Arsenite-Induced Mitochondrial Superoxide Formation: Time and Concentration Requirements for the Effects of the Metalloid on the Endoplasmic Reticulum and Mitochondria.

The present study used human myeloid leukemia U937 cells, a versatile promonocytic cellular system that, based on its endoplasmic reticulum (ER)/mitochondria functional relationships, responds to low micromolar concentrations of arsenite with a single, defined mechanism of superoxide (O2 -.) formation. Under these conditions, we observe an initial Ca2+ mobilization from the ER associated with the mitochondrial accumulation of the cation, which is followed by Ca2+-dependent mitochondrial O2 -. (mitoO2 -.) formation. These events, which were barely detectable after 3 hours, were better appreciated at 6 hours. We found that markedly shorter exposure to arsenite and lower concentrations of arsenite are required to induce extensive O2 - formation in cells supplemented with inositol-1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR) agonists. Indeed, nanomolar arsenite induced maximal O2 -. formation after only 10 minutes of exposure, and this response was uniquely dependent on the enforced mitochondrial Ca2+ accumulation. The dramatic anticipation of and sensitization to the effects of arsenite caused by the IP3R or RyR agonists were accompanied by a parallel significant genotoxic response in the absence of detectable mitochondrial dysfunction and cytotoxicity. We conclude that the prolonged, low-micromolar arsenite exposure paradigm resulting in mitoO2 -. formation is necessary to affect Ca2+ homeostasis and accumulate the cation in mitochondria. The arsenite requirements to promote mitoO2 -. formation in the presence of sufficient mitochondrial Ca2+ were instead remarkably lower in terms of both concentration and time of exposure. These conditions were associated with the induction of extensive DNA strand scission in the absence of detectable signs of toxicity. SIGNIFICANCE STATEMENT: In respiration-proficient cells, arsenite causes mitochondrial Ca2+ accumulation and Ca2+-dependent mitochondrial superoxide formation. We now report that the second event requires remarkably lower concentrations of and time of exposure to the metalloid than the former. Indeed, a brief exposure to nanomolar levels of arsenite produced maximal effects under conditions in which the mitochondrial Ca2+ concentration ([Ca2+]m) was increased by inositol-1,4,5-trisphosphate receptor or ryanodine receptor agonists. Hence, specific substances or conditions enhancing the [Ca2+]m may potentiate the deleterious effects of arsenite by selectively increasing mitochondrial superoxide formation.

The compartmentalised nature of the mechanisms governing superoxide formation and scavenging in cells exposed to arsenite.

In this study, respiration-proficient (RP) and -deficient (RD) cells were exposed to 2.5 or 10 µM arsenite to generate superoxide (O2-.) respectively in the mitochondrial respiratory chain or via NADPH oxidase activation. These treatments, while causing similar, although mitochondrial permeability transition-dependent (RP-cells) or independent (RD-cells), delayed apoptosis, surprisingly generated identical kinetics and levels of dihydrorhodamine oxidation, indicative of O2-. formation. These similarities were attributable to the involvement of a common upstream event resulting in activation of the two O2-.-generating systems, and to intrinsic features of the cells. Both mechanisms required an initial and sequential mobilization of Ca2+ from the inositol-1,4,5-trisphosphate receptor and the ryanodine receptor (RyR), with however different implications. The close contacts existing between the RyR and the mitochondria created optimal conditions for the Ca2+ clearance, and the ensuing formation of O2-. in RP-cell mitochondria. Exposure to low concentrations of l-ascorbic acid (AA) transported by high affinity mechanisms in cells and mitochondria, suppressed O2-. formation. Much more Ca2+, and hence more arsenite, was necessary to promote NADPH oxidase activation in RD-cells, as a consequence of the cytosolic dilution and mitochondrial clearance of Ca2+. For the same reasons, an exposure to high concentrations of AA was required to suppress O2-. formation under these conditions.

The dual role of mitochondrial superoxide in arsenite toxicity: Signaling at the boundary between apoptotic commitment and cytoprotection.

Arsenite toxicity is in numerous cellular systems dependent on the formation of reactive oxygen and or nitrogen species. This is also true in U937 cells in which the metalloid selectively promotes the formation of mitochondrial superoxide (mitoO2-) rapidly converted to diffusible H2O2. We tested the hypothesis that, under the same conditions, mitoO2- also mediates the triggering of a parallel survival signaling. We found that a low concentration of the metalloid causes an early activation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), and a downstream signaling leading to enhanced GSH biosynthesis, via a mechanism sensitive to various treatments/strategies selectively preventing mitoO2- formation. Under the same conditions, the toxic effects mediated by arsenite, leading to delayed mitochondrial permeability transition (MPT)-dependent apoptosis, were also prevented. Additional studies revealed remarkable similarities in the kinetics of mitoO2- formation, MPT induction, Nrf2 activation and GSH biosynthesis, prior to the onset of apoptosis in a small portion of the cells. Importantly, mitoO2- formation, as well as the ensuing toxic events, were significantly potentiated and anticipated under conditions associated with inhibition of de novo GSH biosynthesis triggered by the metalloid through Nrf2 activation. We conclude that, in the arsenite toxicity paradigm under investigation, mitoO2- represents the only trigger of two opposite pathways leading to activation of the Nrf2 signaling and/or to a MPT-dependent apoptotic death. The first pathway, through enhanced GSH biosynthesis, mitigates the extent of further mitoO2- formation, thereby limiting and delaying an otherwise rapid and massive apoptotic death.

Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism.

Exposure of U937 cells to peroxynitrite promotes mitochondrial superoxide formation via a mechanism dependent on both inhibition of complex III and increased mitochondrial Ca2+ accumulation. Otherwise inactive concentrations of the oxidant produced the same maximal effects in the presence of either complex III inhibitors or agents mobilizing Ca2+ from the ryanodine receptor and enforcing its mitochondrial accumulation. l-Ascorbic acid (AA) produced similar enhancing effects in terms of superoxide formation, DNA strand scission and cytotoxicity. However, AA failed to enhance the intra-mitochondrial concentration of Ca2+ and the effects observed in cells supplemented with peroxinitrite, while insensitive to manipulations preventing the mobilization of Ca2+, or the mitochondrial accumulation of the cation, were also detected in human monocytes and macrophages, which do not express the ryanodine receptor. In all these cell types, mitochondrial permeability transition-dependent toxicity was detected in cells exposed to AA/peroxynitrite and, based on the above criteria, these responses also appeared Ca2+-independent. The enhancing effects of AA are therefore similar to those mediated by bona fide complex III inhibitors, although the vitamin failed to directly inhibit complex III, and in fact enhanced its sensitivity to the inhibitory effects of peroxynitrite.

The mitochondrial transporter of ascorbic acid functions with high affinity in the presence of low millimolar concentrations of sodium and in the absence of calcium and magnesium.

We recently reported that U937 cell mitochondria express a functional Na+-dependent ascorbic acid (AA) transporter recognised by anti-SVCT2 antibodies. The present study confirms and extends these observations by showing that this transporter is characterised by a Km and a pH-dependence comparable with that reported for the plasma membrane SVCT2. In isolated mitochondria, Na+ increased AA transport rate in a cooperative manner, revealed by a sigmoid curve and a Hill coefficient of 2, as also observed in intact Raw 264.7 cells (uniquely expressing SVCT2). There was however a striking difference on the Na+ concentrations necessary to reach saturation, i.e., 1 or 100 mM for the mitochondrial and plasma membrane transporters, respectively. Furthermore the mitochondrial, unlike the plasma membrane, transporter was fully active also in the absence of added Ca++ and/or Mg++. Taken together, the results presented in this study indicate that the U937 cell mitochondrial transporter of AA, because of its very low requirement for Na+ and independence for Ca++ and Mg++, displays kinetic characteristics surprisingly similar with those of the plasma membrane SVCT2.

The study of the mechanism of arsenite toxicity in respiration-deficient cells reveals that NADPH oxidase-derived superoxide promotes the same downstream events mediated by mitochondrial superoxide in respiration-proficient cells.

We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O2(.-)) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5µM, arsenite elicited selective formation of O2(.-) in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O2(.-) triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O2(.-) because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O2(.-) mediated by NADPH oxidase. Interestingly, extramitochondrial O2(.-) triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O2(.-) availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis.

Intracellular dehydroascorbic acid inhibits SVCT2-dependent transport of ascorbic acid in mitochondria.

Exposure of U937 cells to low concentrations of L-ascorbic acid (AA) is associated with a prompt cellular uptake and a further mitochondrial accumulation of the vitamin. Under the same conditions, dehydroascorbic acid (DHA) uptake was followed by rapid reduction and accumulation of identical intracellular levels of AA, however, in the absence of significant mitochondrial uptake. This event was instead observed after exposure to remarkably greater concentrations of DHA. Furthermore, experiments performed in isolated mitochondria revealed that DHA transport through hexose transporters and Na(+) -dependent transport of AA were very similar. These results suggest that the different subcellular compartmentalization of the vitamin is mediated by events promoting inhibition of mitochondrial AA transport, possibly triggered by low levels of DHA. We obtained results in line with this notion in intact cells, and more direct evidence in isolated mitochondria. This inhibitory effect was promptly reversible after DHA removal and comparable with that mediated by established inhibitors, as quercetin. The results presented collectively indicate that low intracellular concentrations of DHA, because of its rapid reduction back to AA, are a poor substrate for direct mitochondrial uptake. DHA concentrations, however, appear sufficiently high to mediate inhibition of mitochondrial transport of AA/DHA-derived AA.

A novel biological role of dehydroascorbic acid: Inhibition of Na(+)-dependent transport of ascorbic acid.

A U937 cell clone, in which low micromolar concentrations of ascorbic acid (AA) and dehydroascorbic acid (DHA) are taken up at identical rates, was used to investigate possible interactions between transport systems mediating cellular uptake of the two forms of the vitamin. Results obtained with different experimental approaches showed that DHA potently and reversibly inhibits AA uptake through Na(+)-AA cotransporters. Hence, a progressive increase in extracellular DHA concentrations in the presence of a fixed amount of AA caused an initial decrease in the net amount of vitamin C accumulated, and eventually, at higher levels, it caused an accumulation of the vitamin solely based on DHA uptake through hexose transporters. DHA-dependent inhibition of AA uptake was also detected in various other cell types. Taken together, our results provide evidence of a novel biological effect mediated by concentrations of DHA compatible with those produced at inflammatory sites.

Sodium-dependent transport of ascorbic acid in U937 cell mitochondria.

U937 cells exposed to physiological concentrations of ascorbic acid (AA) accumulate the reduced form of the vitamin in the cytosol and even further in their mitochondria. In both circumstances, uptake was dependent on Na(+) -AA-cotransport, with hardly any contribution of hexose transporters, which might be recruited to transport the oxidized form of the vitamin. There was an identical linear relationship between the mitochondrial accumulation of the vitamin and the extramitochondrial AA concentration, regardless of whether detected in experiments using intact cells or isolated mitochondria. Western blot experiments revealed expression of both SVCT1 and 2 in plasma membranes, whereas SVCT2 was the only form of the transporter expressed at appreciable amounts in mitochondria. These results therefore provide the novel demonstration of SVCT2-dependent mitochondrial transport of AA and hence challenge the present view that mitochondria only take up the oxidized form of the vitamin.

A moderate decline in U937 cell GSH levels triggers PI3 kinase/Akt-dependent Bad phosphorylation, thereby preventing an otherwise prompt apoptotic response.

We report that a moderate decline in GSH levels causes remarkable changes in Bad sub-cellular localization. An about 30% reduction of the GSH pool, regardless of whether mediated by diamide or DL-buthionine-[S,R]-sulfoximine, indeed promoted loss of the fraction of Bad normally associated with the mitochondria of untreated U937 cells via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. Interestingly, inhibition of this pathway was associated with an unexpected delayed lethal response, preceded by the translocation and enforced accumulation of Bad and Bax in the mitochondrial compartment, prevented by inhibitors of mitochondrial permeability transition and characterized by morphological and biochemical features of apoptosis. Collectively, the results herein presented demonstrate that mild redox imbalance associated with a slight reduction of the GSH pool commits U937 cells to apoptosis, however prevented by events leading to PI3K/Akt-dependent mitochondrial loss of Bad.

Differentiation-associated loss of ryanodine receptors: a strategy adopted by monocytes/macrophages to prevent the DNA single-strand breakage induced by peroxynitrite.

Monocytes/macrophages respond to peroxynitrite with the triggering of events leading to prevention of an otherwise prompt lethal response. This survival signaling regulated by molecules of the arachidonate cascade however presents a hypothetical critical limitation. In human promonocytic cell lines, peroxynitrite indeed promotes ryanodine receptor-derived Ca(2+)-dependent mitochondrial formation of H2O2, entirely responsible for the ensuing DNA strand scission. The occurrence of the same events in monocytes/macrophages at the inflammatory sites would therefore enhance the extent of DNA strand scission in viable cells, thereby increasing the rate of mutation and neoplastic transformation. The present study illustrates the details of a novel strategy based on a differentiation-associated loss of expression of ryanodine receptors. These cells simply do not accumulate mitochondrial Ca(2+) in response to peroxynitrite and therefore fail to generate superoxide/H2O2, thereby preserving the integrity of their DNA. We propose that an important component of the overall strategy adopted by monocytes/macrophages to survive to peroxynitrite, with no increased risk of neoplastic transformation, involves down-regulation of ryanodine receptor expression.

Arachidonic acid inhibits neuronal nitric oxide synthase elicited by proinflammatory stimuli and promotes astrocyte survival with both exogenous and endogenous peroxynitrite via different mechanisms.

Cytosolic phospholipase A(2)-inhibited astrocytes respond to the cocktail lipopolysaccharide/interferon-gamma with an immediate formation of peroxynitrite (ONOO(-)) and a delayed lethal response. Low concentrations of arachidonic acid (ARA; i.e., <0.1 microM) cause tyrosine kinase-dependent inhibition of neuronal nitric oxide synthase (nNOS) activity, thereby suppressing formation of ONOO(-) and the ensuing lethal response. ARA promoted its effects only when given to the cultures just prior to, or in parallel with, the proinflammatory mixture. High concentrations of ARA, i.e., >3 microM, promoted cytoprotection when applied to the cultures up to 50 min after the formation of endogenous ONOO(-) had been completed or up to 30 min after addition of exogenous ONOO(-). The mechanism(s) involved in these responses was, however, independent of tyrosine kinase activation and was in fact mediated by ARA metabolites of the lipoxygenase pathway. These results are consistent with a scenario in which astrocytes respond to low or high amounts of ARA with the triggering of different pathways involved in the inflammatory response. Early nNOS inhibition mediated by very low levels of ARA is indeed critical for nuclear factor-kappaB activation, which is otherwise effectively inhibited by constitutive nitric oxide, and for preventing early formation of ONOO(-). Greater ARA concentrations promote survival in astrocytes committed to death by ONOO(-), a species extensively released under inflammatory conditions, via a mechanism dependent on lipoxygenase metabolism and inhibition of downstream events leading to cell demise.

Prostaglandin E2 signals monocyte/macrophage survival to peroxynitrite via protein kinase A converging in bad phosphorylation with the protein kinase C alpha-dependent pathway driven by 5-hydroxyeicosatetraenoic acid.

Monocytes/macrophages committed to death by peroxynitrite nevertheless survive with a signaling response promoting Bad phosphorylation, as well as its cytosolic localization, via upstream activation of cytosolic phospholipase A(2), 5-lipoxygenase, and protein kinase C alpha. We now report evidence for an alternative mechanism converging in Bad phosphorylation when the expression/activity of the above enzymes are suppressed. Under these conditions, also associated with peroxynitrite-dependent severe inhibition of Akt, an additional Bad kinase, Bad dephosphorylation promoted its accumulation in the mitochondria and a prompt lethal response. PGE(2) prevented toxicity via EP(2) receptor-mediated protein kinase A-dependent Bad phosphorylation. This notion was established in U937 cells by the following criteria: 1) there was a strong correlation between survival and cAMP accumulation, both in the absence and presence of phosphodiesterase inhibitors; 2) direct activation of adenylyl cyclase afforded cytoprotection; and 3) PGE(2) promoted loss of mitochondrial Bad and cytoprotection, mimicked by EP(2) receptor agonists, and prevented by EP(2) receptor antagonists or protein kinase A inhibitors. Finally, selected experiments performed in human monocytes/macrophages and in rat peritoneal macrophages indicated that the above cytoprotective pathway is a general response of cells belonging to the monocyte/macrophage lineage to both exogenous and endogenous peroxynitrite. The notion that two different pathways mediated by downstream products of arachidonic acid metabolism converge in Bad phosphorylation emphasizes the relevance of this strategy for the regulation of macrophage survival to peroxynitrite at the inflammatory sites.

Mitochondrial H2O2 limits U937 cell survival to peroxynitrite by promoting ERK1/2 dephosphorylation.

Sequential activation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), critically regulated by extracellular signal-regulated kinase 1 and 2 (ERK1/2)-dependent phosphorylation, mediates U937 cell survival to peroxynitrite. In contrast, a limiting factor is represented by the parallel mitochondrial formation of H2O2 leading to suppression of the survival signaling. We now report that the inhibitory effects of H2O2 are at the level of ERK1/2 phosphorylation and involve activation of orthovanadate-sensitive phosphotyrosine protein phosphatase(s). Under these conditions, the otherwise stimulatory effects of peroxynitrite on ERK1/2 phosphorylation are concealed by phosphatase-dependent dephosphorylation and the activities of cPLA2 and 5-LO are significantly reduced or suppressed, respectively. The ensuing inhibition of downstream events preventing mitochondrial permeability transition rapidly leads these cells to death. Thus, endogenous H2O2 limits U937 cell survival to peroxynitrite via activation of phosphotyrosine protein phosphatase(s) promoting upstream inhibition of the survival signaling critically regulated by the extent of ERK1/2 phosphorylation.

Arachidonic acid: a key molecule for astrocyte survival to peroxynitrite.

Nontoxic concentrations of peroxynitrite (ONOO(-)) nevertheless commit rat astrocytes to mitochondrial permeability transition-dependent toxicity, however prevented by a signaling response driven by arachidonic acid (ARA). The lipid messenger was released upon ONOO(-)-dependent activation of cytosolic phospholipase A(2) and its pharmacological inhibition, or knock-down, was invariably associated with a prompt apoptotic response sensitive to exogenous ARA, but insensitive to other polyunsaturated fatty acids, as eicosapentaenoic or linoleic acid. Interestingly, while microglia also used ARA to cope with ONOO(-), cerebellar granule cells were killed by the same concentrations of ONOO(-) employed in astrocyte/microglia experiments via a mechanism sensitive to inhibition of ARA release. These results collectively support the notion that resistance of glial cells to ONOO(-), a species extensively produced under neuroinflammatory conditions, is largely based on a critical survival signaling triggered by the inflammatory product ARA. In remarkable contrast with these results, the lipid messenger appears to mediate toxicity in neuronal cells.

Prostaglandin E2 Signals Through E Prostanoid Receptor 2 to Inhibit Mitochondrial Superoxide Formation and the Ensuing Downstream Cytotoxic and Genotoxic Effects Induced by Arsenite.

We investigated the effects of prostaglandin E2 (PGE2), an important inflammatory lipid mediator, on the cytotoxicity-genotoxicity induced by arsenite. With the use of a toxicity paradigm in which the metalloid uniquely induces mitochondrial superoxide (mitoO2 -.) formation, PGE2 promoted conditions favoring the cytosolic accumulation of Bad and Bax and abolished mitochondrial permeability transition (MPT) and the ensuing lethal response through an E prostanoid receptor 2/adenylyl cyclase/protein kinase A (PKA) dependent signaling. It was, however, interesting to observe that, under the same conditions, PGE2 also abolished the DNA-damaging effects of arsenite and that this response was associated with an unexpected suppression of mitoO2 -. formation. We conclude that PGE2 promotes PKA-dependent inhibition of mitoO2 -. formation, thereby blunting the downstream responses mediated by these species, leading to DNA strand scission and MPT-dependent apoptosis. These findings are therefore consistent with the possibility that, in cells responding to arsenite with mitoO2 -. formation, PGE2 fails to enhance-but rather decreases-the risk of neoplastic transformation associated with genotoxic events.

Calcium signals between the ryanodine receptor- and mitochondria critically regulate the effects of arsenite on mitochondrial superoxide formation and on the ensuing survival vs apoptotic signaling.

A low concentration of arsenite (6 h), selectively stimulating the intraluminal crosstalk between the inositol-1, 4, 5-triphosphate receptor and the ryanodine receptor (RyR), increased the mitochondrial transport of RyR-derived Ca2+ through the mitochondrial Ca2+ uniporter. This event was characterized in intact and permeabilized cells, and was shown to be critical for mitochondrial superoxide (mitoO2.-) formation. Inhibition of mitochondrial Ca2+ accumulation therefore prevented the effects of arsenite, in both the mitochondrial (e.g., cardiolipin oxidation) and extramitochondrial (e.g., DNA single- strand breakage) compartments, and suppressed the Nrf2/GSH survival signaling. The effects of arsenite on Ca2+ homeostasis and mitoO2.- formation were reversible, as determined after an additional 10 h incubation in fresh culture medium and by measuring long-term viability. A 16 h continuous exposure to arsenite instead produced a sustained increase in the cytosolic and mitochondrial Ca2+ concentrations, a further increased mitoO2.- formation and mitochondrial permeability transition. These events, followed by delayed apoptosis (48 h), were sensitive to treatments/manipulations preventing mitochondrial Ca2+ accumulation. Interestingly, cells remained viable under conditions in which the deregulated Ca2+ homeostasis was not accompanied by mitoO2.-formation. In conclusion, we report that the fraction of Ca2+ taken up by the mitochondria in response to arsenite derives from the RyR. Mitochondrial Ca2+ appears critical for mitoO2.- formation and for the triggering of both the cytoprotective and apoptotic signaling. The effects of arsenite were reversible, whereas its prolonged exposure caused a sustained increase in mitochondrial Ca2+ and mitoO2.- formation, and the prevalence of the apoptotic vs survival signaling.

The arachidonate-dependent survival signaling preventing toxicity in monocytes/macrophages exposed to peroxynitrite.

Cells belonging to the monocyte/macrophage lineage are in general highly resistant to peroxynitrite. Resistance is not dependent on the scavenging of peroxynitrite itself, or of other secondary reactive species, but is rather associated with the prompt activation of a survival signaling leading to the prevention of toxicity in cells otherwise committed to mitochondrial permeability transition (MPT)-dependent necrosis. The signaling pathway is triggered by cytosolic phospholipase A2-released arachidonic acid, leading to the sequential activation of 5-lipoxygenase (5-LO) and protein kinase C alpha, an event associated with the cytosolic accumulation of Bad. Hence, inhibition of 5-LO (or that of any of the aforementioned enzymes involved in the signaling cascade) was associated with the mitochondrial accumulation of Bad and Bax and with a rapid MPT-dependent toxicity. These results contribute to the definition of the mechanism(s) whereby monocytes/macrophages survive to peroxynitrite in inflamed tissues and provide insights for the development of novel anti-inflammatory therapies based on the suppression of inflammatory cell survival.

Melatonin protects hippocampal HT22 cells from the effects of serum deprivation specifically targeting mitochondria.

Neurons contain a high number of mitochondria, these neuronal cells produce elevated levels of oxidative stress and live for a long time without proliferation; therefore, mitochondrial homeostasis is crucial to their health. Investigations have recently focused on mitochondrial dynamics revealing the ability of these organelles to change their distribution and morphology. It is known that mitochondrial fission is necessary for the transmission of mitochondria to daughter cells during mitosis and mitochondrial fragmentation has been used as an indicator of cell death and mitochondrial dysfunction. Oxidative stress is a trigger able to induce changes in the mitochondrial network. The aim of the present study was to determine the effects of melatonin on the mitochondrial network in HT22 serum-deprived cells. Our results showed that serum deprivation increased reactive oxygen species (ROS) content, promoted the activation of plasma membrane voltage-dependent anion channels (VDACs) and affected the expression of pDRP1 and DRP1 fission proteins. Moreover, parallel increases in apoptotic and autophagic features were found. Damaged and dysfunctional mitochondria are deleterious to the cell; hence, the degradation of such mitochondria through mitophagy is crucial to cell survival. Our results suggest that melatonin supplementation reduces cell death and restores mitochondrial function through the regulation of autophagy.

5-Hydroxyeicosatetraenoic acid is a key intermediate of the arachidonate-dependent protective signaling in monocytes/macrophages exposed to peroxynitrite.

Endogenous generation of arachidonic acid via selective activation of cytosolic phospholipase A(2) has been implicated in the mechanism of monocytes/macrophage survival in the presence of peroxynitrite. In particular, the lipid messenger was shown to prevent the otherwise rapid onset of a mitochondrial permeability-transition (MPT)-dependent necrosis by causing the mitochondrial translocation of protein kinase Calpha (PKCalpha) and the ensuing cytosolic accumulation of the Bcl-2-antagonist of cell death (Bad), an event promoting the anti-MPT function of Bcl-2 (or Bcl-X(L)). Here, we show that the effects on PKCalpha are not mediated directly by arachidonate but rather, by downstream products of the enzyme 5-lipoxygenase (5-LO). Peroxynitrite elicited the nuclear membrane translocation of 5-LO and enhanced its enzymatic activity via a mechanism sensitive to low concentrations of inhibitors of 5-LO or the 5-LO-activating protein, as well as to genetic depletion of the latter enzyme. Inhibition of 5-LO activity was invariably associated with the cytosolic localization of PKCalpha, the mitochondrial accumulation of Bad, and a rapid MPT-dependent necrosis. All these events were prevented by nanomolar concentrations of the 5-LO product 5-hydroxyeicosatetraenoic acid.