Liana Cerioni

Inhibition of complex III promotes loss of Ca2+ dependence for mitochondrial superoxide formation and permeability transition evoked by peroxynitrite

In intact U937 cells, peroxynitrite promotes the mitochondrial formation of superoxide via a Ca2+-dependent mechanism involving inhibition of complex III. Superoxide then readily dismutates to H2O2 causing lesions on different biomolecules, including DNA. Here we show that formation of H2O2 and DNA damage are suppressed by inhibition of complex I (by rotenone) or ubisemiquinone formation (by myxothiazol), as well as by a variety of manipulations preventing either the mobilization of Ca2+ or its mitochondrial accumulation. In addition, complex III inhibitors promoted rotenone- or myxothiazol-sensitive formation of H2O2 and DNA strand scission in cells exposed to otherwise inactive concentrations of peroxynitrite. However, under these conditions, the intra-mitochondrial concentration of Ca2+ remained unchanged and the effects of peroxynitrite therefore take place via Ca2+-independent mechanisms. H2O2 formation was paralleled by, and causally linked to, the loss of mitochondrial membrane potential associated with the mitochondrial release of cytochrome c and AIF, and with the mitochondrial accumulation of Bax. These events, although Ca2+ independent, were rapidly followed by death mediated by mitochondrial permeability transition, generally considered a typical Ca2+-dependent event. Thus, enforced inhibition of complex III promotes the loss of Ca2+ dependence of those mitochondrial mechanisms regulating superoxide formation and mitochondrial permeability transition evoked by peroxynitrite.

The Raf/MEK inhibitor PD98059 enhances ERK1/2 phosphorylation mediated by peroxynitrite via enforced mitochondrial formation of reactive oxygen species

Exposure of PC12 cells to 100 μM peroxynitrite promotes phosphorylation of extracellular signal‐regulated kinases 1 and 2 (ERK1/2) sensitive to PD98059 or U0126. At higher concentrations, however, ERK1/2 phosphorylation was prevented by U0126 and increased by PD98059 via a U0126‐sensitive mechanism. PD98059, unlike U0126, enhanced the peroxynitrite‐dependent formation of reactive oxygen species (ROS). These results, along with others obtained using respiratory chain inhibitors and respiration‐deficient cells, lead to the conclusion that PD98059, while effectively inhibiting the peroxynitrite‐induced Raf/MEK signaling leading to ERK1/2 phosphorylation, promotes an enforced mitochondrial formation of ROS inducing ERK1/2 phosphorylation via a Raf‐1‐independent/MEK‐dependent mechanism.

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.

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 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.

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 A2 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 Cα (PKCα) 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‐XL). Here, we show that the effects on PKCα 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 PKCα, 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.

Superoxide dictates the mode of U937 cell ascorbic acid uptake and prevents the enhancing effects of the vitamin to otherwise nontoxic levels of reactive oxygen/nitrogen species

Exposure of U937 cells to low micromolar levels of ascorbic acid or dehydroascorbic acid, while resulting in identical ascorbic acid accumulation, is unexpectedly associated with remarkably different responses to exogenous oxidants. We observed that otherwise nontoxic levels of hydrogen peroxide, tert-butylhydroperoxide or peroxynitrite promote toxicity in cells preloaded with ascorbic acid, whereas hardly any effect was detected in cells pretreated with dehydroascorbic acid. Further experiments performed with peroxynitrite in cells preloaded with ascorbic acid provided evidence for a very rapid nonapoptotic death, preceded by early Bax mitochondrial translocation and by mitochondrial permeability transition. The notion that conversion of extracellular ascorbic acid to dehydroascorbic acid prevents the enhancing effects on oxidant toxicity and nevertheless preserves the net amount of vitamin C accumulated was also established using ascorbate oxidase as well as various sources of superoxide, namely, xanthine/xanthine oxidase or ATP-driven NADPH oxidase activation. These findings suggest that superoxide-dependent conversion of extracellular ascorbic acid to dehydroascorbic acid represents an important component of the overall survival strategy of some cell types to reactive oxygen/nitrogen species.

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.

Mitochondrial ascorbic acid is responsible for enhanced susceptibility of U937 cells to the toxic effects of peroxynitrite.

Otherwise nontoxic levels of peroxynitrite promote toxicity in U937 cells pre-exposed to low micromolar concentrations of l-ascorbic acid (AA). This event was associated with the mitochondrial accumulation of the vitamin and with the early formation of secondary reactive oxygen species and DNA single-strand breaks. The same concentrations of peroxynitrite, however, failed to elicit detectable effects in cells pre-exposed to dehydroascorbic acid (DHA), in which mitochondrial accumulation of vitamin C did not occur despite the identical cytosolic levels. Coherently, oxidation of extracellular AA failed to affect the intracellular concentration of the vitamin, but nevertheless prevented its mitochondrial localization as well as the enhanced response to peroxynitrite. Furthermore, in cells postincubated in vitamin C-free medium, time-dependent loss of mitochondrial AA was paralleled by a progressive decline of susceptibility to peroxynitrite, under the same conditions in which cells retained about half of the initial AA. Using different experimental approaches, we finally showed that the enhancing effects of AA are mediated by events associated with peroxynitrite-dependent superoxide/H2 O2 formation in the mitochondrial respiratory chain. Collectively, these results indicate that mitochondria actively take up vitamin C as AA and respond to otherwise inactive concentrations of peroxynitrite with the mitochondrial formation of secondary species responsible for DNA damage and toxicity. DHA preloading, while leading to the accumulation of identical levels of vitamin C, fails to produce these effects because of the poor mitochondrial accumulation of the vitamin.

Prostaglandin E2 Signals Monocyte/Macrophage Survival to Peroxynitrite via Protein Kinase A Converging in Bad Phosphorylation with the Protein Kinase Cα-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 A2, 5-lipoxygenase, and protein kinase Cα. 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. PGE2 prevented toxicity via EP2 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) PGE2 promoted loss of mitochondrial Bad and cytoprotection, mimicked by EP2 receptor agonists, and prevented by EP2 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.