Evidence that 85 kDa phospholipase A(2) is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC(50)s of 10 and 12 mu M, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A(2) (PLA(2)), and AACOCF(3), an inhibitor of 85 kDa PLA(2), both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF(3) had no effect on A23187-induced PAF formation at concentrations as high as 3 mu M. Further, depletion of 85 kDa PLA(2) using antisense (SB 7111, 1 mu M) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA(2) in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA(2) inhibitor scalaradial blocked PAF synthesis in monocytes (IC(50)s of 2 and 0.5 mu M, respectively), suggesting a key role of 14 kDa PLA(2) in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 mu M) had a greater effect on the production of 1-O-alkyl (- 80%) than of 1-acyl (- 14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA(2), but not rh 85 kDa PLA(2), induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA(2) to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA(2)-like activity, and not 85 kDa PLA(2), is coupled to monocyte CoA-IT-induced PAF production.