Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

“…To test this, we used chemical inhibitors that have been shown previously to have some (although not total) specificity for the different PLA 2 s; concentrations of inhibitors were chosen based on previous reports (18,23). MJ33 inhibits Prdx6 PLA 2 , pBPB is an inhibitor of secreted PLA 2 , BEL inhibits intracellular calciumindependent PLA 2 , and AACOCF 3 inhibits cytosolic PLA 2 .…”

“…Unilamellar liposomes (ϳ100 m) consisting of 3 H-DPPC, egg phosphatidylcholine, phosphatidylglycerol, and cholesterol (0.5, 0.25, 0.1, and 0.15 mol fraction) were prepared by extrusion through a membrane under pressure. Lysates were incubated in Ca 2ϩ -free buffer (50 mM Tris-HCl, 1 mM EGTA) at pH 7.0 for 1 h. The radiolabeled free fatty acid product was extracted, resolved by thin layer chromatography using hexane/ether/acetic acid, and analyzed by scintillation counting (18,19). To evaluate inhibitors, MJ33 was added to the liposomes at 1 mol %; other inhibitors were added to the incubation medium in aqueous solution.…”

“…Cells were then washed and incubated with or without an inhibitor (MJ33 or U01026, both at 10 M) for 30 min, followed by the addition or not of Ang II (10 M) for 1 h. Cells before and after the Ang II incubation were lysed by sonication, extracted with CH 3 /MeOH, and analyzed by thin layer chromatography using CHCl 3 /MeOH/NH 4 OH as the solvent system, and dpm in the [lyso-3 H]PC band was measured by scintillation counting as described previously (18,19). Results are the dpm recovered after the 1-h incubation period minus the "zero time" control and are expressed as dpm/mg of protein in 1 h. Because positional specificity of the lyso-PC product was not determined, this assay reflects both PLA 1 and PLA 2 activities and is called PLA activity.…”

“…PLA 2 Activity-PLA 2 activity was measured in PMVEC that were disrupted by sonication and incubated with radiolabeled liposomes as described previously (18,19). Unilamellar liposomes (ϳ100 m) consisting of 3 H-DPPC, egg phosphatidylcholine, phosphatidylglycerol, and cholesterol (0.5, 0.25, 0.1, and 0.15 mol fraction) were prepared by extrusion through a membrane under pressure.…”

The serpentine path to a novel mechanism-based inhibitor of acute inflammatory lung injury

“…Protein expressed with this clone had activity characteristic of aiPLA 2 (52). After generating antibodies, we found especially high expression in lung epithelial cells and lamellar bodies (1, 51), while "knock out" of the protein in mice resulted in a marked decrease in the ability of their lungs to degrade internalized DPPC (34).…”