We examined the effect of the two
protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and
herbimycin A, on the activation processes of rat basophilic
leukemia (RBL-2H3) cells by cross-linking of
IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal
IgE antibody were stimulated with multivalent
antigen (DNP conjugate of
bovine serum albumin). Analysis of
phosphotyrosine-containing
proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the
tyrosine phosphorylation of several
proteins (32, 42, 56, 66, 72, 92, 150 kDa) including
phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of
inositol 1,4,5-trisphosphate, and the increase in cytosolic
calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of
antigen-dependent
calcium signals in individual cells showed that these two
tyrosine kinase inhibitors inhibited the
calcium influx from the external medium more powerfully than the mobilization of
calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic
calcium ion concentration or the histamine release induced by the
calcium ionophore A23187. Taken together, our results suggest that
tyrosine phosphorylation following
antigen stimulation regulates
phosphatidylinositol hydrolysis and the influx of extracellular
calcium.