Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

Spermidine oxidase‐derived H2O2 regulates pollen plasma membrane hyperpolarization‐activated Ca2+‐permeable channels and pollen tube growth

Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca²⁺ concentration that accompanies pollen tube growth. Using the whole‐cell patch clamp and outside‐out single‐channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization‐activated Ca²⁺ current: the addition of Spd cannot induce the channel open probability increase in excised outside‐out patches, indicating that the effect of Spd in the induction of Ca²⁺ currents is exerted via a second messenger. This messenger is hydrogen peroxide (H₂O₂), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization‐activated Ca²⁺‐permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca²⁺‐permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal‐encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca²⁺‐permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca²⁺ influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd‐derived H₂O₂ signals Ca²⁺ influx, thereby regulating pollen tube growth.     

Response properties of the genetically encoded optical H2O2 sensor HyPer

Reactive oxygen species mediate cellular signaling and neuropathologies. Hence, there is tremendous interest in monitoring (sub)cellular redox conditions. We evaluated the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures. Two days after lipofection, neurons and glia showed sufficient expression levels, and H₂O₂ reversibly and dose-dependently increased the fluorescence ratio of cytosolic HyPer. Yet, repeated H₂O₂ treatment caused progressively declining responses, and with millimolar doses an apparent recovery started while H₂O₂ was still present. Although HyPer should be H₂O₂ specific, it seemingly responded also to other oxidants and altered cell-endogenous superoxide production. Control experiments with the SypHer pH sensor confirmed that the HyPer ratio responds to pH changes, decreasing with acidosis and increasing during alkalosis. Anoxia/reoxygenation evoked biphasic HyPer responses reporting apparent reduction/oxidation; replacing Cl⁻ exerted only negligible effects. Mitochondria-targeted HyPer readily responded to H₂O₂—albeit less intensely than cytosolic HyPer. With ratiometric two-photon excitation, H₂O₂ increased the cytosolic HyPer ratio. Time-correlated fluorescence-lifetime imaging microscopy (FLIM) revealed a monoexponential decay of HyPer fluorescence, and H₂O₂ decreased fluorescence lifetimes. Dithiothreitol failed to further reduce HyPer or to induce reasonable FLIM and two-photon responses. By enabling dynamic recordings, HyPer is superior to synthetic redox-sensitive dyes. Its feasibility for two-photon excitation also enables studies in more complex preparations. Based on FLIM, quantitative analyses might be possible independent of switching excitation wavelengths. Yet, because of its pronounced pH sensitivity, adaptation to repeated oxidation, and insensitivity to reducing stimuli, HyPer responses have to be interpreted carefully. For reliable data, side-by-side pH monitoring with SypHer is essential.     

Epigallocatechin‐3‐gallate induces mesothelioma cell death via H2O2−dependent T‐type Ca2+ channel opening

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin‐3‐gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H₂O₂ release in cell cultures, and exogenous catalase (CAT) abrogated EGCG‐induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3‐loaded, EGCG‐exposed MMe cells showed significant [Ca²⁺]_i rise, prevented by CAT, dithiothreitol or the T‐type Ca²⁺ channel blockers mibefradil and NiCl₂. Cell loading with dihydrorhodamine 123 revealed EGCG‐induced ROS production, prevented by CAT, mibefradil or the Ca²⁺ chelator BAPTA‐AM. Direct exposure of cells to H₂O₂ produced similar effects on Ca²⁺ and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Ca_v3.2 T‐type Ca²⁺ channels in these cells, compared to normal mesothelium. Also, Ca_v3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Ca_v3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T‐type Ca²⁺ channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target.     

Relationship between NaCl- and H2O2-Induced Cytosolic Ca2+ Increases in Response to Stress in Arabidopsis

Salinity is among the environmental factors that affect plant growth and development and constrain agricultural productivity. Salinity stress triggers increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Ca²⁺ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS). It is well established that ROS also triggers increases in [Ca²⁺]_i. However, the relationship and interaction between salinity stress-induced [Ca²⁺]_i increases and ROS-induced [Ca²⁺]_i increases remain poorly understood. Using an aequorin-based Ca²⁺ imaging assay we have analyzed [Ca²⁺]_i changes in response to NaCl and H₂O₂ treatments in Arabidopsis thaliana. We found that NaCl and H₂O₂ together induced larger increases in [Ca²⁺]_i in Arabidopsis seedlings than either NaCl or H₂O₂ alone, suggesting an additive effect on [Ca²⁺]_i increases. Following a pre-treatment with either NaCl or H₂O₂, the subsequent elevation of [Ca²⁺]_i in response to a second treatment with either NaCl or H₂O₂ was significantly reduced. Furthermore, the NaCl pre-treatment suppressed the elevation of [Ca²⁺]_i seen with a second NaCl treatment more than that seen with a second treatment of H₂O₂. A similar response was seen when the initial treatment was with H₂O₂; subsequent addition of H₂O₂ led to less of an increase in [Ca²⁺]_i than did addition of NaCl. These results imply that NaCl-gated Ca²⁺ channels and H₂O₂-gated Ca²⁺ channels may differ, and also suggest that NaCl- and H₂O₂-evoked [Ca²⁺]_i may reduce the potency of both NaCl and H₂O₂ in triggering [Ca²⁺]_i increases, highlighting a feedback mechanism. Alternatively, NaCl and H₂O₂ may activate the same Ca²⁺ permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

Phospholipases AtPLDζ1 and AtPLDζ2 function differently in hypoxia

Besides hydrolyzing different membrane phospholipids, plant phospholipases D and molecular species of their byproducts phosphatidic acids (PLDs/PAs) are involved in diverse cellular events such as membrane‐cytoskeleton dynamics, hormone regulation and biotic and/or abiotic stress responses at cellular or subcellular levels. Among the 12 Arabidopsis PLD genes, PLDζ1 and PLDζ2 uniquely possess Ca²⁺‐independent phox (PX) and pleckstrin (PH) homology domains. Here, we report that mutants deficient in these PLDs, pldζ1 and pldζ2, show differential sensitivities to hypoxia stimulus. In the present study, we used protoplasts of wild type and mutants and compared the hypoxia‐induced changes in the levels of three major signaling mediators such as cytoplasmic free calcium [Ca²⁺_cyt.], hydrogen peroxide (H₂O₂) and PA. The concentrations of cytosolic Ca²⁺ and H₂O₂ were determined by fluorescence microscopy and the fluorescent dyes Fura 2‐AM and CM‐H₂DCFDA, specific for calcium and H₂O₂, respectively, while PA production was analyzed by an enzymatic method. The study reveals that AtPLDζ1 is involved in reactive oxygen species (ROS) signaling, whereas AtPLDζ2 is involved in cytosolic Ca²⁺ signaling pathways during hypoxic stress. Hypoxia induces an elevation of PA level both in Wt and pldζ1, while the PA level is unchanged in pldζ2. Thus, it is likely that AtPLDζ2 is involved in PA production by a calcium signaling pathway, while AtPLDζ1 is more important in ROS signaling.     

The effect of polar auxin transport on adventitious branches formation in Gracilaria lichenoides in vitro

Seaweed tissue culture (STC) is an important micropropagation tool that has been applied for strain improvement, micropropagation and genetic engineering. Because the mechanisms associated with STC are poorly understood, its application to these organisms lags far behind that of tissue culture propagation of higher plants. Auxin, calcium (Ca²⁺) and hydrogen peroxide (H₂O₂) fluxes all play key roles during plant growth and development. In this study, we therefore measured indole‐3‐acetic acid, Ca²⁺ and H₂O₂ fluxes of Gracilaria lichenoides explants during adventitious branches (ABs) formation for the first time using noninvasive micro‐test technology. We confirmed that polar auxin transport (PAT) also occurs in the marine red alga G. lichenoides. We additionally found that N‐1‐naphthylphthalamic acid may suppress auxin efflux via ABCB1 transporters and then inhibit ABs formation from the apical region of G. lichenoides segments. The involvement of Ca²⁺ and H₂O₂ fluxes in PAT‐mediated AB formation in G. lichenoides was also investigated. We propose that complex feedback among Ca²⁺, H₂O₂ and auxin signaling and response systems may occur during ABs polar formation in G. lichenoides explants, similar to that in higher plants. Our results provide innovative insights that should aid future elucidation of mechanisms operative during STC.     

Chlamydomonas carries out fatty acid β‐oxidation in ancestral peroxisomes using a bona fide acyl‐CoA oxidase

Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl‐CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl‐CoAs into trans‐2‐enoyl‐CoA and produced H₂O₂. This result demonstrated that CrACX2 is a genuine acyl‐CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) β‐oxidation spiral. A fluorescent protein‐tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA β‐oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of β‐oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant‐type FA β‐oxidation involving H₂O₂‐producing acyl‐CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: I. Selectivity of Protection Against Oxidative Stress

Abstract: Previous research has suggested that the initial effects of cellular free radical neurotoxic insult involve large increases in intracellular Ca²⁺. However, the exact role of oxidative stress on the various parameters involved in these increases has not been specified. The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 µM H₂O₂ for 30 min in growth medium alone or containing either nifedipine (L-type Ca²⁺ antagonist), conotoxin (N-type antagonist), Trolox (vitamin E analogue), or α-phenyl-n-tert-butylnitrone (nitrone trapping agent; PBN). The concentrations of H₂O₂ were chosen by examining the degree of cell killing induced by exposure to graded concentrations of H₂O₂. The 5 and 25 µM concentrations of H₂O₂ produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 µM concentration produced a moderate degree of cell killing that did not increase between the two times. Fluorescent imaging was used to visualize intracellular Ca²⁺ changes in fura-2-loaded cells. Baseline (pre-30 mM KCI) Ca²⁺ levels were increased significantly by H₂O₂ treatment (e.g., 300 µM, 200%), but the rise in the level of free intracellular Ca²⁺ after KCI stimulation (i.e., peak) was decreased (e.g., 300 µM, 50%) and the cell's ability to sequester or extrude the excess Ca²⁺ (i.e., Ca²⁺ recovery time) after depolarization was decreased significantly. All compounds prevented baseline Ca²⁺ increases and, with the exception of conotoxin, antagonized the peak decreases in Ca²⁺. It is interesting that after 300 µM H₂O₂ exposure, only Trolox was partially effective in preventing these deficits in recovery. Conotoxin increased the decrement recovery in the absence of H₂O₂. However, in cells exposed to 5 or 25 µM H₂O₂, conotoxin as well as the other agents were effective in preventing the deficits in recovery.     

Influence of Ca<Superscript>2+</Superscript> ions on metabolism of active oxygen species in wheat calli cocultured with the bunt pathogen <Emphasis Type="Italic">Tilletia caries</Emphasis>

The effect of Ca²⁺ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O²⁻, H₂O₂, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca²⁺ in the culture medium of calli. The increase of the concentration of Ca²⁺ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.     

Arachidonic acid release by H2O2 mediated proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC and MAPKs‐induced EGFR transactivation

Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H₂O₂‐indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10^?4 M H₂O₂ for 1 h. In addition, H₂O₂ increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen‐activated protein kinase (MAPK), and JNK/SAPK. Moreover, H₂O₂ induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H₂O₂‐induced cytosolic phospholipase A₂ (cPLA₂) activation and AA release. H₂O₂ increased NF‐κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)‐2 proteins. Subsequently, H₂O₂ stimulated PGE₂ synthesis, which was reduced by the inhibition of NF‐κB activation. Moreover, each H₂O₂ or PGE₂ increased DNA synthesis and the number of cells. However, H₂O₂‐induced increase in DNA synthesis was inhibited by the suppression of cPLA₂ pathway. In conclusion, H₂O₂ increased AA release and PGE₂ production by the upregulation of cPLA₂ and COX‐2 via Ca²⁺/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009. © 2009 Wiley‐Liss, Inc.     

Hydrogen peroxide depolarizes mitochondria and inhibits IP3-evoked Ca2+ release in the endothelium of intact arteries

Hydrogen peroxide (H₂O₂) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H₂O₂ regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H₂O₂ on Ca²⁺ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca²⁺ signals and acetylcholine evoked Ca²⁺ increases were inhibited by H₂O₂. H₂O₂ inhibition of acetylcholine-evoked Ca²⁺ signals was reversed by catalase. H₂O₂ exerts its inhibition on the IP₃ receptor as Ca²⁺ release evoked by photolysis of caged IP₃ was supressed by H₂O₂. H₂O₂ suppression of IP₃-evoked Ca²⁺ signalling may be mediated by mitochondria. H₂O₂ depolarized mitochondria membrane potential. Acetylcholine-evoked Ca²⁺ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP₃-evoked Ca²⁺ release by H₂O₂ arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca²⁺ overload during IP₃-linked Ca²⁺ signals by a H₂O₂ mediated negative feedback depolarization of the organelle and inhibition of IP₃-evoked Ca²⁺ release.     

Proteome of plant peroxisomes: new perspectives on the role of these organelles in cell biology

Peroxisomes are cell organelles bounded by a single membrane with a basically oxidative metabolism. Peroxisomes house catalase and H₂O₂‐producing flavin‐oxidases as the main protein constituents. However, since their discovery in early fifties, a number of new enzymes and metabolic pathways have been reported to be also confined to these organelles. Thus, the presence of exo‐ and endo‐peptidases, superoxide dismutases, the enzymes of the plant ascorbate‐glutathione cycle plus ascorbate and glutathione, several NADP‐dehydrogenases, and also L‐arginine‐dependent nitric oxide synthase activity has evidenced the relevant role of these organelles in cell physiology. In recent years, the study of new functions of peroxisomes has become a field of intensive research in cell biology, and these organelles have been proposed to be a source of important signal molecules for different transduction pathways. In plants, peroxisomes participate in seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species (ROS and RNS, respectively). In order to decipher the nature and specific role of the peroxisomal proteins in these processes, several approaches including in vivo and in vitro import assays and generation of mutants have been used. In the last decade, the development of genomics and the report of the first plant genomes provided plant biologists a powerful tool to assign to peroxisomes those proteins which harbored any of the two peroxisomal targeting signals (PTS, either PTS1 or PTS2) described so far. Unfortunately, those molecular approaches could not give any response to those proteins previously localized in plant peroxisomes by classical biochemical and cell biology methods that did not contain any PTS. However, more recently, proteomic studies of highly purified organelles have provided evidence of the presence in peroxisomes of new proteins not previously reported. Thus, the contribution of proteomic approaches to the biology of peroxisomes is essential, not only for elucidation of the mechanisms involved in the import of the PTS1‐ and PTS2‐independent proteins, but also to the understanding of the role of these organelles in the cell physiology of plant growth and development.     

Enhanced Depolarization-Evoked Calcium Signal and Reduced [ATP]/[ADP] Ratio Are Unrelated Events Induced by Oxidative Stress in Synaptosomes

Abstract: Oxidative insult elicited by hydrogen peroxide (H₂O₂) was previously shown to increase the basal intracellular Ca²⁺ concentration in synaptosomes. In the present study, the effect of H₂O₂ on the depolarization-evoked [Ca²⁺] signal was investigated. Pretreatment of synaptosomes with H₂O₂ (0.1–1 mM) augmented the [Ca²⁺] rise elicited by high K⁺ depolarization with essentially two alterations, the sudden sharp rise of [Ca²⁺]_i due to K⁺ depolarization is enhanced and, instead of a decrease to a stable plateau, a slow, steady rise of [Ca²⁺]_i follows the peak [Ca²⁺]_i. H₂O₂ in the same concentration range lowered the ATP level and the [ATP]/[ADP] ratio. When carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (1 µM) or rotenone (2 µM)/oligomycin (10 µM) was applied initially to block mitochondrial ATP production, the lowered [ATP]/[ADP] ratio was further reduced by subsequent addition of 0.5 mM H₂O₂. The decline of the [ATP]/[ADP] ratio was parallel with but could not explain the enhanced K⁺-evoked [Ca²⁺]_i signal, indicated by experiments in which the [ATP]/[ADP] ratio was decreased by FCCP (0.1 µM) or rotenone (2 µM) to a similar value as by H₂O₂ without causing any alteration in the [Ca²⁺]_i signal. These results indicate that H₂O₂-evoked oxidative stress, in its early phase, gives rise to a complex dysfunction in the Ca²⁺ homeostasis and, parallel with it, to an impaired energy status.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-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 (_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⁺]_o) 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. (Mol Cell Biochem 269: 165–173, 2005)