CONFOCAL MICROSCOPY TO ANALYZE CYTOSOLIC AND NUCLEAR CALCIUM IN CULTURED VASCULAR CELLS
CONFOCAL MICROSCOPY TO ANALYZE CYTOSOLIC AND NUCLEAR CALCIUM IN CULTURED VASCULAR CELLS
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DOI：
10.1152/ajpcell.1994.266.4.c1118
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发表时间：
1994-04-01
影响因子：
--
通讯作者：
BRUNNER, HR
中科院分区：
文献类型：
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作者：
BURNIER, M;CENTENO, G;BRUNNER, HR
随着钙敏感荧光染料和视频显微镜成像的发展，一些研究人员已经定位了细胞内钙的变化在细胞质中，在核周区域，并可能在核中。然而，由于来自核周区的荧光干扰了核的荧光，因此通常难以确定核中钙的存在。我们已经使用共聚焦显微镜连同两个钙敏感染料[乙酰氧基甲酯的烟道3（烟道3-AM）和rhod 2（rhod 2-AM）]，以分析研究在休息和刺激后，与受体依赖性（血管紧张素，加压素）和受体非依赖性（KCl）刺激的血管平滑肌和内皮细胞的胞浆和核钙分布。对于flue 3-AM，基线荧光位于细胞质中，但在细胞核中略高。在所有刺激下，两个隔室中的荧光强度都增加，但在细胞核内仍然更明显。然而，校准后，细胞溶质钙浓度大于在休息时的细胞核，并同样高刺激后，表明不同的性质的烟道3在细胞溶质和细胞核中。对于rhod 2-AM，细胞核中的基线荧光较低，细胞质中的基线荧光较高。细胞刺激引起细胞溶质钙的初始增加，细胞核中没有变化，随后两个隔室中的升高。因此，血管细胞的刺激与胞质和核钙的显著增加有关。Flue 3-AM比Rhod 2-AM更能反映细胞核内钙的变化。血管紧张素II和加压素引起的细胞核内钙离子的增加可能与其细胞增殖作用有关。
With the development of calcium-sensitive fluorescent dyes and videomicroscopic imaging, several investigators have located the changes in intracellular calcium in the cytoplasm, in the perinuclear region, and possibly in the nucleus. However, the presence of calcium in the nucleus is often difficult to ascertain because the fluorescence derived from the perinuclear area interferes with that of the nucleus. We have used confocal microscopy together with two calcium-sensitive dyes [acetoxymethyl esters of flue 3 (flue 3-AM) and rhod 2 (rhod 2-AM)] to analyze the cytosolic and nuclear calcium distribution in vascular smooth muscle and endothelial cells studied at rest and after stimulation with receptor-dependent (angiotensin, vasopressin) and receptor-independent (KCl) stimuli. With flue 3-AM, the baseline fluorescence was located in the cytoplasm but was slightly higher in the nucleus. With all stimuli, the fluorescence intensity increased in both compartments but remained more pronounced within the nucleus. Yet, after calibration, the cytosolic calcium concentration was greater than that of the nucleus at rest and was equally high after stimulation, suggesting different properties of flue 3 in the cytosol and in the nucleus. With rhod 2-AM, baseline fluorescence was low in the nucleus and high in the cytosol. Cell stimulation caused an initial increase in cytosolic calcium with no change in the nucleus followed by a rise in both compartments. Thus the stimulation of vascular cells is associated with marked increases in cytosolic and nuclear calcium. Flue 3-AM seems to be a better indicator of nuclear calcium than rhod 2-AM. The increases in nuclear calcium induced by angiotensin II and vasopressin may contribute to their cell proliferative effect.