The effects of a vitamin D deficiency on insulin and glucagon release was determined in the isolated perfused rat pancreas by radioimmunoassay of the secreted proteins. During a 30-minute period of perfusion with glucose and arginine, pancreases from vitamin D-deficient rats exhibited a 48 percent reduction in insulin secretion compared to that for pancreases from vitamin D-deficient rats that had been replenished with vitamin D. Vitamin D status had no effect on pancreatic glucagon secretion. This result, along with the previously demonstrated presence in the pancreas of a vitamin D-dependent calcium-binding protein and cytosol receptor for the hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, indicates an important role for vitamin D in the endocrine functioning of the pancreas.

The vitamin D-dependent calcium-binding protein (CaBP) was localized by immunocytochemistry in the rat and human kidney. In both species 80% of the cells lining the distal convoluted tubules contained CaBP. In the connecting segment and the initial collecting tubule of rat kidney, 50% of the cells was positive; in the outer medullary collecting duct only 15% was positive. In the human kidney, collecting ducts in medullary rays contained 50% positive cells, whereas in the rest of the medulla no positive cells were found. The CaBP-positive cells were identified as principal or clear cells by immunoelectron-microscopy, using the protein A-gold technique. Mitochondria-rich dark cells were negative. In principal cells, CaBP immunoreactive sites were found throughout the cytosol and the nuclear euchromatin. No preferential labeling of cellular membranes was found. The data show that CaBP-positive cells are present in tubular regions that are important in regulating the final excretion of calcium. However, the subcellular distribution of CaBP does not suggest a role in the initial transmembrane transport of Ca2+ but rather indicates an involvement in processes regulating intracellular calcium.

The vitamin D-dependent calcium binding protein (CaBP) of chick duodenum has been localized by immunocytochemistry and by radioimmunoassay. Light microscopically, CaBP was seen to be present in the absorptive cells of the villi while in other cell types of the villi and the crypts, including goblet cells and endocrine cells, no CaBP was seen. At the electron microscopic level, CaBP was shown to be localized in the cytosol and the euchromatin of the nucleus but not in membrane-bounded cytoplasmic compartments. Quantitative evaluation of the immunocytochemical protein A-gold label showed that the terminal web and the cytosol of basal cellular regions were most highly labeled while the brush border was weakly labeled. The radioimmunoassay evaluation of intestinal subcellular fractions indicated that 96% of the homogenate CaBP is in the cytosol high-speed supernatant fraction. Collectively, these results support the hypothesis that the vitamin D-dependent intestinal CaBP may play a role in either regulation of intracellular calcium concentration or movement of calcium across the epithelial cell, rather than directly in the initial step(s) of calcium absorption across the brush border membrane from the gut lumen.The intestinal absorption of calcium is dependent upon continuous access to vitamin D (calciferol) and its daughter metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2Da] (29). 1,25-(OH)2Da functions in a fashion analogous to that of classic steroid hormones to induce the biosynthesis of cellular components essential for an efficient calcium translocation process, including a calcium binding protein (CaBP, references 40,43,44). The steady-state level of intestinal CaBP is known to be affected by the dietary level of calcium and phosphorus (17). There is a good correlation between the absolute level of CaBP (l-3% of the soluble cellular protein) and both the occupancy of the intestinal 1,25(OH)2D3 receptors (2, 20) and the stimulation of intestinal calcium transport (24, 37).Thus, there is little doubt that the vitamin D-dependent intestinal CaBP is associated with intestinal calcium uptake (28, 29) but the mechanism of its action at the cellular and molecular level and its localization within the intestinal mucosa are controversial. It has been suggested by Taylor and Wasserman (42) that CaBP is produced in goblet cells and located in the brush border of the absorptive columnar cells while others (1,21,25,26) have found it to be present inside the absorptive columnar cells.This controversy surrounding the localization and role of the

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Induction of terminal differentiation of leukemic and preleukemic cells is a therapeutic approach to leukemia and preleukemia. The 1 alpha, 25- dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active form of vitamin D3, can induce differentiation and inhibit proliferation of leukemia cells, but concentrations required to achieve these effects cause life-threatening hypercalcemia. Seven new analogs of 1,25(OH)2D3 were discovered to be either equivalent or more potent than 1,25(OH)2D3 as assessed by: (a) inhibition of clonal proliferation of HL-60, EM-2, U937, and patients' myeloid leukemic cells: and (b) induction of differentiation of HL-60 promyelocytes. Furthermore, these analogs stimulated clonal growth of normal human myeloid stem cells. The most potent analog, 1,25-dihydroxy-16ene-23yne-vitamin D3, was about fourfold more potent than 1,25(OH)2D3. This analog decreased clonal growth and expression of c-myc oncogene in HL-60 cells by 50% within ten hours of exposure. Effects on calcium metabolism of these novel analogs in vivo was assessed by intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Each of the analogs mediated markedly less (10 to 200-fold) ICA and BCM as compared with 1,25(OH)2D3. To gain insight into the possible mechanism of action of these new analogs, receptor binding studies were done with 1,25(OH)2–16ene-23yne-D3 and showed that it competed only about 60% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in HL-60 cells and 98% as effective as 1,25(OH)2D3 for receptors present in chick intestinal cells. In summary, we have discovered seven novel vitamin D analogs that are more potent than the physiologic 1,25(OH)2D3 as measured by a variety of hematopoietic assays. In contrast, these compounds appear to have the potential to be markedly less toxic (induction of hypercalcemia). These novel vitamin D compounds may be superior to 1,25(OH)2D3 in a number of clinical situations including leukemia/preleukemia; they will provide a tool to dissect the mechanism of action of vitamin D seco-steroids in promoting cellular differentiation.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes.

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