and TRPA1-deficient mice, we demonstrate that TRPA1 is the principal excitatory receptor for increased [Zn 2؉ ]i in DRG neurons. In conclusion, we have discovered that TRPA1 acts a sensor of intracellular Zn 2؉ , and that Zn 2؉ ionophores, such as CQ and ZnPy, activate TRPA1 by increasing [Zn 2؉ ]i. We also demonstrate that CQ-evoked mechanical hyperalgesia and cold allodynia require TRPA1 in vivo.
AMPA receptor activation potentiates zinc neurotoxicity
Neuron, 1993
Extracellular Zn2+ attenuates NMDA receptor-mediated neurotoxicity and increases AMPA receptor-mediated toxicity. Known electrophysiological effects of Zn*+ predict only the former. We considered the possibility that the latter rather reflects AMPA potentiation of Zn*+ toxicity, perhaps mediated by neuronal depolarization and Zn2+ entry through voltage-gated Ca2+ channels. High K+ or kainate also potentiated Zn2+ toxicity, and AMPA plus Zn2+ toxicity was attenuated by raising extracellular Ca2+, or by Ca2+ channel blockers. AMPA plus Zn2+ exposure induced an increase in fluorescence from neurons loaded with the Zn2+-sensitive dye TS-Q and increased subsequent 45Ca2+ accumulation. The ability of AMPA receptor activation to potentiate Zn2+ toxicity may be relevant to neuronal death associated with intense activation of glutamatergic pathways.
Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity
European Journal of Neuroscience, 2000
Exposure of central neurons to Zn 2+ triggers neuronal death. The routes of Zn 2+ entry were investigated in living cortical neurons from the mouse using the speci®c Zn 2+¯u orescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ), which preferentially detects membrane-bound Zn 2+. Exposure of cortical neurons to increasing concentrations of Zn 2+ (1±100 mM) induced a progressive increase in the¯uorescence of TSQ. This¯uorescence signal was not attenuated by the permeation of plasma membrane with digitonin. Accordingly, the major part of TSQ¯uorescence (two-thirds) was associated to the particulate fraction of cortical neurons exposed to Zn 2+. These results suggest that Zn 2+ detected with TSQ in neurons is mainly bound to membranes. TSQ¯uorescence measured in neurons exposed to 3 mM Zn 2+ was enhanced by Na +-pyrithione, a Zn 2+ ionophore, a-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA) or KCl-induced depolarization. However, in the absence of any treatment, TSQ labelling of neurons exposed to 3 mM Zn 2+ was only decreased by NMDA receptor antagonists, whereas it remained unaltered in the presence of antagonists of AMPA receptors or L-type voltage-gated Ca 2+ channels. Zn 2+ entry through NMDA receptors did not contribute to Zn 2+-induced neuronal death, as it was prevented by antagonists of NMDA receptors only when they were added after the Zn 2+ exposure. Finally, Zn 2+ induced a delayed accumulation of extracellular glutamate which might be responsible for the delayed NMDA receptor activation that leads to neuronal death.
Purinergic …, 2005
In this review, we focus on two attributes of P2X receptor channel function, one essential and one novel. First, we propose that P2X receptors are extracellular sensors as well as receptors and ion channels. In particular, the large extracellular domain (that comprises 70% of the molecular mass of the receptor channel protein) lends itself to be a cellular sensor. Moreover, its exquisite sensitivity to extracellular pH, ionic strength, and multiple ligands evokes the function of a sensor. Second, we propose that P2X receptors are extracellular zinc receptors as well as receptors for nucleotides. We provide novel data in multiple publications and illustrative data in this invited review to suggest that zinc triggers ATP-independent activation of P2X receptor channel function. In this light, P2X receptors are the cellular site of integration between autocrine and paracrine zinc signaling and autocrine and paracrine purinergic signaling. P2X receptors may sense changes in these ligands as well as in extracellular pH and ionic strength and transduce these sensations via calcium and/or sodium entry and changes in membrane potential. Abbreviations: ADP-adenosine diphosphate; ASIC-acid-sensing ion channels; ATP-adenosine 5 ¶-triphosphate; Ca 2+calcium; CF-cystic fibrosis; CFTR-cystic fibrosis transmembrane conductance regulator; Cl j-chloride; COPD-chronic obstructive pulmonary diseases; DEG-degenerin channels; DRASIC-ASIC from Drosophila; ENaC-epithelial Na + channel; FDA-US Food and Drug Administration; G protein-heterotrimeric GTP-binding protein; GPCR-G proteincoupled receptor; H +-proton; IP3-inositol trisphosphate; K(ATP)-ATP-regulated K + channel of sulfonylurea transporters and inwardly rectifying K + channels; K +-potassium; MDCK-MadinYDarby canine kidney cells; Mg 2+-magnesium; Na +-sodium; NMDG-N-methyl-D-glucamine; OTC-over-the-counter; P2X-purinergic receptor channel subfamily; PKD-polycystic kidney disease; PLC-phospholipase C; ppk-pickpocket from Drosophila; rpk-ripped pocket from Drosophila; TRP-Transient receptor potential channels; ZAC-zinc-activated channel; Zip-zinc influx protein transporters; Zn 2+-zinc; ZnT-zinc loading or efflux transporter
Functional Exploration Of T-Type Calcium Channels (Cav3.2 And Cav3.3) And Their Sensitivity To Zinc
The Open Microbiology Journal
Introduction: T-type Ca2+ channels (TTCC) are low Voltage-gated calcium channels, expressed in various tissues such as the brain and heart, and contribute to a variety of physiological functions including neuronal excitability, hormone secretion, muscle contraction, and pacemaker activity. At high concentrations, Zinc (Zn2+) is naturally attached to cell membranes and is therefore considered a reversible inhibitor of calcium. Zinc is also involved in the kinetics of sodium and potassium currents. Zinc is essential for many functions. A low zinc tenor is associated with emotional instability, digestive disorders, slow-growing and alteration of protein synthesis. Material and Methods: For the Cell Culture we used HEK-293/tsA-201, and for transfection, the pCDNA3 plasmid constructs encoding human CaV3.2, and CaV3.3 subunits. Electrophysiological experiments were performed using the whole cell configuration of the patch-clamp technique. T-type currents were recorded using a test pulse f...
Comparative in vitro toxicity of seven zinc-salts towards neuronal PC12 cells
Toxicology in Vitro, 2009
Currently much attention has been given to the neurotoxicity of zinc, yet little is known about the influence of the counterions present. Therefore, we investigated the influence of different Zn 2+-salts (concentrations range 0.05-0.3 mM) on cell viability, ATP and glutathione concentration and caspase activation in differentiated PC12 cells as a model for neuronal cells. Generally, at concentrations of 0.05 mM most Zn 2+-salts were not cytotoxic except for zinc-citrate. At concentrations between 0.1 and 0.3 mM Zn 2+ a significant decrease in GSH and ATP levels preceded cell death induced by all salts, except of zinc-histidinate. Zinc-citrate and zinc-sulphate turned out to be the most toxic salts particularly at low concentrations. Analyses of caspase 3/7 activity showed that dependent on the concentration and the type of the salt used cell death may show more or less signs of both, necrosis and apoptosis. Interestingly, the uptake of Zn 2+ from zinc-sulphate and zinc-citrate was significantly higher than that of other salts, implicating a correlation between uptake and toxicity. In conclusion, Zn 2+-salts could be divided into three categories with high (zinc-citrate, zinc-sulphate), moderate (zinc-orotate, zinc-acetate, zinc-chloride , zinc-gluconate) and low cytotoxicity (zinc-histidinate).
Cells
Chemicals can exhibit significant toxic properties. While for most compounds, unspecific cell damaging processes are assumed, a plethora of chemicals exhibit characteristic odors, suggesting a more specific interaction with the human body. During the last few years, G-protein-coupled receptors and especially chemosensory ion channels of the transient receptor potential family (TRP channels) were identified as defined targets for several chemicals. In some cases, TRP channels were suggested as being causal for toxicity. Therefore, these channels have moved into the spotlight of toxicological research. In this review, we screened available literature in PubMed that deals with the role of chemical-sensing TRP channels in specific organ systems. TRPA1, TRPM and TRPV channels were identified as essential chemosensors in the nervous system, the upper and lower airways, colon, pancreas, bladder, skin, the cardiovascular system, and the eyes. Regarding TRP channel subtypes, A1, M8, and V1 w...
Diverse effects of metal chelating agents on the neuronal cytotoxicity of zinc in the hippocampus
Brain Research, 1998
Abnormal metabolism of metal ions such as zinc may contribute to neuropathology. Complexing zinc could reduce this pathology. Thus, to examine the effectiveness of metal chelating agents in vivo, a model system was used. This involved determining the ability of chelating agents to prevent neuronal death caused by zinc chloride injected into the rat hippocampus. Significant protection against zinc toxicity was obtained with pyrithione, inositol hexakisphosphate, ethylenediamine tetraacetate (EDTA) and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). The affinity of these agents for zinc varied between 106 M-1 and 1018 M-1. Thus, the affinity for zinc within this range does not appear to be a major factor affecting the ability of chelators to provide neuroprotection. While almost complete protection was found with EDTA and TPEN given simultaneously with zinc chloride, poor protection was obtained if TPEN was given before or after zinc chloride. Other agents either did not protect against zinc-induced neuronal death (zincon), or exacerbated zinc toxicity (BTC-5N and about 40% of rats injected with a combination of zinc chloride and diethylenetriamine pentaacetate [DTPA]). Rats showing increased damage after zinc plus BTC-5N or DTPA suffered wet dog-like shakes (WDS), suggesting that these zinc chelate complexes can induce seizures resulting in seizure-related damage. In contrast, in the 60% of rats treated with zinc chloride and DTPA that had no WDS, there was about an 80% reduction in the size of the zinc-induced lesion. The ability of chelators to cross cell membranes was examined by determining whether Timm's staining for vesicular zinc was reduced following the injection of a chelator into the hippocampus. TPEN and pyrithione reduced Timm's staining for zinc. However, cell permeability was not necessary for a chelator to protect against zinc toxicity.
The Essential Toxin: Impact of Zinc on Human Health
International Journal of Environmental Research and Public Health, 2010
Compared to several other metal ions with similar chemical properties, zinc is relatively harmless. Only exposure to high doses has toxic effects, making acute zinc intoxication a rare event. In addition to acute intoxication, long-term, high-dose zinc supplementation interferes with the uptake of copper. Hence, many of its toxic effects are in fact due to copper deficiency. While systemic homeostasis and efficient regulatory mechanisms on the cellular level generally prevent the uptake of cytotoxic doses of exogenous zinc, endogenous zinc plays a significant role in cytotoxic events in single cells. Here, zinc influences apoptosis by acting on several molecular regulators of programmed cell death, including caspases and proteins from the Bcl and Bax families. One organ where zinc is prominently involved in cell death is the brain, and cytotoxicity in consequence of ischemia or trauma involves the accumulation of free zinc. Rather than being a toxic metal ion, zinc is an essential trace element. Whereas intoxication by excessive exposure is rare, zinc deficiency is widespread and has a detrimental impact on growth, neuronal development, and immunity, and in severe cases its consequences are lethal. Zinc deficiency caused by malnutrition and foods with low bioavailability, aging, certain diseases, or deregulated homeostasis is a far more common risk to human health than intoxication.
Silencing of ZnT-1 expression enhances heavy metal influx and toxicity
Journal of Molecular Medicine, 2006
ZnT-1 reduces intracellular zinc accumulation and confers resistance against cadmium toxicity by a mechanism which is still unresolved. A functional link between the L-type calcium channels (LTCC) and ZnT-1 has been suggested, indicating that ZnT-1 may regulate ion permeation through this pathway. In the present study, immunohistochemical analysis revealed a striking overlap of the expression pattern of LTCC and ZnT-1 in cardiac tissue and brain. Using siRNA to silence ZnT-1 expression, we then assessed the role of ZnT-1 in regulating cation permeation through the L-type Ca 2+ channels in cells that are vulnerable to heavy metal permeation. Transfection of cortical neurons with ZnT-1 siRNA resulted in about 70% reduction of ZnT-1 expression and increased Ca 2+ influx via LTCC by approximately fourfold. Moreover, ZnT-1 siRNA transfected neurons showed ∼30% increase in synaptic release, monitored using the FM1-43 dye. An increased cation influx rate, through the LTCC, was also recorded for Zn 2+ and Cd 2+ in cells treated with the ZnT-1 siRNA. Furthermore, Cd 2+ -induced neuronal death increased by approximately twofold after transfection with ZnT-1 siRNA. In addition, ZnT-1 siRNA transfection of the ovarian granulosa cell line, POGRS1, resulted in a twofold increase in Cd 2+ influx rate via the LTCC. Finally, a robust nimodipine-sensitive Cd 2+ influx was observed using a low extracellular Cd 2+ concentration (5 μM) in neurons and EHUD (UDI) OHANA is a graduate student in the laboratory of Israel Sekler, studying the mechanisms of function and regulation of the ZnT proteins and their role in the cellular homeostasis of heavy metals.