Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction.
Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubation with alkyl-[32P]lysoPC. Upon stimulation with the chemotactic peptide, formylMet-Leu-Phe (fMLP), these 32P-labeled cells produce 1-O-alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and, in the presence of ethanol, 1-O-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because the cellular ATP contains no 32P, alkyl-[32P]PA and alkyl-[32P]PEt must be formed from alkyl-[32P]PC by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Analyses of the sn-1 bonds by selective hydrolysis and mass measurements reveal that the PA and PEt formed during stimulation contain both ester and ether bonds with distributions similar to that in the endogenous PC. Furthermore, in neutrophils labeled in alkyl-[32P]PC, the specific activities of the diradyl-PA and diradyl-PEt formed during stimulation are similar to that of diradyl-PC. These results demonstrate that the fMLP-induced PLD utilizes diradyl-PC as the major substrate. It is further concluded that, at early times (30 s), PA and PEt are both formed almost exclusively by PLD. Following stimulation with fMLP, neutrophils double-labeled in alkyl-PC by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC generate [3H]alkyl-DG and [32P]orthophosphate [( 32P]PO4) with superimposable kinetics, indicating degradation of PA by a phosphohydrolase. Generation of [3H]alkyl-DG and [32P]PO4 lags behind PA formation and parallels the decline in PA accumulation. In addition, generation of both [3H]alkyl-PA and [3H]alkyl-DG requires extracellular Ca2+ and cytochalasin B. Furthermore, the phosphohydrolase inhibitor, propranolol, decreases both [3H]alkyl-DG and [32P]PO4 while increasing [3H]alkyl-PA and not altering [3H]alkyl-PEt. Moreover, the decreases in DG are accounted for by increases in PA. These results demonstrate that PLD-derived alkyl-PA is degraded by a phosphohydrolase to produce alkyl-DG. DG formed during stimulation contains both ester and ether-linked species and this DG formation is inhibited completely by propranolol. Upon stimulation, alkyl-[32P]PC-labeled neutrophils do not produce [32P]phosphocholine, suggesting that PC is not hydrolyzed by phospholipase C. In addition, PA is formed in amounts sufficient to account for all of the DG formed during stimulation. It is concluded that the DG formed during fMLP stimulation is derived almost exclusively from PC via the PLD/PA phosphohydrolase pathway.
Billah
M M
MM
Department of Allergy and Inflammation, Schering-Plough Corporation, Bloomfield, New Jersey 07003.
Eckel
S
S
Mullmann
T J
TJ
Egan
R W
RW
Siegel
M I
MI
D004075:Diglycerides / Q000097:blood*; D004789:Enzyme Activation; D005989:Glycerides / Q000097:blood*; D006801:Humans; D006868:Hydrolysis; D008297:Male; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000378:metabolism*; D010711:Phosphatidate Phosphatase / Q000097:blood; D010712:Phosphatidic Acids / Q000097:blood*; D010713:Phosphatidylcholines / Q000097:blood*; D010714:Phosphatidylethanolamines / Q000097:blood; D010739:Phospholipase D / Q000097:blood*; D010740:Phospholipases / Q000097:blood*; D015398:Signal Transduction; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism
D004075:Diglycerides; D005989:Glycerides; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010740:Phospholipases; D010711:Phosphatidate Phosphatase; D010738:Type C Phospholipases; D010739:Phospholipase D