Abstract
Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2α (cPLA2α) or calcium-independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca(2+) uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.
Highlights
Mitochondrial calcium overload triggers permeability transition pore formation, fatty acid release, and necrotic cell death
IMLF␣ϩ/ϩ and IMLF␣Ϫ/Ϫ were treated with the calcium ionophore A23187, which is a well described inducer of necrotic cell death due to cellular calcium overload and mitochondrial permeability transition pore (MPTP) formation resulting in plasma membrane rupture and lactate dehydrogenase (LDH) release [10, 46]
This study describes a cell death pathway induced in mouse lung fibroblasts by A23187 with characteristics of primary necrosis involving rapid formation of MPTP due to calcium overload and plasma membrane rupture resulting in LDH release
Summary
Mitochondrial calcium overload triggers permeability transition pore formation, fatty acid release, and necrotic cell death. Conclusion: Serine hydrolase inhibitors block mitochondrial calcium uptake but do not directly inhibit the enzyme releasing fatty acids during pore formation. Serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. Serine Hydrolase Inhibitors Block Necrotic Cell Death ical function in regulating mitochondrial Ca2ϩ homeostasis, its irreversible opening occurs in response to extreme environmental insults or during disease resulting in mitochondria swelling, depolarization, ATP depletion, and plasma membrane permeabilization [10, 14]. We found that A23187 stimulates a similar cell death pathway in cPLA2␣ wild type and knock-out lung fibroblasts with characteristics of primary necrosis including mitochondrial calcium overload, cyclophilin D-dependent MPTP formation, and regulation by CaMKII. The inhibitors act by blocking calcium loading of the mitochondria but do not directly inhibit fatty acid release that occurs by a novel pathway during MPTP formation (Fig. 12)
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