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

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 microM) had no stimulatory effect but potently enhanced total superoxide production induced by an optimal fMLP (N-formyl-methionyl-leucyl-phenylalanine) concentration (0.1 microM). The maximum increase plateaued with 50-75 nM calyculin A and 2-4 microM 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 IC50 of 60 nM and 2 microM, 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, suboptimal respiratory burst induced by PMA (1-7 nM) was enhanced by calyculin A and okadaic acid. Unprimed and primed respiratory bursts 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.(ABSTRACT TRUNCATED AT 250 WORDS)