The kidneys are thought to be the only organs capable of 1 alpha-hydroxylation of vitamin D and its metabolites. We have examined the in vivo conversion of 3H-(25,26)-25-hydroxyvitamin D3(25OHD3) to 3H-(25,26)-1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] in vitamin D-deficient, pregnant and nonpregnant rats. As expected, nephrectomy of nonpregnant, vitamin D-deficient rats prevented the conversion of 25OHD3 to 1 alpha,25(OH)2D3. In contrast, nephrectomy of pregnant, vitamin D-deficient rats reduced but did not abolish the formation of 1 alpha,25(OH)2D3 from its precursor. The identity of the radioactive metabolite formed from 3H-25OHD3 which circulated in nephrectomized, pregnant rats was established as 1 alpha,25(OH)2D3 by comigration with synthetic 1 alpha,25(OH)2D3 on high-pressure liquid chromatography. The simultaneous absence of 1 alpha,25(OH)2D3 in the fetal kidneys indicated that the site of 1 alpha-hydroxylation after nephrectomy of the pregnant rat was probably extra-renal in origin. Two sites of 1 alpha-hydroxylation of 25OHD3, one renal and the other extra-renal, either fetoplacental or maternal, may exist in the pregnant, vitamin D-deficient rat.

We studied the effect of 17f8-estradiol (E) on the proliferation and alkaline phosphatase activity of cultured UMR106 cells, a clonal osteoblastic cell line. Growth rates were reduced and alkaline phosphatase activity was increased in cells incubated for 2 days in medium containing E (10-8 M). In contrast, E had no effect on the growth rates or alkaline phosphatase of a human fibroblastic cell line, S90E. The effect of E was not observed with low cell density or at confluence. 1,25-Dihydroxyvitamin D3 antagonized the response to E. Preincubation of the cells with dexamethasone, a potent inducer of differentiation, reversed the effect of E or 1,25-dihydroxyvitamin D3. These results indicate that cellular and/or extracellular factors such as cell density, the phase of the cell cycle, the state of differentiation, and the presence or absence of other steroids influenced the response of UMR106 cells to E. Serum was removed from the culture medium to minimize the effect of the steroids, growth factors, and nutrients present in serum. A striking stimulation of alkaline phosphatase by E occurred with serum-free conditions. This stimulation was biphasic over an E concentration from 101-2 to 10-8 M, with the peak response at 10-1' M. The action of E on UMR106 cells was metabolite-specific, since the isomer 17a-estradiol produced no effect on proliferation rates or alkaline phosphatase activity. The cyclic AMP response to parathyroid hormone (residues 1-34) was not altered by E treatment of these cells. In contrast, dexamethasone exposure did increase the cyclic AMP response to parathyroid hormone. These results demonstrate a direct effect of E on an osteoblastic cell line. They also raise the possibility that similar or identical actions of E occur in cultured normal osteoblasts.Estrogens influence the skeleton, as evidenced by the loss of bone density in postmenopausal women (1, 2) and the preventative action of exogenous estrogens on the loss of bone density in ovariectomized subjects (3). Estrogen administration also produces changes related to bone and mineral metabolism, such as increased intestinal absorption of calcium (4) and alterations in the circulating calciotropic hormones (4, 5). The mechanism of estrogens' action on the skeleton is still unknown. Most investigators have postulated an indirect pathway because of the apparent absence of estrogen receptors in bone (6-9). We report the results of experiments showing an effect of 17p-estradiol (E) on the UMR106 cell line, a well-characterized osteoblastic cell model (10-12). MATERIALS AND METHODSMaterials. The culture media and additives were obtained from the Tissue Culture Facility of the Lineberger Cancer Research Center, University of North Carolina. The additives included fetal bovine serum, penicillin, streptomycin, insulin, transferrin, selenium, and trypsin, the latter for release of adherent cells. E and 17a-estradiol (17a-E) and dexamethasone were purchased from Sigma. Parathyroid hormone, as the synthetic fragment consisting of amino acid res...

1. Intestinal phosphate absorption in human subjects was studied by the technique of triple lumen intestinal perfusion in vivo. 2. Ileal phosphate absorption increased as the intraluminal phosphate concentration was increased. 3. Ileal rates of phosphate absorption were lower at any given intraluminal phosphate concentration than previously described jejunal rates. Acidification of the ileal lumen did not increase phosphate absorption. 4. Phosphate absorption was shown in the jejunum to be dependent on the intraluminal sodium concentration. 5. Phosphate absorption in the human small intestine consists of at least two components, one directly proportional to water movement and the second apparently independent of water movement.

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The question of whether the skeleton metabolizes 25-hydroxycholecalciferol [25(OH)D3] to more-polar products was studied. Calvarial cells were dispersed from 18day old chicken embryos by using collagenase and then grown in culture in serum-free medium. Confluent cell cultures were incubated with 7 nM 25(OH3H3 for 2 hr, and the vitamin D metabolites were then extracted. At least four polar metabolites were produced. Based on separation by Sephadex LH-20 chromatography followed by hiNFressure liquid chromatography, two of these metabolites were identified as 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3. These metabolites were also produced by cultured kidney cells but not by liver, heart muscle, or skin cells isolated from the same embryos. The specific activities of the calvarial 1-and 24-hydroxylases were similar in magnitude to those in isolated kidney cells. The specific activity of the calvarial 25(OH)D3:1-hydroxylase was inhibited by an 8-hr preincubation with 1,25(OH)2D3, whereas the 24-hydroxylase was enhanced. It is concluded that (i) vitamin D metabolism by isolated cells is organ-specific, (ii) calvarial cells produce active metabolites of vitamin D in significant amounts, (lii) vitamin D metabolism by calvarial ce is reglated by 1,2Z(OHhD3, and (iv) locally produced, active meta lites could act locally, thereby adding a new dimension to the regulation of mineral metabolism by vitamin D metabolites. la,25-Dihydroxycholecalciferol [1,25(OH)2D3] is a potent mineral homeostatic hormone. It acts on the skeleton to mobilize bone mineral and on the intestine to enhance absorption of calcium and phosphorus (1-3). In addition, it appears to act on the kidney, in concert with parathyroid hormone (PTH), to promote calcium reabsorption (4). It was formerly believed that 1,25(OH)2D3 is synthesized exclusively in the kidney but recent evidence suggests that it may be synthesized in placenta as well (5-7). In this paper, we report that cells isolated from calvaria of chicken embryos and grown in culture metabolize 25-hydroxycholecalciferol [25(OH)D3] to several more-polar metabolites, including 1,25(OH)2D3. These findings suggest that the tissue distribution of 1,25(OH)2D3 production may be wider than previously thought and that locally produced as well as systemically derived hormone may be important in mediating vitamin D action in target organs such as the skeleton.MATERIALS AND METHODS Isolation of Cells. Cells were isolated from calvaria of 15-to 16-day chicken embryos (Gallus gallus, Fors Farms Hatchery, Puyallup, WA) by a modification of the method developed for fetal rat calvaria (8). Briefly, the frontal and parietal bones and intervening suture line area were aseptically dissected, rinsed, and incubated for up to 120 min in Tris-HCI/NaCl, pH 7.4 (four calvaria per ml) containing collagenase (2 mg/ml; Worthington, type II). The cells were recovered as described (8), washed twice in enzyme-free Hanks' buffer (pH 7.4), and diluted in BGJb medium (Fitton-...