Studies on Calciferol Metabolism

Abstract

Abstract It has previously been postulated from studies conducted under in vivo conditions (Tsai, H. C., Wong, R. G., and Norman, A. W. (1972) J. Biol. Chem. 247, 5728–5735) that there may be a cause-and-effect relationship between the appearance of the calciferol (vitamin D) metabolite, 1,25-dihydroxycholecalciferol in the intestinal chromatin fraction, and the initiation of the physiological responses attributed to calciferol in this target tissue. The present report describes efforts to study via in vitro techniques the properties, specificity, and requirements of an intestinal homogenate system capable of effecting the localization of 1,25-dihydroxycholecalciferol in the intestinal chromatin fraction. Incubation of intestinal homogenates in 0.25 m sucrose, 0.02 m KCl, 0.05 m MgCl2, and 0.05 m Tris-Cl, pH 7.5, with 32 to 325 pmoles of 1,25-dihydroxy[3H]cholecalciferol at 0–4° for 30 min resulted in the saturation of the subsequently isolated chromatin fraction at a level of 6.2 pmoles of steroid per chick intestinal chromatin. Previous experiments (Tsai, H. C., Wong, R. C., and Norman, A. W. (1972) J. Biol. Chem. 247, 5728–5735) where 1,25-dihydroxy[3H]cholecalciferol was given intracardially to a cholecalciferol-deficient chick resulted in the binding of 5.2 pmoles of steroid per chick intestinal chromatin fraction. Under identical conditions in vitro only 1.4 pmoles of cholecalciferol, 2.0 pmoles of 25-hydroxycholecalciferol, or 0.3 pmole of estradiol were bound to the intestinal chromatin fraction. Evidence was obtained for the mandatory binding of the 1,25-dihydroxy[3H]cholecalciferol to a cytoplasmic receptor protein prior to the association of this steroid with the intestinal chromatin fraction. The intestinal cytoplasmic receptor was judged to be a macromolecule on the basis of its migration in a sucrose gradient, gel filtration chromatography, and a protein on the basis of its sensitivity to treatment with Pronase, but not RNase or DNase, its heat lability, and its precipitability by ammonium sulfate. The cytoplasmic receptor was highly specific for the binding and transfer of 1,25-dihydroxycholecalciferol to the intestinal chromatin fraction; only a 250-fold excess of 25-hydroxycholecalciferol, but not cholecalciferol, 5,6-trans-cholecalciferol, or dihydrotachysterol effected a significant (50%) reduction of the chromatin localization of 1,25-dihydroxy-[3H]cholecalciferol. Evidence was also obtained from a tissue specificity both for the cytoplasmic receptor and the chromatin binding. Neither liver, kidney, or spleen cytosol were able to transfer effectively 1,25-dihydroxycholecalciferol to intestinal chromatin, nor were liver or kidney nuclei able to accept the steroid from the intestinal cytosol receptor. These results describing the in vitro conditions which permit the sequential binding of 1,25-dihydroxycholecalciferol to first a cytoplasmic and then a nuclear receptor are analogous both to the results obtained in vivo with this steroid and to those reported to occur under in vivo and in vitro conditions for many other steroid hormones.