Interaction of viscotoxins A3 and B with membrane model systems: implications to their mechanism of action

Viscotoxins are small proteins that are thought to interact with biomembranes, displaying different toxic activities against a varied number of cell types, being viscotoxin A(3) (VtA(3)) the most cytotoxic whereas viscotoxin B (VtB) is the less potent. By using infrared and fluorescence spectroscopies, we have studied the interaction of VtA(3) and VtB, both wild and reduced ones, with model membranes containing negatively charged phospholipids. Both VtA(3) and VtB present a high conformational stability, and a similar conformation both in solution and when bound to membranes. In solution, the infrared spectra of the reduced proteins show an increase in bandwidth compared to the nonreduced ones indicating a greater flexibility. VtA(3) and VtB bind with high affinity to membranes containing negatively charged phospholipids and are motional restricted, their binding being dependent on phospholipid composition. Whereas nonreduced proteins maintain their structure when bound to membranes, reduced ones aggregate. Furthermore, leakage experiments show that wild proteins were capable of disrupting membranes whereas reduced proteins were not. The effect of VtA(3) and VtB on membranes having different phospholipid composition is diverse, affecting the cooperativity and fluidity of the membranes. Viscotoxins interact with membranes in a complex way, most likely organizing themselves at the surface inducing the appearance of defects that lead to the destabilization and disruption of the membrane bilayer.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Phospholipid metabolism and prostacyclin synthesis in hypoxic myocytes.

We observed that in hypoxic myocardial cells prostacyclin and arachidonic acid release increased and that during hypoxia phospholipid degradation also occurred. In order to clarify the mechanism of phospholipid degradation, we determined the activity of phospholipases A2 and C. We found that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were markedly decreased and that lysophosphatidylcholine and lysophosphatidylethanolamine were increased. In contrast, there was only slight phosphatidylinositol degradation and no lysophosphatidylinositol elevation was observed. These results show that phospholipase A2 was activated in hypoxic myocytes and had substrate specificity towards PC and PE. To study phospholipase C activity, membrane phospholipids were labeled with [3H]choline, [3H]inositol or [3H]ethanolamine. The release of inositol was observed, but neither choline nor ethanolamine was released. In hypoxia, myocardial-cell phospholipase C has high substrate specificity towards phosphatidylinositol. The activation of phospholipases is closely related to the intracellular Ca2+ concentration; it is though that inositol polyphosphatides may regulate intracellular Ca2+. We determined how Ca2+ influx occurs in hypoxia. beta-Adrenergic blockade and Ca2+ antagonists markedly suppressed Ca2+ influx, phospholipase A2 activity, phospholipase C activity and cell death. However, the alpha 1-adrenergic blockade was less effective in suppressing these phenomena. These results suggest that in hypoxic myocardial cells Ca2+ influx mediated by beta-adrenergic stimulation activates phospholipases A2 and C, and that phospholipid degradation and prostacyclin release then occur.     

麦冬皂苷D对过氧化氢造模的HUVEC保护作用机制研究

目的:研究麦冬皂苷D对HUVEC凋亡相关分子的影响,探讨其作用机制。方法:用H2O2构建凋亡模型,运用MTT及流式细胞仪检测细胞活性,激光共聚焦方法测定粒体膜电位和钙离子浓度。结果:麦冬皂苷D可以稳定线粒体膜电位,减少钙离子内流,增加细胞的活力。结论:麦冬皂苷D对HUVEC有一定的保护作用。     

Role of TRPM2 in H(2)O(2)-Induced Cell Apoptosis in Endothelial Cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.     

Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl

We investigated the mechanism of H(2)O(2) activation of the Ca(2+)-regulated NADPH oxidase NOX5. H(2)O(2) induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H(2)O(2) is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H(2)O(2). H(2)O(2) NOX5 activation was inhibited by extracellular Ca(2+) chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP-c-Abl colocalized with vesicular sites of superoxide production in a Ca(2+)-dependent manner. In contrast to H(2)O(2), the Ca(2+) ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H(2)O(2) treatment. Furthermore, H(2)O(2)-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP-Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca(2+)-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.     

Oxidative stress is involved in the permeabilization of the inner membrane of brain mitochondria exposed to hypoxia/reoxygenation and low micromolar Ca2+

From in vivo models of stroke it is known that ischemia/reperfusion induces oxidative stress that is accompanied by deterioration of brain mitochondria. Previously, we reported that the increase in Ca2+ induces functional breakdown and morphological disintegration in brain mitochondria subjected to hypoxia/reoxygenation (H/R). Protection by ADP indicated the involvement of the mitochondrial permeability transition pore in the mechanism of membrane permeabilization. Until now it has been unclear how reactive oxygen species (ROS) contribute to this process. We now report that brain mitochondria which had been subjected to H/R in the presence of low micromolar Ca2+ display low state 3 respiration (20% of control), loss of cytochrome c, and reduced glutathione levels (75% of control). During reoxygenation, significant mitochondrial generation of hydrogen peroxide (H2O2) was detected. The addition of the membrane permeant superoxide anion scavenger TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) suppressed the production of H2O2 by brain mitochondria metabolizing glutamate plus malate by 80% under normoxic conditions. TEMPOL partially protected brain mitochondria exposed to H/R and low micromolar Ca2+ from decrease in state 3 respiration (from 25% of control to 60% of control with TEMPOL) and permeabilization of the inner membrane. Membrane permeabilization was obvious, because state 3 respiration could be stimulated by extramitochondrial NADH. Our data suggest that ROS and Ca2+ synergistically induce permeabilization of the inner membrane of brain mitochondria exposed to H/R. However, permeabilization can only partially be prevented by suppressing mitochondrial generation of ROS. We conclude that transient deprivation of oxygen and glucose during temporary ischemia coupled with elevation in cytosolic Ca2+ concentration triggers ROS generation and mitochondrial permeabilization, resulting in neural cell death.     

Hydrogen peroxide removes TRPM4 current desensitization conferring increased vulnerability to necrotic cell death

Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H(2)O(2) induces a sustained activity of TRPM4, a Ca(2+)-activated, Ca(2+)-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H(2)O(2) was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys(1093) residue is crucial for the H(2)O(2)-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H(2)O(2) elicited necrosis as well as apoptosis. H(2)O(2)-mediated necrosis but not apoptosis was abolished by replacement of external Na(+) ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H(2)O(2)-induced necrotic cell death. In addition, HeLa cells exposed to H(2)O(2) displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown.     

植物体细胞胚发生中抗氧化系统代谢动态和程序性细胞死亡

在胚性细胞分化过程中ＳＯＤ活性明显高于对照，同时ＳＯＤ活性与胚性细胞分化密切相关，表明胚性细胞具有较强的抗氧化能力。低浓度的Ｈ２Ｏ２对胚性细胞的分化有促进作用，并认为Ｈ２Ｏ２可能是作为一种细胞信号物，通过细胞信号系统调节相关基因表达，或通过提高细胞内Ｃａ＾２＋浓度而促进细胞分化。在胚性细胞分化和发育过程中存在程序性细胞死亡（ｐｒｏｇｒａｍｍｅｄ ｃｅｌｌ ｄｅａｔｈ，ＰＣＤ）。活性氧在诱发植物ＰＣ     

Comparative study of hydrogen peroxide- and 4-hydroxy-2-nonenal-induced cell death in HT22 cells

Several studies have indicated that lipid peroxidation often occurs in response to oxidative stress, and that many aldehydic products including 4-hydroxy-2-nonenal (HNE) are formed when lipid hydroperoxides break down. In order to clarify the mechanism of oxidative stress-induced neuronal death in the nervous system, we investigated H(2)O(2)- and HNE-induced cell death pathways in HT22 cells, a mouse hippocampal cell line, under the same experimental conditions. Treatment with H(2)O(2) and HNE decreased the viability of these cells in a time- and concentration-dependent manner. In the cells treated with H(2)O(2), significant increases in the immunoreactivities of DJ-1 and nuclear factor-kappaB (NF-kappaB) subunits (p65 and p50) were observed in the nuclear fraction. H(2)O(2) also induced an increase in the intracellular concentration of Ca(2+), and cobalt chloride (CoCl(2)), a Ca(2+) channel inhibitor, suppressed the H(2)O(2)-induced cell death. In HNE-treated cells, none of these phenomena were observed; however, HNE adduct proteins were formed after exposure to HNE, but not to H(2)O(2). N-Acetyl-L-cysteine (NAC) suppressed both HNE-induced cell death and HNE-induced expression of HNE adduct proteins, whereas H(2)O(2)-induced cell death was not affected. These findings suggest that the mechanisms of cell death induced by H(2)O(2) different from those induced by HNE in HT22 cells, and that HNE adduct proteins play an important role in HNE-induced cell death. It is also suggested that the pathway for H(2)O(2)-induced cell death in HT22 cells does not involve HNE production.     

Accidental cell death and generation of reactive oxygen intermediates in human lymphocytes induced by thionins from Viscum album L.

The cytotoxic mechanisms of thionins from Viscum album L., the viscotoxins, were investigated in human granulocytes and lymphocytes. The time course of viscotoxin effects indicate accidental cell death, i.e. membrane permeabilization, degradation of cytoplasm and chromatin, swelling of mitochondria with loss of their cristae, and generation of reactive oxygen intermediates within 1-2 h, followed by secondary apoptosis-associated events. The viscotoxin homologue purothionin from whole-wheat flour and viscotoxin B, however, did not induce cell death in cultured lymphocytes. Cytotoxicity of cationic and amphipathic viscotoxin was prevented only by cleavage of its disulphide bridges.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

The Galpha protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gbeta subunits

Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca2+-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (beta-NAD+) to the MutT motif. Arachidonic acid and Ca2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFalpha-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death.     

Glucocorticoid-induced apoptosis of thymocytes: requirement of proteasome-dependent mitochondrial activity

Thymocytes undergo negative and positive selection during development in the thymus. During this selection process, the majority of thymocytes are eliminated by apoptosis through signaling via TCR or die by neglect, possibly mediated through glucocorticoids. In this study, we report that thymocytes require molecular oxygen to undergo apoptosis induced by dexamethasone (DEX), a synthetic glucocorticoid, and treatment with N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibits thymocyte apoptosis in vivo as well as ex vivo. We detected elevated intracellular levels of hydrogen peroxide (H(2)O(2)) during DEX-induced apoptosis, which is reduced by NAC treatment, indicating that the elevated levels of intracellular H(2)O(2) are proapoptotic. We also show that loss of mitochondrial membrane potential, cytochrome c release, as well as caspase-3 activation induced by DEX are attenuated by NAC treatment. We identified the production site for H(2)O(2) as the ubiquinone cycle at complex III of mitochondria by using various inhibitors of the mitochondrial electron transport chain, and we show that the cell death events mediated by mitochondria are also significantly reduced when the inhibitors were used. Through inhibition of the proteasome, we also show that the production of H(2)O(2) and the cell death events mediated by mitochondria are regulated by proteosomal activities in DEX-induced thymocyte apoptosis. We conclude that in DEX-treated thymocytes, the increased production of H(2)O(2) originates from mitochondria and is proapoptotic for cell death mediated by mitochondria. We also conclude that all the apoptotic events mediated by mitochondria are regulated by proteasomes.     

NADPH-dependent H2O2 generation catalyzed by thyroid plasma membranes. Studies with electron scavengers

Hog thyroid plasma membrane preparations containing a Ca2+-regulated NADPH-dependent H2O2-generating system were studied. The Ca2+-dependent reductase activities of ferricytochrome c, 2,6-dichloroindophenol, nitroblue tetrazolium, and potassium ferricyanide were tested and the effect of these scavengers on H2O2 formation, NADPH oxidation and O2 consumption were measured, with the following results. 1. Thyroid plasma membrane Ca2+-independent cytochrome c reduction was not catalyzed by the NADPH-dependent H2O2-generating system. This activity was superoxide-dismutase-insensitive. 2.Of the three other electron scavengers tested, only K3Fe(CN)6 was clearly, but partially reduced in a Ca2+-dependent manner. 3. Though the NADPH-dependent reduction of nitroblue tetrazolium was very low and superoxide-dismutase-insensitive, nitroblue tetrazolium inhibited O2 consumption, H2O2 formation and NADPH oxidation, indicating that nitroblue tetrazolium inhibits the H2O2-generating system. We conclude that the thyroid plasma membrane H2O2-generating system does not or liberate O2- and that Ca2+ controls the first step (NADPH oxidation) of the H2O2-generating system.     

Blockade of 5-HT(3) receptor with MDL7222 and Y25130 reduces hydrogen peroxide-induced neurotoxicity in cultured rat cortical cells

The present study was performed to examine the neuroprotective effects of 5-hydroxytryptamine (5-HT)(3) receptor antagonists against hydrogen peroxide (H(2)O(2))-induced neurotoxicity using cultured rat cortical neurons. Pretreatment of 5-HT(3) receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1 and 1 microM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.5 and 5 microM), significantly inhibited the H(2)O(2) (100 microM)-induced neuronal cell death as assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. The protective effects of MDL72222 (1 microM) and Y25130 (5 microM) were completely blocked by the simultaneous treatment with 100 microM 1-phenylbiguanide, a 5-HT(3) receptor agonist, indicating that the protective effects of these compounds were due to 5-HT(3) receptor blockade. In addition, MDL72222 (1 microM) and Y25130 (5 microM) inhibited the H(2)O(2) (100 microM)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) and glutamate release, generation of reactive oxygen species (ROS), and caspase-3 activity. These results suggest that the activation of the 5-HT(3) receptor may be partially involved in H(2)O(2)-induced neurotoxicity, by membrane depolarization for Ca(2+) influx. Therefore, the blockade of 5-HT(3) receptor with MDL72222 and Y25130 may ameliorate the H(2)O(2)-induced neurotoxicity by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.     

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.     

Cadmium induces hydrogen peroxide production and initiates hydrogen peroxide-dependent apoptosis in the gill of freshwater crab, Sinopotamon henanense

Cadmium (Cd) is a well-known toxic heavy metal that accumulates in the aquatic environment. Cd has been reported to induce oxidative damage and apoptosis. We investigated the regulation mechanism of hydrogen peroxide (H(2)O(2)) on Cd-induced apoptosis. We show that in the gills of the freshwater crab Sinopotamon henanense Cd induced apoptosis, in a time- and concentration-dependent manner, as confirmed by DNA fragmentation analysis and transmission electron microscopy. Additionally, Cd caused production of H(2)O(2) after 2h of treatment at 58mg L(-1) Cd, and significantly increased the caspase-3/8/9 activity in crabs relative to the control group. Pre-treatment with the scavenger for H(2)O(2), dimethylthiourea (DMTU) and antioxidant, N-acetyl cysteine (NAC), effectively inhibited the activities of caspase-3 and caspase-9, eventually blocked Cd-induced DNA fragmentation and the appearance of markers for apoptotic cell death. These results suggest that Cd might induce intracellular H(2)O(2) generation to trigger the crab apoptotic processes by regulating the activities of caspase enzymes.     

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.     

Modes of membrane interaction of a natural cysteine-rich peptide: viscotoxin A3

Among the very homologous family of α- and β-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from other members because it is cytotoxic against tumoral cells but weakly hemolytic. We studied the interactions between the most active of these toxins, viscotoxin A3 (VA3), and model membranes made of phosphatidylcholine and phosphatidylserine (PS), the major zwitterionic and acidic phospholipids found in eukaryotic cells. Monolayer studies showed that electrostatic forces are essential for the interaction and are mainly involved in modulating the embedding of the toxin in the PS head group region. This in turn induces membrane stiffening, as shown by fluorescence polarization assays with 1,6-diphenyl-1,3,5-hexatriene and its derivatives. Moreover, vesicle permeabilization analyses showed that there are two modes of interaction, which are directly related to the stiffening effect and depend on the amount of VA3 bound to the surface of the vesicles. We propose an interaction model in which the embedding of VA3 in the membrane induces membrane defects leading to the gradual release of encapsulated dye. When the surfaces of the vesicles are saturated with the viscotoxin, complete vesicle destabilization is induced which leads to bilayer disruption, all-or-none encapsulated dye release and rearrangement of the vesicles.