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 microM 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.
Seeds
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MC
Department of Internal Medicine/Section on Pulmonary and Critical Care, Bowman Gray School of Medicine, Wake Forest University, Medical Center Blvd., Winston-Salem, NC 27157, USA. mseeds@bgsm.edu
Nixon
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AB
Wykle
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Bass
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D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D013582:Synovial Fluid / Q000201:enzymology
C011439:1,2-diacylglycerol; D004075:Diglycerides; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C038237:1-oleoyl-2-acetylglycerol; D010741:Phospholipases A; D054467:Phospholipases A2