Specific binding of 1alpha,25-dihydroxycholecalciferol to nuclear components of chick intestine.

Specific binding of 1alpha,25-dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D-deficient chicks. The nuclear 1alpha,25-dihydroxycholecalciferol-macromolecule complex was isolated on sucrose density gradients and sediments at 3.7 S in the presence of 0.3 M KCl. Agarose gel filtration of the nuclear component indicated an apparent molecular weight of 47,000. The nuclear receptor complexes could not be distinguished from previously described cytoplasmic 1alpha,25-dihydroxycholecalciferol-binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3-H-sterol with the nuclear component is thermolabile and is destroyed by treatment with pronase, but not by nucleases; the receptor component is therefore presumed to be a protein. The macromolecular-1alpha,25-dihydroxycholecalciferol complex formed in vivo or in vitro at 25 degrees can be extracted from intestinal nuclei by 0.3 M KCl, but not by low salt buffers. Smaller amounts of the 3.7 S binding component can be detected in isolated purified chromatin or after incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin at 0 degrees. Following incubation of the labeled hormone with reconstituted cytosol-chromatin at 0 degrees, 1alpha,25-dihydroxy[3-H]cholecalciferol is primarily associated with the cytoplasmic receptor, After shifting the incubation temperature to 25 degrees, a progressive increase in the concentration of the nuclear receptor complex and a concomitant decrease in the concentration of the cytoplasmic binding component occur. Thus the 1alpha,25-dihydroxycholecalciferol binding molecules appear to exist primarily in the cytoplasm, where they presumably function to transport the hormone into the nucleus. Experiments employing incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin from nontarget tissues indicate a requirement for both intestinal cytosol and chromatin for maximal formation of the nuclear hormone-receptor complex. These results suggest that the nuclear-binding component arises from hormone-dependent transfer of the cytoplasmic 1alpha,25-dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.     

1 alpha,25-Dihydroxycholecalciferol and a human myeloid leukaemia cell line (HL-60).

Human promyelocytic leukaemia cells (HL-60) can be induced to differentiate into mature granulocytes in vitro by 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], the active form of cholecalciferol. The differentiation-associated properties, such as phagocytosis and C3 rosette formation, were induced by as little as 0.12 nM-1 alpha,25(OH)2D3, and, at 12 nM, about half of the cells exhibited differentiation on day 3 of incubation. Concomitantly the viable cell number was decreased to less than half of the control. Among various derivatives of cholecalciferol examined, 1 alpha,25(OH)2D3 and 1 alpha,24R-dihydroxycholecalciferol were the most potent in inducing differentiation, followed successively by 1 alpha,24S-dihydroxycholecalciferol, 1 alpha-hydroxycholecalciferol, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol. A cytosol protein specifically bound to 1 alpha,25 (OH)2D3 was found in HL-60 cells. Its physical properties closely resembled those found in such target tissues as intestine and parathyroid glands. 1 alpha,25(OH)2D3 bound to the cytosol receptor was transferred quantitatively to the chromatin fraction. The specificity of various derivatives of cholecalciferol in inducing differentiation was well correlated with that of their association with the cytosol receptor. These results are compatible with the hypothesis that the active form of cholecalciferol induces differentiation of human myeloid leukaemia cells by a mechanism similar to that proposed for the classical concept of steroid hormone action.     

Structural changes in parathyroid glands exposed to vitamin D metabolites in vitro

Isolated parathyroid glands from normal adult Mongolian gerbils were incubated for 10 minutes to 16 1/2 hours at high, medium or low concentrations of Ca2+, with or without added 1,25-dihydroxycholecalciferol (1.25-DHCC) or 25-hydroxycholecalciferol (25-HCC), after which they were studied electron microscopically. 1,25-DHCC appeared to potentiate the inhibitory action of high calcium on parathyroid activity, whereas 25-HCC had no structurally detectable effect.     

A simple method for generation of antibodies with specificity for 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol

A simple method for production of antisera with high affinity and selectivity for 1 alpha, 25-dihydroxyergocalciferol and 1 alpha, 25-dihydroxychole-calciferol is described. 1 alpha-Hydroxy-25,26,27-trisnorcholecalciferol-24-oic acid was coupled directly to bovine serum albumin. Rabbits immunized with this conjugate rapidly produced antibodies that bound 3H-1 alpha,-25-dihydroxycholecalciferol with high affinity and demonstrated nearly equal reactivity with 1 alpha, 25-dihydroxyergocalciferol and poor reactivity with 25-hydroxycholecalciferol; 24,25-dihydroxycholecalciferol; 25,26-dihydroxycholecalciferol; and 1 beta,25-dihydroxycholecalciferol. The use of one of these antisera has led to the development of a specific assay for 1 alpha,25-dihydroxyergocalciferol and 1 alpha,25-dihydroxycholecalciferol in human serum.     

A high-affinity cytosol binding protein for 1 alpha,25-dihydroxycholecalciferol in the uterus of Japanese quail.

Cytosol fractions prepared from the uterine mucosa of egg-laying Japanese Quail were analysed for binding of the metabolites of cholecalciferol. When the uterus was incubated at 37 degrees C with various radioactive metabolites of cholecalciferol, the nuclear fraction incorporated only 1 alpha,25-dihydroxy[3H]cholecalciferol. When the uterus was incubated at 0 degree C with 1 alpha,25-dihydroxy[3H]cholecalciferol, most of the radioactivity was found in the cytosol. Translocation of 1 alpha,25-dihydroxy[3H]cholecalciferol from the cytosol to the nucleus was temperature-dependent. The addition of 100-fold excess amounts of unlabelled 1 alpha-25-dihydroxycholecalciferol significantly diminished the nuclear binding of 1 alpha,25-dihydroxy[3H]cholecalciferol. The cytosol fraction contained a 3.5 S macromolecule that specifically binds 1 alpha,25-dihydroxy[3H]cholecalciferol. The dissociation constant was 0.39 nM and the maximal binding was 55 fmol/mg of protein. These results strongly suggest that the uterus in egg-laying birds is a target organ or 1 alpha,25-dihydroxycholecalciferol.     

Inhibitory effects of 1 alpha, 25-dihydroxycholecalciferol on parathyroid hormone secretion in rats

In an attempt to study the influence of vitamin D metabolites on PTH secretion, serum calcium and urinary excretion of cAMP were sequentially measured in conscious perfused rats, and the effects of a single iv injection of the metabolites on these parameters were examined. Four hours after the administration of 0.25 microgram/kg (0.6 nmol/kg, probably a physiological dose) of 1 alpha, 25-dihydroxycholecalciferol [1 alpha, 25 (OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized rats (approximately 60% of the initial value; P less than 0.05) and this level was sustained for nearly 24 h. Serum concentrations of calcium (total and ionized) did not change. In parathyroidectomized rats which were continuously infused with bovine PTH (1 U/h), the vitamin D metabolite had no significant effect on the urinary excretion of cAMP. 24 R, 25-dihydroxcholecalciferol (12.5 microgram/kg) had no significant effect either on the urinary excretion of cAMP or on serum calcium. These results suggest that in rats, a physiological dose of 1 alpha, 25(OH)2D3 inhibits PTH secretion without causing a significant rise iu serum calcium, reflecting a feed-back mechanism between active vitamin D metabolite, 1 alpha, 25(OH)2D3 and the parathyroid glands.     

Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation

The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.     

Inhibitory effects of active vitamin D preparations on PTH secretion in rats

To study the effects of various vitamin D preparations on PTH secretion, serum calcium and urinary excretion of cAMP were monitored in conscious perfused rats, and the influences of a bolus iv injection of the preparations on these parameters were examined. Three hours after the administration of 0.25 microgram/kg (0.6 nmol/kg) of 1 alpha, 24(R)-dihydroxycholecalciferol [1 alpha, 24(OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized (PTX) rats (50% of initial value; p less than 0.05) with no change in the concentration of serum calcium (total and ionized). In PTX rats supplemented with bovine PTH (1 U/h), the vitamin D preparation showed no significant effects either on the urinary excretion of cAMP or on serum calcium. These effects were rather specific for active vitamin D preparations, i.e. 1 alpha, 25(OH)2D3 (0.25 micrograms/kg) and 1 alpha OHD3 (1.25-6.25 micrograms/kg). However, 24,25(OH)2D3 (up to 25 micrograms/kg) had no significant effect on these parameters. These results suggest that, in rats, active vitamin D preparations specifically inhibit PTH secretion without causing a significant increase in the serum calcium concentration, reflecting a direct feedback mechanism between active vitamin D metabolite and the parathyroid glands.     

1 alpha, 25-dihydroxycholecalciferol and human histiocytic lymphoma cell line (U-937): the presence of receptor and inhibition of proliferation

Human histiocytic lymphoma cells (U-937) contain high affinity binding protein for 1 alpha, 25-dihydroxycholecalciferol. The Kd (0.2 nM) and sedimentation coefficient on sucrose gradients (3.7 S) are similar to those reported for 1 alpha, 25-dihydroxycholecalciferol receptor in other tissues. This secosteroid added to the culture was able to inhibit cell proliferation in a dose dependent manner. Differentiation-associated properties such as phagocytic ability, C3 rosette formation, positive reaction for alpha- naphtyl acetate esterase as well as electron microscopy examination indicate that 1 alpha, 25-dihydroxycholecalciferol induces in vitro monocyte-macrophage differentiation in this monoblast-like cell line.     

Stimulation of creatine kinase BB activity by 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol in rat tissues.

Vitamin D metabolites stimulate creatine kinase BB activity in organs of vitamin D-deficient rats. In epiphyses of long bones, creatine kinase BB activity increases 2.6-fold 24 h after injection of 24R,25-dihydroxycholecalciferol but not of 1 alpha,25-dihydroxycholecalciferol. Contrariwise, 1 alpha,25-dihydroxycholecalciferol, but not 24R,25-dihydroxycholecalciferol, increases creatine kinase BB activity in diaphyses and in kidney. Neither metabolite affects creatine kinase activity in duodenal mucosa.     

The effects of 24R,25-dihydroxycholecalciferol and of 1 alpha,25-dihydroxycholecalciferol on ornithine decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat bone and in the duodenum.

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.     

Intranuclear localization and receptor proteins for 1,25-dihydroxycholecalciferol in chick intestine

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.     

The effect of 1 alpha, 25-dihydroxycholecalciferol on iron metabolism

Chronic administration of hypercalcemic doses of 1 alpha, 25-dihydroxycholecalciferol to intact, vitamin-D repleted rats for 4 weeks, enhanced net intestinal absorption of iron and liver iron stores. Daily net iron and calcium absorptions were found to be significantly correlated in both control and treated rats. In duodenal loop experiments, pretreatment with 1 alpha, 25-dihydroxycholecalciferol reversed the adverse effect of high Ca/Fe ratio on iron absorption. The increased intestinal absorption of iron did not result in a change of serum iron levels nor of total iron binding capacity due to the enhanced incorporation of absorbed iron into liver ferritin. The curve of uptake of 59Fe into circulating red cells of treated rats suggested retarded release of the isotope from stores. The hypothesis is advanced that the systemic metabolic defect (tissue hypoxia, raised erythropoietin levels) produced by 1 alpha, 25-dihydroxycholecalciferol is responsible for the disruption of the physiological coordination between iron stores and intestinal absorption.     

Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from human term decidua

Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from term human decidua were studied. The production of 1 alpha,25-dihydroxycholecalciferol (calcitriol) was linear with time for up to 6 h and was directly proportional to the number of cells up to 20 X 10(6)/dish at a substrate concentration of 100 nM. Under these conditions the apparent Km was 88 nM and the Vmax 3.0 pmol/10(6) cells. The production of [3H]calcitriol was inhibited by 0.1 nM (P less than 0.01) and 1 nM (P less than 0.005) unlabeled calcitriol. Unlike the kidney enzyme and for reasons that remain unclear, neither inorganic phosphate salts nor parathyroid hormone had any acute effect on the calcitriol production. Further studies are required to delineate the regulatory mechanism of this enzyme.     

Vitamin D metabolism and its possible role in the developing chick embryo.

The relationship between bone formation and vitamin D metabolism was investigated in the developing chick embryo. Fertilized White Leghorn eggs were incubated at 38 degrees C in an incubator for 21 days. The fresh weight and calcium content of embryonic tibiae began to increase at day 12 and attained maximal values at day 19. Bone alkaline phosphatase and citrate decarboxylation activities, both of which represent osteoblastic activity, also began to increase at days 10-12, reached maximal values at day 19 and sharply declined thereafter. Both bone enzyme activities were highly correlated with CA2+-binding activity in the chorioallantoic membrane measured by the Chelex 100 assay. When mesonephric and metanephric homogenates were incubated with 25-hydroxy[3H]cholecalciferol, a marked and concomitant increase occurred in the metanephric 1 alpha- and 24-hydroxylase activity after day 14. The production of 1 alpha, 25-dihydroxycholecalciferol attained a maximal value at day 19 and decreased thereafter, whereas that of 24,25-dihydroxycholecalciferol continued to increase until hatching. The production rate of 1 alpha, 25-dihydroxycholecalciferol by the metanephros coincided with the changes in Ca2+-binding activity in the chorioallantoic membrane and osteoblastic activity. Since both intestinal calcium absorption and bone mineral mobilization do not occur in embryonic life, these results support the idea that 1 alpha, 25-dihydroxycholecalciferol may be involved directly in bone formation or induction of a calcium-binding protein in the chorioallantoic membrane.     

Binding and action of aldosterone, dexamethasone, 1-25(OH)2D3, and estradiol along the nephron

The localization of specific binding sites of four steroids--aldosterone, dexamethasone, 1-25-dihydroxycholecalciferol and estradiol--is described along the nephron. This localization has been determined by using an autoradiographic method on dry films, applied to intact microdissected tubular segments. Aldosterone binds specifically to nuclei of the distal and cortical collecting tubule. No specific labeling was observed in the cytoplasm. This localization corresponds to the known sites of action of aldosterone on sodium reabsorption. Specific nuclear binding of dexamethasone is present all along the tubule except the proximal tubule. In this latter part of the nephron, a specific cytoplasmic labeling is observed, in the absence of nuclear labeling. This cytoplasmic binding could correspond to physiological effects in this structure, via non-genomic mechanisms. 1-25(OH)2D3 nuclear binding is located mainly in the loop of Henle and medullary collecting tubule, which are the sites of synthesis of calcium-binding proteins. No specific binding of estradiol is present in any part of the nephron.     

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.     

Selective analogs of 1alpha,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1alpha,25-dihydroxyvitamin D(3) synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3'-propylidene-19-nor-(20S or 20R))-1alpha,25-dihydroxyvitamin D(3) and 2-methylene-19-21-dinor-1alpha,25-dihydroxyvitamin D(3). Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD), the 2alpha-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1alpha-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1alpha,25-dihydroxyvitamin D(3). These latter compounds do not elevate serum calcium until doses of the order of >300 microg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

Production of prostaglandins by dispersed cells and fragments from bovine parathyroid glands

We studied the production of prostaglandins by fragments and dispersed cells from bovine parathyroid glands. Fragments released 138 +/- 19 (SE), 132 +/- 21, 4.3 +/- 0.5, and 13 +/- 6.6 pg/mg/h of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and thromboxane B2, respectively (n = 7 - 26), while dispersed cells released 414 +/- 110, 22 +/- 7.3, 27 +/- 3.8, and 29 +/- 11 pg/10(6) cells/h, respectively, of the same compounds (n = 6 - 25). Indomethacin (1 microgram/ml) inhibited the release of 6-keto-PGF1 alpha by 80-90% in fragments and cells, while mellitin stimulated release of this prostaglandin, suggesting de novo synthesis of prostaglandins in these preparations. Calcium stimulated production of 6-keto-PGF1 alpha by 1.3-fold in cells and 2.6-fold in fragments and also enhanced production of PGF2 alpha by 1.9-fold in fragments. Isoproterenol, on the other hand, had no effect on production of 6-keto-PGF1 alpha in either preparation. These results demonstrate that parathyroid tissue as well as parathyroid cells per se produce a variety of prostaglandins. We have previously shown that PGE2 and PGF2 alpha modulate cAMP accumulation and PTH release in dispersed bovine parathyroid cells. The role of the endogenous production of prostaglandins by the parathyroid gland in the acute or chronic regulation of parathyroid function, however, remains to be determined.     

Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats.

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.