Abstract
NADPH oxidase, the respiratory burst enzyme of human neutrophils, is a multi-component complex that is assembled and activated during stimulation of the cells by inflammatory or phagocytic stimuli. The signal mechanisms leading to activation of the enzyme are unclear, but it is likely that phospholipases are involved. Recent work has shown that phosphatidic acid, the initial product of phospholipase D activation, is a weak activator of NADPH oxidase in a cell-free system. We now show that diacylglycerol enhances the cell-free activation of NADPH oxidase activation by phosphatidic acid. 1,2-Didecanoyl phosphatidic acid (10:0-PA) and 1,2-dioctanoylglycerol (8:0-DG) each increased levels of NADPH oxidase activity in mixtures of membrane and cytosolic fractions about 2-fold. The combination of both lipids increased NADPH oxidase activity approximately 12-fold, indicative of a synergistic response. Fatty acid and neutral lipid metabolites of 10:0-PA or 8:0-DG were ineffective, suggesting activation is directly mediated by phosphatidic acid and diacylglycerol. Activation was time- and concentration-dependent with maximum activation at 30-60 min and a sharp peak of maximal activity at 10 microM 10:0-PA and 30 microM 8:0-DG. In lipid specificity studies, activity of PA or DG decreased with increasing acyl chain length but was restored by introducing unsaturation in the acyl chain. Natural forms of PA stimulated levels of activity comparable to that seen with 10:0-PA. Synthetic and natural phosphatidylserines, but not other phospholipids, could replace phosphatidic acid in the synergistic response. These studies provide direct evidence for a synergistic interaction between phosphatidic acid and diacylglycerol in mediating a cellular function: the assembly and activation of NADPH oxidase. Our results support the concept that the generation of second messenger lipids by phospholipase D is a key step in activation of the respiratory burst enzyme.
Highlights
NADPH oxidase, the respiratory burst enzyme of hu- ease (CGD),l a family of diseases in which various defects in man neutrophils, is a multi-component complex that is the protein components of the oxidase result in a loss of the assembled and activated during stimulation of the cells respiratory burst and an inabiolifttyhe patientsto resist many by inflammatory or phagocyticstimuli.The signal infections [1,3]
We show that diacylglycerol enhances the cell-free activation ofNADPH oxidase activation by phosphatidic acid.1,2-Didecanoyl phosphatidic acid(100-PA) and 1,2-dioctanoylglycerol (80-DG)each increased levels of NADPH oxidase activity in mixtures of membrane and cytosolic fractions about 2-fold.Thecombination of brane andthat it requires at least five polypeptides during the assembly and activationprocess (Refs. 7-15;reviewed in Refs. 1-6)
Fatty acid and through receptor-mediated activation of a phosphatidylinositol neutral lipid metabolites of 100-PAor 80-DG were inef- 4,5-bisphosphate-specificg,uanine nucleotide regulatory profective, suggesting activation is directly mediated by tein-dependent phospholipase C [25,26,27] and, indirectly, phosphatidic acid and diacylglycerol.Activationwas through the activation of a phosphatidylcholine-specific phostime- and concentration-dependent with maximum ac- pholipase Dto form PA that is converted to DG byPA phosphotivation at 30-80 min and a sharp peak of maximal ac- hydrolase [28].PA has multiple pathwafyosr its synthesis, tivity at 10 JIM 100-PAand 30 JIM 80-DG
Summary
Materials-Heparin at 1000unitdml was obtainedfrom Elkins-Sinn, Alz(SOJ3 + NaF, and maximal activation was achieved in -30 min. Phenylmethylsulfonyl DG Kinase Assay-The levels of8:O-DG and 10:O-DG derived from fluoride and PMA were stored as stock solutions in MezSO at -20 “C 1O:O-PA were measured using the DG kinase assay as described by and were added to the appropriate buffer or reaction mixture immedi- Preiss et al [50].Incubations were performedexactlyas for the NADPH ately before use. (BCA)protein assay reagents were obtained from Pierce Chemical Co. trols included the lipids incubated for the same period of time in the Potassium phosphate (&PO,) and NaF were obtained from Fisher. Venousblood was obtained fromtwo patients with CGD where [50]. B. was the patient tories, Woburn, MA) at 1PMwas included during the preincubation to of Dr Richard Reese at Bassett Hospital (Cooperstown,NY).Blood from prevent conversion of8:O-DG to 8:O-PA. Obtained from Serdary included 1,2-didecanoyl-3-sn-phosphatidiacid (lO:O-PA), 1,2-dipalmitoyl-3-sn-phosphatidicacid, 1,2-dioleoy1-3-sn-
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