Protein phosphorylation in alveolar macrophages after stimulation with heavy metal-coated silica particles

The aim of our study was to determine agonist- and time-dependent phosphorylation of distinct proteins in bovine alveolar macrophages after activation with quartz dusts and heavy metal-coated silica particles. Silica particles were coated with vanadium oxide or cadmium oxide. Separation of proteins was achieved by one-dimensional and optimized horizontal two-dimensional polyacrylamide gel electrophoresis. Protein phosphorylation was either determined by [32P] labeling and subsequent autoradiography or Western blotting using monoclonal antibodies against phosphotyrosine. One of the phosphorylated proteins was identified as the heat shock protein HSP-27. Stimulation with cadmium oxide-coated silica particles and PMA led to a significant increase of phosphorylated HSP-27. Additionally cadmium oxide-coated silica particles induced tyrosine phosphorylation of six main proteins. Stimulation with vanadium oxide-coated silica particles drastically increased the amount of tyrosine phosphorylated proteins while this effect was not observed with uncoated silica particles.