ATP-induced apoptosis involves a Ca2+-independent phospholipase A2 and 5-lipoxygenase in macrophages

Abstract Macrophages express P2X 7 and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP e )-induced P2X 7 -dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X 7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1β secretion. We investigated signaling pathways involved in the induction of cell death by ATP e in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP. Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect on apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca 2+ were chelated during the induction period. Mepacrine, a generic PLA 2 inhibitor and BEL, an inhibitor of Ca 2+ -independent PLA 2 (iPLA 2 ) blocked apoptosis, while pBPB and AACOOPF 3 , inhibitors of secretory and Ca 2+ -dependent PLA 2 respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX −/− macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA 2 and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca 2+ -independent step involving an iPLA 2 and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X 7 in macrophages.