title: Serine hydrolase inhibitors block necrotic cell death by preventing calcium overload of the mitochondria and permeability transition pore formation.
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.
authors.affiliation: From the Department of Pediatrics, National Jewish Health, Denver, Colorado 80206.
authors.forename: Bogeon
authors.initials: B
authors.lastname: Yun
authors.forename: HeeJung
authors.initials: H
authors.lastname: Lee
authors.forename: Moumita
authors.initials: M
authors.lastname: Ghosh
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.forename: Ku-Lung
authors.initials: KL
authors.lastname: Hsu
authors.forename: Joseph V
authors.initials: JV
authors.lastname: Bonventre
authors.forename: Heather
authors.initials: H
authors.lastname: Ewing
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
authors.forename: Christina C
authors.initials: CC
authors.lastname: Leslie
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D002614:Chelating Agents / Q000494:pharmacology; D016572:Cyclosporine / Q000493:pharmacokinetics* / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D054513:Group IV Phospholipases A2 / Q000037:antagonists & inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D009336:Necrosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D064801:Phospholipase A2 Inhibitors / Q000494:pharmacology*; D011759:Pyrrolidines / Q000494:pharmacology*; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: C520323:1-oleoyl-2-acetoyl-sn-glycerol; D002614:Chelating Agents; D004075:Diglycerides; D007527:Isoenzymes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D064801:Phospholipase A2 Inhibitors; D011759:Pyrrolidines; C455981:pyrrophenone; D016718:Arachidonic Acid; D004533:Egtazic Acid; D019284:Thapsigargin; D016572:Cyclosporine; D007770:L-Lactate Dehydrogenase; C409676:lactate dehydrogenase 2; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054513:Group IV Phospholipases A2; D002118:Calcium
keywords: Arachidonic Acid; CaMKII; Calcium; Cyclosporin A; Fatty Acid; Mitochondria; Mitochondrial Permeability Transition; Phospholipase A; Pyrrophenone; Serine Hydrolase