Trypanosoma rangeli: A possible role for ecto-phosphatase activity on cell proliferation

Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by β-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes.     

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H⁺-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H⁺-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H⁺-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of K_m = 1.387 ± 0.1674 mM Pi and V_max = 198.6 ± 10.23 Pi × h^?1 × mg protein^?1 hence reflecting a low affinity Pi transport. H⁺-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H⁺-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H⁺-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H⁺-dependent Pi transport was five-fold higher than the Na⁺-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H⁺-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na⁺-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.     

Trypanosoma rangeli: Differential expression of ecto-phosphatase activities in response to inorganic phosphate starvation

In this work, we showed that living cells of Trypanosoma rangeli express different ecto-phosphatase activities in response to different inorganic phosphate (Pi) concentrations in the culture medium. The ecto-phosphatase activity from T. rangeli grown at low-Pi concentration was inhibited by the increase of the pH, while the ecto-phosphatase of the cells grown at high Pi concentration was not modulated by the change of the pH of the medium. Okadaic acid inhibited only the ecto-phosphatase activity from cells grown at low-Pi concentration but not the ecto-phosphatase activity from cells grown at high-Pi concentration. Accordingly, phosphatase activity from T. rangeli grown at low Pi concentration was able to hydrolyze P-serine and P-threonine at high rate but not P-tyrosine. The phosphatase activity from T. rangeli grown at high-Pi concentration was able to hydrolyze P-serine, P-threonine and P-tyrosine with the same rate. The addition of anterior midgut homogenate of Rhodnius prolixus on the epimastigotes suspension inhibited the enzyme activity of T. rangeli grown at low-Pi concentration. On the other hand, anterior midgut homogenate had no effect in the ecto-phosphatase of T. rangeli maintained at high-Pi concentration. Altogether, the results described here indicate that ecto-phosphatase activities hydrolyzing phosphorylated compounds present in the extracellular medium of T. rangeli are regulated by the external Pi concentration.     

Cocoa polyphenols attenuate hydrogen peroxide-induced inhibition of gap-junction intercellular communication by blocking phosphorylation of connexin 43 via the MEK/ERK signaling pathway

Cocoa, a good source of dietary antioxidative polyphenols, exhibited anticarcinogenic activity in animal models, but the molecular mechanisms of the chemopreventive potential of cocoa remain unclear. Inhibition of gap-junction intercellular communication (GJIC) is strongly related to tumorigenesis. Cocoa polyphenol extracts (CPE) dose dependently attenuated hydrogen peroxide (H₂O₂)-induced inhibition of GJIC in rat liver epithelial (RLE) cells. CPE inhibited the H₂O₂-induced phosphorylation and internalization of connexin 43, which is a regulating protein of GJIC in RLE cells. The H₂O₂-induced accumulation of reactive oxygen species and activation of extracellular signal-regulated kinase were inhibited by CPE treatment. However, CPE did not block H₂O₂-induced phosphorylation of p38 mitogen-activated protein kinase. An ex vivo kinase assay demonstrated that CPE inhibited the H₂O₂-induced mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 activity in RLE cell lysates. Ex vivo pull-down assay data revealed that CPE directly bound with MEK1 to inhibit MEK1 activity. These results indicate that CPE protects against the H₂O₂-induced inhibition of GJIC through antioxidant activity and direct inhibition of MEK activity, which may contribute to its chemopreventive potential.     

Selenium Protects Bone Marrow Stromal Cells Against Hydrogen Peroxide-Induced Inhibition of Osteoblastic Differentiation by Suppressing Oxidative Stress and ERK Signaling Pathway

Osteoporosis is a bone disease that leads to an increased risk of fracture. Oxidative stress may play a major role in the development of osteoporosis in part by inhibiting osteoblastic differentiation of bone marrow stromal cells (MSCs). Some evidence suggested that antioxidant selenium could prevent osteoporosis, but the underlying mechanism remains unclear. In this work, the effect of sodium selenite on H₂O₂-induced inhibition of osteoblastic differentiation of primary rat bone MSCs and the related mechanisms were examined. Pretreatment with selenite inhibited the adverse effect of H₂O₂ on osteoblastic differentiation of MSCs, based on alkaline phosphatase activity, gene expression of type I collagen and osteocalcin, and matrix mineralization. In addition, selenite pretreatment also suppressed the activation of extracellular signal-regulated kinase (ERK) induced by H₂O₂. The above effects were mediated by the antioxidant effect of selenite. Selenite enhanced the gene expression and activity of glutathione peroxidase, reversed the decreased total antioxidant capacity and reduced glutathione, and suppressed reactive oxygen species production and lipid peroxidation level in H₂O₂-treated MSCs. These results showed that selenite protected MSCs against H₂O₂-induced inhibition of osteoblastic differentiation through inhibiting oxidative stress and ERK activation, which provided, for the first time, the mechanistic explanation for the negative association of selenium status and risk of osteoporosis in terms of bone formation.     

Cathepsin B links oxidative stress to the activation of NLRP3 inflammasome

Oxidative stress-mediated activation of NLRP3 inflammasome in microglia is critical in the development of neurodegerative diseases such as Alzheimer's disease (AD), Parkinson disease (PD). However, the mechanism underlying oxidative stress activates NLRP3 inflammasome remains exclusive. Here we demonstrated cathepsin B (CTSB) as a regulator of the activation of NLRP3 inflammasome by H₂O₂·H₂O₂ induced IL-1β secretion in NLRP3 inflammasome-dependent manner·H₂O₂ treatment increased CTSB activity, which in turn activated NLRP3 inflammasome, and subsequently processed pro-caspase-1 cleavage into caspase-1, resulting in IL-1 β secretion. Genetic inhibition or pharmacological inhibition of CTSB blocked the cleavage of pro-caspase-1 into caspase-1 and subsequent IL-1 β secretion induced by H₂O₂. Importantly, CTSB activity, IL-1β levels and malondialdehyde (MDA) were remarkably elevated in plasma of AD patients compared to healthy controls, while glutathione was significantly lower than healthy controls. Correlation analyses showed that CTSB activity was positively correlated with IL-1β and MDA levels, but negatively correlated with GSH levels in plasma of AD patients. Taken together, our results indicate that oxidative stress activates NLRP3 through upregulating CTSB activity. Our results identify an important biological function of CTSB in neuroinflammation, suggesting that CTSB is a potential target in AD therapy.     

Reactive oxygen species generated in different compartments induce cell death,survival, or senescence

Although reactive oxygen species (ROS) are well-established mediators of oxidative damage and cell demise, the mechanisms by which they trigger specific cell death modalities and the temporal/spatial requirements underlying this phenomenon are largely unknown. Yet, it is well established that most anticancer therapies depend on ROS production for efficient tumor eradication. Using several non-small-cell lung cancer cell lines, we have dissected how the site of ROS production and accumulation in various cell compartments affect cell fate. We demonstrate that high levels of exogenously generated H₂O₂ induce extensive DNA damage, ATP depletion, and severe cytotoxicity. Although these effects were independent of caspase activity, they could—at least in part—be prevented by RIP1 kinase inhibition. In contrast, low levels of exogenously produced H₂O₂ triggered a modest drop in ATP level, delayed toxicity, G2/M arrest, and cell senescence. Mitochondrially produced H₂O₂ induced a reversible ATP drop without affecting cell viability. Instead, the cells accumulated in the G1/S phase of the cell cycle and became senescent. Concomitant inhibition of glycolysis was found to markedly sensitize cells to death in the presence of otherwise nontoxic concentrations of H₂O₂, presumably by the inhibition of ATP-restoring mechanisms. Combined, our data provide evidence that ROS might dictate different cellular consequences depending on their overall concentration at steady-state levels and on their site of generation.     

Characterization of an ecto-phosphatase activity in malpighian tubules of hematophagous bug Rhodnius prolixus

We have characterized a phosphatase activity present on the external surface of intact Malpighian tubules in Rhodnius prolixus. This phosphatase hydrolyses the substrate p-nitrophenyl phosphate at a rate of 3.38 +/- 0.07 nmol Pi x mg(-1) x min(-1). Phosphatase activity decreased with the increase of the pH from 6.4 to 7.6 pH, a range in which tubules cellular integrity was maintained for at least 1 h. Classical inhibitors of acid phosphatase, such as ammonium molybdate, fluoride, vanadate, mpV-PIC, and bpV-PHEN, caused different patters of inhibition. The ecto-phosphatase present an apparent Km of 1.67 +/- 0.34 mM and Vmax of 5.71 +/- 0.37 nmol Pi x mg(-1) x min(-1) for p-NPP. Zinc chloride inhibited 78.2% of ecto-phosphatase activity, with Ki of 0.35 mM. Such inhibition was reversed by incubation with cysteine and GSH, but not DTT, serine, and GSSG, showing that cysteine residues are important for enzymatic activity. Phosphatase activity increased 141% three days after blood meal, and returned to basal levels 2 days later. These results suggest that ecto-phosphatase activity could be involved in a diuretic mechanism, essential in the initial days after a blood meal for the control of Rhodnius homeostasis.     

Rapid breakdown of exogenous extracellular hydrogen peroxide by lichens

All organisms, even highly stress‐tolerant lichens, produce a variety of reactive oxygen species (ROS) during and after stress. Furthermore, the cell walls of some lichens in Suborder Peltigerineae contain laccases, and therefore can produce quinone radicals that can break down to yield ROS. While the extracellular ROS produced by these enzymes probably play important roles in the biology of these lichens, they may also be potentially harmful and need to be rapidly broken down. To test this, rates of breakdown of exogenously supplied H₂O₂ were measured in a range of lichen species. Considerable diversity existed in rates of H₂O₂ breakdown but rates were on average almost double in members of Suborder Peltigerineae. While all lichens tested appeared to lack extracellular peroxidases and catalases, enzymes normally involved in breaking down H₂O₂, extracellular tyrosinase activity could be readily detected in the Peltigerineae. A role for tyrosinases in H₂O₂ breakdown was supported by the results from experiments involving inhibitors, and demonstration of the simultaneous release into an incubation solution of tyrosinase activity and the ability to breakdown H₂O₂. Rates of breakdown were very high, and tyrosinase appeared to break down H₂O₂ by a catalase‐like mechanism. However, significant rates of breakdown of H₂O₂ also occurred in species that did not possess cell wall redox enzymes. These species probably took up the exogenously supplied H₂O₂ intracellularly and then broke it down by the usual catalases and peroxidases. The importance of H₂O₂ degradation is discussed in terms of its possible role in defence against the harmful effects of ROS.     

Active oxygen species production in tobacco cells elicited by cryptogein*

We analyse the relationship between active oxygen species (AOS) production and pH changes induced in tobacco cells by cryptogein, a fungal proteinaceous elicitor of defence mechanisms in plants. When tobacco cells were treated with cryptogein, an intracellular acidification, an alkalinization of the extracellular medium and a transient burst of AOS (H₂O₂) were observed. Treatment of elicited cells with either diphenyleneiodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, or Tiron, which scavenges O₂^˙? abolished AOS production. These data suggest the involvement of a NADPH oxidase-like enzyme leading to H₂O₂ production through O₂^˙? dismutation. Although H₂O₂ production could be, per se, the origin of the pH changes observed, we showed that it was not the main cause, since DPI and Tiron did not inhibit extracellular alkalinization. On the other hand, cryptogein-induced changes in pH could be abolished using fusicoccin (FC), which is known to stimulate the plasmalemma H⁺ ATPase. Consequently, the observed changes in pH induced by cryptogein could be mainly due to the inhibition of the plasmalemma H⁺-ATPase activity. Furthermore, changes in extracellular pH were shown to modulate the intensity of AOS production by elicited cells. The possible regulation of the NAD(P)H oxidase activity of plant cells by changes in pH is further discussed.     

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5–25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H₂O₂, did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H₂O₂ signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H₂O₂ in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.     

Hydrogen Peroxide-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells: Possible Involvement of Ca2+-Dependent Protein Tyrosine Kinase

Abstract: The mechanism for hydrogen peroxide (H₂O₂)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled PC12 cells. In the presence of butanol, H₂O₂ caused a great accumulation of [³H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H₂O₂ of cell lysates exerted no effect on PLD activity. Treatment with H₂O₂ had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H₂O₂-induced PLD activity. Thus, H₂O₂-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H₂O₂-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H₂O₂ treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca²⁺ abolished H₂O₂-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca²⁺ potentiated H₂O₂-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca²⁺-dependent protein tyrosine kinase(s) somehow participates in H₂O₂-induced PLD activation in PC12 cells.     

Salinity induced the changes of root growth and antioxidative responses in two wheat cultivars

This study aimed to investigate the inhibitory mechanism of root growth and to compare antioxidative responses in two wheat cultivars, drought-tolerant Ningchun and drought-sensitive Xihan, exposed to different NaCl concentrations. Ningchun exhibited lower germination rate, seedling growth, and lipid peroxidation than Xihan when exposed to salinity. The loss of cell viability was correlated with the inhibition of root growth induced by NaCl stress. Moreover, treatments with H₂O₂ scavenger dimethylthiourea and catalase (CAT) partly blocked salinity-induced negative effects on root growth and cell viability. Besides, the enhancement of superoxide radical and H₂O₂ levels, and the stimulation of CAT and diamine oxidase (DAO) as well as the inhibition of glutathione reductase (GR) were observed in two wheat roots treated with salinity. However, hydroxyl radical content increased only in Xihan roots under NaCl treatment, and the changes of soluble peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and cell-wall-bound POD activities were different in drought-tolerant Ningchun and drought-sensitive Xihan exposed to different NaCl concentrations. In conclusion, salinity might induce the loss of cell viability via a pathway associated with extracellular H₂O₂ generation, which was the primary reason leading to the inhibition of root growth in two wheat cultivars. Here, it was also suggested that increased H₂O₂ accumulation in the roots of drought-tolerant Ningchun might be due to decreased POD and GR activities as well as enhanced cell-wall-bound POD and DAO ones, while the inhibition of APX and GR as well as the stimulation of SOD and DAO was responsible for the elevation of H₂O₂ level in drought-sensitive Xihan roots.     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

Hydrogen peroxide release by rat peritoneal macrophages in the presence and absence of tumor cells

The ability of mineral oil-elicited rat peritoneal macrophages to release hydrogen peroxide (H₂O₂) to the extracellular medium was measured in the presence and absence of rat lymphoma cells grown in tissue culture, and in the presence of phorbol myristate acetate (PMA). Horseradish peroxidase (HRP)-catalyzed oxidation of scopoletin or phenol red was used to measure H₂O₂ release during incubation of cells in monolayer culture for periods up to 24 h. Macrophages appeared to release H₂O₂ with or without PMA, although PMA greatly increased the amount of H₂O₂ released in short (1 to 4 h) incubations. Tumor cells did not replace PMA as a triggering agent for H₂O₂ release. Instead, tumor cells inhibited H₂O₂ release. The probable basis for inhibition was competition between macrophages and tumor cells for the supply of oxygen (O₂). Tumor cells did not inhibit H₂O₂ release when the O₂ concentration was held constant. The rates at which macrophages took up O₂ and released H₂O₂ were proportional to the O₂ concentration, as measured with the O₂ electrode. Rates of H₂O₂ release could be calculated from the difference in the rate constants for O₂ uptake measured in the presence of two different extracellular H₂O₂-consuming systems (HRP-scopoletin vs catalase). PMA-stimulated uptake of O₂ and release of H₂O₂ were highest in a small subpopulation of macrophages, obtained at the lowest-density position on gradients of bovine serum albumin. These cells also released H₂O₂ in the absence of PMA. Tumor cells had no effect on the rate constants for O₂ uptake and H₂O₂ release by the unfractionated macrophages or the macrophage subpopulations.     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

Mechanism for proliferation inhibition by various selenium compounds and selenium-enriched broccoli extract in rat glial cells

The objective of this study was to investigate the differential effects of various selenium (Se) compounds and Se-enriched broccoli extracts on cell proliferation and the possible mechanism responsible for the Se-induced growth inhibition. C6 rat glial cells were incubated with graded concentrations up to 1000 nM of selenite, selenate, selenomethionine (SeM), Se-methyl-selenocysteine (SeMCys), high-Se broccoli (H-SeB) extract or low-Se broccoli (L-SeB) extract for 24 and 48 h. MTT results indicated that all Se sources and levels examined inhibited C6 cell proliferation at 48 h. The results from cell cycle progression and apoptosis analysis indicated that SeM, SeMCys, H-SeB or L-SeB treatments at the concentration of 1000 nM reduced the cell population in G₀/G₁ phase, but induced G₂/M phase arrest and increased apoptosis and secondary necrosis in C6 cells at 24 h. The populations of apoptotic cells and secondary necrotic cells were increased by all Se sources examined. The COMET assay indicated that there was no significant DNA single-strand break found for all Se treatments in C6 cells for 48 h. In addition, the Se-induced proliferation inhibition may involve a hydrogen peroxide (H₂O₂)-dependent mechanism with elevated cellular glutathione peroxidase (cGPX) activity. Both H-SeB and L-SeB inhibited C6 cell proliferation but H-SeB was less inhibitory than L-SeB. The proliferation inhibition by H-SeB in C6 cells is apparently related to the increased H₂O₂ with the elevated cGPX activity, but the inhibition by L-SeB was H₂O₂-independent without change in cGPX activity.     

H2O2 is involved in the dormancy-breaking effect of hydrogen cyanamide in grapevine buds

Hydrogen cyanamide (HC) is widely used to induce the breakage of endodormancy (ED) in grape and other deciduous fruit crop, though its mechanism of action is poorly understood. Applications of HC to grapevine buds produce oxidative stress and transient respiratory disturbances which are related to the breakage of ED. Moreover, since the expression and activity of catalase (Cat) is inhibited by HC, enhancements in the levels of H₂O₂ have also been associated to the breakage of ED in grapevine buds. Here, we reported that increases in H₂O₂ level in HC-treated grapevine buds are due to the inhibition of Cat activity and enhancement of the respiratory activity of buds. In addition, exogenous applications of H₂O₂ partially reproduced the inducing effect of HC in the breakage of ED, thus providing further support for the hypothesis that H₂O₂ mediates the effects of HC. On the other hand, Mit isolated from both control and HC-treated buds respired equally well when NADH was used as a respiratory substrate, but when succinate was used as an electron donor Mit respiration was non-detected, suggesting that the stimulatory effect of HC on bud respiration is related to metabolic alterations leading to increase of the concentration of NADH rather than to changes in Mit functionality.