ACCELERATED COMMUNICATION U937 Cell Necrosis Mediated by Peroxynitrite Is Not Caused by Depletion of ATP and Is Prevented by Arachidonate via an ATP-Dependent Mechanism

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 12fold higher concentration of the oxidant, in the absence of additional treatments, caused remarkably greater DNA singlestrand 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. Cell death, a process of paramount importance in a variety of physiological and pathological processes, is mediated by different mechanisms in which apoptosis and necrosis represent the two extremes on a continuum (Leist and Nicotera, 1997). Because apoptosis requires energy-dependent events, the choice among these different modes of cell death may be determined on the basis of the ATP availability (Nicotera and Melino, 2004). In the presence of ATP, a toxic treatment leads to apoptosis, whereas in its absence, cell death would take place via a passive mechanism (i.e., necrosis). In support of this notion are the following observations: 1) toxicity paradigms that cause necrosis are associated with depletion of the ATP pool (Lelli et al., 1998), which is, however, wellpreserved using lower concentrations of the toxic agent under conditions in which apoptosis, but not necrosis, is concomitantly induced; 2) depletion of ATP, early in the apoptotic process, switches the predominant form of cell death from apoptosis to necrosis (Leist et al., 1997); and 3) activation of poly(ADP-ribose)polymerase leads to ATP depletion and necrosis, whereas inhibition of its activity prevents both ATP depletion and necrosis and eventually switches cells into apoptosis (Ha and Snyder, 1999). Thus, the above results define an important role for ATP in the regulation of cell death and imply that necrosis takes place in damaged cells unable to perform the highly energy-demanding processes involved in apoptosis. However, necrosis may also represent the primary mode of cell death induced by an otherwise apoptotic treatment when This work was supported by a grant from Ministero dell’Università e della Ricerca Scientifica e Tecnologica, Progetti di Ricerca di Interesse Nazionale (to O.C.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.009498. ABBREVIATIONS: MPT, mitochondrial permeability transition; AACOCF3, arachidonyl trifluoromethyl ketone; cPLA2, cytosolic phospholipase A2; DHR, dihydrorhodamine-123, ETYA, 5,8,11,14-eicosatetraynoic acid. 0026-895X/05/6705-1399–1405$20.00 MOLECULAR PHARMACOLOGY Vol. 67, No. 5 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 9498/1198149 Mol Pharmacol 67:1399–1405, 2005 Printed in U.S.A. 1399 at A PE T Jornals on A ril 8, 2017 m oharm .aspeurnals.org D ow nladed from parameters other than ATP are affected (e.g., when caspases are inhibited) (Hirsch et al., 1997; Nicotera and Melino, 2004). Furthermore, it is becoming increasingly clear that necrosis may also represent a primary mode of cell death in a variety of physiopathological conditions (Proskuyakov et al., 2003), and several early events described in the apoptotic process are also critical in the regulation of necrosis (Leist and Nicotera, 1997; Proskuyakov et al., 2003). A good example is given by mitochondrial permeability transition (MPT), which may trigger both apoptosis and necrosis (Kroemer et al., 1998) via mechanisms that are similarly regulated by proteins of the Bcl-2 family (Single et al., 2001; Proskuyakov et al., 2003). Although severe depletion of ATP was described in toxicity paradigms resulting in primary necrosis (Barros et al., 2001; Proskuyakov et al., 2003), whether ATP depletion does always occur and is in fact the most prominent cause of this lethal response remains to be established. This is an important question, because a general requirement for ATP depletion would demonstrate that necrosis is always a passive response, regardless of whether it is primarily induced or it takes place as a consequence of the inability of the cells to perform the apoptotic process. The present study was designed to investigate whether depletion of ATP is needed in primary necrosis resulting from a toxicity paradigm that has been extensively characterized in our laboratory. We reported that exposure of U937 cells to peroxynitrite promotes an MPT-dependent necrosis within minutes, followed by immediate cell lysis (Sestili et al., 2001). An initial event triggered by peroxynitrite (i.e., inhibition of complex III of the mitochondrial respiratory chain), was responsible for the time-dependent formation of H2O2 that was essential for the occurrence of cell death (Tommasini et al., 2002a). We also showed that otherwise nontoxic concentrations of peroxynitrite nevertheless commit cells to MPT-dependent necrosis, which is, however, prevented by a cytoprotective signaling driven by arachidonic acid released by the cytosolic phospholipase A2 isoform (cPLA2) (Tommasini et al., 2002b, 2004a). We do not know whether arachidonate itself or some downstream product of the cyclooxygenase or lipoxygenase pathways is responsible for the survival signaling. Thus, although this toxicity paradigm represents a model of “severe primary necrosis” in which extensive ATP depletion may occur, because both peroxynitrite and H2O2 potently inhibit the respiratory chain and glycolysis (Hyslop et al., 1988; Souza and Radi, 1998), it nevertheless presents some features of a highly regulated event. The present study demonstrates a requirement for ATP in the above cytoprotective signaling and indicates that the ATP pool is well-preserved when the necrotic response, which takes place via a highly regulated mechanism independent of the damage accumulated, is elicited by suppression of the protective signaling. Materials and Methods Chemicals. 5,8,11,14-Eicosatetraynoic acid (ETYA), arachidonic acid, 2-deoxy-D-glucose, catalase, N-acetyl-L-cysteine, and most of the reagent grade chemicals were obtained from Sigma-Aldrich (Milan, Italy). Arachidonyl trifluoromethyl ketone (AACOCF3), cyclosporin A, and dihydrorhodamine-123 (DHR) were purchased from Calbiochem (San Diego, CA), Novartis (Bern, Switzerland), and Molecular Probes Europe (Leiden, The Netherlands), respectively. Cell Culture and Treatment Conditions. U937 or THP-1 cells were cultured in suspension in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), penicillin (50 U/ml), and streptomycin (50 g/ml; SeraLab Ltd., Crawley Down, UK), at 37°C in T-75 tissue culture flasks (Corning Glassworks, Corning, NY) gassed with an atmosphere of 95% air/5% CO2. Peroxynitrite was synthesized by the reaction of nitrite with acidified H2O2 as described by Radi et al. (1991), with minor modifications (Tommasini et al., 2002a). Treatments were performed in 2 ml of prewarmed saline A (8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/l NaHCO3, and 0.9 g/l glucose) containing 5 10 5 cells. Viability Assay. Cytotoxicity was determined with the trypan blue exclusion assay. In brief, an aliquot of the cell suspension was diluted 1:1 (v/v) with 0.4% trypan blue, and the viable cells were counted with a hemocytometer. Alkaline Halo Assay. DNA single-strand breakage was determined using the alkaline halo assay as described previously (Sestili and Cantoni, 1999). After treatments, the cells were resuspended at 2.0 10 cells/100 l in 1.5% low-melting agarose in phosphatebuffered saline (8 g/l NaCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4, and 0.2 g/l KCl), containing 5 mM EDTA and immediately sandwiched between an agarose-coated slide and a coverslip. After complete gelling, the coverslips were removed, and the slides were immersed in an alkaline buffer (0.1 M NaOH/1 mM EDTA, pH 12.5), washed, and stained for 5 min with 10 g/ml ethidium bromide. The ethidium bromide-labeled DNA was visualized using a confocal laser microscope (DVC 250; Bio-Rad, Hercules, CA), and the resulting images were taken and processed with a chilled chargecoupled device camera (5985; Hamamatsu Italy S.P.A., Milan, Italy) coupled with a Macintosh computer (Apple Computer, Cupertino, CA) using the NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nih-