Alternative oxidase impacts the plant response to biotic stress by influencing the mitochondrial generation of reactive oxygen species

Previously, we showed that inoculation of tobacco with Pseudomonas syringae incompatible pv. maculicola results in a rapid and persistent burst of superoxide (O₂^‐) from mitochondria, no change in amount of mitochondrial alternative oxidase (AOX) and induction of the hypersensitive response (HR). However, inoculation with incompatible pv. phaseolicola resulted in increased AOX, no O₂^‐ burst and no HR. Here, we show that in transgenic plants unable to induce AOX in response to pv. phaseolicola, there is now a strong mitochondrial O₂^‐ burst, similar to that normally seen only with pv. maculicola. This interaction did not however result in a HR. This indicates that AOX amount is a key determinant of the mitochondrial O₂^‐ burst but also that the burst itself is not sufficient to induce the HR. Surprisingly, the O₂^‐ burst normally seen towards pv. maculicola is delayed in plants lacking AOX. This delay is associated with a delayed HR, suggesting that the burst does promote the HR. A O₂^‐ burst can also be induced by the complex III inhibitor antimycin A (AA), but is again delayed in plants lacking AOX. The similar mitochondrial response induced by pv. maculicola and AA suggests that electron transport is a target during HR‐inducing biotic interactions.     

Leishmania donovani Attachment Stimulates PKC-Mediated Oxidative Events in Bone Marrow-Derived Macrophages

We investigated activation signaling events in bone marrow-derived macrophages after infection with Leishmania donovani, an intracellular parasite of macrophages. Leishmania donovani infection caused a general suppression of activation parameters like O₂^- and NO production. However, conditions which allow parasite attachment and prevent entry resulted in triggering of O₂^- and NO production and stimulation of O₂ consumption. Optimal NO and O₂^- production occurred when bone marrow-derived macrophages and Leishmania ratio was 1:100. The activation signal for O₂^- production was initiated 15 min after parasite attachment, whereas augmentation of NO production started 6 h after attachment. Activation of O₂^- and NO generation by L. donovani attachment was inhibited by staurosporine as well as by prolonged treatment of phorbol myristate acetate suggesting a protein kinase C-dependent mechanism. Translocation studies showed that protein kinase C activity in cell membrane fraction rapidly and transiently increased following parasite attachment. No such protein kinase C translocation event occurred in L. donovani infected bone marrow-derived macrophages. Phorbol myristate acetate was found to stimulate membrane translocation of protein kinase C in parasite attached cells whereas it was impaired in infected cells. However, both attachment and infection induced a similar shift of phorbol receptors from cytosolic to membrane fraction indicating that in infected cells the translocation of protein kinase C protein was not impaired but the activity of the membrane associated enzyme was somehow inhibited. These results suggest that although internalization of intracellular parasites like L. donovani caused inhibition of nitrite and superoxide release, mere attachment on macrophage surface resulted in an activation of protein kinase C-mediated downstream oxidative events.     

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O₂^•-, H₂O₂, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death^1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response⁷. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O₂^•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells⁸. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others^9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O₂^•- that are important in the host defense mechanism^11,12. Although paracrine-derived O₂^•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca²⁺ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O₂^•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O₂^•- lead to calcium signaling in adjacent endothelial cells.     

Mitochondria Regulate Neutrophil Activation by Generating ATP for Autocrine Purinergic Signaling

Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y₂ receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca²⁺ signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca²⁺ signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca²⁺ signaling via P2X receptor-mediated Ca²⁺ influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.     

The oxidative burst in plant defense: Function and signal transduction

The rapid production and accumulation of active oxygen species (AOS), the oxidative burst, has been shown to occur in a variety of plant/pathogen systems. In particular, two species, hydrogen peroxide (H₂O₂) and the superoxide radical anion O₂^? have received considerable attention. H₂O₂ and O₂^?, while acting directly as antimicrobial agents, may also serve as second messengers or catalysts in plants to activate a more diverse set of defense responses. Some of the better studied downstream responses promoted by AOS are (1) the cross-linking of cell wall proteins, (2) the induction of defense-related genes, (3) the stimulation of phytoalexin biosynthesis and (4) promotion of the hypersensitive response (HR). A useful model for studying the oxidative burst in plants is the neutrophil NADPH ox-idase complex, the primary source of AOS production in mammals. Several of the subunits of the neutrophil NADPH oxidase complex have been immunologically identified in plants. Furthermore, many of the components known to be involved in the signal transduction pathway in neutrophils have also been found to play a role in the oxidative burst in plants. Just as various ligands activate the oxidase complex in neutrophils, several ligands (elicitors or pathogens) also lead to induction of the oxidative burst in plant cells. The similarities between the neutrophil and plant oxidative bursts will be elaborated in this review. Following stimulation with elicitors, different signal transduction pathways are activated in plants, depending on the source of elicitor used. While the identities and chronologies of the major intermediates in these pathways remain largely unknown, there is strong evidence at least for participation of phospholipases, H⁺/K⁺ exchange, Ca²⁺ influxes, protein kinases and phosphatases, and GTP binding proteins. In an effort to integrate these various signaling events into a single scheme, we have constructed a hypothetical model that proposes how different elicitors might induce the oxidative burst in the same cell by different pathways.     

Superoxide-Driven Aconitase FE–S Center Cycling

O^- ₂ produced by the autoxidation of respiratory chain electron carriers, and other cellular reductants, inactivates bacterial and mammalian iron-sulfur-containing (de)hydratases including the citric acid cycle enzyme aconitase. Release of the solvent-exposed iron atom and oxidation of the [4Fe-4S]²⁺ cluster accompanies loss of catalytic activity. Rapid reactivation is achieved by iron-sulfur cluster reduction and Fe²⁺ insertion. Inactivation-reactivation is a dynamic and cyclical process which modulates aconitase and (de)hydratase activities in Escherichia coli and mammalian cells. The balance of inactive and active aconitase provides a sensitive measure of the changes in steady-statO^- ₂ levels occuring in living cells and mitochondria under stress conditions. Aconitases are also inactivated by other oxidants including O₂, H₂O₂, NO^., and ONOO^– which are associated with inflammation, hyperoxia and other pathophysiological conditions. Loss of aconitase activity during oxidant stress may impair energy production, and the liberation of reactive iron may further enhance oxidative damage. Iron-sulfur center cycling may also serve adaptive functions by modulating gene expression or by signaling metabolic quiescence.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria

We hypothesized that NO^• is generated in isolated cardiac mitochondria as the source for ONOO⁻ production during oxidative stress. We monitored generation of ONOO⁻ from guinea pig isolated cardiac mitochondria subjected to excess Ca²⁺ uptake before adding succinate and determined if ONOO⁻ production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl₂ and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O₂^•−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO⁻ to form dityrosine (diTyr) in proportion to the ONOO⁻ present. We found that exposing mitochondria to excess CaCl₂ before succinate resulted in an increase in diTyr and amplex red fluorescence (H₂O₂) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa²⁺ and succinate, increased mitochondrial ONOO⁻ production via NO^• and O₂^•−. Changes in mitochondrial ONOO⁻ production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO^• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO⁻ production, PTIO decreased production of ONOO⁻, and TEMPOL decreased ONOO⁻ levels by converting more O₂^•− to H₂O₂. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO⁻ into the buffer is dependent both on O₂^•− released from mitochondria and NO^• derived from a mtCa²⁺-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.     

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O₂^•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O₂^•− in a hydrogen peroxide (H₂O₂)-dependent manner. However, this model is incomplete since the source of the initially required H₂O₂ could not be explained. Based on the recently proposed role for H₂O₂-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA.     

Ozone mediates the anticancer effect of air plasma by triggering oxidative cell death caused by H2O2 and iron

Cold atmospheric plasmas and plasma-treated solutions (PTSs) have emerged as promising approaches in cancer treatment because of their tumor-selective actions. While oxidative stress is critical for their effects, the precise mechanisms, including chemical mediators, remain obscure. Previously, we reported that air plasma-activated medium (APAM) exhibited tumor-selective anticancer activity. The fragmentation of mitochondria and their asymmetrical assembly around the peripheral regions of the damaged nucleus, namely, monopolar perinuclear mitochondrial clustering (MPMC), proceed to the effect. Subsequently, we found that APAM had a substantial amount of O₃ in addition to hydrogen peroxide (H₂O₂), nitrile (NO₂^-), and nitrate (NO₃^-). In the present study, we investigated the possible role of O₃ in the anticancer effect. For this purpose, we created a nitrogen oxide-free ozonated medium ODM. ODM exhibited potent cytotoxicity against various cancer but not nonmalignant cells. ODM also increased MPMC, hydroxyl radicals, lipid peroxides, and their shifts to perinuclear sites in cancer cells. Catalase and iron chelation prevented these events and cytotoxicity. ODM also decreases the intracellular labile irons while increasing those within mitochondria. ODM had substantial H₂O_2, but this oxidant failed to cause MPMC and cytotoxicity. These results show that ODM can mimic the effects of APAM, including MPMC and tumor-selective anticancer effects. The findings suggest that O₃ is critical in mediating the anticancer effects of APAM by triggering oxidative cell death caused by H₂O₂ and iron.     

Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production

Abstract

The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC₅₀ for aconitase inhibition ranging from 7 to 24 μM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient (> 0.95). Aconitase-specific activity varied widely among tissues examined from ?60 mU/mg in liver to 321 mU/mg in kidney at 21% O₂. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O₂ reflecting aconitase inactivation by oxygen-derived oxidants at 21% O₂. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O₂^●? and H₂O₂ formation by the respiratory chain.     

α-lipoic acid induces apoptosis in human colon cancer cells by increasing mitochondrial respiration with a concomitant O<Subscript>2</Subscript><Superscript>−.</Superscript>-generation

The antioxidant -lipoic acid (ALA) has been shown to affect a variety of biological processes associated with oxidative stress including cancer. We determined in HT-29 human colon cancer cells whether ALA is able to affect apoptosis, as an important parameter disregulated in tumour development. Exposure of cells to ALA or its reduced form dihydrolipoic acid (DHLA) for 24 h dose dependently increased caspase-3-like activity and was associated with DNA-fragmentation. DHLA but not ALA was able to scavenge cytosolic O₂^–. in HT-29 cells whereas both compounds increased O₂^{– .}-generation inside mitochondria. Increased mitochondrial O₂^{– .}-production was preceded by an increased influx of lactate or pyruvate into mitochondria and resulted in the down-regulation of the anti-apoptotic protein bcl-X_L. Mitochondrial O₂^–.-generation and apoptosis induced by ALA and DHLA could be prevented by the O₂^{– .}-scavenger benzoquinone. Moreover, when the lactate/pyruvate transporter was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoate, ALA- and DHLA-induced mitochondrial ROS-production and apoptosis were blocked. In contrast to HT-29 cells, no apoptosis was observed in non-transformed human colonocytes in response to ALA or DHLA addition. In conclusion, our study provides evidence that ALA and DHLA can effectively induce apoptosis in human colon cancer cells by a prooxidant mechanism that is initiated by an increased uptake of oxidizable substrates into mitochondria.     

Nicotinamide Nucleotide Transhydrogenase (Nnt) Links the Substrate Requirement in Brain Mitochondria for Hydrogen Peroxide Removal to the Thioredoxin/Peroxiredoxin (Trx/Prx) System

Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H₂O₂) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H₂O₂ catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP⁺, provides the link between mitochondrial respiration and H₂O₂ detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H₂O₂ in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H₂O₂, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H₂O₂ catabolism, (b) decreased NADPH and increased NADP⁺ levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H₂O₂ and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H₂O₂ antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.     

Unravelling mitochondrial retrograde regulation in the abiotic stress induction of rice ALTERNATIVE OXIDASE 1 genes

Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress‐induced MRR studies. We found that the induction mediated by the superoxide ion (O_2·^‐)‐generating chemical methyl viologen was stronger than that of hydrogen peroxide (H₂O₂). The addition of reactive oxygen species (ROS) scavengers demonstrated that the stress induction was reduced by eliminating O_2·^‐. Furthermore, the stress induction did not rely on chloroplast‐ or cytosol‐derived O_2·^‐. Next, we generated transgenic plants overexpressing the superoxide dismutase (SOD) gene at different subcellular locations. The results suggest that only the mitochondrial SOD, OsMSD, attenuated the stress induction of OsAOX1a/b specifically. Therefore, our findings demonstrate that abiotic stress initiates the MRR on OsAOX1a/b and that mitochondrial O_2·^– is involved in the process.     

High rates of extracellular superoxide production by lichens in the suborder Peltigerineae correlate with indices of high metabolic activity

Rates of extracellular superoxide radical (O₂^{· ?}) formation were measured in 34 species of lichens from different taxonomic groupings and contrasting habitats before and after desiccation stress. All 21 species from the suborder Peltigerineae produce O₂^{· ?} extracellularly at high rates, even when they are not stressed. In addition, some species show a burst of O₂^{· ?} production during rehydration following desiccation. Extracellular production of O₂^{· ?} is almost absent in the species from other lichen groups. In general, production of high levels of O₂^{· ?} and the existence of an inducible oxidative burst are best developed in species growing in wet microhabitats. Rates of O₂^{· ?} production are also positively correlated to previously published indices of lichen metabolic activity. Preliminary studies on the identity of the O₂^{· ?} producing enzymes suggest that they do not possess the classical characteristics of those suggested to produce reactive oxygen species in higher plants. Patterns of O₂^{· ?} production are discussed in terms of the strategies used by different lichens groups in their defence against pathogenic fungi and bacteria.     

Protective Effects of the Synthetic Cannabinoids CP55,940 and JWH-015 on Rat Brain Mitochondria upon Paraquat Exposure

The effects of cannabinoids in mitochondria after acute oxidative stress insult are not fully established. We investigated the ability of CP55,940 and JWH-015 to scavenge reactive oxygen species and their effect on mitochondria permeability transition (MPT) in either a mitochondria-free superoxide anion generation system, intact rat brain mitochondria or in sub-mitochondrial particles (SMP) treated with paraquat (PQ). Oxygen consumption, mitochondrial membrane potential (Δψ_m) and MPT were determined as parameters of mitochondrial function. It is found that both cannabinoids effectively attenuate mitochondrial damage against PQ-induced oxidative stress by scavenging anion superoxide radical (O₂ ^∙−) and hydrogen peroxide (H₂O₂), maintaining Δψ_m and by avoiding Ca²⁺-induced mitochondrial swelling. Understanding the mechanistic action of cannabinoids on mitochondria might provide new insights into more effective therapeutic approaches for oxidative stress related disorders.     

Fine-tuning of eTRPM8 expression and activity conditions keratinocyte fate

Recently, we reported the cloning and characterization of short isoform of the icilin-activated cold receptor TRPM8 channel in keratinocytes, dubbed eTRPM8. We demonstrated that eTRPM8 via fine tuning of the endoplasmic reticulum (ER) – mitochondria Ca²⁺ shuttling regulates mitochondrial ATP and superoxide (O₂^?-) production and, thereby, mediates control of epidermal homeostasis by mild cold. Here, we provide additional information explaining why eTRPM8 suppression and TRPM8 stimulation both inhibit keratinocyte growth. We also demonstrate that stimulation of eTRPM8 with icilin may give rise to sustained oscillatory responses. Furthermore, we show that ATP-induced cytosolic and mitochondrial Ca²⁺ responses are attenuated by eTRPM8 suppression. This suggests positive interplay between eTRPM8 and purinergic signaling pathways, what may serve to facilitate the ER-mitochondria Ca²⁺ shuttling. Finally, we demonstrate that cold (25°C) induces eTRPM8-dependent superoxide-mediated necrosis of keratinocytes. Altogether, these results are in line with our model of eTRPM8-mediated cold-dependent balance between keratinocyte proliferation and differentiation.     

Rac-dependent feedforward autoactivation of NOX2 leads to oxidative burst

NADPH oxidase 2 (NOX2) produces the superoxide anion radical (O₂⁻), which has functions in both cell signaling and immune defense. NOX2 is a multimeric-protein complex consisting of several protein subunits including the GTPase Rac. NOX2 uniquely facilitates an oxidative burst, which is described by initially slow O₂⁻ production, which increases over time. The NOX2 oxidative burst is considered critical to immune defense because it enables expedited O₂⁻ production in response to infections. However, the mechanism of the initiation and progression of this oxidative burst and its implications for regulation of NOX2 have not been clarified. In this study, we show that the NOX2 oxidative burst is a result of autoactivation of NOX2 coupled with the redox function of Rac. NOX2 autoactivation begins when active Rac triggers NOX2 activation and the subsequent production of O₂⁻, which in turn activates redox-sensitive Rac. This activated Rac further activates NOX2, amplifying the feedforward cycle and resulting in a NOX2-mediated oxidative burst. Using mutagenesis-based kinetic and cell analyses, we show that enzymatic activation of Rac is exclusively responsible for production of the active Rac trigger that initiates NOX2 autoactivation, whereas redox-mediated Rac activation is the main driving force of NOX2 autoactivation and contributes to generation of ∼98% of the active NOX2 in cells. The results of this study provide insight into the regulation of NOX2 function, which could be used to develop therapeutics to control immune responses associated with dysregulated NOX2 oxidative bursts.