Studies on the role of 1,25-dihydroxyvitamin D in chick embryonic development

Vitamin D-deficient laying hens were repleted with 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3. Egg production returned to normal for both groups of hens by the third week. Eggs from hens fed either 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3 contained 1,25-dihydroxy[26,27-3H]vitamin D3. Eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 contained substantial amounts of 25-hydroxy[26,27-3H]vitamin D3, while those from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained none. Plasma from 18-day embryos from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained little or no 1,25-dihydroxy[26,27-3H]vitamin D3, while that from 18-day embryos from hens given 25-hydroxy[26,27-3H]vitamin D3 had normal levels of 1,25-dihydroxy[26,27-3H]vitamin D3. No eggs from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 hatched, while eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 achieved a hatchability of 90%. It appears that embryos from hens maintained on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D are essentially vitamin D deficient.     

Stabilization of 1,25-dihydroxyvitamin D-3 receptor in the human leukemia cell line, HL-60, with diisopropylfluorophosphate

We have investigated the reason for the lack of specific 1,25-dihydroxyvitamin D-3 binding activity in extracts of ATCC HL-60 cells. Although intact ATCC HL-60 cells specifically and saturably take up 1,25-dihydroxy[3H]vitamin D-3, whole cell extracts have little or no specific binding of 1,25-dihydroxyvitamin D-3. The absence of specific binding can now be explained by the action of a serine proteinase in these cells. When diisopropylfluorophosphate (DFP), a potent inhibitor of serine proteinase, is added to the buffer used for extraction, specific binding of 1,25-dihydroxy[3H]vitamin D-3 in the extract is observed. The loss of specific binding could not be prevented by hydrolyzed DFP or other serine proteinase inhibitors, such as phenylmethylsulfonylfluoride, benzamidine and aprotinin. The proteolytic activity from ATCC cells also destroyed specific 1,25-dihydroxy[3H]vitamin D-3 binding in high-salt extracts from pig intestinal nuclei or from another HL-60 cell line (LG HL-60 cells). However, the proteinase did not affect the levels of the specific binding in these preparations if the receptor was occupied with 1,25-dihydroxy[3H]vitamin D-3 prior to exposure to the proteinase. The binding and sedimentation characteristics of the receptors from various sources were not changed by the presence of DFP. The Kd of the receptor in ATCC HL-60 cells is 1.2.10(-10) M, which is identical to that in the LG HL-60 cells. The 1,25-dihydroxy[3H]vitamin D-3 receptor complex from the ATCC cells sediments as a single 3.5 S component and elutes from DNA-Sephadex column in two peaks at 0.09 and 0.15 M KCl. The material eluting at 0.15 M KCl has the same DNA-binding activity as preparations from pig intestine or LG HL-60 cells. Immunoprecipitation studies demonstrated that monoclonal antibodies to the pig receptor, IVG8C11, quantitatively precipitate the 1,25-dihydroxy[3H]vitamin D-3-binding activity from ATCC HL-60 cells as well as that from LG HL-60 cells or pig intestinal nuclei. Therefore, the previous failure to demonstrate the 1,25-dihydroxyvitamin D-3 receptor in ATCC HL-60 cells is because of the presence of a potent serine proteinase and not because of an abnormal or absent receptor.     

The plasma binding protein for vitamin D is a site of discrimination against vitamin D-2 compounds by the chick

The binding of 25-hydroxy-[26,27-3H]vitamin D-3 and 25-hydroxy-[26,27-3H]vitamin D-2 to the vitamin D binding protein in the plasma of both rats and chicks has been studied. In the case of rats, sucrose density gradient analysis, competitive displacement, and Scatchard analysis demonstrate that 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-2 are bound equally well to the vitamin D binding protein. In contrast, 25-hydroxyvitamin D-2 is poorly bound, while 25-hydroxyvitamin D-3 is tightly bound to the vitamin D binding protein in chick plasma. On the other hand, the chick intestinal receptor binds 1,25-dihydroxyvitamin D-2 and 1,25-dihydroxyvitamin D-3 equally well with a KD of 7.10(-11) M for both compounds. These results strongly suggest that the failure of the plasma transport protein in chicks to bind the vitamin D-2 compounds may be responsible for their relative ineffectiveness in these animals.     

Studies on the site of 1,25-dihydroxyvitamin D3 synthesis in vivo

Anephric, vitamin D-deficient male rats were injected with a physiologic dose of 25-hydroxy[26,27-3H]vitamin D3 (specific activity of 160 Ci/mmol), and 18-20 h later, intestine, bone, and serum were analyzed by high performance liquid chromatography for 1,25-dihydroxy-[26,27-3H]vitamin D3. Identical studies were carried out using sham-operated rats and rats with ligated ureters. No 1,25-dihydroxy[26,27-3H]vitamin D3 was detected in the tissues from anephric rats, while large amounts were detected in sham-operated and ureteric ligated controls. This result demonstrates that in the nonpregnant rat, 1,25-dihydroxyvitamin D3 is either not synthesized or is synthesized in vanishingly small amounts in bone and intestine in vivo, casting considerable doubt of the physiological importance of reports of in vitro synthesis of 1,25-dihydroxyvitamin D3 by cells in culture derived from bone and elsewhere.     

Metabolism of 1,25-dihydroxyvitamin D3: evidence for side-chain oxidation.

Approximately 7% of a 650-pmol dose of 25-hydroxyl[26,27-14C]vitamin D3 and 25% of a 325-pmol dose of 1,25-dihydroxyl[26,27-14C]vitamin D3 are metabolized to 14CO2 by vitamin D deficient rats. Nephrectomy prevents the metabolism of 25-hydroxy[26,27-14C]vitamin D3 to 14CO2 but not that of 1,25-dihydroxy[26,27-14C]vitamin D3. Less than 5% of the 14C from 24,25-dihydroxy[26,27-14C]vitamin D3 is metabolized to 14CO2. Feeding diets high in calcium and supplemented with vitamin D3 markedly diminishes the amount of 14CO2 formed from 25-hydroxy[26,27-14C]vitamin D3 but not that from 1,25-dihydroxyl[26,27-14C]vitamin D3. These results provide strong evidence that only 1-hydroxylated vitamin D compounds and especially 1,25-dihydroxyvitamin D3 undergo side-chain oxidation and cleavage to yield an unknown metabolite and CO2.     

Synthesis of 25-hydroxy-[26,27-3H]vitamin D2, 1,25-dihydroxy-[26,27-3H]vitamin D2 and their (24R)-epimers

Synthesis of a C-24-epimeric mixture of 25-hydroxy-[26,27-3H]vitamin D2 and a C-24-epimeric mixture of 1,25-dihydroxy-[26,27-3H]vitamin D2 by the Grignard reaction of the corresponding 25-keto-27-nor-vitamin D2 and 1 alpha-acetoxy-25-keto-27-nor-vitamin D3 with tritiated methyl magnesium bromide is described. Separation of epimers by high-performance liquid chromatography afforded pure radiolabeled vitamins of high specific activity (80 Ci/mmol). The identities and radiochemical purities of 25-hydroxy-[26,27-3H[vitamin D2 and 1,25-dihydroxy-[26,27-3H]vitamin D2 D2 were established by cochromatography with synthetic 25-hydroxyvitamin D2 or 1,25-dihydroxyvitamin D2. Biological activity of 25-hydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the rat plasma binding protein for vitamin D compounds, and by its in vitro conversion to 1,25-dihydroxy-[26,27-3H]vitamin D2 by kidney homogenate prepared from vitamin D-deficient chickens. The biological activity of 1,25-dihydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3.     

Metabolism and biologica.

24R,24,25-Dihydroxyvitamin D3 is capable of inducing a minimal intestinal calcium transport response in chicks when compared to an equal amount of 25-hydroxyvitamin D3. 1,24,25-Trihydroxyvitamin D3 is also less active than 1,25-dihydroxyvitamin D3, and its activity is much shorter lived than that of 1,25-dihydroxyvitamin D3. A comparison of the metabolism of 25-hydroxy[26,27-3H]vitamin D3 and 24,25-dihydroxy[26,27-3H]vitamin D3 in the rat and chick shows that 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 disappear at least 10 times more rapidly from the blood and intestine of chicks. Furthermore, examination of the excretory products from both of these species demonstrates that chicks receiving a single dose of 24,25-dihydroxy[26,27-3H]vitamin D3 excrete 66% of the total radioactivity by 48 hours, whereas rats receiving the same dose excrete less than one-half that amount. These results demonstrate that 24,25-dihydroxyvitamin D3 is considerably less biologically active in the chick than in the rat, probably due to more rapid metabolism and excretion.     

A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase

A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy[26,27-3H]vitamin D3 and 1,25-dihydroxy[26,27-3H]vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase.     

Isolation and characterization of 1 alpha-hydroxy-23-carboxytetranorvitamin D: a major metabolite of 1,25-dihydroxyvitamin D3.

The in vivo side-chain oxidation of 1 alpha,25-dihydroxyvitamin D3 was investigated by using a double-label radiotracer technique. Rats dosed with 1 alpha,25-dihydroxy-[3 alpha-3H]vitamin D3 and 1 alpha,25-dihydroxy[26,27-14C]vitamin D3 produced compounds with a high 3H/14C ratio. These compounds were found in sizable quantities in intestine and liver within 3 h after dosing. The major side-chain oxidized metabolite migrated as an acid on DEAE-Sephadex chromatography and contained no 14C. Methyl esterification of this compound with diazomethane proceeded in good yield and rendered the compound more amenable to chromatographic purification. The metabolite was isolated in several steps from rats dosed with 1 microgram of 1 alpha,25-dihydroxy[3 alpha-3H]vitamin D3. The metabolite was obtained in pure form as the methyl ester and was positively identified as 1 alpha,3 beta-dihydroxy-24-nor-9,10-seco-5,7,10(19)cholatrien-23-oic acid. The trivial name calcitroic acid is proposed for this major side-chain oxidized metabolite of 1,25-dihydroxyvitamin D3.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells

Ketoconazole (an inhibitor of vitamin D-24 hydroxylase) was used to study the role of self-induced 1,25-dihydroxyvitamin D3 (1,25-D3) metabolism on cellular responsiveness to 1,25-D3. Eighteen hours of treatment with 1,25-dihydroxy-[26,27-methyl-3H]vitamin D3 (1,25-[3H]D3) increased total 1,25-D3 receptors (VDR) from 60 to 170 fmol mg/protein. In cells treated with both 1,25-[3H]D3 and ketoconazole, up-regulation of VDR was increased by 40% over that observed with cells receiving 1,25-[3H]D3 alone. Ketoconazole alone had no agonistic activity. Treatment of cells with 1 nM 1,25-[3H]D3 plus increasing doses of ketoconazole (0-30 microM) resulted in a dose-dependent increase in occupied VDR and total VDR. This up-regulation was associated with reduced 1,25-[3H]D3 catabolism. 1,25-[3H]D3-induced up-regulation of VDR typically peaked at 14 h and declined thereafter. Ketoconazole lengthened the time to reach peak VDR up-regulation to 20 h. The ability of ketoconazole to increase cell responsiveness (VDR up-regulation) was the result of both increased and prolonged occupancy of VDR by 1,25-[3H]D3. The t1/2 of occupied VDR was 2 h in the absence of ketoconazole and greater than 7 h when ketoconazole was present. Collectively, these results suggested that self-induced catabolism of 1,25-D3 is an important regulator of VDR occupancy and therefore cellular responsiveness to hormone. These data also demonstrate the usefulness of ketoconazole as an inhibitor of vitamin D hydroxylases in intact cells.     

A new tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase

A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-[1 alpha-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 3H2O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 3H2O formed when 25-hydroxy-[1 beta-3H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-3H]vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 3H2O produced from 25-hydroxy-[1 alpha-3H]vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

Effects of 1,25-dihydroxyvitamin D-3 on phospholipid metabolism in chick myoblasts

1,25-Dihydroxyvitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels of myoblasts. Recent studies have suggested that the metabolite stimulates the methylation of phosphatidylethanolamine into phosphatidylcholine. In addition, the sterol increases the arachidonate content of phosphatidylcholine. Experiments were carried out to identify the steps of muscle cell lipid metabolism affected by 1,25-dihydroxyvitamin D-3. Primary cultures of chick embryo myoblasts pretreated with physiological concentrations of 1,25-dihydroxyvitamin D-3 were labelled with [14C]ethanolamine. The sterol increased the incorporation of precursor into dimethylphosphatidylethanolamine and phosphatidylcholine, whereas it decreases the labelling of phosphatidylethanolamine. Prior treatment with cycloheximide and actinomycin D blocked these changes. 1,25-Dihydroxyvitamin D-3 also stimulated the incorporation of [14C]ethanolamine into CDP-ethanolamine. In addition, the sterol increased the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction but did not affect the incorporation of [14C]palmitic acid. The incorporation of labelled fatty acids into diacylglycerol was not changed by the sterol, whereas it stimulated incorporation of both precursors into triacylglycerol. The data indicate that 1,25-dihydroxyvitamin D-3 enhances the synthesis of phosphatidylcholine through a stimulation of de novo synthesis and methylation of phosphatidylethanolamine via a nuclear mechanism. The sterol may also increase the polyunsaturated fatty acid content of phosphatidylcholine by means of an activation of its deacylation-reacylation cycle.     

End product inhibition of hepatic 25-hydroxyvitamin D production in the rat: specificity and kinetics

The role of vitamin D metabolites in the regulation of hepatic 25-hydroxyvitamin D production was investigated by examining the effects of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D on the synthesis of [25-3H]hydroxyvitamin D by rachitic rat liver homogenates. Production of [25-3H]hydroxyvitamin D was inhibited by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, but not by 24,25-dihydroxyvitamin D. 25-Hydroxyvitamin D increased the Km of the vitamin D-25-hydroxylase enzyme(s), while 1,25-dihydroxyvitamin D decreased the Vmax with a Ki of 88.7 ng/ml. Inhibition of hepatic 25-hydroxyvitamin D production by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D may be another control mechanism to regulate circulating vitamin D levels.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.     

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

Pituitary 1,25-dihydroxyvitamin D3 receptors in hyperthyroid- and hypothyroid-rats

The binding of 1 alpha,25-dihydroxy (26,27-methyl-[3H]) cholecalciferol ([3H]1,25-(OH)2D3) to its receptor in cytosol of the anterior pituitary cells was examined in hyperthyroid- and hypothyroid rats, as well as in normal rats. The binding capacity increased by 41% in L-Thyroxine-treated hyperthyroid rats and decreased by 49% in propylthiouracil-ingested hypothyroid rats as compared with normal control rats, whereas the affinity of the receptor for [3H]-1,25(OH)2D3 showed no difference among these 3 animal groups. These findings indicate that the number of 1,25(OH)2D3 receptors in the pituitary may be regulated by thyroid hormone, and further suggest that 1,25-(OH)2D3 may play some role in regulating functions of the anterior pituitary.