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

Abstract

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 A 2. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA 2) and secretory (sPLA 2) phospholipase A 2s. We hypothesized that AAG as a protein kinase activator would activate cPLA 2 via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA 2. 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 cPLA 2 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 cPLA 2 that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA 2. Both AAG and EAG directly caused significant secretion of neutrophil sPLA 2. EAG also increased the release of sPLA 2 in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA 2 and stimulated secretion of sPLA 2. In contrast, EAG did not activate cPLA 2, but directly activated secretion of sPLA 2. We also demonstrated that human synovial fluid sPLA 2 increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB 4, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA 2) or phosphorylation independent (e.g. secretion of sPLA 2) events.