Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP₃). Maximal concentrations of EGF (316 ng/ml; ~ 50 nM), bradykinin (1 μM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K_0.5 values for stimulation were approximately 10 nM, 3 nM, and 10 μM for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid accumulation of the two isomers of InsP₃, Ins(1,3,4)P₃, and Ins(1,4,5)P₃ as determined by high performance liquid chromatography analysis; maximal accumulation of Ins(1,4,5)P₃ occurred within 15 s. EGF and bradykinin also stimulated a rapid (maximal levels attained within 30 s after addition of hormone) and a sustained 4- and 6-fold rise, respectively, in cytosolic free Ca²⁺ levels as measured by Fura-2 fluorescence. EGF and bradykinin also produced a rapid, although transient, 3- and 5-fold increase, respectively, in cytosolic free Ca²⁺ after chelation of extracellular Ca²⁺ with 3 mM EGTA. These data are consistent with the idea that EGF elevates intracellular Ca²⁺ levels in A431 cells, at least in part, as a result of the rapid formation of Ins(1,4,5)P₃ and the consequential release of Ca²⁺ from intracellular stores.