Waybill M M, Yelamarty R V, Zhang Y L, Scaduto R C, LaNoue K F, Hsu C J, Smith B C, Tillotson D L, Yu F T, Cheung J Y
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Am J Physiol. 1991 Jul;261(1 Pt 1):E49-57. doi: 10.1152/ajpendo.1991.261.1.E49.
Ca2+ concentrations ([Ca2+]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca2+] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca2+ (210 +/- 15 nM) was 1.6- to 2-fold higher than nuclear Ca2+ (128 +/- 12 nM). This difference in Ca2+ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca2+; however, the nuclear-to-cytosolic Ca2+ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca2+ permeability barriers and Ca2+ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca2+ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca(2+)-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.
利用三维光学切片显微镜测定了用fura - 2负载的培养大鼠肝细胞胞质溶胶和细胞核中的Ca2 +浓度（[Ca2 +]）。在确定了荧光显微镜耦合数字视频成像系统的经验性三维点扩散函数后，使用最近邻方法（NNA）算法通过去卷积去除了感兴趣平面上方和下方光学平面产生的杂散光。尽管去卷积在单波长激发图像中荧光强度测定的准确性方面有显著提高，并且细胞区室之间的边界也更清晰，但复杂的数学过程并没有显著提高从比率（双波长激发比率）图像得出的[Ca2 +]值的精度。在静息肝细胞中，胞质溶胶Ca2 +（210±15 nM）比细胞核Ca2 +（128±12 nM）高1.6至2倍。在原始比率图像和用NNA算法处理的图像中均检测到两个区室之间Ca2 +的这种差异。添加精氨酸加压素或表皮生长因子导致胞质溶胶和细胞核Ca2 +均显著增加（2至3倍）；然而，在用有丝分裂原刺激的肝细胞中，细胞核与胞质溶胶的Ca2 +梯度得以保留。我们得出结论，肝细胞核膜含有可能对激素敏感的Ca2 +通透性屏障和Ca2 +转运机制。我们推测细胞核Ca2 +的增加可能在有丝分裂原诱导的细胞增殖调节中起重要作用，可能是通过激活Ca（2 +）依赖性核酸内切酶、核钙调蛋白或核蛋白激酶C来实现的。
