Nemere I, Leathers V, Norman A W
J Biol Chem. 1986 Dec 5;261(34):16106-14.
A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.
多种肠道细胞细胞器和蛋白质被认为可介导1,25 - 二羟基维生素D3（1,25-(OH)2D3）刺激的钙吸收。在本研究中，进行了生化分析，以确定在实验前15小时给维生素D缺乏的雏鸡注射1.3 nmol的1,25-(OH)2D3或载体后，体内钙转运后45Ca的亚细胞定位。通过差异沉降，随后在Percoll梯度中离心，并评估适当的标记酶活性，实现了上皮匀浆中高尔基体、线粒体、基底外侧膜和溶酶体组分的分离。维生素D缺乏和1,25-(OH)2D3处理的雏鸡在溶酶体组分中的45Ca比活性水平最高。溶酶体也是唯一显示1,25-(OH)2D3介导的钙含量差异的细胞器，增加到对照的138%。从1,25-(OH)2D3处理的雏鸡制备的溶酶体中免疫反应性钙结合蛋白-D28k（钙结合蛋白）的水平也最高。测试了已知会干扰溶酶体功能的药物氯喹，发现它能抑制1,25-(OH)2D3刺激的肠道钙吸收。1,25-(OH)2D3和氯喹均不影响[3H]2O转运。在另外的实验中，微粒体膜（105,000×g沉淀）进行梯度离心。在1,25-(OH)2D3处理的雏鸡的材料中，45Ca比活性和钙结合蛋白的最高水平出现在比内质网密度更高的组分中，可能代表内吞小泡。在对暴露于肠腔Ca2+或Ca2+加氯喹30分钟后分级分离的1,25-(OH)2D3处理的鸟类的肠黏膜研究中，发现相对于对照，45Ca在溶酶体和假定的内吞小泡中积累。提出了一种涉及小泡流动的机制来解释1,25-(OH)2D3介导的肠道钙转运。Ca2+的内吞内化、小泡与溶酶体的融合以及在基底外侧膜的胞吐作用完成了转运过程。
