Gel-permeation high-performance liquid chromatography as a powerful technique for rapid analysis of calcitriol (1,25-dihydroxycholecalciferol) receptors.
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ABSTRACT:
SUBMITTER: Danan JL
PROVIDER: S-EPMC1154075 | biostudies-other | 1983 Jan
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.
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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.
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Project description:The synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3] from 25-hydroxycholecalciferol [25(OH)D3] has previously been shown to occur in cells isolated from bone. The main findings of the present study are that the enzyme systems which catalyse these syntheses are: (1) active at 'in vitro' substrate concentrations over the range of 2-50 nM; (2) regulatable in a complex way by 1,25(OH)2D3, 24,25(OH)2D3, 25,26-dihydroxycholecalciferol and 25(OH)D3, but not by cholecalciferol ('vitamin D3'); and (3) have relatively short half-lives (approx. 5 h).
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