Measurement of receptor-operated Ca²⁺ influx by microspectrofluometry combined with the whole-cell patch clamp technique.

Abstract

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 μM 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 bradykinininduced 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.