The binding of 1,25-dihydroxy[3H]cholecalciferol (1,25(OH)2[3H]D3) and 25-hydroxy-[3H]cholecalciferol (25(OH)[3H]D3) in vitamin D target and non-target tissues from the fetal rat has been compared using two incubation conditions, each followed by charcoal adsorption and sucrose gradient centrifugation. In intact tissue incubations, equilibrium with the ligand was established overnight at 4 °C before cell disruption. In pre-prepared cytosol incubations, cytosol was prepared from the tissue before incubation with ligand. With both ligands, more sterol was bound during pre-prepared cytosol incubation despite the use of fourfold lower ligand concentrations. With 1,25(OH)2[3H]D3, intact tissue incubation led to most marked binding with calvaria, which was preferentially displaced by unlabelled 1,25(OH)2D3; radioactivity sedimented almost entirely as a 3·2–3·7S peak (peak I) which was completely displaced by 100-fold excess 1,25(OH)2D3 but only partially by 25(OH)D3. Fetal small intestine, another putative target tissue, also showed displaceable binding of 1,25(OH)2D3; however, this was much less marked, and was not accompanied by a significant peak on sucrose gradients. Other fetal tissues (large intestine, kidney, skin, brain and heart) did not show significant displaceable binding of 1,25(OH)2D3 in intact tissue. In contrast, when intact calvaria, kidney, brain and heart were incubated with 25(OH)D3 they all showed significant displaceable binding to a 5·0–5·7S peak (peak II).
Incubations with pre-prepared cytosol confirmed ubiquitous binding of both sterols to peak II with the notable exception of that from small intestine; this peak II binding was preferential for 25(OH)D3, and is believed to be to the plasma vitamin D-binding protein (DBP). Only calvaria showed an additional peak I under these conditions, and even here it was dwarfed by peak II. We conclude that, at least in fetal bone, intact tissue incubations selectively detect receptor (peak I) binding of 1,25(OH)2D3 in the presence of the DBP. The substantial increase in binding of both sterols to peak II in pre-prepared cytosol compared with that in the same fetal tissues incubated intact suggests that some DBP is intracellular and therefore relatively inaccessible to extracellular sterol. The apparent absence of both peak I and peak II binding of cytosol from fetal small intestine merits further investigation.
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Print ISSN: 0022-0795
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