Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils. Evidence for different arachidonate pools.
The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0-10 micro m did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 approximately 2.5 micro m). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell-free assay systems, 10 micro m AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities. [3H]AA labeling studies indicated that the specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism.
Fonteh
Alfred N
AN
Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA. afonteh@hmri.org
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism*; D001095:Arachidonic Acids / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D007476:Ionophores / Q000494:pharmacology; D007975:Leukotriene B4 / Q000096:biosynthesis; D015289:Leukotrienes / Q000378:metabolism; D050356:Lipid Metabolism; D016859:Lipoxygenase Inhibitors; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D010972:Platelet Activating Factor / Q000096:biosynthesis
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D007476:Ionophores; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D010743:Phospholipids; D010972:Platelet Activating Factor; C081565:arachidonyltrifluoromethane; D007975:Leukotriene B4; D016718:Arachidonic Acid; D000001:Calcimycin; D001094:Arachidonate 5-Lipoxygenase; D000217:Acyltransferases; C039766:arachidonyl transacylase; D010741:Phospholipases A; D054467:Phospholipases A2