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.

We have studied the relationships existing between delayed formation of H2O2 and activation of cytosolic phospholipase A2 (cPLA2), events respectively promoting toxicity or survival in U937 cells exposed to peroxynitrite. The outcome of an array of different approaches using phospholipase A2 inhibitors, or cPLA2 antisense oligonucleotides, as well as specific respiratory chain inhibitors and respiration-deficient cells led to the demonstration that H2O2 does not mediate toxicity by producing direct molecular damage. Rather, the effects of H2O2 were found to be upstream to the arachidonic acid (AA)-mediated cytoprotective signalling and in fact causally linked to inhibition of cPLA2. 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 cPLA2-dependent release of AA. A rapid necrotic response, however, takes place when high concentrations of peroxynitrite promote formation of H2O2 at levels impairing the cPLA2 cytoprotective signalling.

Peroxynitrite stimulates the activity of cytosolic phospholipase A2 in U937 cells: the extent of arachidonic acid formation regulates the balance between cell survival or death.

Peroxynitrite stimulates in U937 cells release of arachidonic acid (AA) sensitive to various phospholipase A(2) (PLA(2)) inhibitors, including arachidonyl trifluoromethyl ketone (AACOCF(3)), which specifically inhibits cytosolic PLA(2) (cPLA(2)). This response linearly increases using non toxic concentrations of the oxidant, and reaches a plateau at levels at which toxicity becomes apparent. Three separate lines of evidence are consistent with the notion that AA generated by cPLA(2) promotes survival in cells exposed to peroxynitrite. Firstly, toxicity was suppressed by nanomolar levels of exogenous AA, or by AA generated by the direct PLA(2) activator melittin. Secondly AACOCF(3), or other PLA(2) inhibitors, promoted cell death after exposure to otherwise non toxic concentrations of peroxynitrite; exogenous AA abolished the enhancing effects mediated by the PLA(2) inhibitors. Finally, U937 cells transfected with cPLA(2) antisense oligonucleotides were killed by concentrations of peroxynitrite that were non-toxic for cells transfected with nonsense oligonucleotides. This lethal response was insensitive to AACOCF(3) and prevented by exogenous AA.

Peroxynitrite promotes mitochondrial permeability transition-dependent rapid U937 cell necrosis: survivors proliferate with kinetics superimposable on those of untreated cells.

A short term exposure to peroxynitrite promotes a time- and concentration-dependent lethal response in U937 cells. The mode of cell death was necrosis and rapid (within minutes) cell lysis was found to occur via a mechanism involving mitochondrial permeability transition. Apoptosis was not detected in cells exposed to low levels of peroxynitrite, or in cells which survived a treatment with toxic amounts of peroxynitrite, neither after the 60 min exposure nor following increasing time intervals of growth in fresh culture medium. Rather, cells treated with peroxynitrite concentrations which were not immediately lethal, as well as the survivors of treatments with toxic levels of peroxynitrite, proliferated with kinetics superimposable on those observed in untreated cells.

Essential role of the mitochondrial respiratory chain in peroxynitrite-induced strand scission of genomic DNA.

A large body of experimental evidence suggests that DNA damage and cytotoxicity mediated by peroxynitrite are linked by a causal relationship and important events in various pathological conditions. In the present study, we investigated the mechanism whereby peroxynitrite causes DNA single strand breakage in intact cells and found that the respiratory chain plays a pivotal role in this response. In particular, peroxynitrite mediates inhibition of complex III and, under these conditions, electrons are directly transferred from ubisemiquinone to molecular oxygen. Hydrogen peroxide produced by the dismutation of superoxides is the species mediating the peroxynitrite-dependent DNA cleavage.

8-aminoquinolines as anticoccidials - Part III.

Analogues of the antimalarial pentaquine, 1, in which the nature of the side-chain on the 8-amino position was varied, were prepared and evaluated for anticoccidial activity both in vitro and in vivo. Specifically, both the inter-nitrogen distance and the nature of the terminal amino group were investigated. Novel analogues of equal or improved efficacy in vitro and in vivo to pentaquine were discovered.

8-Aminoquinolines as anticoccidials--II.

During a chemistry program aimed at finding a novel analogue of pentaquine with improved in vivo activity, a number of hypotheses concerning the way this drug acts in the chicken were investigated. Consideration of the products of monoamine oxidase metabolism of pentaquine suggested that pentaquine aldehyde is the likely active metabolite. Although isolation of this unstable compound was not possible, oxime and cyclic acetal and ketal derivatives were obtained and shown to possess in vitro anticoccidial activity.