Arachidonic Acid Released by Phospholipase A2 Activation Triggers Ca2+-dependent Apoptosis through the Mitochondrial Pathway

Abstract

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 Ca(2+) ([Ca(2+)](c)), which returned close to the resting level within about 40 s. The [Ca(2+)](c) rise was immediately followed by phospholipid hydrolysis, which could be inhibited by aristolochic acid or by pretreatment with thapsigargin in Ca(2+)-free medium, indicating that the Ca(2+)-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 cPLA(2), PTP opening, and cell death, all events but the [Ca(2+)](c) rise were prevented by aristolochic acid. The number of cells killed by A23187 was doubled by treatment with 0.5 microm MK886 and 5 microm 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 cPLA(2) 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 Ca(2+) homeostasis and the effector mechanisms of cell death.

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis

“…4 Here, we have investigated the mechanisms whereby p66Shc primes T cells to death by the Ca 2 þ ionophore A23187, a potent inducer of apoptosis that causes PTP opening in other systems. 17 We show that p66Shc promotes T cell apoptosis both by triggering cytochrome c release and mitochondrial membrane depolarization, and by impairing Ca 2 þ homeostasis due to downregulation of plasma membrane ATPases and defective Ca 2 þ extrusion. These two independent activities, which both require Ca 2 þ -dependent p66Shc phosphorylation at S36, are likely to synergize in mediating p66Shc-dependent apoptosis.…”

“…19 The pharmacological evidence presented suggests that PTP opening does not play a major role in p66Shc-dependent mitochondrial membrane depolarization and triggering of the intrinsic apoptotic pathway in A23187-treated T cells. This was somewhat surprising, given that A23187 activates cPLA 2 resulting in arachidonic acid production, 17 and that p66Shc increases ROS production, 9,10 events which both contribute to increase the probability of PTP opening. We cannot, however, fully exclude PTP involvement in mitochondrial depolarization, particularly in the light of the ultrastructural changes detectable after treatment with A23187.…”

“…We have previously shown that in hepatoma cells A23187 triggers Ca 2 þ -dependent PTP opening through activation of cytosolic phospholipase A2 (cPLA 2 ) and production of arachidonic acid. 17 We therefore tested whether A23187-induced mitochondrial depolarization in p66Shc-expressing cells could be inhibited by the cPLA 2 inhibitor aristolochic acid or potentiated by inhibition of 5-lipoxygenase by MK886 and cyclooxygenases by indomethacin, a treatment that results in arachidonic acid accumulation. 17 These treatments did not affect the mitochondrial membrane potential response to A23187 (Figure 2e and data not shown).…”

Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549

“…Recently, some studies have reported that CrTX has anti-tumor effects. However, the potential mechanism is unclear [2][3][4][5] .…”

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

“…There is evidence that calciumindependent PLA 2 (iPLA 2 ), which localizes to mitochondria, regulates MPTP formation based on the use of the iPLA 2 inhibitor bromoenolactone and using mitochondria from iPLA 2 ␥ knock-out mice (28 -31). The Group IVA cytosolic PLA 2 (cPLA 2 ␣) is also suggested to regulate MPTP formation and cell death by releasing arachidonic acid (32,33). Calcium regulates cPLA 2 ␣ by promoting its translocation from the cytosol to intracellular membranes although there is no evidence that it localizes to mitochondria (34,35).…”