Phosphatase activity regulates superoxide anion generation and intracellular signaling in human neutrophils

Abstract

Phosphorylation of components of the neutrophil NADPH oxidase plays a critical role in activation and maintenance of superoxide anion (O 2 −) generation. To investigate the role of dephosphorylation by phosphatases in regulating O 2 − 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 O 2 − production. However, neutrophils exposed to 50 nM calyculin A and the chemotactic peptide formyl-met-leu-phe (FMLP, 100 nM) displayed markedly enhanced O 2 − production in comparison to cells stimulated with FMLP alone (28.63±7.00 versus 8.69±3.69 nmol O 2 −/1.5×10 6 neutrophils/5 min, respectively, n=18, p<0.001), with an increased duration of O 2 − production. In contrast, phosphatase-inhibition decreased oxidative responsiveness to phorbol myristate acetate (PMA, ≥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 O 2 − production. Further, it appears that phospholipase D activity and PA generation represent important steps in the pathway for NADPH activation triggered by FMLP.