Inhibition of Ca²⁺ inflow causes an abrupt cessation of growth-factor-induced repetitive free Ca²⁺ transients in single NIH-3T3 cells

In single NIH-3T3 fibroblasts loaded with fura-2, bombesin induced one of three patterns of increase in the concentration of intracellular free Ca²⁺ ([Ca²⁺](i)): a single transient increase, a sustained increase, or repetitive transient increases in [Ca²⁺](o). Foetal-calf serum and ATP also gave these three patterns of response, although a lower proportion of cells gave repetitive Ca²⁺ transients in response to ATP. An increase in the concentration of bombesin from 1 to 25 nM increased the proportion of cells which exhibited repetitive Ca²⁺ transients. At 25 nM-bombesin, the proportion of cells which exhibited repetitive Ca²⁺ transients increased as the extracellular Ca²⁺ (Ca²⁺(o)) concentration was increased from 1 to 5 mM. Removal of Ca²⁺(o) by addition of EGTA, or inhibition of Ca²⁺ inflow by treatment of cells incubated in the presence of Ca²⁺(o) with verapamil or an activator of protein kinase C, abruptly terminated repetitive Ca²⁺ transients, with only one transient observed after the cessation of Ca²⁺ inflow. Repetitive Ca²⁺ transients were not observed in cells incubated in the absence of Ca²⁺(o) and in the presence of EGTA. Addition of Ca²⁺(o) to cells previously incubated in the presence of EGTA caused a resumption of repetitive Ca²⁺ transients. Addition of thapsigargin alone induced a large transient increase in [Ca²⁺](i), whereas much smaller transient increases in [Ca²⁺](i) were induced in about 30% of cells tested by caffeine or carbonyl cyanide m-chlorophenylhydrazone (CCCP) plus oligomycin. Thapsigargin or the combination of CCCP plus oligomycin completely inhibited bombesin-induced repetitive Ca²⁺ transients, whereas caffeine had no effect. It is concluded from the studies of the role of Ca²⁺(o) that NIH-3T3 cells differ from other cell types in the anatomical or chemical links between extracellular Ca²⁺ and the intracellular stores involved in the generation of Ca²⁺ transients, whereas the results of the experiments with inhibitors indicate that the generation of repetitive Ca²⁺ transients in NIH3T3 cells is unlikely to involve Ca²⁺-induced Ca²⁺ release from caffeine-sensitive stores.