The Principle Investigator's interest has been the subject of calcium and phosphate transport across the intestinal epithelium during maturation. Initial studies using in vivo perfusion techniques and in vitro everted gut sacs has defined the overall picture of transport. During the tenure of this grant, we have validated and characterized plasma membranes (brush border and basolateral membranes) for the use in transport studies during development. These studies have elucidated the developmental processes by which calcium and phosphate enter the intestinal epithelial cell across the brush border membranes, and how they exit across the basolateral membranes. The role of vitamin D in regulating these processes has been probed. Because calcium concentration in the cell is in the micromolar range, a buffering mechanism must exist within the cell to allow transcytosolic calcium homeostasis. The mechanisms of both transcytosolic calcium movement and maintenance of cytosolic calcium homeostasis in intestinal epithelial cells remain largely unknown. It is generally thought that the mitochondrion, Golgi, lysosomes, endoplasmic reticulum, and calcium binding proteins, play significant roles in cytosolic calcium storage and buffering. The appearance of calcium binding proteins (CaBP) in adult animals has been shown to depend on binding of 1,25(OH)2D3 to a nuclear receptor coding for mRNA to synthetize CaBP. Our studies, and those of others, have shown no role of vitamin D in the process of calcium and phosphate entry across the brush border membranes. Moreover, CaBP does not appear in response to vitamin D in suckling rats. We hypothesize that subcellular organelles (i.e., Golgi apparatus, endoplasmic reticulum, mitochondria and lysosomes) play a major role in transcytosolic movement and storage of calcium during maturation. Moreover, the role of these organelles in phosphate homeostasis is not known. The current proposal is designed to extend our studies on plasma membranes to subcellular movement of calcium and phosphate during maturation. These studies will utilize established techniques to isolate subcellular fractions and define the role of these organelles in Ca++ and Pi transcytosolic movement and its regulation by vitamin D. The proposal is a logical extension of in vivo transport studies which were extended to plasma membranes and now will be extended to the level of the intracellular organelles.
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