Monck J R, Reynolds E E, Thomas A P, Williamson J R
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Biol Chem. 1988 Apr 5;263(10):4569-75.
The kinetics of agonist-induced increases in cytosolic free Ca2+ have been measured in single A10 vascular smooth muscle cells and rat hepatocytes using fluorescent videomicroscopy with fura-2 as a Ca2+ indicator. At high agonist concentrations there was no difference in the kinetics of the Ca2+ transient measured in vasopressin-stimulated single A10 cells or in cell populations. However, stimulation of single A10 cells with concentrations of vasopressin below 0.5 nM produced characteristic Ca2+ transients composed of two distinct peaks. The two peaks appeared to represent a temporal separation between release of intracellular Ca2+ and influx of extracellular Ca2+. The double transient was not observed in single rat hepatocytes stimulated with low concentrations of vasopressin or phenylephrine. In both A10 cells and hepatocytes, the initial rate of increase in Ca2+ concentrations in response to submaximal agonist concentrations was faster in single cells than in cell populations. This difference was due to asynchrony of the cellular response, where there was a latent period of variable length before onset of a rapid increase in Ca2+ concentration. The duration of the latent period was dependent on the agonist concentration, higher concentrations of agonist giving a reduced latent period. The hormone-stimulated Ca2+ transient measured in single hepatocytes with fura-2 was different from the series of transient spikes as previously reported using aequorin as the Ca2+ indicator, suggesting that fura-2 and aequorin may report different aspects of the Ca2+ response in stimulated cells. Collectively, these results demonstrate that measurement of Ca2+ transients in single cells provide novel information concerning the nature of the Ca2+ transient that is not apparent from studies with cell populations.
使用以fura - 2作为Ca²⁺指示剂的荧光视频显微镜，在单个A10血管平滑肌细胞和大鼠肝细胞中测量了激动剂诱导的胞质游离Ca²⁺增加的动力学。在高激动剂浓度下，在血管加压素刺激的单个A10细胞或细胞群体中测量的Ca²⁺瞬变动力学没有差异。然而，用低于0.5 nM浓度的血管加压素刺激单个A10细胞会产生由两个不同峰值组成的特征性Ca²⁺瞬变。这两个峰值似乎代表了细胞内Ca²⁺释放和细胞外Ca²⁺内流之间的时间间隔。在用低浓度血管加压素或去甲肾上腺素刺激的单个大鼠肝细胞中未观察到双瞬变。在A10细胞和肝细胞中，响应次最大激动剂浓度时Ca²⁺浓度的初始增加速率在单个细胞中比在细胞群体中更快。这种差异是由于细胞反应的异步性，即在Ca²⁺浓度快速增加开始之前存在可变长度的潜伏期。潜伏期的持续时间取决于激动剂浓度，较高浓度的激动剂导致潜伏期缩短。用fura - 2在单个肝细胞中测量的激素刺激的Ca²⁺瞬变与先前使用水母发光蛋白作为Ca²⁺指示剂报道的一系列瞬态尖峰不同，这表明fura - 2和水母发光蛋白可能报告了受刺激细胞中Ca²⁺反应的不同方面。总的来说，这些结果表明，在单个细胞中测量Ca²⁺瞬变提供了有关Ca²⁺瞬变性质的新信息，而这些信息从细胞群体研究中并不明显。
