INTESTINAL CA2+ TRANSPORT: VITAMIN D-DEPENDENT MECHANISM

在以往的维生素D依赖性钙转运机制的研究中， 我们从大鼠小肠中制备了膜囊泡， 上皮细胞（肠上皮细胞），并观察到高尔基膜囊泡 与外侧-基底（L-B）相比， 和微绒毛（MV）膜囊泡。 这种Ca 2+摄取是特异性的， 依赖于维生素D的活性代谢物1，25（OH）2D 3。 最近 由该基金支持的研究表明，这种Ca 2+吸收是由于 与膜中非酯化脂肪酸（NEFA）结合， 与磷脂酶A活性增加有关。 制备的膜 分离的肠上皮细胞的Ca 2+结合明显降低， 证明了维生素D依赖的ATP需要的Ca 2+摄取，特别是 十二指肠绒毛细胞（与隐窝细胞相比）。 追求 这些发现进一步我们提出：1）定义NEFA的作用， 磷脂和磷脂酶的分子机制 在肠道吸收过程中螯合钙，2）净化 L-B膜Ca ~（2+）-ATP酶可能是ATP依赖性Ca ~（2+） 摄取，以及3）研究1，25（OH）2D 3 影响Ca 2 +-ATP酶功能的基因组机制或通过 酶的翻译后激活。 我们还将尝试确定 如果1，25-（OH）2D 3刺激高尔基体中蛋白质的合成或出现， L-B膜对脂质代谢有重要作用。 1，25（OH）2D 3的作用 对Ca ~（2+）-ATP酶mRNA的影响。 这些参数的变化 将强调与肠上皮细胞分化相关的。 由于 是1，25（OH）2D 3依赖性钙转运的证据，事实上， 依赖于肠上皮细胞的分化，它似乎是一个理想的系统， 增加我们对分化和组织结构的理解， 与特定肠道功能的关系。 调节肠道 钙转运是钙稳态的基础。 因此这些 研究将有助于理解骨质疏松症的病理生理学， 肾结石和肠道疾病对钙吸收的影响 和钙代谢。

In previous studies on the mechanism of vitamin D-dependent Ca2+ transport across the intestine, we prepared membrane vesicles from rat intestinal epithelial cells (enterocytes) and observed that Golgi membrane vesicles demonstrated the highest Ca2+ uptake when compared with lateral-basal (L-B) and microvillus (MV) membrane vesicles. This Ca2+ uptake was specifically dependent on 1,25(OH)2D3, the active metabolite of vitamin D. Recent studies, supported by this grant, have shown that this Ca2+ uptake was due to binding to non-esterified fatty acids (NEFAs) in the membranes and was associated with an increased phospholipase A activity. Membranes prepared from isolated enterocytes had markedly decreased Ca2+ binding and better demonstrated a vitamin D-dependent ATP-requiring Ca2+ uptake, particularly for duodenal villus cells (as contrasted with crypt cells). To pursue these findings further we propose: 1) to define the role of NEFAs, phospholipids and phospholipases in the molecular mechanisms of sequestering calcium during its absorption by the intestine, 2) to purify the Ca2+-ATPase of L-B membrane presumed responsible for ATP-dependent Ca2+ uptake, and 3) to study the molecular mechanisms by which 1,25(OH)2D3 effects Ca2+-ATPase function by either a genomic mechanism or through a post-translational activation of the enzyme. We will also try to determine if 1,25-(OH)2D3 stimulates the synthesis or appearance of proteins in Golgi and L-B membrane important to lipid metabolism. The effect of 1,25(OH)2D3 on the mRNA for Ca2+-ATPase will be studied. Changes in these parameters as related to enterocyte differentiation will be emphasized. Since there is evidence that 1,25(OH)2D3-dependent calcium transport is, in fact, dependent on enterocyte differentiation, it seems an ideal system for increasing our understanding of differentiation and tissue organization in relationship to a specific intestinal function. Regulation of intestinal calcium transport is fundamental to calcium homeostasis. Therefore, these studies will help in understanding the pathophysiology of osteoporosis, kidney stones, and the effects of intestinal diseases on calcium absorption and calcium metabolism.