Role of Tyrosine Phosphorylation in the Signalling of Superoxide Anion Generation in Platelet-Activating-Factor-Stimulated Peritoneal Macrophages

Abstract

The signalling pathway(s) involved in the activation of superoxide anion (O^–₂) generation by platelet-activating factor (PAF) are not well understood, But elevations of sn-l,2-diacylglycerol and intracellular calcium contribute to activation by stimulating protein kinase C activity. Since recent studies have suggested that tyrosine phosphorylation may also be a component of the signalling of O^–₂ generation in human neutrophils, we have investigated whether PAF stimulates tyrosine phosphorylation in guinea pig and rat macrophages. PAF (10 nM) stimulated tyrosine phosphorylation within 10–30 s, coinciding with the onset of the O^-₂ generation in guinea pig peritoneal macrophages. At 30 min after exposure of guinea pig macrophages to PAF, the phosphorylation levels were similar to those of unstimulated cells. In contrast, in rat peritoneal macrophages, PAF did not stimulate tyrosine phosphorylation of high molecular weight proteins, nor did it elicit the generation of O^–₂. PAF-induced O^–₂ generation was inhibited by pre-treatment of guinea pig peritoneal macrophages with the protein tyrosine kinase inhibitors methyl 2, 5-dihydroxymethylcinnamate and tyrphostin. The qualitative difference between PAF-induced phosphorylation patterns in rat and guinea pig macrophages and the inhibitory effects of protein tyrosine kinase inhibition are consistent with an important role for protein tyrosine kinase(s) in O^–₂ generation.