Goldman W F, Bova S, Blaustein M P
Department of Physiology, University of Maryland School of Medicine, Baltimore.
Cell Calcium. 1990 Feb-Mar;11(2-3):221-31. doi: 10.1016/0143-4160(90)90073-4.
A rise in cytosolic free Ca2+ is the immediate trigger for contraction in vascular smooth muscle (VSM). We employed the fluorescent Ca2(+)-indicator, Fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in cultured A7r5 cells and the changes evoked by activation with 5-HT. Several methodological considerations that affect the temporal and spatial resolution of Ca2+ images have been addressed. These include: cytoplasmic distribution of Fura-2, wavelength selection for ratio imaging, signal:noise ratio measurement and the effect of [Ca2+] on the limits of detectability under conditions in which [Ca2+] is changing. The distribution of apparent free Ca2+, [Ca2+]App, in A7r5 cells was heterogeneous. This reflects, in part, different pools of intracellular Ca2+. [Ca2+]App was lowest in the nucleus (113 +/- 14 nM; n = 20 cells) and highest in the organelle-rich perinuclear region (228 +/- 12; n = 20), while the surrounding cytoplasmic area (containing relatively few organelles) had intermediate [Ca2+]app levels (150 +/- 13; n = 20). 5-HT (1 microM) evoked transient increases in [Ca2+]App that began within 11 s as relatively modest elevations of [Ca2+]App in the periphery, near the sarcolemma, and subsequently spread to the entire cell, reaching a peak within 18-24 s. At the peak of the Ca2+ transients, [Ca2+]App was highest in the perinuclear region where it sometimes exceeded the maximal detectable levels of the system (1.9 microM). The average peak Ca2+ transient amplitude in the non-nuclear cytoplasm was 1083 +/- 208 nM (1 microM 5-HT; n = 20 cells). Despite the continued presence of 5-HT following the Ca2+ transients, [Ca2+]App then returned to pre-stimulation levels within 5 min. These observations indicate that digital imaging microscopy enables the study of subcellular regulation of intracellular Ca2+ in VSM. The results provide new insights into the role of localized changes in Ca2+ in the regulation of VSM contractility.
胞质游离Ca2+浓度升高是血管平滑肌（VSM）收缩的直接触发因素。我们使用荧光Ca2+指示剂Fura-2和数字成像显微镜来研究培养的A7r5细胞内Ca2+的空间分布以及5-羟色胺（5-HT）激活所引起的变化。我们还探讨了影响Ca2+图像时间和空间分辨率的几个方法学因素。这些因素包括：Fura-2在细胞质中的分布、比率成像的波长选择、信噪比测量以及在Ca2+浓度变化条件下Ca2+对可检测极限的影响。A7r5细胞中表观游离Ca2+（[Ca2+]App）的分布是不均匀的。这部分反映了细胞内不同的Ca2+池。[Ca2+]App在细胞核中最低（113±14 nM；n = 20个细胞），在富含细胞器的核周区域最高（228±12；n = 20），而周围细胞质区域（细胞器相对较少）的[Ca2+]App水平处于中间值（150±13；n = 20）。5-HT（1 microM）引起[Ca2+]App的瞬时升高，在11秒内开始，最初是肌膜附近外周区域[Ca2+]App相对适度的升高，随后扩散到整个细胞，在18 - 24秒内达到峰值。在Ca2+瞬变的峰值时，[Ca2+]App在核周区域最高，有时超过系统的最大可检测水平（1.9 microM）。非核细胞质中Ca2+瞬变的平均峰值幅度为1083±208 nM（1 microM 5-HT；n = 20个细胞）。尽管在Ca2+瞬变后5-HT持续存在，但[Ca2+]App随后在5分钟内恢复到刺激前水平。这些观察结果表明，数字成像显微镜能够研究VSM中细胞内Ca2+的亚细胞调节。这些结果为Ca2+局部变化在VSM收缩性调节中的作用提供了新的见解。
