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.