Activation of the human neutrophil nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C.

A variety of phagocytosable and soluble agonists stimulate the human neutrophil respiratory burst enzyme, NADPH-oxidase, an activity required for normal microbicidal function. Of these agonists, the phorbol esters, which stimulate diverse systems by their ability to substitute for diacylglycerol to activate protein kinase C (the major phorbol ester receptor), have now been shown to directly stimulate NADPH-oxidase through this same receptor. Almost 90% of the specific receptors for phorbol 12,13-dibutyrate (PDBu) were found in the cytosol upon subcellular fractionation. The dissociation constant for 3HPDBu was 1.2 nM. No significant difference was found in the distribution of the receptor between subcellular fractions from resting as compared with phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils. On the basis of these binding studies, we were able to establish a reconstituted system in which PMA activated dormant NADPH-oxidase in a light membrane fraction when cytosol, NADPH, phosphatidylserine, or phosphatidylinositol and ATP were added. The calcium chelator, EGTA, inhibited the activation, which suggested a requirement for calcium at low concentrations. The half-maximally effective PMA dose was 1.1 nM, as predicted from the receptor content in these preparations. Reconstitution of oxidase activity was rapid, peaking within 1 min of incubation. Purified protein kinase C was able to substitute for the cytosol fraction, and accounted for 80% of the cytosol activity. These studies demonstrate that phorbol esters stimulate the neutrophil respiratory burst through activation of cytosolic protein kinase C, which in turn activates either a regulatory constituent or the NADPH-oxidase directly in the plasma membrane to generate an active O-2-generating system.