The effect of a ferric iron complex on isolated rat-liver mitochondria. III. Mechanistic aspects of iron-induced calcium efflux

Addition of iron(III)-gluconate complex to isolated rat liver mitochondria induced a net efflux of Ca2+ which was not inhibited by ruthenium red. This process resulted in the enhancement of Ca2+ cycling and a consequent membrane potential drop. Under these experimental conditions the content of mitochondrial glutathione did not appear to be critically modified, whereas an extensive oxidation of mitochondrial pyridine nucleotides was parallelly detected. Iron failed to induce appreciable changes in the oxidation level of pyridine nucleotides in mitochondria isolated from rats fed a selenium deficient diet, a condition in which mitochondrial glutathione peroxidase resulted inhibited by 80%. The iron-induced Ca2+ release in Se-deficient mitochondria appeared largely delayed and the membrane potential of these mitochondrial did not present gross alterations. Iron was also found to induce a transient increase in the mitochondrial cyanide-insensitive oxygen consumption. This effect was largely prevented by the addition of the hydrogen peroxide scavenger catalase. It was concluded that iron induced the activation of a specific Ca2+ efflux pathway via the oxidation of pyridine nucleotides due to the hydrogen peroxide metabolism by glutathione enzyme system.     

Role for an episulfonium ion in S-(2-chloroethyl)-DL-cysteine-induced cytotoxicity and its reaction with glutathione

The cysteine S conjugate of 1,2-dichloroethane, S-(2-chloroethyl)-DL-cysteine (CEC), is hepatotoxic, nephrotoxic, and mutagenic. To determine the cellular and chemical mechanisms involved in CEC-induced toxicity and to assess the role of an episulfonium ion, the effect of CEC on the viability of isolated rat hepatocytes was studied. CEC addition resulted in both a time- and concentration-dependent loss of cell viability. Depletion of intracellular glutathione concentrations (greater than 70%) and inhibition of microsomal Ca2+ transport and Ca2+-ATPase activity preceded the loss of cell viability, and initiation of lipid peroxidation paralleled the loss of viability. The depletion of glutathione concentrations was partially attributable to a reaction between glutathione and CEC to form S-[2-(DL-cysteinyl)ethyl]glutathione, which was identified by NMR and mass spectrometry. N-Acetyl-L-cysteine, vitamin E, and N,N'-diphenyl-p-phenylenediamine protected against the loss of cell viability. N,N'-Diphenyl-p-phenylenediamine inhibited CEC-initiated lipid peroxidation but did not protect against cell death at 4 h, indicating that lipid peroxidation was not the cause of cell death. The analogues S-ethyl-L-cysteine, S-(3-chloropropyl)-DL-cysteine, and S-(2-hydroxyethyl)-L-cysteine, which cannot form an episulfonium ion, were not cytotoxic, thus demonstrating a role for an episulfonium ion in the cytotoxicity associated with exposure to CEC and, possibly, 1,2-dichloroethane. These results show that an alteration in Ca2+ homeostasis and the generation of an electrophilic intermediate may be involved in the mechanism of cell death.     

Thiol depletion induces lethal cell injury in cultured cardiomyocytes.

Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.     

Calcium supplementation modulates arsenic-induced alterations and augments arsenic accumulation in callus cultures of Indian mustard (Brassica juncea (L.) Czern.)

In the present study, the effect of arsenate (AsV) exposure either alone or in combination with calcium (Ca) was investigated in callus cultures of Brassica juncea (L.) Czern. cv. Pusa Bold grown for a period up to 24 h. The AsV?(250 μM) + Ca (10 mM) treatment resulted in a significantly higher level of As (464 μg g^?1 dry weight (DW)) than AsV without Ca (167 μg g^?1 DW) treatment at 24 h. Furthermore, AsV + Ca-treated calli had a higher percent of AsIII (24–47%) than calli subjected to AsV treatment (12–14%). Despite this, AsV + Ca-treated calli did not show any signs of hydrogen peroxide (H₂O₂) accumulation or cell death upon in vivo staining, while AsV-exposed calli had increased H₂O₂, shrinkage of cytoplasmic contents, and cell death. Thus, AsV treatment induced oxidative stress, which in turn elicited a response of antioxidant enzymes and metabolites as compared with control and AsV + Ca treatment. The positive effects of Ca supplementation were also correlated to an increase in thiolic constituents', viz., cysteine, reduced glutathione, and glutathione reductase in AsV + Ca than in AsV treatment. An analysis of selected signaling related genes, e.g., mitogen-activated protein kinases (MAPK3 and MAPK6) and jasmonate ZIM-domain (JAZ3) suggested that AsV and AsV + Ca followed variable pathways to sense and signal the As stress. In AsV-alone treatment, jasmonate signaling was seemingly activated, while MAPK3 was not involved. In contrast, AsV + Ca treatment appeared to specifically inhibit jasmonate signaling and activate MAPK3. In conclusion, Ca supplementation may hold promise for achieving increased As accumulation in plants without compromising their tolerance.     

Transport routes of metalloids into and out of the cell: a review of the current knowledge

Except for their extra- and intra-cellular interfaces, cell membranes are hydrophobic and inhibit the transport of hydrophilic molecules. Metalloids in aqueous solutions form chemical species with oxygen and hydroxyl groups and, therefore, exist as hydrophilic neutral polar solutes or as hydrophilic anions. This characteristic of metalloids introduces a large barrier for their passage through the cell membrane via unaided diffusion. The necessity for an uptake mechanism for metalloids arises from the requirement of these species for the maintenance of life, such as the need of boron for plant cells. Conversely, the transport of these species out of the cell is necessary because some metalloids are toxic, such as arsenic and antimony, and their entrance into the cell is undesirable. The undesired uptake of these toxic species is possible via pathways designed for the uptake of other structurally and chemically similar essential compounds. Therefore, the extrusion of arsenic and antimony out of the cell is an example of a detoxification mechanism. As a consequence of the hydrophobic character of the cell membrane in all living systems, the main route for the uptake and efflux of metalloids is facilitated by transmembrane proteins, driven either by concentration gradients or by energy-fueled pumps. However, metalloids forming or embedded in nano-sized particles escape the need to cross the cell membrane because these particles can be taken into the cell by endocytosis. Here, we review the uptake and efflux pathways of boron, silicon, arsenic, and antimony through the cell membranes of different organisms and the protein channels involved in these processes. In particular, passive diffusion via aquaglyceroporins, active transport via primary and secondary ion pumps, extrusion into vacuoles of metalloid-thiol conjugates via ATP-binding cassette, the efflux of methylated metalloids, and endocytosis are summarized.     

Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells

Glutathione (GSH) constitutes the single most important antioxidant in neurons, whereas iron causes oxidative stress that leads to cell damage and death. Although GSH and iron produce opposite effects on redox cell status, no mechanistic relationships between iron and GSH metabolism are known. In this work, we evaluated in SH-SY5Y neuroblastoma cells the effects of iron accumulation on intracellular GSH metabolism. After 2 d exposure to increasing concentrations of iron, cells underwent concentration-dependent iron accumulation and a biphasic change in intracellular GSH levels. Increasing iron from 1 to 5 microM resulted in a marked increase in intracellular oxidative stress and increased GSH levels. Increased GSH levels were due to increased synthesis. Further increases in iron concentration led to significant reduction in both reduced (GSH) and total (GSH + (2 x GSSG)) glutathione. Cell exposure to high iron concentrations (20-80 microM) was associated with a marked decrease in the GSH/GSSG molar ratio and the GSH half-cell reduction potential. Moreover, increasing iron from 40 to 80 microM resulted in loss of cell viability. Iron loading did not change GSH reductase activity but induced significant increases in GSH peroxidase and GSH transferase activities. The changes in GSH homeostasis reported here recapitulate several of those observed in Parkinson's disease substantia nigra. These results support a model by which progressive iron accumulation leads to a progressive decrease in GSH content and cell reduction potential, which finally results in impaired cell integrity.     

Mitochondrial glutathione status during Ca2+ ionophore-induced injury to isolated hepatocytes

In this study the Ca2+ ionophore, A23187, was used to determine the effects of disrupted Ca2+ homeostasis on cellular thiols. Isolated rat hepatocytes were incubated with varying concentrations of extracellular Ca2+ and A23187 to induce accumulation or loss of cellular Ca2+. These treatments resulted in loss of mitochondrial and cytosolic glutathione (GSH), loss of protein-thiols, and cell injury. This injury was dependent on the concentrations of ionophore and extracellular Ca2+. A correlation was found between cell injury and the loss of mitochondrial GSH, while the loss of cytosolic glutathione preceded both these events. The time course of protein-thiol loss appeared secondary to the loss of non-protein thiols. In the absence of extracellular Ca2+, the antioxidants alpha-tocopherol and diphenyl-p-phenylenediamine both totally prevented A23187-induced cell injury and loss of mitochondrial GSH, and thus protected the cells from the effects of mobilization of intracellular Ca2+. In the presence of extracellular Ca2+, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. The mitochondrial Ca2+ channel blocker, ruthenium red, protected hepatocytes from A23187-induced injury in the absence of extracellular Ca2+. Leupeptin, an inhibitor of Ca2+-activated proteases, and dibucaine, a phospholipase inhibitor, did not affect cytotoxicity. Our results indicate that the level of mitochondrial GSH may be important for cell survival during ionophore-induced perturbation of cellular Ca2+ homeostasis.     

Role of the Multidrug Resistance Protein 1 in protection from heavy metal oxyanions: investigations in vitro and in MRP1-deficient mice

The Multidrug Resistance Protein 1 (MRP1) is a membrane pump that mediates the efflux of a wide variety of xenobiotics, including arsenical and antimonial compounds, as demonstrated by the study of MRP1-transfected cell lines. We have previously shown that mrp1(-/-) cells are hypersensitive to sodium arsenite, sodium arsenate, and antimony potassium tartrate. We now report that the retroviral vector-mediated overexpression of MRP1 and of the two subunits of gamma-GCS (heavy and light) resulted in higher intracellular glutathione levels and in a greater level of resistance to sodium arsenite and antimony potassium tartrate, compared to the overexpression of MRP1 and gamma-GCS heavy alone. These observations further demonstrate that glutathione is an important component of MRP1-mediated cellular resistance to arsenite and antimony. However, the constitutive expression of MRP1 did not protect mice from the lethality of sodium arsenite and antimony potassium tartrate nor reduced the tissue accumulation of arsenic in mice injected i.p. with sodium arsenite. It is conceivable that, in vivo, other pump(s) effectively vicariate for MRP1-mediated transport of heavy metal oxyanions.     

Glutamate-induced cell death in HT22 mouse hippocampal cells is attenuated by paxilline, a BK channel inhibitor

In the present study, we show that the large conductance calcium-activated potassium channel (BK(Ca) channel) inhibitor paxilline protects neuronal cells against glutamate-induced cell death. In our studies, we used HT22 mouse hippocampal cells as an experimental model and observed that the effect of paxilline was dose-dependent. We also found that other inhibitors of BK(Ca) channels, iberiotoxin and charybdotoxin, were not cytoprotective. Paxillinol, which is a structural analog of paxilline but does not inhibit BK(Ca) channel, also protected HT22 cells against glutamate-induced toxicity. These data suggest that the observed cytoprotection was not related to BK(Ca) channel inhibition by paxilline. In addition, paxilline neither restored glutathione levels nor reduced the amount of reactive oxygen species upon glutamate treatment. Our results suggest that paxilline protects neuronal HT22 cells against glutamate-induced cell death independently of BK(Ca) channel activity and oxidative stress induced by glutamate treatment.     

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells

The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH-SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase-3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase-3-like, but not calpain, activity. Pan-caspase inhibitors protected against EGTA-, but not CaCl2-, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase-3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death-mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase-3-like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.     

As(III) and Sb(III) uptake by GlpF and efflux by ArsB in Escherichia coli

The toxicity of the metalloids arsenic and antimony is related to uptake, whereas detoxification requires efflux. In this report we show that uptake of the trivalent inorganic forms of arsenic and antimony into cells of Escherichia coli is facilitated by the aquaglyceroporin channel GlpF and that transport of Sb(III) is catalyzed by the ArsB carrier protein; everted membrane vesicles accumulated Sb(III) with energy supplied by NADH oxidation, reflecting efflux from intact cells. Dissipation of either the membrane potential or the pH gradient did not prevent Sb(III) uptake, whereas dissipation of both completely uncoupled the carrier protein, suggesting that transport is coupled to either the electrical or the chemical component of the electrochemical proton gradient. Reciprocally, Sb(III) transport via ArsB dissipated both the pH gradient and the membrane potential. These results strongly indicate that ArsB is an antiporter that catalyzes metalloid-proton exchange. Unexpectedly, As(III) inhibited ArsB-mediated Sb(III) uptake, whereas Sb(III) stimulated ArsB-mediated As(III) transport. We propose that the actual substrate of ArsB is a polymer of (AsO)(n), (SbO)(n), or a co-polymer of the two metalloids.     

Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells

Sodium iodate (SI) is a widely used oxidant for generating retinal degeneration models by inducing the death of retinal pigment epithelium (RPE) cells. However, the mechanism of RPE cell death induced by SI remains unclear. In this study, we investigated the necrotic features of cultured human retinal pigment epithelium (ARPE-19) cells treated with SI and found that apoptosis or necroptosis was not the major death pathway. Instead, the death process was accompanied by significant elevation of intracellular labile iron level, ROS, and lipid peroxides which recapitulated the key features of ferroptosis. Ferroptosis inhibitors deferoxamine mesylate (DFO) and ferrostatin-1(Fer-1) partially prevented SI-induced cell death. Further studies revealed that SI treatment did not alter GPX4 (glutathione peroxidase 4) expression, but led to the depletion of reduced thiol groups, mainly intracellular GSH (reduced glutathione) and cysteine. The study on iron trafficking demonstrated that iron influx was not altered by SI treatment but iron efflux increased, indicating that the increase in labile iron was likely due to the release of sequestered iron. This hypothesis was verified by showing that SI directly promoted the release of labile iron from a cell-free lysate. We propose that SI depletes GSH, increases ROS, releases labile iron, and boosts lipid damage, which in turn results in ferroptosis in ARPE-19 cells.Subject terms: Disease model, Cell death     

Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes

Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H2O2. The present study shows that upon activation of death response by H2O2, a dose- and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca2+ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na+ and mitochondrial Ca2+. Partial inhibition of cytosolic Ca2+ increase achieved by chelating extracellular or intracellular Ca2+ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca2+ is an additive result of release of Ca2+ from intracellular stores as well as by influx of extracellular Ca2+ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.     

Glutathione depletion, lipid peroxidation, DNA double-strand breaks and the cytotoxicity of 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone in rat renal cortical cells.

The mechanisms involved in the cytotoxicity of 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone, a model compound for hydroquinone derived mercapturic acids, were investigated in rat renal proximal tubule cells. 2-Bromo-3-(N-acetylcystein-S-yl)hydroquinone induced a time- and concentration-dependent decrease in cell viability and in the levels of cellular glutathione. Antioxidants such as N,N'-diphenyl-p-phenylene diamine and ascorbic acid and the iron chelator desferrioxamine very efficiently protected the cells from 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone without influencing glutathione depletion. The acetoxymethyl ester of the Ca2+ chelator Quin-2, the inhibitor of the Ca(2+)- and Mg(2+)-dependent endonucleases, aurintricarboxylic acid and the poly(ADP-ribose)-polymerase inhibitor 3-aminobenzamide also ameliorated 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone cytotoxicity. Moreover, 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone depleted Ca2+ from isolated kidney mitochondria, increased the amount of malondialdehyde in rat kidney cells and induced DNA double-strand breaks in renal cells in culture. These results suggest that renal cells oxidize 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone to the corresponding quinone; this soft electrophile reacts rapidly with glutathione, thus depleting cellular glutathione concentrations as indicated by the tentative identification of a 2-bromo-3-(N-acetylcystein-S-yl)hydroquinone thioether in the incubation medium of renal cells treated with the mercapturate. As a result of the massive glutathione depletion, peroxidative mechanisms then cause an elevation of the cytosolic concentrations of ionized calcium; impairment of the ability of the mitochondria to sequester Ca2+ plays an important role in the elevation of the Ca2+ concentration. Finally, activation of Ca(2+)- and Mg(2+)-dependent endonucleases results in DNA damage and cell death.     

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.     

Accumulation of Ca2+ induced by cytotoxic levels of menadione in the isolated, perfused rat liver

Previous studies have indicated that the presence of cytotoxic levels of menadione (2-methyl-1,4-naphthoquinone) causes rapid changes in intracellular thiol and Ca2+ homeostasis in isolated rat hepatocytes. The present investigation was undertaken to examine these effects in the intact liver. Rat livers were therefore perfused with Krebs-Henseleit buffer containing 1.3 mM Ca2+ using a single-pass mode, and the perfusate Ca2+ level was monitored with an on-line Ca2+-selective electrode. Infusion of menadione elicited an increased O2 uptake by the liver, followed by a dose-dependent decrease in the perfusate level of Ca2+. Hepatic accumulation of Ca2+ was accompanied by stimulation of cytosolic phosphorylase a activity. Cessation of menadione infusion resulted in gradual recovery of perfusate Ca2+ to base levels. Ca2+ uptake was not accompanied by decreases in reduced pyridine nucleotide or ATP levels in the liver as evidenced by measurements either during maximal Ca2+ uptake or after recovery. However, Ca2+ uptake was correlated with decreased glutathione and increased glutathione disulfide levels in the liver, both of which reversed during recovery from Ca2+ uptake. Moreover, depletion of hepatic glutathione by pretreatment with diethylmaleate resulted in increased Ca2+ uptake during menadione infusion. The amount of protein-bound mixed disulfides showed a particularly striking relationship to Ca2+ uptake, reaching a maximal level during Ca2+ uptake and reversing toward normal value during recovery from Ca2+ accumulation. The present findings suggest that menadione-induced Ca2+ uptake is due to plasma membrane dysfunction as a result of loss of protein thiol groups critical for maintaining the plasma membrane Ca2+ extrusion mechanism. Our model offers a particularly useful opportunity to study mechanisms underlying toxic disturbances in Ca2+ homeostasis in the intact liver, since Ca2+ fluxes can be monitored under conditions in which cellular control mechanisms are not obliterated by excessive toxicity.     

Association of blebbing with assembly of cytoskeletal proteins in ATP-depleted EL-4 ascites tumour cells.

ATP depletion in EL-4 ascites tumour cells rapidly induced the changes in cell morphology (blebbing), cytoskeletal protein assembly and finally resulted in cell death. After 1 hr of incubation with 2 microM rotenone (inhibitor of respiration) in glucose-free medium, when ATP level was 4% of the initial level, there were increases in triton-insoluble actin and vinculin levels (2.5-fold and 2.8-fold, respectively) and 44% of cells showed blebs; such treatment damaged cells irreversibly. Ca2+ removal did not diminish the effect of ATP depletion on cytoskeleton, blebbing and cell death, although the elevation of free intracellular Ca2+ in rotenone-treated cells was prevented. The role of ATP in maintaining cytoskeleton and cell shape is discussed.     

Cytosolic-free Ca2+ and cell killing in hepatoma 1c1c7 cells exposed to chemical anoxia

Exposure of cultured hepatoma 1c1c7 cells to KCN and iodoacetate, to produce chemical anoxia, caused a rapid and sustained increase in cytosolic-free Ca2+ concentration, which was associated with depletion of intracellular ATP and glutathione. These changes occurred before the loss of cell viability and were accompanied by the appearance of plasma membrane blebs. Pretreatment of the cells with the Ca2+ chelators Quin 2 or BAPTA markedly delayed both the onset of blebbing and loss of cell viability, but did not affect KCN- and iodoacetate-induced loss of ATP and glutathione. Together, these results strongly suggest that a sustained increase in cytosolic Ca2+ concentration plays an important role in killing of hepatoma cells by chemical anoxia.     

Paroxetine-induced Ca2+ movement and death in OC2 human oral cancer cells

The effect of the antidepressant paroxetine on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1,000 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 microM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining suggests that apoptosis plays a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine (up to 50 microM) induced cell death in a Ca2+-independent manner.     

Calpain activates caspase-3 during UV-induced neuronal death but only calpain is necessary for death

While caspases have been strongly implicated in delayed neuronal death in a variety of experimental paradigms, other proteases such as calpain can also contribute to neuronal death. To evaluate the relative roles of caspase and calpain, we used a model system wherein UV treatment induced moderate or severe delayed cortical neuronal death, as quantified by propidium iodide and calcein AM. UV treatment led to increases in both caspase and calpain activation. Calpain inhibitor III (MDL-28170) reduced caspase activation, suggesting that caspase activation was mediated by calpain. Calpain contributed to neuronal death, as indicated by strong neuroprotection provided by calpain inhibitor III, calpeptin, or Ca2+-free medium. In contrast, caspase inhibitors were not neuroprotective. These results suggest that UV neurotoxicity is mediated by a loss of Ca2+ homeostasis which leads to a calpain-dependent, caspase-independent cell death. That calpain, but not caspase, may mediate death in instances involving the activation of both proteases may have relevance to other neuronal death models.