Catalase Inhibitor Aminotriazole Research Articles

Transcriptomic evidence for involvement of reactive oxygen species in Rhizoctonia solani AG1 IA sclerotia maturation.

Rhizoctonia solani AG1 IA is a soil-borne fungal phytopathogen that can significantly harm crops resulting in economic loss. This species overwinters in grass roots and diseased plants, and produces sclerotia that infect future crops. R. solani AG1 IA does not produce spores; therefore, understanding the molecular mechanism of sclerotia formation is important for crop disease control. To identify the genes involved in this process for the development of disease control targets, the transcriptomes of this species were determined at three important developmental stages (mycelium, sclerotial initiation, and sclerotial maturation) using an RNA-sequencing approach. A total of 5,016, 6,433, and 5,004 differentially expressed genes (DEGs) were identified in the sclerotial initiation vs. mycelial, sclerotial maturation vs. mycelial, and sclerotial maturation vs. sclerotial initiation stages, respectively. Moreover, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses showed that these DEGs were enriched in diverse categories, including oxidoreductase activity, carbohydrate metabolic process, and oxidation-reduction processes. A total of 12 DEGs were further verified using reverse transcription quantitative PCR. Among the genes examined, NADPH oxidase 1 (NOX1) and superoxide dismutase (SOD) were highly induced in the stages of sclerotial initiation and maturation. In addition, the highest reactive oxygen species (ROS) production levels were detected during sclerotial initiation, and enzyme activities of NOX1, SOD, and catalase (CAT) matched with the gene expression profiles. To further evaluate the role of ROS in sclerotial formation, R. solani AG1 IA was treated with the CAT inhibitor aminotriazole and H2O2, resulting in the early differentiation of sclerotia. Taken together, this study provides useful information toward understanding the molecular basis of R. solani AG1 IA sclerotial formation and maturation, and identified the important role of ROS in these processes.

Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the glioprotective action of Pituitary adenylate cyclase-activating polypeptide (PACAP) against H2 O2 -induced astrocyte damages and cell apoptosis in cultured rat astrocytes. PACAP, through activation of its receptor, PAC1-R, and the protein kinase A (PKA), protein kinase C (PKC), and MAP-kinases signaling pathways, prevents accumulation of ROS andinhibition of SOD and catalase activities. This allows the preservation of mitochondrial membrane integrity and the reduction in caspase-3 activation induced by H2 O2 . These data may lead to the development of new strategies for cerebral injury treatment. Cat, catalase; Cyt. C, cytochrome C; SOD, superoxide dismutase.

Endogenous hydrogen peroxide in the hypothalamic paraventricular nucleus regulates neurohormonal excitation in high salt-induced hypertension

Reactive oxygen species (ROS) in the brain plays an important role in the progression of hypertension and hydrogen peroxide (H2O2) is a major component of ROS. The aim of this study is to explore whether endogenous H2O2 changed by polyethylene glycol-catalase (PEG-CAT) and aminotriazole (ATZ) in the hypothalamic paraventricular nucleus (PVN) regulates neurotransmitters, renin-angiotensin system (RAS), and cytokines, and whether subsequently affects the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in high salt-induced hypertension. Male Sprague–Dawley rats received a high-salt diet (HS, 8% NaCl) or a normal-salt diet (NS, 0.3% NaCl) for 10 weeks. Then rats were treated with bilateral PVN microinjection of PEG-CAT (0.2 i.u./50nl), an analog of endogenous catalase, the catalase inhibitor ATZ (10nmol/50nl) or vehicle. High salt-fed rats had significantly increased MAP, RSNA, plasma norepinephrine (NE) and pro-inflammatory cytokines (PICs). In addition, rats with high-salt diet had higher levels of NOX-2, NOX-4 (subunits of NAD(P)H oxidase), angiotensin-converting enzyme (ACE), interleukin-1beta (IL-1β), glutamate and NE, and lower levels of gamma-aminobutyric acid (GABA) and interleukin-10 (IL-10) in the PVN than normal diet rats. Bilateral PVN microinjection of PEG-CAT attenuated the levels of RAS and restored the balance of neurotransmitters and cytokines, while microinjection of ATZ into the PVN augmented those changes occurring in hypertensive rats. Our findings demonstrate that ROS component H2O2 in the PVN regulating MAP and RSNA are partly due to modulate neurotransmitters, renin-angiotensin system, and cytokines within the PVN in salt-induced hypertension.

Hydrogen Sulfide Prevents Advanced Glycation End-Products Induced Activation of the Epithelial Sodium Channel.

Advanced glycation end-products (AGEs) are complex and heterogeneous compounds implicated in diabetes. Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal nephron plays an important role in diabetic hypertension. Here, we report that H2S antagonizes AGEs-induced ENaC activation in A6 cells. ENaC open probability (P O) in A6 cells was significantly increased by exogenous AGEs and that this AGEs-induced ENaC activity was abolished by NaHS (a donor of H2S) and TEMPOL. Incubating A6 cells with the catalase inhibitor 3-aminotriazole (3-AT) mimicked the effects of AGEs on ENaC activity, but did not induce any additive effect. We found that the expression levels of catalase were significantly reduced by AGEs and both AGEs and 3-AT facilitated ROS uptake in A6 cells, which were significantly inhibited by NaHS. The specific PTEN and PI3K inhibitors, BPV(pic) and LY294002, influence ENaC activity in AGEs-pretreated A6 cells. Moreover, after removal of AGEs from AGEs-pretreated A6 cells for 72 hours, ENaC P O remained at a high level, suggesting that an AGEs-related “metabolic memory” may be involved in sodium homeostasis. Our data, for the first time, show that H2S prevents AGEs-induced ENaC activation by targeting the ROS/PI3K/PTEN pathway.

Azide-dependent nitric oxide emission from the water fern Azolla pinnata

Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling molecule. To date, plants have been reported to produce NO following exposure to nitrite (N O2−) the amino acid L-arginine, hydroxylamine, or polyamines. Here we demonstrate azide-dependent NO production in plants. The water fern Azolla pinnata emitted NO into air upon exposure to sodium azide (NaN3). The NO production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN). Incubation of A. pinnata with the catalase inhibitor 3-aminotriazole (3-AT) abolished the azide-dependent NO production. Although nitrite-dependent NO production was inhibited by sodium azide, azide-dependent NO production was not affected by nitrite. These results indicate that A. pinnata enzymatically produces NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the action of catalase as previously reported in animals.

Emerging Roots Alter Epidermal Cell Fate through Mechanical and Reactive Oxygen Species Signaling

A central question in biology is how spatial information is conveyed to locally establish a developmental program. Rice (Oryza sativa) can survive flash floods by the emergence of adventitious roots from the stem. Epidermal cells that overlie adventitious root primordia undergo cell death to facilitate root emergence. Root growth and epidermal cell death are both controlled by ethylene. This study aimed to identify the signal responsible for the spatial control of cell death. Epidermal cell death correlated with the proximity to root primordia in wild-type and ADVENTITIOUS ROOTLESS1 plants, indicating that the root emits a spatial signal. Ethylene-induced root growth generated a mechanical force of ~18 millinewtons within 1 h. Force application to epidermal cells above root primordia caused cell death in a dose-dependent manner and was inhibited by 1-methylcyclopropene or diphenylene iodonium, an inhibitor of NADPH oxidase. Exposure of epidermal cells not overlying a root to either force and ethylene or force and the catalase inhibitor aminotriazole induced ectopic cell death. Genetic downregulation of the reactive oxygen species (ROS) scavenger METALLOTHIONEIN2b likewise promoted force-induced ectopic cell death. Hence, reprogramming of epidermal cell fate by the volatile plant hormone ethylene requires two signals: mechanosensing for spatial resolution and ROS for cell death signaling.

Oxidative stress provokes distinct transcriptional responses in the stress-tolerant atr7 and stress-sensitive loh2 Arabidopsis thaliana mutants as revealed by multi-parallel quantitative real-time PCR analysis of ROS marker and antioxidant genes

The Arabidopsis thaliana atr7 mutant is tolerant to oxidative stress induced by paraquat (PQ) or the catalase inhibitor aminotriazole (AT), while its original background loh2 and wild-type plants are sensitive. Both, AT and PQ, which stimulate the intracellular formation of H₂O₂ or superoxide anions, respectively, trigger cell death in loh2 but do not lead to visible damage in atr7. To study gene expression during oxidative stress and ROS-induced programmed cell death, two platforms for multi-parallel quantitative real-time PCR (qRT-PCR) analysis of 217 antioxidant and 180 ROS marker genes were employed. The qRT-PCR analyses revealed AT- and PQ-induced expression of many ROS-responsive genes mainly in loh2, confirming that an oxidative burst plays a role in the activation of the cell death in this mutant. Some of the genes were specifically regulated by either AT or PQ, serving as markers for particular types of ROS. Genes significantly induced by both AT and PQ in loh2 included transcription factors (ANAC042/JUB1, ANAC102, DREB19, HSFA2, RRTF1, ZAT10, ZAT12, ethylene-responsive factors), signaling compounds, ferritins, alternative oxidases, and antioxidant enzymes. Many of these genes were upregulated in atr7 compared to loh2 under non-stress conditions at the first time point, indicating that higher basal levels of ROS and higher antioxidant capacity in atr7 are responsible for the enhanced tolerance to oxidative stress and suggesting a possible tolerance against multiple stresses of this mutant.

Catalase activity prevents exercise-induced up-regulation of vasoprotective proteins in venous tissue

Physical activity induces favourable changes of arterial gene expression and protein activity, although little is known about its effect in venous tissue. Although our understanding of the initiating molecular signals is still incomplete, increased expression of endothelial nitric oxide synthase (eNOS) is considered a key event. This study sought to investigate the effects of two different training protocols on the expression of eNOS and extracellular superoxide dismutase (ecSOD) in venous and lung tissue and to evaluate the underlying molecular mechanisms. C57Bl/6 mice underwent voluntary exercise or forced physical activity. Changes of vascular mRNA and protein levels and activity of eNOS, ecSOD and catalase were determined in aorta, heart, lung and vena cava. Both training protocols similarly increased relative heart weight and resulted in up-regulation of aortic and myocardial eNOS. In striking contrast, eNOS expression in vena cava and lung remained unchanged. Likewise, exercise up-regulated ecSOD in the aorta and in left ventricular tissue but remained unchanged in lung tissue. Catalase expression in lung tissue and vena cava of exercised mice exceeded that in aorta by 6.9- and 10-fold, respectively, suggesting a lack of stimulatory effects of hydrogen peroxide. In accordance, treatment of mice with the catalase inhibitor aminotriazole for 6 weeks resulted in significant up-regulation of eNOS and ecSOD in vena cava. These data suggest that physiological venous catalase activity prevents exercise-induced up-regulation of eNOS and ecSOD. Furthermore, therapeutic inhibition of vascular catalase might improve pulmonary rehabilitation.

A study of the relative importance of the peroxiredoxin-, catalase-, and glutathione-dependent systems in neural peroxide metabolism

Cells are endowed with several overlapping peroxide-degrading systems whose relative importance is a matter of debate. In this study, three different sources of neural cells (rat hippocampal slices, rat C6 glioma cells, and mouse N2a neuroblastoma cells) were used as models to understand the relative contributions of individual peroxide-degrading systems. After a pretreatment (30min) with specific inhibitors, each system was challenged with either H2O2 or cumene hydroperoxide (CuOOH), both at 100μM. Hippocampal slices, C6 cells, and N2a cells showed a decrease in the H2O2 decomposition rate (23–28%) by a pretreatment with the catalase inhibitor aminotriazole. The inhibition of glutathione reductase (GR) by BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) significantly decreased H2O2 and CuOOH decomposition rates (31–77%). Inhibition of catalase was not as effective as BCNU at decreasing cell viability (MTT assay) and cell permeability or at increasing DNA damage (comet test). Impairing the thioredoxin (Trx)-dependent peroxiredoxin (Prx) recycling by thioredoxin reductase (TrxR) inhibition with auranofin neither potentiated peroxide toxicity nor decreased the peroxide-decomposition rate. The results indicate that neural peroxidatic systems depending on Trx/TrxR for recycling are not as important as those depending on GSH/GR. Dimer formation, which leads to Prx2 inactivation, was observed in hippocampal slices and N2a cells treated with H2O2, but not in C6 cells. However, Prx-SO3 formation, another form of Prx inactivation, was observed in all neural cell types tested, indicating that redox-mediated signaling pathways can be modulated in neural cells. These differences in Prx2 dimerization suggest specific redox regulation mechanisms in glia-derived (C6) compared to neuron-derived (N2a) cells and hippocampal slices.

Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5'-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H(2)O(2) was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1-312), we found that mutation of cysteine 299 to alanine diminished the ability of H(2)O(2) to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H(2)O(2) on kinase activity. Similar to the results obtained with H(2)O(2)-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H(2)O(2) are increased. These results demonstrate that physiologically relevant concentrations of H(2)O(2) can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.

Modulation of SCFβ-TrCP-dependent IκBα Ubiquitination by Hydrogen Peroxide

Reactive oxygen species are known to participate in the regulation of intracellular signaling pathways, including activation of NF-kappaB. Recent studies have indicated that increases in intracellular concentrations of hydrogen peroxide (H(2)O(2)) have anti-inflammatory effects in neutrophils, including inhibition of the degradation of I kappaB alpha after TLR4 engagement. In the present experiments, we found that culture of lipopolysaccharide-stimulated neutrophils and HEK 293 cells with H(2)O(2) resulted in diminished ubiquitination of I kappaB alpha and decreased SCF(beta-TrCP) ubiquitin ligase activity. Exposure of neutrophils or HEK 293 cells to H(2)O(2) was associated with reduced binding between phosphorylated I kappaB alpha and SCF(beta-TrCP) but no change in the composition of the SCF(beta-TrCP) complex. Lipopolysaccharide-induced SCF(beta-TrCP) ubiquitin ligase activity as well as binding of beta-TrCP to phosphorylated I kappaB alpha was decreased in the lungs of acatalasemic mice and mice treated with the catalase inhibitor aminotriazole, situations in which intracellular concentrations of H(2)O(2) are increased. Exposure to H(2)O(2) resulted in oxidative modification of cysteine residues in beta-TrCP. Cysteine 308 in Blade 1 of the beta-TrCP beta-propeller region was found to be required for maximal binding between beta-TrCP and phosphorylated I kappaB alpha. These findings suggest that the anti-inflammatory effects of H(2)O(2) may result from its ability to decrease ubiquitination as well as subsequent degradation of I kappaB alpha through inhibiting the association between I kappaB alpha and SCF(beta-TrCP).

Neuroprotective effect of adenoviral catalase gene transfer in cortical neuronal cultures

Reduced availability of reactive oxygen species is a key component of neuroprotection against toxic stimuli. Recently, we have shown that the hydrogen peroxide scavenger catalase plays a central role in delayed preconditioning induced by the mitochondrial ATP‐sensitive potassium channel opener BMS‐191095 (Gáspár et al. JCBFM 28 (6): 1090‐1103, 2008). The aim of our present experiments was to investigate the neuroprotective effect of catalase in vitro using an adenoviral catalase gene transfer protocol. To induce catalase overexpression, cultured rat cortical neurons were incubated with the adenoviral vector Ad5CMVcatalase and control cells with Ad5CMVntLacZ for 24 h. Gene transfer increased catalase protein levels and activity, but did not influence other antioxidants. Catalase transfection did not protect against glutamate excitotoxicity and oxygen‐glucose deprivation, but conferred a dose‐dependent neuroprotection against 6 h exposure to 80 μM hydrogen peroxide (viability: control, 20.70±1.96%; LacZ 10 pfu/cell, 15.00± 1.66%; catalase 1 pfu/cell, 57.60±5.46%*; catalase 2 pfu/cell, 104.90±1.74%*; catalase 10 pfu/cell, 111.20±1.92%*; *p<0.05 vs. control; mean±SEM). Finally, the protection could be antagonized using the catalase inhibitor 3‐aminotriazole. Our results support the view that enhancing cellular antioxidant capacity may play a crucial role in neuroprotective strategies.

Neuroprotective effect of adenoviral catalase gene transfer in cortical neuronal cultures

Reduced availability of reactive oxygen species is a key component of neuroprotection against various toxic stimuli. Recently we showed that the hydrogen peroxide scavenger catalase plays a central role in delayed preconditioning induced by the mitochondrial ATP-sensitive potassium channel opener BMS-191095. The purpose of the experiments discussed here was to investigate the neuroprotective effect of catalase in vitro using a recombinant adenoviral catalase gene transfer protocol. To induce catalase overexpression, cultured rat cortical neurons were infected with the adenoviral vector Ad5CMVcatalase and control cells were incubated with Ad5CMVntLacZ for 24 h. Gene transfer effectively increased catalase protein levels and activity, but did not influence other antioxidants tested. Ad5CMVcatalase, with up to 10 plaque forming units (pfu) per neuron, did not affect cell viability under control conditions and did not protect against glutamate excitotoxicity or oxygen–glucose deprivation. In contrast, catalase overexpression conferred a dose-dependent protection against exposure to hydrogen peroxide (viability: control, 33.02 ± 1.09%; LacZ 10 pfu/cell, 32.85 ± 1.51%; catalase 1 pfu/cell, 62.09 ± 4.17%⁎; catalase 2 pfu/cell, 98.71 ± 3.35%⁎; catalase 10 pfu/cell, 99.68 ± 1.99%⁎; ⁎ p < 0.05 vs. control; mean ± SEM). Finally, the protection could be antagonized using the catalase inhibitor 3-aminotriazole. Our results support the view that enhancing cellular antioxidant capacity may play a crucial role in neuroprotective strategies.

Abstract 227: Activity of Soluble Guanylyl Cyclase, the Nitric Oxide Receptor, is Modulated by Cysteines Redox

The enzyme soluble guanylyl cyclase (sGC) increases cellular level of the second messenger cGMP upon stimulation by nitric oxide (NO), whose role is exemplified in the cardiovascular system where it governs smooth muscle cell proliferation, vascular relaxation and platelet aggregation. Various pathologies like diabetes, hypertension and hypercholesterolemia are associated with enhanced oxidative stress with increased formation of reactive oxygen (ROS) and nitrogen species (RNS). We recently showed that cysteines beta1C243 and beta1C122 are S-nitrosylated in sGC leading to its desensitization. We have now identified by Mass Spectrometry, two additional cysteines (beta1C571 and alpha1C516) as potentially modified. Our hypothesis is that loss in vascular reactivity in oxidative pathologies is mediated, in part, by desensitization of sGC via thiols oxidation. We studied the effect of oxidative stress on sGC in primary vascular smooth muscle cells (VSMC) by directly exposing them to hydrogen peroxide (H2O2) in the absence or presence of a catalase inhibitor Amino-Triazole (AT). H2O2 induced inhibition of NO-stimulated sGC activity in VSMC and in COS 7 cells transiently transfected with sGC. Desensitization of sGC was further increased in VSMC treated with both H2O2 and AT. Next, we conducted mutational analysis of the 4 cysteines to assess their involvement in redox sensing. Replacement of C571 and C122 eliminated the oxidative-dependent desensitization observed in the wild-type. In addition, peroxynitrite, a strong oxidant, inhibited the NO-stimulated sGC activity in a purified system without oxidation of the heme. In conclusion, our results suggest that sGC activity is directly modulated by the redox state of the cells. Moreover, the identified 4 cysteines can be modified by both ROS and RNS suggesting a complex modulation. Ultimately, our goal is to determine whether loss of vascular reactivity in oxidative pathophysiologies is in part mediated by desensitization of sGC and what are the involved cysteines. This work was supported by NIH GM067640, HL089771.

Hydrogen Peroxide Potentiates the EDHF Phenomenon by Promoting Endothelial Ca 2+ Mobilization

The purpose of this study was to test the hypothesis that H(2)O(2) contributes to the EDHF phenomenon by mobilizing endothelial Ca(2+) stores. Myograph studies with rabbit iliac arteries demonstrated that EDHF-type relaxations evoked by the SERCA inhibitor cyclopiazonic acid (CPA) required activation of K(Ca) channels and were potentiated by exogenous H(2)O(2) and the thiol oxidant thimerosal. Preincubation with a submaximal concentration of CPA unmasked an ability of exogenous H(2)O(2) to stimulate an EDHF-type response that was sensitive to K(Ca) channel blockade. Imaging of cytosolic and endoplasmic reticulum [Ca(2+)] in rabbit aortic valve endothelial cells with Fura-2 and Mag-fluo-4 demonstrated that H(2)O(2) and thimerosal, which sensitizes the InsP(3) receptor, both enhanced CPA-evoked Ca(2+) release from stores, and that the potentiating effect of H(2)O(2) was suppressed by the cell-permeant thiol reductant glutathione monoethylester. CPA-evoked relaxations were attenuated by exogenous catalase and potentiated by the catalase inhibitor 3-aminotriazole, and were abolished by the connexin-mimetic peptide (43)Gap26, which interrupts intercellular communication via gap junctions constructed from connexin 43. H(2)O(2) can enhance EDHF-type relaxations by potentiating Ca(2+) release from endothelial stores, probably via redox modification of the InsP(3) receptor, leading to the opening of hyperpolarizing endothelial K(Ca) channels and an electrotonically-mediated relaxant response.

ROS-Independent Preconditioning in Neurons via Activation of mitoKATP Channels by BMS-191095

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3beta. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.

Catalase characterization and implication in bleaching of a symbiotic sea anemone

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.

Glutathione peroxidase 1 and a high cellular glutathione concentration are essential for effective organic hydroperoxide detoxification in astrocytes

Organic hydroperoxides are produced in the eicosanoid metabolism and by lipid peroxidation. To examine the contribution of glutathione peroxidase-1 (GPx1) and glutathione (GSH) in the disposal of organic hydroperoxides in brain astrocytes, primary astrocyte cultures from wild type or GPx1-deficient (GPx1(-/-)) mice were exposed to cumene hydroperoxide (CHP). After application of 100 microM CHP, the peroxide disappeared quickly from the incubation medium of wild type cells with a half-life of 9 min, whereas CHP clearance was strongly retarded in GPx1(-/-) astrocytes. Depletion of GSH by pre-incubation with buthionine sulfoximine (BSO) significantly slowed CHP clearance by wild type astrocytes, while almost completely preventing peroxide disposal by GPx1(-/-) cells. In contrast, the catalase inhibitor 3-aminotriazole (3AT) had no effect on CHP clearance. Application of CHP to wild type astrocytes was followed by a rapid and transient accumulation of GSSG, whereas in GPx1(-/-) cells no increase in the GSSG content was detected. Astrocytes from both mouse lines remained viable for up to 24 h following CHP exposure, however depletion of cellular GSH by pre-treatment with BSO compromised the viability of astrocytes, an effect that was stronger in GPx1(-/-) than in wild type cells. This cell death was almost completely prevented by iron chelators, whereas pre-incubation with iron increased CHP toxicity. These novel data demonstrate that the toxicity of organic hydroperoxides in astrocytes is iron-mediated, and that an intact GSH system is required for the effective removal of organic hydroperoxides and for protection from these peroxides.

Identification and Characterization of a Stress-Inducible and a Constitutive Small Heat-Shock Protein Targeted to the Matrix of Plant Peroxisomes

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL>) and a functional PTS2 (RLx5HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one- and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions.

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.