UTILITY OF STAUROSPORINE IN UNCOVERING DIFFERENCES IN THE SIGNAL TRANSDUCTION PATHWAYS FOR SUPEROXIDE PRODUCTION IN NEUTROPHILS

Neutrophils exhibit an intense phosphorylation of a 47 kDa protein and release large quantities of superoxide (O2 -) upon stimulation with phorbol 12-myristate 13-acetate (PMA) or fMet-Leu-Phe (fMLP). Antagonists of protein kinases (e.g., 200 μM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); 15 nM staurosporine) inhibited these phenomena when the stimulus was PMA (Badwey, J.A. et al. (1989) J. Biol. Chem. 264, 14947-14953). In this paper, we now report that while neutrophils treated with 15 nM staurosporine and PMA release little O2 -, cells in the presence of these compounds can be stimulated to release near normal quantities of O2 - by the subsequent addition of fMLP. Surprisingly, staurosporine (15 nM) reduced the incorporation of 32P into the 47 kDa protein in fMLP stimulated cells at least as effectively as H-7, yet, while the staurosporine treated cells released substantial amounts of O2 -, the cells treated with H-7 did not. These data suggest that a stimulatory pathway exists in neutrophils that contains a protein kinase 'distinct' from that which is activated when PMA is the stimulus and that this pathway may enable the O2 - producing system to become functional with little or no phosphorylation of the 47 kDa protein. They further suggest that the steps which are sensitive to H-7 in the signal-transduction pathways utilized by PMA and fMLP may be different. © 1990.