Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems.
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ABSTRACT:
SUBMITTER: Puzas JE
PROVIDER: S-EPMC1148126 | biostudies-other | 1987 Jul
REPOSITORIES: biostudies-other
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Project description:The location of intestinal cells taken from the base of the crypt to the tip of the villus responsive to calcitriol (1,25-dihydroxycholecalciferol) and the distribution of [3H]calcitriol within the intestinal epithelium has been determined in vitamin D-deficient rats. The calcitriol responses examined were CaBP (Ca2+-binding protein) levels as measured by immunodiffusion and alkaline phosphatase levels as determined cytochemically. Calcitriol had no effect on villus structure or on enterocyte kinetics. This made it possible to compare levels of CaBP and alkaline phosphatase activity in enterocytes at different ages in rats at known times after hormone injection. Cells from both the crypt and villus synthesized CaBP in response to calcitriol. Alkaline phosphatase activity was not detectable in crypt cells, although a pool of precursor was produced in these cells in response to calcitriol. Enzyme activity was increased in all villus cells in response to calcitriol, but the quantitative description of this effect was very different from that found for calcitriol effects on CaBP synthesis. Calcitriol injected into vitamin D-deficient rats was detected, within 2h, in all cells of the crypt and villus. Most of the binding was to sites having a high affinity for the injected hormone.
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Project description:(23S)-23,25-Dihydroxycholecalciferol was converted into a polar metabolite in a calciferol-deficient chick kidney homogenate. The metabolite was identified as (23S)-1,23,25-trihydroxycholecalciferol by absorbance spectroscopy and mass spectrometry, and by formation of derivatives. (23S)-1,23,25-Trihydroxycholecalciferol was also observed as a 1,25-dihydroxycholecalciferol metabolite in intestinal cells isolated from 1,25-dihydroxycholecalciferol-treated rat. The trihydroxy metabolite was 50-fold less potent than 1,25-dihydroxycholecalciferol in the chick intestinal 1,25-dihydroxycholecalciferol receptor assay.
| S-EPMC1152235 | biostudies-other
Project description:Many factors influence the production of 1,25(OH)2D3 (1,25-dihydroxycholecalciferol) by the kidney. One important factor seems to be feedback regulation by 1,25(OH)2D3 itself. Administration of 1,25(OH)2D3 to vitamin D-deficient chicks abolishes renal 25(OH)D3(25-hydroxycholecalciferol)1-hydroxylase activity and induces the appearance of 25(OH)D3 24-hydroxylase activity. It is likely that these effects are mediated via a nuclear effect, as they are prevented by pretreatment with actinomycin D and alpha-amanitin. Further, 1,25(OH)2D3 has a marked effect on gene transcription in the kidney cell, as assessed by measurement of RNA polymerase activities. RNA polymerase I and II activities are 80-90% inhibited by 12.5nmol of 1,25(OH)2D3 within 30min of subcutaneous administration, indicating an immediate and massive decrease in total gene transcription. By 4h RNA polymerase II activity has returned to control values, but RNA polymerase I activity is markedly enhanced. These results are consistent with the view that regulation of cholecalciferol metabolism in the kidney is associated with an effect of the active metabolite on the kidney nucleus.
| S-EPMC1164760 | biostudies-other
Project description:A specific 1,25-dihydroxycholecalciferol-binding protein has been detected in high-salt cytosols prepared from human medullary thyroid carcinomas. The binding protein had the same equilibrium dissociation constant (Kd = 0.17 +/- 0.05 nM; n = 4) and sedimentation coefficient on sucrose gradients (3.7S) as than seen in established vitamin D target tissues. This protein was not detected in normal thyroid cytosols, which may reflect the low proportion of C-cells within the gland.
| S-EPMC1158568 | biostudies-other
Project description:O2-dependent CA2+ uptake by rat duodenal discs has been characterized and used in a revised assay for 1,25-dihydroxycholecalciferol-induced intestinal Ca2+ transport. Although both muscle and mucosal surfaces are exposed in this free-floating-disc assay, the Ca2+ influx across the muscle surface is small, not O2- or vitamin D-dependent, and can be subtracted out. Depriving the animals of food for 9-14 h before assay increases the O2-dependent uptake by about 75%. Half-saturation values for O2-dependent Ca2+ uptake as determined with this assay are: 0.8mM-Ca2+ (fed) and 0.5mM-Ca2+ (food-deprived) for vitamin D-deficient rats, and 0.9mM-Ca2+ (fed) and 1.5mM-Ca2+ (food-deprived) for rats dosed with 1,25-dihydroxycholecalciferol. The maximum velocity of uptake varies from 6.7nmol of Ca2+ per cm2/min (fed) to 7.0nmol of Ca2+ per cm2/min (food-deprived) for vitamin D-deficient rats and 16.7nmol of Ca2+ per cm2/min (fed) to 29 nmol of Ca2+ per cm2/min (food-deprived) for 1,25-dihydroxycholecalciferol-treated rats. By using a 5 min preincubation and 15 min incubation with 1.0mM-Ca2+, duodenal tissue taken from vitamin D-treated rats shows about a 3-fold increase in O2-dependent Ca2+ uptake when compared with tissue taken from vitamin D-deficient animals. The calcium ionophore A23187, depending on concentration, either has no significant effect on or inhibits the O2-dependent uptake, rather than increasing it. Actinomycin D, at a dose of 2 micrograms/g, inhibits the O2-dependent uptake in intestinal discs from both vitamin D-deficient and vitamin D-treated rats by 58 and 80% respectively, when administered in vivo 3 1/2 h before assay.
| S-EPMC1162731 | biostudies-other
Project description:1. The intranuclear distribution of cholecalciferol and its metabolites was studied in the intestine of rachitic chicks. 2. At high doses of cholecalciferol the nuclei contain the vitamin and its 25-hydroxy metabolite, but over 80% of this is localized on the nuclear membranes. The hormone, 1,25-dihydroxycholecalciferol, is found within the cell nuclei irrespective of the intake of cholecalciferol, but significant amounts could not be found with chromatin isolated free of nuclear membranes. 3. 1,25-Dihydroxycholecalciferol is associated in the nucleus with an acidic protein. Since one of the actions of 1,25-dihydroxycholecalciferol is to control the synthesis of mRNA for calcium-binding protein it was to be expected that the hormone would be bound to chromatin, as with the other steroid hormones. It is suggested that the hormone-receptor complex exists as part of an equilibrium mixture of the complex bound to the DNA and in a free form. 4. A protein extract of nuclei was obtained, which when incubated at 4 degrees C for 1h took up the 1,25-dihydroxycholecalciferol. The nature of this binding was studied. 5. There appear to be two nuclear proteins able to bind the hormone one of which is the intestinal nuclear receptor. The binding sites on this protein are saturable with the hormone, have an association constant of 2x10(9)m(-1) and show a high chemical specificity for the 1,25-dihydroxycholecalciferol. The number of nuclear binding sites for the hormone provided by this receptor is similar to the maximum intestinal hormone concentration so far observed. Its sedimentation coefficient is 3.5S, and is very close to that observed for the nuclear protein to which is attached the 1,25-dihydroxycholecalciferol formed in vivo from vitamin D. 6. The cytoplasmic protein has an association constant of 1x10(9)m(-1)and a sedimentation coefficient of 3.0S, but its relation with the nuclear receptor is not yet clear.
| S-EPMC1168536 | biostudies-other
Project description:Stimulation of intestinal calcium transport by the hormone 1,25-dihydroxycholecalciferol appears to involve RNA transcriptions and the synthesis of new proteins. Although one of these proteins has been identified as calcium-binding protein, no RNA molecules specifically induced by the hormone in the nucleus have been identified. Nuclear RNA from intestine of vitamin D-deficient chicks before and at various time intervals after treatment with the hormone or cholecalciferol was tested for its ability to code for calcium-binding protein in a cell-free system. Calcium-binding-protein mRNA could only just be detected in the intestinal nuclei 2h after dosing with these steroids which is the same time that it was first observed in the polyribosomes. Thus 1,25-dihydroxycholecalciferol induces the production of new calcium-binding protein by stimulating the formation and rapid release from the nucleus of new mRNA molecules for this protein. Polyribosomal translation of the mRNA continued only as long as it was being synthesized, and the maximum rate of synthesis following a pulse dose of 125ng of the hormone was the same as that observed after prolonged stimulation with cholecalciferol. The possibility that other 1,25-dihydroxycholecalciferol-dependent events may be occurring in the nucleus in the lag period between accumulation of the hormone in the intestine and the appearance of active calcium-binding-protein mRNA, and that these may ultimately control the synthesis of that mRNA, is discussed.
| S-EPMC1186171 | biostudies-other
Project description:Specific high-affinity receptors for 1,25-dihydroxycholecalciferol [1,25-(OH)(2)D(3)] have been described recently in broken-cell preparations of several cultured human breast cancer cell lines including the T47 D line. It was necessary to determine whether intact breast cancer cells in culture would bind 1,25-(OH)(2)D(3) specifically and whether the next step in the proposed scheme of action, i.e. nuclear translocation, occurred. The following results were obtained. (1) Specific uptake of 1,25-(OH)(2)D(3) by T47 D cells occurs in intact cells in culture. (2) The rate of uptake is proportional to medium 1,25-(OH)(2)D(3) concentration but is slow compared with that of other steroid hormones, e.g., oestradiol, under identical conditions. Even at 0.5nm-1,25-(OH)(2)D(3) in the medium, at least 4h are required to reach maximum compared with less than 1h for oestradiol binding. (3) Estimation of binding characteristics by Scatchard analysis indicates a single class of binding sites with K(d) of 68pm and 11800 binding sites/cell, which are similar results to those obtained with broken-cell preparations. (4) Inclusion of various vitamin D metabolites in the incubation medium decreased specific binding of 1,25-(OH)(2)D(3) by the intact cells in a manner identical with their effects in the broken-cell preparation and with potencies similar to their potency on Ca(2+) transport and bone resorption in vivo. Order of potency was 1,25-(OH)(2)D(3)>(24R)-1,24,25-trihydroxycholecalciferol >>25-hydroxycholecalciferol>(25R)-24,25-dihydroxycholecalciferol >>(25R)-25,26-dihydroxycholecalciferol. (5) In the 1,25-(OH)(2)D(3)-depleted state, 80% of the 1,25(OH)(2)D(3) receptor is found in the cytosol fraction of the cells even when the subcellular fractionation is performed under low-salt conditions. By contrast after incubation with [(3)H]1,25-(OH)(2)D(3), 59% of the specific 1,25-(OH)(2)D(3) binding is found in the partially purified nuclei fraction. These data indicate that nuclear translocation of the receptor-hormone complex takes place in the intact T47 D cell. The results also support the hypothesis that the 1,25-(OH)(2)D(3) receptor is functional in this cultured breast cancer cell line, which may provide a useful model for further study of the early biochemical events in 1,25-(OH)(2)D(3) action.
| S-EPMC1163537 | biostudies-other
Project description:1. A simple technique has been developed to obtain subcellular fractions of chick bone. The method yielded 60-70% of total DNA in the nuclear debris fraction and 80-90% of total (14)C recovered in bone after a dose of radioactive vitamin D. 2. After a dose of [4-(14)C,1,2-(3)H(2)]cholecalciferol (0.5mug) was given to vitamin D-deficient chicks, the time-course of total (14)C radioactivity in the epiphysis, metaphysis and diaphysis of proximal tibiae was measured. The maximum concentrations were reached at 6h, corresponding to a similar peak of radioactivity in blood, decreasing until 24h and indicating the dependence on the circulating (14)C and on the blood supply of the three bone components. 3. The (14)C radioactivity of cholecalciferol and 25-hydroxycholecalciferol (expressed per mg of DNA) followed the pattern of incorporation of total (14)C radioactivity in all three bone components. The more polar metabolite fraction reached a peak of radioactivity at 6-9h and maintained its concentration over the 24h period studied in all anatomical bone components. 4. After a dose of [4-(14)C,1-(3)H]cholecalciferol (0.5mug) was given to vitamin D-deficient chicks, the subcellular distribution was studied. At 24h after dosing, the nuclear fraction contained 27% and the supernatant fraction had 67% of total (14)C recovered in the bone filtrate. When the (14)C in the residual bone fragments was included, the nuclear fraction contained up to 35% of the total radioactivity in the bone. 5. The subcellular distribution pattern of individual vitamin D metabolites indicated that the purified nuclear fraction concentrated the polar metabolite, which lost (3)H at C-1, so that 77% of the radioactivity could be accounted for by 1,25-dihydroxycholecalciferol. The supernatant fraction contained smaller amounts of 1,25-dihydroxycholecalciferol (9%), with 66% of 25-hydroxycholecalciferol forming the major metabolite, corresponding to its concentration found in blood at 24h. 6. The preferential accumulation of 1,25-dihydroxycholecalciferol in the nuclear fraction and the overall pattern of other metabolites, found previously in intestinal tissue, suggests a similar mechanism of action in bone to that postulated for the intestinal cell in calcium translocation.
| S-EPMC1178034 | biostudies-other
Project description:A radioligand immunoassay for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] receptors was developed utilizing a specific, high-affinity (Kd = 1.8 X 10(-11 M) monoclonal antibody (9A7 gamma) obtained from suspension cultures of rat spleen X mouse myeloma hybrid SP2/0-9A7. A standard curve was established, based on the competition between 1,25(OH)2[3H]D3-receptor (18 fmol/tube) and increasing concentrations of radioinert 1,25(OH)2D3-receptor (0-240 fmol/tube) for the binding site on 9A7 gamma. Samples, prepared in identical buffer, contained 0-100 fmol of receptor/tube. After an equilibrium incubation of 1,25(OH)2[3H]D3-receptor with either standard or sample (16 h at 4 degrees C), antibody-bound receptor was immunoprecipitated with rabbit anti-(rat immunoglobulin) prelinked to Staphylococcus aureus and quantified. The assay is statistically sensitive to 2 fmol of receptor/tube, with intra- and inter-assay variations of 7 and 12% respectively. Occupied, unoccupied and denatured receptor were observed to compete equally in the assay. This quantitative technique has been successfully applied to the characterization of receptors after fractionation by sedimentation analysis and DNA-cellulose chromatography. Finally, the measurement of total receptor by this assay, in conjunction with 1,25(OH)2D3 binding assays, has revealed that rachitic, normal and 1,25(OH)2D3-injected chicks have respectively 13, 20, and 56% of receptor in the occupied form. From these results we consider that this radioligand immunoassay will be a useful tool in further research focusing on quantifying 1,25(OH)2D3 receptors in tissue and cell extracts.
| S-EPMC1144011 | biostudies-other