Apoptosis induced by chelation of intracellular zinc is associated with depletion of cellular reduced glutathione level in rat hepatocytes

Zn(2+) has multiple implications in cellular metabolism, including free radicals metabolism and cell death by apoptosis. In the present study, we examined the role of Zn(2+) in the regulation of apoptosis in cultured rat hepatocytes. The chelation of Zn(2+) by a membrane permeable metal ion chelator, N, N, N', N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), induced apoptosis. Addition of ZnSO(4) prevented TPEN-induced apoptosis. Unlike the effect of TPEN, a membrane impermeable metal ion chelator, diethylenetriamine pentaacetic acid (DTPA), did not induce apoptosis, indicating that chelation of intracellular Zn(2+) was required to trigger apoptosis. Caspase-3-like proteolytic activity, a general biochemical mediator of apoptosis in a variety of cells and tissues, was also activated with the treatment of TPEN but not DTPA. TPEN treatment, but not DTPA, also resulted in the depletion of intracellular reduced glutathione (GSH) but addition of Zn(2+) recovered the GSH level. N-acetyl-L-cysteine (NAC), a thiol antioxidant, prevented TPEN-induced apoptosis. These results taken together suggest that intracellular Zn(2+) interfere with the apoptosis process, possibly through the regulation of cellular redox potential involving GSH.     

Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes

Matrix vesicles released by epiphyseal growth plate chondrocytes are known to contain a significant quantity of labile Zn(2+). Zonal analysis of chicken metatarsal bones showed that the resting/proliferative region of the growth plate contained high levels of Zn(2+) with significantly lower levels in the hypertrophic cartilage suggesting a loss of cellular Zn(2+) as the chondrocytes mature. Intracellular labile Zn(2+) was measured in primary cultures of growth plate chondrocytes by assay with the fluorescent Zn-chelator toluenesulfonamidoquinoline (TSQ) and imaged by multi-photon laser scanning microscopy (MPLSM) with the TSQ derivative zinquin. Short-term exposure to Zn(2+), both in the presence and absence of pyrithione resulted in significant increases in cytosolic Zn(2+). Treatment with the membrane-permeant Zn(2+) chelator TPEN rapidly reduced the levels of labile Zn(2+) and triggered apoptosis. Cytosolic Zn(2+) levels were significantly reduced following 24-h incubations with known inducers of chondrocyte apoptosis. The loss of intracellular Zn(2+) was accompanied by a significant reduction in the cytosolic metal-binding protein metallothionein. Examination of Zn(2+)-treated cells with MPLSM showed uniformly higher zinquin fluorescence. Treatment of Zn(2+)-loaded cells with TPEN quenched zinquin fluorescence confirming that the observed fluorescence in chondrocytes is due to the presence of intracellular Zn(2+). A dose-dependent increase in zinquin fluorescence was observed in cells treated with a range of Zn(2+) concentrations. Short-term treatment of cultured chondrocytes with apoptosis-inducing chemicals resulted in transient increases in intracellular labile Zn(2+). These results indicate that Zn(2+) is mobilized from intracellular binding sites in the early stages of chondrocyte apoptosis and is subsequently lost from the cells. The early mobilization of Zn(2+) provides a mechanism for its movement to matrix vesicles and the extracellular matrix.     

Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes

Cytosolic alterations of calcium ion concentrations are an integral part of signal transduction. Similar functions have been hypothesized for other metal ions, in particular zinc (Zn(2+)), but this still awaits experimental verification. Zn(2+) is important for multiple cellular functions, especially in the immune system. Among other effects, it influences formation and secretion of pro-inflammatory cytokines, including TNF-alpha. Here we demonstrate that these effects are due to a physiological signaling system involving intracellular Zn(2+) signals. An increase of the intracellular zinc ion concentration occurs upon stimulation of human leukocytes with Escherichia coli, LPS, Pam(3)CSK(4), TNF-alpha, or insulin, predominantly in monocytes. Chelating this zinc signal with the membrane permeable zinc-specific chelator TPEN (N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine) completely blocks activation of LPS-induced signaling pathways involving p38 MAPK, ERK1/2, and NF-kappaB, and abrogates the release of proinflammatory cytokines, including TNF-alpha. This function of Zn(2+) is not limited to monocytes or even the immune system, but seems to be another generalized signaling system based on intracellular fluctuations of metal ion concentrations, acting parallel to Ca(2+).     

Poly(ADP-ribosyl)ation of p53 Contributes to TPEN-Induced Neuronal Apoptosis

Depletion of intracellular zinc by N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.     

Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn(2+) binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink". Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn(2+) chelating agents including N,N,N',N'-(2-pyridylethyl)ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2-3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK(1) cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn(3)-Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn(3)-Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn(3)-Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK(1) cells to TPEN for 24h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn(2+).     

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.     

LPS-induced decrease in intracellular labile zinc, [Zn]i, contributes to apoptosis in cultured sheep pulmonary artery endothelial cells

A role in signal transduction for a vanishingly small labile pool of intracellular zinc ([Zn](i)) has been inferred by the sensitivity of various physiological pathways to zinc chelators such as N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and/or associations with changes in nonprotein-bound zinc-sensitive fluorophores. Although we (44) reported that LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) was exacerbated by TPEN, 1) we did not detect acute (30 min) changes in [Zn](i), and 2) it is unclear from other reports whether LPS increases or decreases [Zn](i) and whether elevations or decreases in [Zn](i) are associated with cell death and/or apoptosis. In the present study, we used both chemical (FluoZin-3 via live cell epifluorescence microscopy and fluorescence-activated cell sorting) and genetic (luciferase activity of a chimeric reporter encoding zinc-sensitive metal-response element and changes in steady-state mRNA of zinc importer, SLC39A14 or ZIP14) techniques to show that LPS caused a delayed time-dependent (2-4 h) decrease in [Zn](i) in SPAEC. A contributory role of decreases in [Zn](i) in LPS-induced apoptosis (as determined by caspase-3/7 activation, annexin-V binding, and cytochrome c release) in SPAECs was revealed by mimicking the effect of LPS with the zinc chelator, TPEN, and inhibiting LPS- (or TPEN)-induced apoptosis with exogenous zinc. Collectively, these are the first data demonstrating a signaling role for decrease in [Zn](i) in pulmonary endothelial cells and suggest that endogenous levels of labile zinc may affect sensitivity of pulmonary endothelium to the important and complex proapoptotic stimulus of LPS.     

Release of intracellular Zn<Superscript>2+</Superscript> in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn²⁺ release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn²⁺ release and subsequent cell death. Cortical neurons were loaded with the Zn²⁺ selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn²⁺ levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn²⁺. Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn²⁺ induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn²⁺ release appeared to peak at 1 μM spermine NONOate (123.8 ± 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn²⁺ levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn²⁺ in cortical neurons. The possibility that release of intracellular Zn²⁺ in response to NO could play a role in intracellular signaling is discussed.     

High intracellular Zn<Superscript>2+</Superscript> ions modulate the VHR,ZAP-70 and ERK activities of LNCaP prostate cancer cells

Malignant prostate tissues have markedly reduced zinc (Zn(2+)) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn(2+) level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn(2+) (10 mug/ml) over 5 weeks. The intracellular Zn(2+) level increased in the Zn(2+)-treated cells, and there was a marked increase in the presence of zincosomes, a Zn(2+)-specific intracellular organelle. The proliferation rate of the Zn(2+)-treated cells was markedly reduced. There was also a significant increase (36.6% +/- 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn(2+), reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn(2+)-treated LNCaP cell proliferation to a rate comparable to that of the non Zn(2+)-treated cells. These results highlight the importance of a high intracellular Zn(2+) content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.     

Biphasic modulation of ryanodine receptors by sulfhydryl oxidation in rat ventricular myocytes

To understand better the modulation of ryanodine receptors (RyRs) during oxidative stress, the effect of 4,4'-dithiodipyridine (DTDP), a cell-permeant and thiol-reactive oxidant, on global Ca(2+) signal and spontaneous Ca(2+) sparks of rat ventricular myocytes was investigated. It was shown that a brief Ca(2+) transient was elicited by DTDP, when its concentration was raised to 100 microM DTDP. In addition a dose-dependent increase of cytoplasmic free Zn(2+) concentration was induced by DTDP. An increase of the frequency of spontaneous Ca(2+) sparks appeared at 3 microM DTDP, whereas higher concentration of DTDP caused a biphasic change of the frequency in both intact and permeabilized myocytes. Consistent with the biphasic effect, caffeine-induced Ca(2+) transients were similarly affected. Because DTDP did not reduce the free Ca(2+) concentration in the sarcoplasmic reticulum lumen, it is likely that the effects of DTDP on the frequency and caffeine-induced Ca(2+) transients are due mainly to sulfhydryl oxidation-induced activation and subsequent inactivation of RyRs. Unlike the frequency, the spatio-temporal properties of Ca(2+) sparks were not influenced by DTDP. The finding that DTDP does not affect the duration of Ca(2+) sparks is inconsistent with that the DTDP-induced increase of the open time of reconstituted RyR channels. The mechanism underlying this discrepancy, especially the possible role of the interaction between arrayed RyRs in myocytes, is discussed. This study suggests that, even if oxidative stress is mild enough not to cause intracellular Ca(2+) accumulation, it may affect signaling pathways through directly modulating the RyR or its complex and in turn changing the frequency of spontaneous Ca(2+) sparks. Thus, the functional importance of moderate oxidative stress should not be overlooked.     

Dysregulation of intracellular calcium homeostasis is responsible for neuronal death in an experimental model of selective hippocampal degeneration induced by trimethyltin

Trimethyltin (TMT) intoxication is considered a suitable experimental model to study the molecular basis of selective hippocampal neurodegeneration as that occurring in several neurodegenerative diseases. We have previously shown that rat hippocampal neurons expressing the Ca(2+)-binding protein calretinin (CR) are spared by the neurotoxic action of TMT hypothetically owing to their ability to buffer intracellular Ca(2+) overload. The present study was aimed at determining whether intracellular Ca(2+) homeostasis dysregulation is involved in the TMT-induced neurodegeneration and if intracellular Ca(2+)-buffering mechanisms may exert a protective action in this experimental model of neurodegeneration. In cultured rat hippocampal neurons, TMT produced time- and concentration-dependent [Ca(2+)](i) increases that were primarily due to Ca(2+) release from intracellular stores although Ca(2+) entry through Ca(v)1 channels also contributed to [Ca(2+)](i) increases in the early phase of TMT action. Cell pre-treatment with the Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (2 muM) significantly reduced the TMT-induced neuronal death. Moreover, CR(+) neurons responded to TMT with smaller [Ca(2+)](i) increases. Collectively, these data suggest that the neurotoxic action of TMT is mediated by Ca(2+) homeostasis dysregulation, and the resistance of hippocampal neurons to TMT (including CR(+) neurons) is not homogeneous among different neuron populations and is related to their ability to buffer intracellular Ca(2+) overload.     

Calcium signalling in squid olfactory receptor neurons

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca(2+) imaging techniques to determine if changes in intracellular Ca(2+) were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca(2+) from intracellular stores while betaine did not change internal Ca(2+) concentrations. Application of 10 mM caffeine also released Ca(2+) from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 microM thapsigargin revealed the presence of a Ca(2+) store depletion-activated Ca(2+) influx. The influx of Ca(2+) through the store-operated channel was reversibly blocked by 10 mM Cd(2+). Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca(2+) release from intracellular stores. Copyright Copyright 1999 S. Karger AG, Basel     

Glutamate mobilizes [Zn2+] through Ca2+ -dependent reactive oxygen species accumulation

Liberation of zinc from intracellular stores contributes to oxidant-induced neuronal injury. However, little is known regarding how endogenous oxidant systems regulate intracellular free zinc ([Zn(2+)](i)). Here we simultaneously imaged [Ca(2+)](i) and [Zn(2+)](i) to study acute [Zn(2+)](i) changes in cultured rat forebrain neurons after glutamate receptor activation. Neurons were loaded with fura-2FF and FluoZin-3 to follow [Ca(2+)](i) and [Zn(2+)](i), respectively. Neurons treated with glutamate (100 microM) for 10 min gave large Ca(2+) responses that did not recover after termination of the glutamate stimulus. Glutamate also increased [Zn(2+)](i), however glutamate-induced [Zn(2+)](i) changes were completely dependent on Ca(2+) entry, appeared to arise entirely from internal stores, and were substantially reduced by co-application of the membrane-permeant chelator TPEN during the glutamate treatment. Pharmacological maneuvers revealed that a number of endogenous oxidant producing systems, including nitric oxide synthase, phospholipase A(2), and mitochondria all contributed to glutamate-induced [Zn(2+)](i) changes. We found no evidence that mitochondria buffered [Zn(2+)](i) during acute glutamate receptor activation. We conclude that glutamate-induced [Zn(2+)](i) transients are caused in part by [Ca(2+)](i)-induced reactive oxygen species that arises from both cytosolic and mitochondrial sources.     

N-acetyl-L-cysteine in the presence of Cu<Superscript>2+</Superscript> induces oxidative stress and death of granule neurons in dissociated cultures of rat cerebellum

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1 mM) in the presence of Cu²⁺ (0.0005-0.001 mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn²⁺ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn²⁺ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4 h with Cu²⁺ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu²⁺. Incubation of cultures in a balanced salt solution in the presence of 0.01 mM Cu²⁺ caused neuronal death already after 1 h if the NAC concentration in the solution was within 0.005–0.05 mM. NAC at higher concentrations (0.1–1 mM) together with 0.01mM Cu²⁺ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.     

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems including immunodeficiency and sensory dysfunctions. Previous studies had shown that Zn availability was also important for tumor growth and progression. The aim of the present study was to investigate the potential mechanisms of N,N,N,N-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (a membrane permeable zinc chelator) induced pancreatic cancer cell death. The text of inductively coupled plasma-mass spectrometry (ICP-MS) showed in human pancreatic cancer samples that the zinc content in cancer was higher than that in adjacent tissues. The pancreatic cancer cell lines Panc-1, 8988T, BxPc-3, and L3.6 were used in this study. Our results indicated that TPEN markedly induced cell death, via increasing reactive oxygen species (ROS) and restraining autophagy. Our data also indicated that TPEN-stimulated mitochondrial metabolism produced much ROS. Meanwhile, TPEN reduced the levels of glutathione (GSH) and triggered ROS outbreak, which were the main causes of cell death. In addition, cell autophagy was significantly depressed in Panc-1 cells treated by TPEN, which was due to the ability of disrupting lysosomal by TPEN. Thus, we thought zinc depletion by TPEN was a potential therapeutic strategy for pancreatic cancer.     

Visualization of labile zinc and its role in apoptosis of primary airway epithelial cells and cell lines

The respiratory epithelium is vulnerable to noxious substances, resulting in the shedding of cells and decreased protection. Zinc (Zn), an antioxidant and cytoprotectant, can suppress apoptosis in a variety of cells. Here we used the novel Zn-specific fluorophore Zinquin to visualize and quantify labile intracellular Zn in respiratory epithelial cells. Zinquin fluorescence in isolated ciliated tracheobronchial epithelial cells and intact epithelium from sheep and pigs revealed an intense fluorescence in the apical and mitochondria-rich cytoplasm below the cilia. Zinquin fluorescence was quenched by the Zn chelator N,N,N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and increased by the Zn ionophore pyrithione. We also assessed whether changes in intracellular labile Zn would influence susceptibility of these cells to apoptosis by hydrogen peroxide. Our results confirm that Zn deficiency enhanced hydrogen peroxide-induced caspase activation from 1.24 +/- 0.12 to 2.58 +/- 0.53 units. microg protein(-1). h(-1) (P 相似文献   

Role for zinc in a cellular response mediated by protein kinase C in human B lymphocytes

Recent studies have suggested a role for Zn2+, distinct from that of Ca2+, in the subcellular distribution and activation of protein kinase C (PKC). Here we show that Zn2+ is required for a cellular response mediated by PKC, the rapid loss of expression of a human B cell receptor MER, detected by rosetting with mouse erythrocytes. Zn2+, in the presence of the Zn2+ ionophore pyrithione, caused rapid inhibition of MER rosetting at concentrations which induce the translocation and activation of PKC. This required cellular uptake of Zn2+ and was blocked by 1,10-phenanthroline and TPEN which chelate Zn2+ but not Ca2+. Gold, a metal with similar properties, also induced translocation of PKC and inhibition of MER. By contrast, Ca2+ ionophores A23187 and ionomycin, which induce a different pathway of translocation of PKC, had no effect on MER. Phenanthroline and TPEN also blocked the inhibition of MER induced by the PKC activators phorbol ester and sodium fluoride, suggesting that endogenous cellular Zn2+ is required. We propose that some cellular actions of PKC require a Zn(2+)-dependent event and that these may be a target for gold during chrysotherapy in rheumatoid arthritis.     

The neurosteroids, allopregnanolone and progesterone, induce autophagy in cultured astrocytes

Recent studies have suggested that neurosteroids such as pregnenolone, progesterone (PG) and their derivatives, have a role in activating autophagy in addition to diverse other functions. In our previous studies, we demonstrated that cellular free Zn(2+) is involved in oxidative stress-induced autophagy and autophagic cell death in astrocytes. In the present study, we examined the possibility that neurosteroids, allopregnanolone (Allo) and PG, also activate autophagy in cultured mouse astrocytes through modulation of intracellular Zn(2+). Exposure of astrocytes to 250 nM Allo or 500 nM PG caused cytosolic vacuoles to appear within a few hours of treatment onset. Live-cell confocal microscopy of astrocytes transfected with red fluorescent protein-conjugated LC3 (RFP-LC3), a marker for autophagic vacuoles (AVs), as well as transmission electron microscopy, revealed that these vacuoles were AVs. In addition, Western blots showed increases in LC3-II levels. Interestingly, mTOR and Akt were concurrently activated, and their blockade further increased LC3-II levels and caused some cell death. These results indicate that co-activation of mTOR and Akt may act to limit neurosteroid-induced autophagy and thus inhibit autophagic cell death. As in other cases of autophagy, cellular Zn(2+) levels increased after treatment with neurosteroids. The neurosteroid-induced increase in LC3-II levels was inhibited by addition of the Zn(2+) chelator TPEN. Both the increase in LC3-II levels and activation of Akt and mTOR by neurosteroids were all mediated by PG receptors, as the effects were blocked by the addition of RU-486, a PG receptor antagonist. Moreover, mutant huntingtin (mHtt) aggregates in GFP-mHttQ74-transfected astrocytes were substantially reduced by neurosteroid treatment, indicating that neurosteroid-induced autophagy may be functional. Present results demonstrate that Allo and PG activate autophagy in astrocytes. Notably, unlike several other autophagy inducers that, in excess, may cause autophagic cell death, Allo and PG are relatively non-toxic, possibly because of concurrent Akt and mTOR activation. Thus, as natural endogenous brain substances, Allo and PG may have a potential as therapeutic agents in neurodegenerative conditions in which abnormal protein aggregates are involved.     

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.     

The use of the zinc-fluorophore, Zinpyr-1, in the study of zinc homeostasis in Arabidopsis roots

* The usefulness of the zinc (Zn)-fluorophore, Zinpyr-1, to examine the localization of Zn in the roots of Arabidopsis has been investigated. * In wild-type roots Zinpyr-1 fluorescence was predominantly in the xylem. The fluorescence signal was abolished by the application of the Zn-chelator, N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and was increased by increasing exogenous Zn in the medium, indicating that fluorescence reflected relative Zn concentrations. * In the hma2, hma4 double mutant, which is deficient in root to shoot Zn translocation, Zinpyr-1 fluorescence was low in the xylem and high in the adjacent pericycle cells in which HMA2 and HMA4 are specifically expressed in a wild type. Zinpyr-1 fluorescence was also increased in the endodermis. * These results show that Zinpyr-1 can be used to examine the effects of mutations in Zn transporters on the localization of Zn in Arabidopsis roots and should be a useful addition to the tools available for studying Zn homeostasis in plants.