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 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.     

Effect of I,25-dihydroxycholecalciferol in renal osteodystrophy.

A 23-year-old man with medullary cystic disease had been undergoing hemodialysis for 5 years and had become confined to a wheelchair because of renal osteodystrophy. He was treated with 125-dihydroxycholecalciferol, 2.0 mug (later 1.0 mug) three times a week, administered by way of the venous end of the dialysis machine. Within 1 month bone pain lessened and his ability to stand and walk improved. By 3 months he was walking short distances and by 5 months, long distances. Calcium balance was near zero before treatment and was strongly positive during treatment. Bone mineral content in the lower femur, measured by photon absorptiometry, increased at a rate of 32.2% per year. In contrast, 26 other patients on long-term hemodialysis had a mean loss of bone mineral content of 14.0% per year. Radiographs taken during treatment showed a decrease in subperiosteal bone resorption and healing of a pseudofracture. A significant decrease in the mean serum alkaline phosphatase value was noted during treatment, but no significant changes in mean serum calcium or phosphorus values were seen.     

Structural analogs of 1alpha,25-dihydroxycholecalciferol: preparation and biological assay of 1alpha-hydroxypregnacalciferol.

The synthesis of 1alpha-hydroxypregnacalciferol, a side chain analog of 1alpha,25-dihydroxycholecalciferol (1alpha,25-dihydroxyvitamin D3), is described. Pregnenolone acetate was converted in five steps to 5-pregnen-1alpha,3beta-diol. Conversion of the diol to pregna-5,7-diene-1alpha,3beta diol diacetate followed by ultraviolet irradiation gave the corresponding previtamin derivative. Thermal isomerization, hydrolysis and chromatography then furnished the desired analog, 1alpha-hydroxypregnacalciferol. The compound was tested in vivo for its effect on intestinal calcium transport, serum calcium and phosphate levels and bone calcification, and in vitro for its effect on bone resorption. When given to intact rats, either as a single dose or in repeated daily doses, the analog even at high dose levels, exhibited no biological activity. The compound stimulated bone resorption in vitro, but only at high concentrations.     

Early stimulation of alkaline phosphatase activity in response to 1 alpha, 25-dihydroxycholecalciferol.

Alkaline phosphatase activity (APA) stimulation in response to 1,25-dihydroxycholecalciferol (1,25 (OH)₂D₃) has been studied in vitamin-D-deficient rat intestinal brush borders prepared from $\text{ex}$-$\text{vivo}$-perfused duodeno-jejunal segments. Basal APA in intestines perfused with ethanol remained constant throughout the experiments. APA was significantly increased when intestines were perfused with 1,25 (OH)₂D₃ (3 nM) for 30, 45 or 60 min. A dose-effect response was observed when 1,25 (OH)₂D₃ increased in the perfusion medium. The maximal alkaline phosphatase activity after a 45-min perfusion (2404 ± 379 m^TU/mg prot.) was observed when 1,25 (OH)₂D₃ concentration was 6 nM. Cholecalciferol had no effect in this system.     

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.     

Peptide-chain initiation in duodenal mucosa of rachitic chicks after 1 alpha-,25-dihydroxycholecalciferol administration.

The post-ribosomal fraction of chick duodenal mucosa contains Met-tRNAMetf-binding protein(s) that behaves like the eukaryotic initiation factor (eIF-2) in protein synthesis. The binding activity of cytosol protein can be measured by retention of the radioactive complex formed on a nitrocellulose membrane. Complex-formation requires Met-tRNAMetf and GTP or guanosine [beta, gamma-methylene] triphosphate, and is inhibited by aurintricarboxylic acid. The ternary initiation complex thus formed can bind to ribosomal particles from chick intestine. By sucrose-density-gradient centrifugation, [35S]Met-tRNAMetf was found to bind exclusively to 40S and not to 60S ribosomal subunit particles. In the duodenal mucosa of rachitic chicks the ability of the cytosol proteins to promote the binding of Met-tRNAMetf to ribosomal particles via ternary-complex formation is detectably increased by 3 h after injection of 1 alpha,25-dihydroxycholecalciferol, the active form of vitamin D. Cholecalciferol and ergocalciferol under the same experimental conditions failed to stimulate Met-tRNAMetf-binding activity.     

The mechanism of end-organ resistance to 1 alpha,25-dihydroxycholecalciferol in the common marmoset.

The common marmoset, a New World monkey, requires a large amount of cholecalciferol (110 i.u./day per 100g body wt.) to maintain its normal growth. In a previous report, we demonstrated that the circulating levels of 1 alpha, 25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] in the marmosets are much higher than those in rhesus monkeys and humans, but the marmosets are not hypercalcaemic [Shinki, Shiina, Takahashi, Tanioka, Koizumi & Suda (1983) Biochem. Biophys. Res. Commun. 14, 452-457]. To compare the effect of the daily intake of cholecalciferol, two rhesus monkeys were given a large amount of cholecalciferol (900 i.u./day per 100g body wt). Their serum levels of calcium, 25-hydroxycholecalciferol and 24R,25-dihydroxycholecalciferol were markedly elevated, but the serum 1 alpha,25(OH)2D3 levels remained within a range similar to those in the rhesus monkeys fed the normal diet (intake of cholecalciferol 5 i.u./day per 100g body wt). Intestinal cytosols prepared from both monkeys contained similar 3.5 S macromolecules to which 1 alpha,25(OH)2D3 was bound specifically. However, the cytosols from the marmosets contained only one-sixth as many 1 alpha,25(OH)2D3 receptors as those from the rhesus monkeys. Furthermore, the activity of the 1 alpha,25(OH)2D3-receptor complex in binding to DNA-cellulose was very low in the marmosets. These results suggest that the marmoset possesses an end-organ resistance to 1 alpha,25(OH)2D3 and is a useful animal model for studying the mechanism of vitamin D-dependent rickets, type II.     

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.     

Effects of 1 alpha,25-dihydroxycholecalciferol on sodium-ion translocation across chick intestinal brush-border membrane.

By utilizing isolated brush-border vesicles, Na+ transport across the luminal membrane of chick small intestine was found to be a composite of (i) a saturable (Km 10mM-Na+) amiloride-sensitive Na+/H+ antiport and (ii) a potential-sensitive conductive pathway. No evidence was obtained for the existence of a Na+/Cl- symport system. With the exception of the duodenum, luminal Na+ transfer in the entire small intestine was subject to regulation by vitamin D. Repletion of vitamin D-deficient chicks with 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] significantly decreased net Na+ uptake by isolated membrane vesicles (by approximately 30%). The sterol suppresses the conductive pathway (25-45% inhibition) as well as the Na+/H+ antiport system. Kinetic analysis of the latter revealed that 1 alpha,25(OH)2D3 altered Vmax (from 12.9 to 4.8 nmol of Na+/20s per mg of protein), but did not change Km. Diminution of Na+ transfer, entailing an increase in the electrochemical transmembrane Na+ gradient, provides an explanation of the simultaneously observed stimulatory action of 1 alpha,25(OH)2D3 on Na+-gradient-driven solute transport in chick small intestine. Indirect evidence was obtained that the luminal plasma membrane of chick small intestine displays a definite H+ permeability that is positively affected by 1 alpha,25(OH)2D3.     

Binding properties of serum vitamin D transport proteins in vertebrates for 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol in vitro.

1. The affinities of the specific vitamin D plasma transport proteins for 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol were studied in 34 vertebrate species. 2. Fish plasma proteins bound 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol and 24S, 25-dihydroxycholecalciferol with equal efficiency. 3. Vitamin D transport proteins in birds and a monotreme bound 25-hydroxycholecalciferol more efficiently than 24R, 25-dihydroxycholecalciferol; in one bird the two seco-steroids were bound with equal efficiency. 4. Transport proteins from marsupial and placental mammals bound 24R, 25-dihydroxycholecalciferol more efficiently than 24S, 25-dihydroxycholecalciferol. 5. Twelve mammal transport proteins bound 25-hydroxycholecalciferol and 24R, 25-dihydroxycholecalciferol with equal efficiency, however, in six mammals 25-hydroxycholecalciferol was more efficiently bound.     

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.     

24, 25-dihydroxycholecalciferol but not 25-hydroxycholecalciferol suppresses apolipoprotein A-I gene expression

AimsLigands for the vitamin D receptor (VDR) regulate apolipoprotein A-I (apo A-I) gene expression in a tissue-specific manner. The vitamin D metabolite 24, 25-dihydroxycholecalciferol (24, 25-(OH)₂D₃) has been shown to possess unique biological effects. To determine if 24, 25-(OH)₂D₃ modulates apo A-I gene expression, HepG2 hepatocytes and Caco-2 intestinal cells were treated with 24, 25-(OH)₂D₃ or its precursor 25-OHD₃.Main methodsApo A-I protein levels and mRNA levels were measured by Western and Northern blotting, respectively. Changes in apo A-I promoter activity were measured using the chlorampenicol acetytransferase assay.Key findingsTreatment with 24, 25-(OH)₂D₃, but not 25-OHD₃, inhibited apo A-I secretion in HepG2 and Caco-2 cells and apo A-I mRNA levels and apo A-I promoter activity in HepG2 cells. To determine if 24, 25-(OH)₂D₃ represses apo A-I gene expression through site A, the nuclear receptor binding element that is essential for VDRs effects on apo A-I gene expression, HepG2 cells were transfected with plasmids containing or lacking site A. While the site A-containing plasmid was suppressed by 24, 25-(OH)₂D₃, the plasmid lacking site A was not. Likewise, treatment with 24, 25-(OH)₂D₃ suppressed reporter gene expression in cells transfected with a plasmid containing site A in front of a heterologous promoter. Finally, antisense-mediated VDR depletion failed to reverse the silencing effects of 24, 25-(OH)₂D₃ on apo A-I expression.SignificanceThese results suggest that the vitamin D metabolite 24, 25-(OH)₂D₃ is an endogenous regulator of apo A-I synthesis through a VDR-independent signaling mechanism.     

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.     

In vitro stimulation of articular chondrocyte differentiated function by 1,25-dihydroxycholecalciferol or 24R,25-dihydroxycholecalciferol

The effects of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) (10(-13)M-10(-8) M) and 24R ,25-dihydroxycholecalciferol ( 24R ,25-(OH)2D3) (10(-12)M-10(-7) M) on cell proliferation and proteoglycan deposition were examined in our newly developed multilayer culture system for rabbit and human articular chondrocytes. The cells are embedded in an extracellular matrix similar to that seen in vivo and maintain their in vivo phenotype. We extracted and purified native proteoglycans and degraded material from three culture compartments: the medium, intercellular matrix, and cells. Proteoglycan synthesis and deposition were analyzed by measuring 35SO4 incorporation, hexuronic acid, and galactose contents. In both rabbit and human chondrocyte cultures, chronic 1,25-(OH)2D3 treatment inhibited chondrocyte proliferation and stimulated proteoglycan synthesis and accumulation in the three compartments at 10(-12)-10(-8) M; maximal effect was at 10(-10)M. Cell proliferation was reduced by 55% and the content of hexuronic acid (or galactose) was increased to about three times that of controls in all compartments. 1,25-(OH)2D3 did not alter the proteoglycan composition. Chronic 24R ,25-(OH)2D3 treatment induced comparable effects with a maximum at 10(-8)M. When human dermal fibroblasts were treated as above both vitamin D metabolites increase mitosis. 1,25-(OH)2D3 mainly reduced the pericellular deposition of proteoglycans, while 24R ,25-(OH)2D3 appeared to reduce their synthesis and deposition in both medium and pericellular compartments. These results suggest that both 1,25-(OH)2D3 and 24R ,25-(OH)2D3 act specifically on articular chondrocytes to promote phenotype expression.     

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.