Bradykinin-induced cytosolic Ca2+ oscillations and inositol tetrakisphosphate-induced Ca2+ influx in voltage-clamped ras-transformed NIH/3T3 fibroblasts.

Microspectrofluorometry (fura-2) was combined with the whole-cell patch-clamp technique to study bradykinin-activated calcium (Ca2+) influx in single control or v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts. Application of bradykinin on DT cells, but not on control NIH/3T3 cells, evoked cytosolic Ca2+ oscillations in the presence of extracellular Ca2+, but not in the absence of external Ca2+. This effect of zero external Ca2+ concentration could be mimicked by holding at depolarized membrane potentials. Cytosolic Ca2+ oscillations observed at holding potentials of -20 to -80 mV were terminated by holding at -10 mV or more depolarized potentials. The frequency of Ca2+ oscillations increased with membrane hyperpolarization. Bradykinin significantly enhanced the hyperpolarization-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) upon membrane hyperpolarization only in DT cells but not in control cells. No [Ca2+]i increase upon hyperpolarization was observed in bradykinin-stimulated DT cells in the absence of external Ca2+, suggesting that bradykinin activates Ca2+ influx. [Ca2+]i increased upon application of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) into control and DT cells in an extracellular Ca(2+)-dependent manner, indicating that NIH/3T3 fibroblasts have an Ins(1,3,4,5)P4-gated Ca2+ influx pathway. Ins-(1,3,4,5)P4, however, produced the sustained [Ca2+]i increase in DT cells, but not in control NIH/3T3 cells, suggesting that ras may lock the Ca2+ influx pathway at the activated state. Cytosolic Ca2+ oscillations, bradykinin-enhanced Ca2+ influx, and Ins(1,3,4,5)P4-induced Ca2+ influx were all similar in that activity was increased by membrane hyperpolarization. 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.