EFFECTS OF HERBIMYCIN-A AND ST638 ON FC-EPSILON RECEPTOR-MEDIATED HISTAMINE-RELEASE AND CA2-2H3) CELLS( SIGNALS IN RAT BASOPHILIC LEUKEMIA (RBL)

We examined the effect of the two protein tyrosine kinase inhibitors, ano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimy cin A, on the activation processes of rat basophilic leukemia (RBL-2H3 ) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized wi th DNP-specific monoclonal IgE antibody were stimulated with multivale nt antigen (DNP conjugate of bovine serum albumin). Analysis of phosph otyrosine-containing proteins in their lysates by SDS-PAGE and immunob lotting revealed that these two inhibitors efficiently inhibited the t yrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 1 50 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inosit ol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion conc entration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individua l cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobi lization of calcium ion from internal stores. In contrast, the inhibit ors did not affect the increase in the cytosolic calcium ion concentra tion or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation fol lowing antigen stimulation regulates phosphatidylinositol hydrolysis a nd the influx of extracellular calcium.