[Measurement of receptor-operated Ca2+ influx by microspectrofluometry combined with the whole-cell patch clamp technique].
Signal transduction from mouse bradykinin B2-receptors to Ca2+ influx was studied in single control or v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts. Microspectrofluometry (fura-2) was combined with the whole-cell patch-clamp technique to study bradykinin-activated Ca2+ influx. Cytosolic Ca2+ oscillations observed at holding potentials of -20 to -80 mV were terminated by holding at -10 mV or more depolarized potentials. Bradykinin significantly enhanced the hyperpolarization-induced increases in the intracellular free Ca2+ concentration upon membrane hyperpolarization only in DT cells but not in control cells. Internal application of 10 microM inositol 1,3,4,5-tetrakisphosphate (InsP4) mimicked membrane potential-dependent Ca2+ entry. Activation of B2-receptors resulted in a decrease of cellular fluorescence at the excitation wavelength of 340 or 360 nm after MnCl2 application in DT cells. This Mn2+ entry through the Ca2+ influx pathway increased with membrane hyperpolarization below -20 mV. The results suggest that bradykinin-induced cytosolic Ca2+ oscillations in ras-transformed NIH/3T3 cells are maintained by bradykinin-activated continuous Ca2+ influx, which may use Ins(1,3,4,5)P4 as an intracellular messenger.
Hashii
M
M
Department of Biophysics, Kanazawa University School of Medicine, Japan.
Higashida
H
H
D000818:Animals; D001920:Bradykinin / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D011905:Genes, ras; D007295:Inositol Phosphates / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
D007295:Inositol Phosphates; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; C081655:inositol-1,3,4,5-tetrakisphosphate; D001920:Bradykinin; D002118:Calcium