Calcium entry-dependent oscillations of cytoplasmic calcium concentration in cultured endothelial cell monolayers.

Bovine endothelial cell monolayers grown to confluence and stimulated with bradykinin responded with periodic fluctuations in intracellular Ca2+ concentration ([Ca2+]i) when exposed to K(+)-free Hepes-buffered saline. The fluctuations in [Ca2+]i measured with fura-2 were synchronized among the population of cells observed and were sensitive to extracellular Ca2+ concentration ([Ca2+]o). Thapsigargin, which inhibits the endoplasmic reticular Ca2(+)-ATPase, did not inhibit the [Ca2+]i oscillations. Removal of extracellular Ca2+ or inhibition of Ca2+ entry by using La3+ or 1-(beta- [3-(4-methoxyphenyl)proproxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365) abolished the [Ca2+]i oscillations in endothelial cell monolayers. The fluctuations in [Ca2+]i were therefore dependent on Ca2+ influx rather than Ca2+ mobilization from intracellular stores. Simultaneous measurements of membrane potential (Em) using the potential-sensitive bisoxonol dye bis(1,3-dibutylbarbituric acid)trimethine oxonol [Di-BAC4(3)] and [Ca2+]i using fura-2 showed that Em oscillated at the same frequency as the fluctuations in [Ca2+]i. The peak depolarization signal coincided with the maximum rate of increase in the [Ca2+]i signal. Oscillations in the Em signal were inhibited by removal of Ca2+ or by addition of 1 mM Ni2+ to the external solution. Taken together, these observations suggest that the change in Em is the consequence of oscillatory changes in a membrane conductance that also allows Ca2+ to enter the cell. Oscillations in the DiBAC4(3) signal may reflect a rhythmic entry of Ca2+ through nonselective cation channels. IMAGES:

生长至汇合态的牛内皮细胞(bovine endothelial cell)单层经缓激肽(bradykinin)刺激后，暴露于无钾Hepes缓冲盐溶液时，会出现细胞内钙离子浓度([Ca²⁺]i)的周期性波动。采用弗拉-2(fura-2)检测得到的[Ca²⁺]i波动在观测的细胞群体中呈现同步性，且对细胞外钙离子浓度([Ca²⁺]o)敏感。毒胡萝卜素(thapsigargin)可抑制内质网Ca²⁺-ATP酶(endoplasmic reticular Ca2(+)-ATPase)，但并未抑制[Ca²⁺]i振荡。移除细胞外Ca²⁺，或使用镧离子(La³⁺)、1-(β-[3-(4-甲氧基苯氧基)丙氧基]-4-甲氧基苯乙基)-1H-咪唑盐酸盐(SKF 96365)抑制Ca²⁺内流，均可消除内皮细胞单层中的[Ca²⁺]i振荡。由此可见，[Ca²⁺]i波动依赖于Ca²⁺内流，而非细胞内钙库的钙动员。
采用电位敏感性染料双(1,3-二丁基巴比妥酸)三甲川氧杂蒽[Di-BAC4(3)]同时检测膜电位(Em)，并以弗拉-2(fura-2)检测[Ca²⁺]i，结果显示Em的振荡频率与[Ca²⁺]i波动的频率完全一致。膜电位的去极化峰值与[Ca²⁺]i信号的最大上升速率相重合。Em信号的振荡可通过移除细胞外Ca²⁺，或向外部溶液中添加1 mM Ni²⁺(镍离子)得以抑制。
综合上述观测结果，本研究认为膜电位的变化是膜电导振荡性改变的结果，而该膜电导同时允许Ca²⁺进入细胞。Di-BAC4(3)信号的振荡或许反映了Ca²⁺通过非选择性阳离子通道(nonselective cation channels)的节律性内流。
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