Differential activation of human neutrophil cytosolic phospholipase A2 and secretory phospholipase A2 during priming by 1,2-diacyl- and 1-O-alkyl-2-acylglycerols

We have shown previously that both 1,2-diacylglycerol (AAG) and 1-O-alkyl-2-acylglycerol (EAG) prime neutrophil release of arachidonic acid via uncharacterized phospholipases A₂. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA₂) and secretory (sPLA₂) phospholipase A₂s. We hypothesized that AAG as a protein kinase activator would activate cPLA₂ via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA₂. Neutrophils were primed with either AAG or EAG and then stimulated with fMLP. When neutrophils were primed with 5–20 μM 1,2-diacylglycerol, a shift was observed in cPLA₂ migration on SDS-PAGE gels, consistent with phosphorylation of the protein. This gel shift was not seen after exposure to EAG. AAG also caused a parallel increase in enzymatic activity of cPLA₂ that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA₂. Both AAG and EAG directly caused significant secretion of neutrophil sPLA₂. EAG also increased the release of sPLA₂ in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA₂ and stimulated secretion of sPLA₂. In contrast, EAG did not activate cPLA₂, but directly activated secretion of sPLA₂. We also demonstrated that human synovial fluid sPLA₂ increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB₄, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA₂) or phosphorylation independent (e.g. secretion of sPLA₂) events.