Relation of human neutrophil phorbol ester receptor occupancy and NADPH-oxidase activity

Abstract

Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 ± 2.1 and 41.7 ± 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 105 sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50's for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.