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+]i. 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 NIH-3T3 cells is unlikely to involve Ca(2+)-induced Ca2+ release from caffeine-sensitive stores.
Polverino
A J
AJ
Department of Medical Biochemistry, School of Medicine, Flinders University, Australia.
Hughes
B P
BP
Barritt
G J
GJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001839:Bombesin / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D005312:Fetal Blood; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006133:Growth Substances / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D009840:Oligomycins / Q000494:pharmacology; D010085:Oxidative Phosphorylation / Q000187:drug effects; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D005456:Fluorescent Dyes; D006133:Growth Substances; D009840:Oligomycins; D013729:Terpenes; D002110:Caffeine; D004533:Egtazic Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D001839:Bombesin; D002118:Calcium; D016257:Fura-2