Contrasting effects of calyculin A and okadaic acid on the respiratory burst of human neutrophils

Abstract

The involvement of serine/threonine protein-phosphatases in the production of superoxide (respiratory burst) by human neutrophils was investigated using calyculin A, a potent inhibitor of both protein phosphatases type 1 and 2A, and okadaic acid, which preferentially inhibits protein phosphatase type 2A. Treatment of neutrophils with calyculin A (25–75 nM) or okadaic acid (1–4 μM) had no stimulatory effect but potently enhanced total superoxide production induced by an optimal fMLP ( N-formyl-methionyl-leucoyl-phenylalanine) concentration (0.1 μM). The maximal increase platacuaed with 50–75 nM calyculin A and 2–4 μM okadaic acid, reaching approximately 120 and 200% of control values, respectively. Unlike calyculin A, okadaic acid also primed the initial rate of superoxide production, suggesting that protein phosphatases may down-regulate both initiation and termination of respiratory burst. Optimal stimulation of the respiratory burst by PMA (160 nM) was inhibited by calyculin A and okadaic acid, with an IC 50 of 60 nM and 2 μM, respectively, although both drugs caused protein hyperphosphorylation. The inhibition was partially prevented by a nonstimulatory concentration of A23187, indicating a role of calcium in the inhibitory effects of the drugs. Unlike the optimal respiratory burst, suoptimal respiratory burst induced by PMA (1–7 nM) was enhanced by calyculin A and okadaic acid. Unprimed and primed respiratory burst were depressed by a selective antagonist of protein kinase C (GF 109203X), indicating positive regulation of these responses by protein kinase C. Thus, the use of calyculin A and okadaic acid distinguishes two regulatory processes of superoxide production. The respiratory burst induced by low PMA concentrations of fMLP was up-regulated by both calyculin A and okadaic acid, in keeping with a down-regulatory role of protein phosphatases in these responses. By contrast, intense protein kinase C activation by PMA triggered a respiratory burst which was depressed by both drugs, pointing to positive regulation of the respiratory burst by protein phosphatases.