Pivotal role of superoxides generated in the mitochondrial respiratory chain in peroxynitrite-dependent activation of phospholipase A2.
Exposure of PC12 cells to reagent peroxynitrite promotes the release of arachidonic acid (AA) mediated by activation of phospholipase A(2) [Guidarelli, Palomba and Cantoni (2000) Br. J. Pharmacol. 129, 1539-1542]. We now present experimental evidence consistent with the notion that this response is not directly triggered by peroxynitrite but, rather, by reactive oxygen species generated at the level of complex III of the mitochondrial respiratory chain. In particular, superoxide (and not hydrogen peroxide) has a pivotal role in peroxynitrite-dependent activation of phospholipase A(2). This observation was confirmed by results showing that superoxide, or peroxynitrite, promotes release of AA in isolated mitochondria. Consistently, the release of AA elicited by either peroxynitrite or A23187 in intact cells was shown to be calcium-dependent and differentially affected by phospholipase A(2) inhibitors with different levels of specificity. In particular, the effects of peroxynitrite, unlike those of A23187, were both sensitive to low concentrations of two general phospholipase A(2) inhibitors and insensitive to arachidonyltrifluoromethyl ketone, which shows some selectivity towards cytosolic phospholipase A(2). In addition, peroxynitrite and A23187 synergistically enhanced the release of AA. Collectively, the above results demonstrate that peroxynitrite causes inhibition of complex III, followed by enforced formation of superoxides that stimulate the activity of a calcium-dependent PLA(2) isoform, probably localized in the mitochondria.
Guidarelli
Andrea
A
Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino, Via S. Chiara 27, 61029 Urbino (PU), Italy.
Cantoni
Orazio
O
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D016716:PC12 Cells; D030421:Peroxynitrous Acid / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D020033:Protein Isoforms; D011796:Quinacrine / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species
D004791:Enzyme Inhibitors; D007476:Ionophores; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D015117:5,8,11,14-Eicosatetraynoic Acid; D030421:Peroxynitrous Acid; D016718:Arachidonic Acid; D000001:Calcimycin; D010741:Phospholipases A; D054467:Phospholipases A2; D011796:Quinacrine; D010100:Oxygen