Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides.
The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.
Mullmann
T J
TJ
Department of Allergy and Inflammation, Schering-Plough Corporation, Bloomfield, NJ 07003.
Siegel
M I
MI
Egan
R W
RW
Billah
M M
MM
D000478:Alkylation; D002118:Calcium / Q000494:pharmacology; D015936:Complement C5a / Q000494:pharmacology*; D003571:Cytochalasin B / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009504:Neutrophils / Q000201:enzymology*; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D011433:Propranolol / Q000494:pharmacology; D011994:Recombinant Proteins
D004075:Diglycerides; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011994:Recombinant Proteins; D003571:Cytochalasin B; D015936:Complement C5a; D011433:Propranolol; D010740:Phospholipases; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D; D002118:Calcium