Dynamic digital fluorescence ratio imaging of cell calcium in vascular endothelial cells.

C Y Kwan, T K Kwan
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Abstract

Aim: To study the spatial and temporal distribution of intracellular Ca2+ concentration in cultured bovine pulmonary artery endothelial (BPAE) cells.

Methods: Cultured BPAE cells were loaded with Fura-2 and observed under an inverted microscope coupled to a microfluorimeter, which enables pixel-to-pixel ratio imaging of the BPAE cells in real time.

Results: Addition of Ca2+ 1-2 mmol.L-1 to BPAE cells, which were exposed to Ca(2+)-free medium containing egtazic acid, resulted in a transient elevation of cytosolic Ca2+ concentration, which rapidly returned to the resting level. Biphasic elevation (a larger transient phase followed by a smaller sustained phase) of intracellular Ca2+ concentration was observed upon the addition of ATP (via activation of surface membrane receptor). 4-Chloro-3-ethyl phenol (CEP; an activator of Ca(2+)-induced Ca2+ channels) potently induced elevation of Ca2+ level. Cyclopiazonic acid (CPA; an inhibitor of endoplasmic reticulum Ca(2+)-ATPase pump) offered a more sustained elevation of Ca2+. In most cases, the highest level of Ca2+ elevation was observed around the cell peripheries, sometimes at rest and particularly upon stimulation. Ca2+ elevation associated with nuclear complex seemed to be higher compared to that in the cytosolic compartment.

Conclusion: Changes of cell Ca2+ upon stimulation by various agents that acted at different intracellular sites were found to be temporarily and spatially heterogenous among BPAE cells. At the single cell level, Ca2+ elevation seemed to occur initially near the peripheral region followed by the nuclear region. This study raised the possibility that nuclear Ca2+ and cytosolic Ca2+ might be regulated independently in BPAE cells.

血管内皮细胞钙的动态数字荧光比成像。
目的:研究培养的牛肺动脉内皮(BPAE)细胞内Ca2+浓度的时空分布。方法:将培养的BPAE细胞负载Fura-2,在倒置显微镜下与微荧光仪耦合观察,实现BPAE细胞的实时像素比成像。结果:添加Ca2+ 1-2 mmol。L-1至BPAE细胞暴露于含egtaic酸的无Ca(2+)培养基中,导致胞质Ca2+浓度瞬间升高,并迅速恢复到静息水平。在添加ATP(通过激活表面膜受体)后,观察到细胞内Ca2+浓度的双相升高(较大的短暂期随后较小的持续期)。4-氯-3-乙基苯酚;一种Ca(2+)诱导的Ca2+通道的激活剂)能诱导Ca2+水平的升高。环吡唑酸;一种内质网Ca(2+)- atp酶泵抑制剂)提供了更持续的Ca2+升高。在大多数情况下,在细胞周围观察到最高水平的Ca2+升高,有时在休息时,特别是在刺激时。与核复合体相关的Ca2+升高似乎高于细胞质室。结论:在BPAE细胞中,受作用于细胞内不同部位的各种药物的刺激,细胞Ca2+的变化具有暂时性和空间异质性。在单细胞水平,Ca2+升高似乎首先发生在外周区域附近,然后是核区域。本研究提出了核Ca2+和胞质Ca2+可能在BPAE细胞中独立调节的可能性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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