Bradykinin B2 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 B2 receptors to calcium influx was studied in ras-transformed NIH/3T3 (DT) fibroblasts. DT cells were preloaded with fura-2 and whole-cell voltage-clamped. Activation of B2 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 (InsP4), but not of inositol 1,4,5-trisphosphate, mimicked membrane potential-dependent Mn2+ entry. Bradykinin- and InsP4-induced Ca2+ influx was blocked by 10-100 microM genistein, a tyrosine kinase inhibitor. B2 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 B2 receptor-activated signal pathways, protein tyrosine phosphorylation and formation of InsP4, merge at the Ca2+ influx process in ras-transformed NIH/3T3 fibroblasts.
Department of Cortical Function Disorder, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
M
M
Hashii
S
S
Nakashima
S
S
Yokoyama
K
K
Enomoto
Y
Y
Minabe
Y
Y
Nozawa
H
H
Higashida
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011905:Genes, ras; D019833:Genistein; D007295:Inositol Phosphates / Q000378:metabolism*; D007529:Isoflavones / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism*; D015398:Signal Transduction
D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D007529:Isoflavones; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D002118:Calcium