In vitro synthesis of 1 alpha,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by isolated calvarial cells.

The question of whether the skeleton metabolizes 25-hydroxycholecalciferol [25(OH)D3] to more-polar products was studied. Calvarial cells were dispersed from 16-day old chicken embryos by using collagenase and then grown in culture in serum-free medium. Confluent cell cultures were incubated with 7 nM 25(OH)[3H]D3 for 2 hr, and the vitamin D metabolites were then extracted. At least four polar metabolites were produced. Based on separation by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography, two of these metabolites were identified as 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3]. These metabolites were also produced by cultured kidney cells but not by liver, heart muscle, or skin cells isolated from the same embryos. The specific activities of the calvarial 1- and 24-hydroxylases were similar in magnitude to those in isolated kidney cells. The specific activity of the calvarial 25(OH)D3:1-hydroxylase was inhibited by an 8-hr preincubation with 1,25(OH)2D3, whereas the 24-hydroxylase was enhanced. It is concluded that (i) vitamin D metabolism by isolated cells is organ-specific, (ii) calvarial cells produce active metabolites of vitamin D in significant amounts, (iii) vitamin D metabolism by calvarial cells is regulated by 1,25(OH)2D3, and (iv) locally produced, active metabolites could act locally, thereby adding a new dimension to the regulation of mineral metabolism by vitamin D metabolites.     

Veratridine stimulation of calcium uptake by chick embryonic heart cells in culture

Veratridine (2 X 10(-5) to 1 X 10(-4)M) stimulated Ca uptake into monolayer cultures of chick embryonic heart cells in a dose-dependent manner. The stimulation of veratridine was independent of the age of the chick embryos from which the heart cells were taken, and was inhibited by tetrodotoxin (3 microM). The half-maximal effect of veratridine was 37 microM. Since young embryonic hearts have few or no functional fast Na channels (Sperelakis, N. (1981) Cardiac Toxicol 1, 39-108), the present results indicate that the action of veratridine is not dependent solely on fast Na channels, but could be related to other properties of the surface membrane, such as increasing the resting Na permeability and/or opening of the slow Na channels. The increased Ca influx could then be a consequence of the Ca0:Nai exchange reaction.     

Rapid stimulation of calcium uptake and protein phosphorylation in isolated cardiac muscle by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 has been shown to induce rapid changes in calcium fluxes in skeletal muscle and other target tissues independently of gene activation. The possibility that the hormone would produce similar effects in heart where 1,25-dihydroxyvitamin D3 receptors and activities have been shown, was studied. A significant increase of 45Ca uptake by left ventricular slices from vitamin D-deficient chicks was observed upon incubation for 1-10 min with physiological doses of 1,25-dihydroxyvitamin D3. This stimulation was dose-dependent and specific for the hormone when compared with vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 and could not be associated to changes in lipid synthesis as assessed by measurements of [3H]glycerol incorporation into cardiac tissue lipids. The Ca channel blockers nifedipine (30 microM) and verapamil (10 microM) abolished the increase in Ca uptake produced by 1,25-dihydroxyvitamin D3. The rapid effects of the hormone on heart Ca influx were accompanied by a stimulation of the phosphorylation of two microsomal proteins of 43 kDa and 55 kDa. These results further support a direct action of 1,25-dihydroxyvitamin D3 in the regulation of cardiac muscle Ca metabolism which may involve activation of Ca channels.     

Response of shell-less cultured chick embryos to exogenous parathyroid hormone and 1,25-dihydroxycholecalciferol

Chick embryos maintained in shell-less culture up to a total age of 14 days and 11-day-old embryos developed in ovo were injected either with 20 USP units parathyroid hormone or with 200 pmol 1,25-dihydroxycholecalciferol. In all cases, a significant increase in the concentration of calcium in blood was observed. Since in the cultured embryos the shell is absent, no calcium can reach the embryo through the chorioallantoic membrane; the present results are thus interpreted as meaning that both parathyroid hormone and 1,25-dihydroxycholecalciferol can produce their effect by acting on target organs other than the chorioallantoic membrane.     

Inhibitors of protein and RNA synthesis and 1,25-dihydroxycholecalciferol formation in vitro

Formation of 1,25-dihydroxycholecalciferol (1,25 (OH)₂CC) from 25-hydroxycholecal-ciferol (25 OHCC) was studied in isolated renal tubules prepared from vitamin D-deflcient chicks. Cycloheximide and puromycin inhibited the rate of formation of 1,25 (OH)₂CC, giving an apparent initial half-life for the activity of the 25 OHCC-1-hydroxylase enzyme in this system of $\text{3}\text{1}\text{2}\text{–4}\text{h}$. 6 h pre-incubation with actinomycin D or α-amanitin did not inhibit the enzyme's activity, suggesting that the half-life of the messenger RNA responsible for the synthesis of the enzyme is considerably greater than 6 h in the chick. Neither cycloheximide nor actinomycin D prevented the stimulation of the enzyme's activity by dibutyryl cAMP, showing that this stimulation is independent of new protein synthesis.     

In vitro uptake of IgA complexes by Kupffer cells

ABSTRACT— Cultured murine Kupffer cells were tested for their ability to bind TNP-sensitized erythrocytes coated with IgA myeloma proteins (MOPC 315 & 460) which have high affinity for TNP ligands, and to ingest TNP-enzyme-IgA soluble immune complexes. In these experimental conditions, 6–18% of Kupffer cells were found to express surface binding sites for IgA, a feature they share with lymphocytes and granulocytes. This functional property, also displayed by circulating monocytes, is thus maintained in liver-residing macrophages, and may contribute to the regulation of immune response to gut-derived antigens.     

Regulatory effect of 1,25-dihydroxycholecalciferol on calcium fluxes in thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2-D3) on the regulation of calcium fluxes in rat thyroid FRTL-5 cells. The ATP-induced uptake of 45Ca2+ was decreased in cells pretreated with 1,25(OH)2D3 for 48 h. No effect was seen on basal uptake of 45Ca2+. At least a 24 h incubation period was required for the effect of 1,25(OH)2D3 to be expressed. Pretreatment with 1,25(OH)2D3 for 48 h did not change resting intracellular Ca2+ ([Ca2+]i) in fura-2-loaded FRTL-5 cells. However, the ATP-induced increase in [Ca2+]i was significantly enhanced in cells preincubated with 1,25(OH)2D3. The effect of 1,25(OH)2D3 was abolished in Ca(2+)-free buffer. No difference in the ionomycin-induced increase in [Ca2+]i was observed between control cells and cells pretreated with 1,25(OH)2D3. However, in Ca(2+)-free buffer the ionomycin response was decreased in cells incubated with 1,25(OH)2D3. The ATP-induced change in [Ca2+]i was decreased when ATP was added after ionomycin to cells treated with 1,25(OH)2D3. The results suggest that 1,25(OH)2D3 has a regulatory effect on Ca2+ fluxes in FRTL-5 cells, possibly by acting on Ca2+ sequestration.     

Autoradiographic demonstration of target cells for 1,25-dihydroxycholecalciferol in the chick embryo chorioallantoic membrane,duodenum, and parathyroid glands

Sixteen-day-old chick embryos were injected with tritiated 1,25-dihydroxycholecalciferol (1,25-DHCC) and sacrificed 2 hr later. Portions of the chorioallantoic membrane, duodenum, yolk sac, and parathyroid glands were frozen and processed for thaw-mount autoradiography. High concentration of radioactivity was found in nuclei of the chorionic epithelium; their spatial distribution suggests, although does not prove, that these nuclei correspond to “calcium-absorbing” cells. Radioactivity was also demonstrated in nuclei of the duodenal epithelial cells and the cords of parathyroid glands. No localization was found in the wall of the yolk sac after 150 days of exposure. The injection of nonradioactive 1,25-DHCC 30 min before the administration of the radiocctive hormone prevented in all cases the nuclear concentration, probably by saturating receptors. The results are interpreted as demonstrating the presence of target cells for the hormone in chorionic epithelium, duodenum, and parathyroid glands, structures all believed to be involved in calcium metabolism in the embryo.     

Short-term effects of 1,25-dihydroxycholecalciferol on disordered calcium metabolism of renal failure

The short-term effects of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) on disordered calcium metabolism were studied in 15 trials in patients with advanced renal failure and, for comparison, in 20 trials in normal persons. The steroid was given orally in doses of 0.027, 0.14, 0.68 and 2.7 μg/day for 7 to 15 days. Calcium absorption and urinary calcium increased after the administration of 0.14 to 2.7 μg/day in normal subjects, but 0.68 to 2.7 μg/day was needed to augment calcium absorption in the patients with uremia. Fecal calcium decreased significantly during metabolic balance studies. Serum calcium increased markedly only in the patients with uremia, and hypercalcemia occurred at a dosage level of 2.7 μg/day. With the increase in serum calcium, the blood levels of immunoreactive parathyroid hormone (iPTH) decreased. Urinary calcium increased substantially only in normal subjects. The steroid corrected the “vitamin D resistance” of uremia, suggesting that the renal production of 1,25-(OH)₂D₃ in uremia is defective.     

Prolactin but not growth hormone stimulates 1,25-dihydroxyvitamin D3 production by chick renal preparations in vitro

We studied the effect of PRL from two species (bovine and turkey) and GH from two species (bovine and turkey) on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production by two whole cell preparations from vitamin D-deficient chick kidneys (slices and tubules). We observed that 8 ng/ml turkey PRL stimulated 1,25(OH)2D3 production by renal tubules and slices. Ovine PRL had a similar effect on 1,25(OH)2D3 production but at higher concentrations. In contrast, neither bovine GH nor turkey GH stimulated 1,25(OH)2D3 production appreciably at doses up to 1000 ng/ml. The effect of PRL on 1,25(OH)2D3 production by renal tubules required a 3-h preincubation, although its effect on 1,25(OH)2D3 production by renal slices was immediate. We conclude that PRL, but not GH, directly stimulates 1,25(OH)2D3 production by the chick kidney.     

Enhancement of human immunodeficiency virus 1 replication in monocytes by 1,25-dihydroxycholecalciferol.

Human immunodeficiency virus (HIV) expression and replication are under tight regulatory control. We demonstrate that 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances the replication of monocyte- and lymphocyte-tropic strains of HIV-1 up to 10,000-fold in monocyte cell lines, peripheral blood monocytes, and unfractionated peripheral blood mononuclear cells. 1,25(OH)2D3 is therefore one of the most potent regulators of HIV-1 replication described to date. Precursors of 1,25(OH)2D3 enhance HIV-1 replication in proportion to their affinity for the 1,25(OH)2D3 intracellular receptor, suggesting that 1,25(OH)2D3 influences HIV-1 replication by mechanisms involving this receptor. These studies may have important implications for the design of effective therapy of HIV-1 infection.     

Highly specific binding of 1,25-dihydroxycholecalciferol in bone cytosol.

A method developed initially for the detection of high-affinity binding of glucocorticoids in the cytosol from foetal rat calvaria has been adapted for metabolites of vitamin D. Consistent displacement of [3H]1,25-dihydroxycholecalciferol ([3H]1,25(OH)2D3) by unlabelled 1,25(OH)2D3 was obtained with an apparent dissociation constant (Kd) of 2.3 x 10(-9) mol/l. The specificity of this binding was examined by competition experiments. Displacement of labelled 1,25(OH)2D3 by a 100-fold excess of unlabelled metabolites, expressing the fall with unlabelled 1,25(OH)2D3 as 100%, was as follows: 25-hydroxycholecalciferol (25(OH)D3), 61%; 24,25-dihydroxycholecalciferol, 29%; cholecalciferol, 3%. These are similar to results for the chick mucosa nuclear 1,25(OH)2D3 receptor. No displacement was obtained with corticosterone, testosterone, oestradiol or progesterone. When [3H]25(OH)D3 was used as ligand, a displacement curve with unlabelled 25(OH)D3 indicated only binding with a greater Kd (approximately 10(-7) mol/l). These data suggest a direct action of 1,25(OH)2D3 on bone which is similar to that of steroid hormones on their target tissues.     

Comparison of serum 1,25-dihydroxycholecalciferol concentrations in rheumatoid arthritis and osteoarthrosis.

We have previously compared 25-hydroxycholecalciferol levels in the serum of patients with osteoarthrosis and rheumatoid arthritis, finding no significant difference between the circulating levels of this hormone. We have now estimated 1,25-dihydroxycholecalciferol levels on stored sera from the same groups of patients and found no significant difference in the levels of this hormone between the 2 groups. The osteopenia that distinguishes rheumatoid arthritis from osteoarthrosis is not the result of altered levels of systemic 25OHD3 or of 1,25(OH)2D3. Local factors may be more important in its pathogenesis.     

Demonstration of a 1,25-dihydroxycholecalciferol cytoplasmic receptor-like binder in mouse kidney.

Isolated mouse renal tubule cells have been employed to demonstrate the presence of a specific high affinity cytoplasmic binding protein for 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in kidney. This receptor-like macromolecule sedimented at 3.2 S in hypertonic sucrose density gradients. Scatchard analysis of [3H]1,25-(OH)2D3 binding at O C revealed an apparent Kd of 0.2 nM and a concentration of binding sites of 50 fmol/mg cytosol protein. In competition experiments, the binder exhibited a low affinity for other vitamin D3 metabolites; the order of potency was 1,25(OH)2D3 greater than 250HD3 greater than u alpha OHD3 greater than 24R,25(OH)2D3. The sedimentation properties, binding affinity, and specificity of this 1,25(OH)2D3 binding protein are strikingly similar to the receptors in rat intestine, mouse bone, and human intestine. The demonstration of a renal receptor-like binder adds further support to the concept that the kidney is a 1,25(OH)2D3 target organ.     

Role of 1,25-dihydroxycholecalciferol in growth-plate cartilage: inhibition of terminal differentiation of chondrocytes in vitro and in vivo.

The effects of vitamin D metabolites on alkaline phosphatase [ALPase; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] activity, a marker of terminal differentiation, in chondrocyte cultures and growth plates in vivo were examined. In cultures of pelleted rabbit growth-plate chondrocytes, 1,25-dihydroxycholecalciferol (1,25-dihydroxyvitamin D3) increased the contents of DNA and macromolecules containing uronic acid (proteoglycans). It also decreased ALPase activity with an ED50 of less than 1 nM. Other vitamin D3 metabolites, such as 24,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol, had little effect on these biochemical parameters. In rachitic growth plates, the uronic acid content was half that in normal growth plates, whereas ALPase activity was 2.5 times that in normal growth plates. Administration of 1,25-dihydroxycholecalciferol at a low dose (0.1 micrograms per kg of body weight) to rachitic rats increased the uronic acid content 1.4-fold and decreased ALPase activity by 40%. This compound, like 24,25-dihydroxycholecalciferol (10 micrograms per kg of body weight), increased the calcium level of the blood. However, administration of 24,25-dihydroxycholecalciferol had little effect on the uronic acid and ALPase contents in growth plates. These observations suggest that 1,25-dihydroxycholecalciferol is a bioactive form of vitamin D that plays an important role in the control of chondrocyte terminal differentiation.     

In vitro production of 1,25-dihydroxyvitamin D3 by rat placental tissue.

Weanling female rats were fed a vitamin D-deficient diet for 4 months until they reached maturity. They were mated with normal, vitamin D-replete male rats and, at 20 days of pregnancy, the female rats were killed and their placentae were removed, homogenzied, and incubated with 25-hydroxyvitamin D3. The incubation mixtures were extracted and the extracts were subjected to Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. The 1,25-dihydroxyvitamin D3 region of the high-pressure liquid chromatogram was recycled to purity and the structure of the product was identified as 1,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry and by mass spectrometry. Thus it is now evident that placenta, in addition to renal tissue, is capable of converting 25-hydroxyvitamin D3 to the hormonal form of vitamin D, 1,25-dihydroxyvitamin D3.     

In vitro stimulation of primate hemoglobin synthesis by L-thyroxine.

Bone marrow cells from adult and abortus primates (marmosets) were incubated in vitro to determine their responsiveness to L-thyroxine. 3H-leucine incorporation into purified globin chains was the parameter assayed to determine responsiveness. Bone marrow from spontaneously aborted animals consistently was stimulated by the presence of physiologic levels of L-thyroxine. Bone marrow cells from adult animals were unaffected by the hormone.     

Vitamin D metabolism and the response to 1,25-dihydroxycholecalciferol in Osteoporosis.

The metabolism of isotopically-labelled cholecalciferol and the response to small doses of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in a group of women with osteoporosis presenting with crush vertebral fracture. No abnormality of vitamin D metabolism was detected. The administration of 1 microgram 1,25-(OH)2D3 for between 8 and 20 days was associated with an increased intestinal absorption and urinary excretion of calcium but caused no improvement in calcium balance. There was a small but significant rise in serum calcium and phosphorus and significant reduction in immunoassayable parathyroid hormone levels during treatment. It is concluded that 1,25-(OH)2D3 is unlikely to be of value in the management of osteoporosis.