A chemiluminescence procedure has been developed to determine superoxide anion (O
2-) generation and myeloperoxidase (MPO) release from human activated neutrophils. By using this procedure, the role of protein kinase C (PKC) and cytosolic calcium ion ([Ca
2+]i) for O
2- generation and MPO release was examined. Activation of FcγR on neutrophils with IgG-coated zymosan (IgGZ) caused a transient rise of [Ca
2+]i, followed by O
2- generation and MPO release. A PKC inhibitor suppressed completely the O
2- generation and slightly the MPO release. Direct activation of PKC by a specific PKC activator, phorbol myristate acetate (PMA), induced a remarkable O
2- generation and a small MPO release, indicating that PKC may regulate entirely O
2- synthesis and partially MPO degranulation. Influx of extracellular Ca
2+ induced by the calcium ionophore A23187 provoked MPO release only. Complete inhibition of this MPO release with a Ca
2+/calmodulin (CaM)-coupling inhibitor and a CaM inhibitor provides evidence that [Ca
2+]i may regulate MPO degranulation through direct activation of CaM, but not PKC. The Ca
2+/CaM inhibitors significantly prevented IgGZ-induced O
2- generation and MPO release, while they did not affect PMA-induced O
2- generation and MPO release. These results suggest that in FcγR-stimulated neutrophils, [Ca
2+]i activates CaM, which in turn mediates not only activation of PKC-induced O
2- synthesis and MPO degranulation, but also MPO degranulation without PKC intermediate .
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