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The localization of 1,25-dihydroxycholecalciferol in bone cell nuclei of rachitic chicks.

ABSTRACT: 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.

SUBMITTER: Weber JC

PROVIDER: S-EPMC1178034 | biostudies-other | 1971 Nov

REPOSITORIES: biostudies-other

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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.

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The localization of 1,25-dihydroxycholecalciferol in bone cell nuclei of rachitic chicks.

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