Bradykinin B-2 receptor-induced and inositol tetrakisphosphate-evoked Ca2+ entry is sensitive to a protein tyrosine phosphorylation inhibitor in ras-transformed NIH/3T3 fibroblasts

Signal transduction from mouse bradykinin B-2 receptors to calcium influx was studied in las-transformed NIH/3T3 (DT) fibroblasts. DT cells were preloaded with fura-2 and whole-cell voltage-clamped. Activation of B-2 receptors resulted in a decrease of cellular fluorescence at the excitation wavelength of 340, or 360 nm after MnCl2 application, in both the presence and absence of external Ca2+ in DT cells, at a holding potential of -40 mV. This Mn2+ entry through the Ca2+ influx pathway increased with membrane hyperpolarization. Internal application of inositol 1,3,4,5-tetrakisphosphate (InsP(4)), but not of inositol 1,4,5-trisphosphate, mimicked membrane potential-dependent Mn2+ entry. Bradykinin- and InsP(4)-induced Ca2+ influx was blocked by 10-100 mu M genistein, a tyrosine kinase inhibitor. B-2 receptor activation induced time-dependent tyrosine phosphorylation of mitogen-activated protein kinase and 120 kDa protein, which was dose-dependently inhibited by genistein. Bradykinin was unable to induce Ca2+ oscillations in genistein-treated DT cells. Our results show that bradykinin-induced Ca2+ influx and oscillations depend upon protein tyrosine phosphorylation. The results suggest that two bradykinin B-2 receptor-activated signal pathways, protein tyrosine phosphorylation and formation of InsP(4), merge at the Ca2+ influx process in ras-transformed NIH/3T3 fibroblasts.