Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid.
The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.
Erickson
R W
RW
Department of Immunology, Genentech, Inc., South San Francisco, California 94080, USA.
Langel-Peveri
P
P
Traynor-Kaplan
A E
AE
Heyworth
P G
PG
Curnutte
J T
JT
D002474:Cell-Free System; D019852:Diacylglycerol Kinase / Q000037:antagonists & inhibitors; D004075:Diglycerides / Q000378:metabolism* / Q000494:pharmacology; D004357:Drug Synergism; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010712:Phosphatidic Acids / Q000378:metabolism* / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D052999:Quinazolinones; D015290:Second Messenger Systems; D013481:Superoxides / Q000378:metabolism
D004075:Diglycerides; D010712:Phosphatidic Acids; D010880:Piperidines; D011799:Quinazolines; D052999:Quinazolinones; C084527:dioctanoylphosphatidic acid; D013481:Superoxides; C058544:R 59949; D019255:NADPH Oxidases; D019852:Diacylglycerol Kinase