Polverino A J, Hughes B P, Barritt G J
Department of Medical Biochemistry, School of Medicine, Flinders University, Australia.
Biochem J. 1991 Sep 15;278 ( Pt 3)(Pt 3):849-55. doi: 10.1042/bj2780849.
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.
在负载fura-2的单个NIH-3T3成纤维细胞中，蛙皮素可诱导细胞内游离Ca2+（[Ca2+]i）浓度出现三种增加模式之一：单次瞬时增加、持续增加或[Ca2+]i的重复瞬时增加。胎牛血清和ATP也会产生这三种反应模式，不过对ATP产生重复Ca2+瞬变反应的细胞比例较低。将蛙皮素浓度从1 nM增加到25 nM，会增加表现出重复Ca2+瞬变的细胞比例。在25 nM蛙皮素时，随着细胞外Ca2+（Ca2+o）浓度从1 mM增加到5 mM，表现出重复Ca2+瞬变的细胞比例也增加。通过添加EGTA去除Ca2+o，或用维拉帕米或蛋白激酶C激活剂处理在有Ca2+o存在的情况下孵育的细胞来抑制Ca2+内流，会突然终止重复的Ca2+瞬变，在Ca2+内流停止后仅观察到一个瞬变。在无Ca2+o且有EGTA存在的情况下孵育的细胞中未观察到重复的Ca2+瞬变。向先前在有EGTA存在的情况下孵育的细胞中添加Ca2+o会导致重复的Ca2+瞬变恢复。单独添加毒胡萝卜素会诱导[Ca2+]i出现大幅瞬时增加，而咖啡因或羰基氰化物间氯苯腙（CCCP）加寡霉素在约30%的测试细胞中诱导的[Ca2+]i瞬时增加要小得多。毒胡萝卜素或CCCP加寡霉素的组合完全抑制了蛙皮素诱导的重复Ca2+瞬变，而咖啡因则无作用。从对Ca2+o作用的研究得出结论，NIH-3T3细胞在细胞外Ca2+与参与Ca2+瞬变产生的细胞内储存之间的解剖学或化学联系上与其他细胞类型不同，而抑制剂实验结果表明，NIH-3T3细胞中重复Ca2+瞬变的产生不太可能涉及从对咖啡因敏感的储存中Ca(2+)诱导的Ca2+释放。
