TYROSINE PHOSPHORYLATION IS INVOLVED IN THE RESPIRATORY BURST OF ELECTROPERMEABILIZED HUMAN NEUTROPHILS AT A STEP BEFORE DIACYLGLYCEROL FORMATION BY PHOSPHOLIPASE-C

We studied a step where tyrosine phosphorylation is involved in a sign aling pathway for the activation of the superoxide (O2-)-generating NA DPH oxidase using electropermeabilized human neutrophils. The permeabi lized cells produced O2- by the addition of a protein tyrosine phospha tase inhibitor, vanadate, as well as N-formyl-methionyl-leucyl-phenyla lanine (fMLP) and protein kinase C (PKC) activators such as phorbol my ristate acetate (PMA) and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol (OA G). The O2- production by the stimulants was completely inhibited by P KC inhibitors such as calphostin C and staurosporine and was not affec ted by 1% ethanol, a metabolic modulator of phospholipase D (PLD). Fur thermore, the O2- production by vanadate and fMLP, but not by OAG and PMA, was inhibited by both an inhibitor of phospholipase C (PLC), neom ycin, and an inhibitor of tyrosine kinase, ST-638. These findings sugg est that tyrosine phosphorylation is involved in the activation of the oxidase at a step before diacylglycerol formation by PLC, and that PL D may not be involved in the signaling pathway in permeabilized cells.