Hydrogen peroxide generated at the level of mitochondria in response to peroxynitrite promotes U937 cell death via inhibition of the cytoprotective signalling mediated by cytosolic phospholipase A2

Abstract

We have studied the relationships existing between delayed formation of H 2 O 2 and activation of cytosolic phospholipase A 2 (cPLA 2 ), events respectively promoting toxicity or survival in U937 cells exposed to peroxynitrite. The outcome of an array of different approaches using phospholipase A 2 inhibitors, or cPLA 2 antisense oligonucleotides, as well as specific respiratory chain inhibitors and respiration-deficient cells led to the demonstration that H 2 O 2 does not mediate toxicity by producing direct molecular damage. Rather, the effects of H 2 O 2 were found to be upstream to the arachidonic acid (AA)-mediated cytoprotective signalling and in fact causally linked to inhibition of cPLA 2 . Thus, it appears that U937 cells exposed to nontoxic concentrations of peroxynitrite are nevertheless committed to death, which however is normally prevented by the activation of parallel pathways resulting in cPLA 2 -dependent release of AA. A rapid necrotic response, however, takes place when high concentrations of peroxynitrite promote formation of H 2 O 2 at levels impairing the cPLA 2 cytoprotective signalling.

Differentiation-Associated Loss of Ryanodine Receptors: A Strategy Adopted by Monocytes/Macrophages to Prevent the DNA Single-Strand Breakage Induced by Peroxynitrite

“…Products of either 5-lipoxygenase (5-7) or cyclooxygenase (9), extensively released at the inflammatory sites, initiate a cascade of events culminating in Bad phosphorylation and thus in its cytosolic accumulation. In addition, under these conditions, Bax is also retained in the cytosol and the cells preserve their ATP pool (27), thereby surviving regardless of the damage accumulated (3)(4)(5)(6)(7)9). In particular, our work performed in U937 cells provided evidence of survival of cells with extensive DNA strand scission (27,28).…”

“…We showed that the resistance phenotype is dependent on the ability of these cells to respond to peroxynitrite with the triggering of a survival signaling in which cytosolic phospholipase A 2 , 5-lipoxygenase, and protein kinase C␣ are sequentially involved (3)(4)(5)(6), leading to Bad phosphorylation and to its cytosolic accumulation (5)(6)(7). Loss of mitochondrial Bad creates optimal conditions for the antimitochondrial permeability transition function of Bcl-2/Bcl-x L (8).…”

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

“…The question we ask has an additional important implication because complex III inhibitors promote rapid necrotic U937 cell death after exposure to otherwise non-toxic concentrations of peroxynitrite (Tommasini et al, 2002a;Tommasini et al, 2004a), thus suggesting that cell demise might also take place via a Ca 2+ -independent mechanism. This would be an interesting observation especially if necrosis takes place via a mitochondrial permeability transition (MPT)-dependent mechanism, as previously noticed using intrinsically toxic levels of peroxynitrite (Sestili et al, 2001).…”

Peroxynitrite damages U937 cell DNA via the intermediate formation of mitochondrial oxidants

“…macrophages), surviving to peroxynitrite regardless from the damage accumulated (20,26). DNA damage mediated by peroxynitrite in these cells under inflammatory conditions is indeed of importance in carcinogenesis (27).…”

“…In this direction, it is important to point out that these cells are resistant to peroxynitrite as they are able to respond with a signaling pathway, sequentially involving cytosolic phospholipase A 2 , 5-lipoxygenase and PKCa, promoting Bad phosphorylation (11,(20)(21)(22)(23). In other words, these cells, as well as cells belonging to the monocyte/macrophage lineage, including human monocytes and macrophages (23)(24)(25)(26), while committed to mitochondrial permeability transition by peroxynitrite, nevertheless survive by promoting the cytosolic localization of Bad, an event creating optimal conditions for the cytoprotective effects of Bcl-2/BclX L .…”