Abstract

Exposure of U937 cells to an otherwise nontoxic concentration of peroxynitrite promotes a rapid necrotic response in the presence of pharmacological inhibitors of phospholipase A₂. A 12-fold higher concentration of the oxidant, in the absence of additional treatments, caused remarkably greater DNA single-strand breakage, delayed formation of H₂O₂, and depletion of reduced glutathione but an identical level of toxicity. Cell death was prevented in both circumstances by nanomolar levels of arachidonic acid or by cyclosporin A via mechanisms unrelated to elimination of the above effects and was causally linked to prevention of mitochondrial permeability transition. Treatment with a high dose of peroxynitrite for 30 min caused an approximately 40% decline in ATP, both in the absence and presence of arachidonate, whereas only a small, arachidonic acid-sensitive reduction of the ATP pool was detected in cells treated with the low dose of peroxynitrite and the phospholipase A₂ inhibitor. ATP-predepleted cells, however, were hypersensitive to peroxynitrite, and under these conditions, toxicity was not prevented by arachidonate. The above findings were reproduced in another promonocytic cell line, THP-1 cells. We concluded that the rapid necrotic response triggered by peroxynitrite in monocytes is mediated by a regulated process, not by ATP depletion, associated with reduced arachidonate availability. Supplementation of exogenous arachidonic acid always rescued cells via an ATP-dependent survival pathway.