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

Richard W Erickson,Paola Langel-Peveri,Alexis E Traynor-Kaplan,Paul G Heyworth,John T Curnutte

doi:10.1074/jbc.274.32.22243

Richard W Erickson, Paola Langel-Peveri + Show 3 more

Open Access

https://doi.org/10.1074/jbc.274.32.22243

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Abstract

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.

Highlights

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
We show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system
We describe for the first time the potent activation of the neutrophil NADPH oxidase in a cell-free system by physiologically attainable concentrations of phosphatidic acid (PA) or DAG, in the absence of other agonists

Summary

ACTIVITY OF DIACYLGLYCEROL IS DEPENDENT ON ITS CONVERSION TO PHOSPHATIDIC ACID*

(Received for publication, February 23, 1999, and in revised form, May 14, 1999). Richard W. We show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. 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. In response to a variety of stimuli, the human neutrophil is capable of the potent and sustained production of superoxide anion (O2.) This is achieved through the single-electron reduction of molecular oxygen in a reaction catalyzed by a multicomponent NADPH oxidase system, : NADPϩ Once fϩorm2Oed. While there is a wealth of data from studies with intact (34 – 43) and electropermeabi-

Activation of NADPH Oxidase by PA or DAG

EXPERIMENTAL PROCEDURES

RESULTS

Superoxide production

Membrane vesicles

DISCUSSION