CONTRASTING EFFECTS OF CALYCULIN-A AND OKADAIC ACID ON THE RESPIRATORY BURST OF HUMAN NEUTROPHILS

The involvement of serine/threonine protein-phosphatases in the produc tion of superoxide (respiratory burst) by human neutrophils was invest igated using calyculin A, a potent inhibitor of both protein phosphata ses type 1 and 2A, and okadaic acid, which preferentially inhibits pro tein phosphatase type 2A. Treatment of neutrophils with calyculin A (2 5-75 nM) or okadaic acid (1-4 mu M) had no stimulatory effect but pote ntly enhanced total superoxide production induced by an optimal fMLP ( N-formyl-methionyl-leucyl-phenylalanine) concentration (0.1 mu M) The maximal increase plateaued with 50-75 nM calyculin A and 2-4 mu M okad aic acid, reaching approximately 120 and 200% of control values, respe ctively. Unlike calyculin A, okadaic acid also primed the initial rate of superoxide production, suggesting that protein phosphatases may do wn-regulate both initiation and termination of respiratory burst. Opti mal 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 mu M, re spectively, although both drugs caused protein hyperphosphorylation. T he inhibition was partially prevented by a nonstimulatory concentratio n of A23187, indicating a role of calcium in the inhibitory effects of the drugs. Unlike the optimal respiratory burst, suboptimal respirato ry burst induced by PMA (1-7 nM) was enhanced by calyculin A and okada ic acid. Unprimed and primed respiratory bursts were depressed by a se lective antagonist of protein kinase C (GF 109203X), indicating positi ve regulation of these responses by protein kinase C. Thus, the use of calyculin A and okadaic acid distinguishes two regulatory processes o f superoxide production. The respiratory burst induced by low PMA conc entrations or fMLP was up-regulated by both calyculin A and okadaic ac id, in keeping with a down-regulatory role of protein phosphatases in these responses. By contrast, intense protein kinase C activation by P MA triggered a respiratory burst which was depressed by both drugs, po inting to positive regulation of the respiratory burst by protein phos phatases.