Tyrosine phosphorylation is involved in the respiratory burst of electropermeabilized human neutrophils at a step before diacylglycerol formation by phospholipase C

Abstract

We studied a step where tyrosine phosphorylation is involved in a signaling pathway for the activation of the superoxide (O⁻ ₂)-generating NADPH oxidase using electropermeabilized human neutrophils. The permeabilized cells produced O⁻ ₂ by the addition of a protein tyrosine phosphatase inhibitor, vanadate, as well as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and protein kinase C (PKC) activators such as phorbol myristate acetate (PMA) and l-α- 1 -oleoyl-2-acetoyl-sn-3-glycerol (OAG). The O⁻ ₂ production by the stimulants was completely inhibited by PKC inhibitors such as calphostin C and staurosporine and was not affected by 1 % ethanol, a metabolic modulator of phospholipase D (PLD). Furthermore, the O⁻ ₂ production by vanadate and fMLP, but not by OAG and PMA, was inhibited by both an inhibitor of phospholipase C (PLC), neomycin, and an inhibitor of tyrosine kinase, ST-638. These findings suggest that tyrosine phosphorylation is involved in the activation of the oxidase at a step before diacylglycerol formation by PLC, and that PLD may not be involved in the signaling pathway in permeabilized cells.