Phosphatase activity regulates superoxide anion generation and intracellular signaling in human neutrophils.
Phosphorylation of components of the neutrophil NADPH oxidase plays a critical role in activation and maintenance of superoxide anion (O2-) generation. To investigate the role of dephosphorylation by phosphatases in regulating O2- production, human neutrophils were treated with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, prior to stimulation. Calyculin A alone did not stimulate O2- production. However, neutrophils exposed to 50 nM calyculin A and the chemotactic peptide formyl-met-leu-phe (FMLP, 100 nM) displayed markedly enhanced O2- production in comparison to cells stimulated with FMLP alone (28.63 +/- 7.00 versus 8.69 +/- 3.69 nmol O2-/1.5 x 10(6) neutrophils/5 min, respectively, n = 18, p < 0.001), with an increased duration of O2- production. In contrast, phosphatase-inhibition decreased oxidative responsiveness to phorbol myristate acetate (PMA, > or = 16 nM). We next examined the effect of calyculin A on products of the phosphatidylcholine-specific phospholipase D (PLD) pathway by assaying the mass levels of phosphatidic acid (PA), choline and diacylglycerol (DAG). Calyculin A increased both PA and choline production to 224 +/- 28% and 315 +/- 61% of FMLP-stimulated controls, respectively (p < 0.01, n = 7) without significantly increasing DAG. Also, membrane protein kinase C activity increased more than 10-fold in FMLP-stimulated cells exposed to calyculin A but decreased in cells stimulated with PMA following calyculin A pre-treatment. These results suggest that phosphatases exert variable and stimulus-dependent effects on pathways leading to O2- production. Further, it appears that phospholipase D activity and PA generation represent important steps in the pathway for NADPH activation triggered by FMLP.
Gay
J C
JC
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232-2574, USA.
Raddassi
K
K
Truett
A P
AP
Murray
J J
JJ
D000328:Adult; D006801:Humans; D008387:Marine Toxins; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000187:drug effects / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008387:Marine Toxins; D010080:Oxazoles; D010712:Phosphatidic Acids; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; C059041:calyculin A; D019255:NADPH Oxidases; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate