Arachidonic acid released by phospholipase A2 activation triggers Ca2+-dependent apoptosis through the mitochondrial pathway

We studied the effects of the divalent cation ionophore A23187 on apoptotic signaling in MH1C1 cells. Addition of A23187 caused a fast rise of cytosolic Ca2+ ([Ca2+](c)), which returned close to the resting level within about 40 s. The [Ca2+](c) rise was immediately followed by phospholipid hydrolysis, which could be inhibited by aristolochic acid or by pretreatment with thapsigargin in Ca2+-free medium, indicating that the Ca2+-dependent cytosolic phospholipase A(2) (cPLA(2)) was involved. These early events were followed by opening of the mitochondrial permeability transition pore (PTP) and by apoptosis in about 30% of the cell population. In keeping with a cause-effect relationship between addition of A23187, activation of cPLA2, PTP opening, and cell death, all events but the [Ca2+](c) rise were prevented by aristolochic acid. The number of cells killed by A23187 was doubled by treatment with 0.5 muM MK886 and 5 muM indomethacin, which inhibit arachidonic acid metabolism through the 5-lipoxygenase and cyclooxygenase pathway, respectively. Consistent with the key role of free arachidonic acid, its levels increased within minutes of treatment with A23187; the increase being more pronounced in the presence of MK886 plus indomethacin. Cell death was preceded by cytochrome c release and cleavage of caspase 9 and 3, but not of caspase 8. All these events were prevented by aristolochic acid and by the PTP inhibitor cyclosporin A. Thus, A23187 triggers the apoptotic cascade through the release of arachidonic acid by cPLA2 in a process that is amplified when transformation of arachidonic acid into prostaglandins and leukotrienes is inhibited. These findings identify arachidonic acid as the causal link between A23187-dependent perturbation of Ca2+ homeostasis and the effector mechanisms of cell death.