Is 24,25-dihydroxycholecalciferol a calcium-regulating hormone in man?

Small doses (1-10 microgram daily) of 24,25-dihydroxycholecalciferol (24,25-(OH)2D3), a renal metabolite of vitamin D of uncertain function, increased intestinal absorption of calcium in normal people and in patients with various disorders or mineral metabolism, including anephric subjects. In five of six patients studied, calcium balance increased, but, unlike 1,25-dihydroxycholecalciferol, 24,25-(OH)2D3 did not increase plasma or urinary calcium concentrations. These results suggest that 24,25-(OH)2D3 may be an important regulator of skeletal metabolism in man with potential value as a therapeutic agent.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Seasonal fluctuations in serum concentrations of vitamin D metabolites in normal subjects.

Serum concentrations of 25-hydroxycholecalciferol (25-OHD), 24,25-dihydroxycholecalciferol (24,25-(OH)2D), and 1,25-dihydroxycholecalciferol (1,25-(OH)2D) were measured at monthly intervals throughout the year in eight normal subjects. 25-OHD was measured by competitive protein-binding assay after Sephadex LH 20 chromatography, 24,25-(OH)2D by competitive protein-binding assay after Sephadex LH 20 and high-pressure chromatography, and 1,25-(OH)2D by radioimmunoassay after the same separation procedure as for 24,25-(OH)2D. A seasonal variation, apparently dependent on exposure to ultraviolet light, was found for all three metabolites. A study in six other normal subjects showed that there was no diurnal rhythm in any of the metabolites. Oral administration of 2 microgram 1,25-(OH)2D caused a sharp rise in serum concentrations of 1,25-(OH)2D and no change in the concentrations of the two other metabolites, but by 12 hours the 1,25-(OH)2D concentration had returned to the basal value. The concentrations of all three metabolites studied vary according to the season. Thus to interpret these concentrations in any subject the normal range for the particular season must be referred to.     

Effects of 1,25-dihydroxycholecalciferol administration on the rat renal vitamin K-dependent carboxylating system

We have shown previously that the in vitro activity of the renal vitamin K-dependent gamma-glutamyl carboxylase toward synthetic oligopeptide substrates is stimulated by administration of either parathyroid hormone (PTH) or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] to rats [(1983) J. Biol. Chem. 258, 12783-12786]. Here we report that administration of 1,25(OH)2D3 to rats increases their levels of endogenous carboxylase substrate as well. Rats fed a vitamin D-deficient diet had highly elevated serum PTH levels while vitamin D-replete animals had undetectable levels. Furthermore, since PTH increases 1,25(OH)2D3 levels by stimulating renal 25-hydroxyvitamin D-1 alpha-hydroxylase, it is very likely that the stimulatory effects of PTH on the renal vitamin K-dependent carboxylating system are mediated by 1,25(OH)2D3.     

1,25-Dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation

Human foreskin keratinocytes in culture produce 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25-(OH)2D3) from 25-hydroxycholecalciferol (25-(OH)D3). The production of 1,25-(OH)2D3 by these cells correlated with the early events of differentiation such as expression of transglutaminase activity and the levels of a precursor protein for the cornified envelopes, involucrin. In contrast, the increased production of 24,25-(OH)2D3, as 1,25-(OH)2D3 production declined, correlated with the terminal differentiation marker, cornified envelope formation. Exogenous 1,25-(OH)2D3 (10(-11)-10(-9) M) inhibited the 1-alpha-hydroxylase at all stages of growth of these cells. Keratinocytes in culture expressed receptors for 1,25-(OH)2D3 which had similar sedimentation behavior in sucrose density gradients as chick intestinal cytosol receptors. Cells in early stages of growth (preconfluent and confluent) contained higher numbers of receptors (26-27 fmol/mg protein) than post-confluent cells. The dissociation constant (237-278 pM) of these receptors for 1,25-(OH)2D3 was not consistently altered by differentiation. Since 1,25-(OH)2D3 is a potent stimulator of cell differentiation in a variety of systems including the epidermis, our results suggest the possibility that endogenous 1,25-(OH)2D3 production may participate in the differentiation of keratinocytes in culture and, perhaps, in vivo.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

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.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

24-Hydroxylase: potential key regulator in hypervitaminosis D3 in growing dogs

A group of growing dogs supplemented with cholecalciferol (vitamin D(3); HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D(3)/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of P(i) and growth-regulating hormones between groups and no signs of vitamin D(3) intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] was increased in HVitD vs. CVitD (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased renal 1alpha-hydroxylase gene expression), plasma 1,25(OH)(2)D(3) levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)(2)D(3) (demonstrated by [(3)H]1,25(OH)(2)D(3) and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D(3) toxicosis.     

Cultured human keratinocytes cannot metabolize vitamin D3 to 25-hydroxyvitamin D3.

The metabolism of [3H]vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H]vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25-OH-D3, 24,25(OH)2D3 and 1,25(OH)2D3 were added to the extracts, as markers, prior to HPLC. HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25-OH-D3 showed a 10 +/- 4% conversion to [3H]1,25(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25-OH-D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25-OH-D3.     

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.     

Kinetics of subcellular distribution in rat intestine of 1,25-dihydroxycholecalciferol administered in vivo. Evidence for concentration within 5 min into purified nuclei.

To better understand the initial steps in the induction of intestinal Ca2+ transport by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], we studied the early subcellular localization of 1,25(OH)2D3 in rat intestine. Vitamin D-deficient rats received 300 pmol of 1,25(OH)2[3H]D3 intravenously at 5 min to 4h before being killed. Cells homogenized in buffer of I = 90 mmol/litre were fractionated by centrifugation into a crude nuclear pellet, purified nuclei, Golgi and basal-lateral membranes, cytosol and a post-nuclear pellet. Nuclear purification was established by biochemical and morphological criteria and gave a yield of 32 +/- 2% (mean +/- S.E.M.; n = 21). Although re-establishment of Ca2+ uptake by Golgi is one of the earliest reported intestinal responses to 1,25(OH)2D3, no direct localization of 1,25(OH)2D3 to Golgi was detected. Purified nuclei had the highest specific radioactivity at all times studied, with nuclear localization detectable at 5 min and peak nuclear uptake at 1 h. Relative specific radioactivity of nuclei to cytosol increased from 5 min to 30 min, at which time equilibrium between cytosol and nucleus appeared to be attained. Nuclear uptake occurred in all cells from villus to crypt. Of total nuclear binding 10% was resistant to high ionic strength buffer (I = 365 mmol/litre); peak nuclear uptake was observed at 30 min in this buffer. This tight binding may represent the active fraction of 1,25(OH)2D3. These results indicate that localization of 1,25(OH)2D3 to rat intestinal nuclei precedes the observed Golgi-membrane effects and suggest the existence of high-affinity nuclear 1,25(OH)2D3-binding sites.     

Vitamin D3 metabolites in rat epididymis: high 24,25-dihydroxy vitamin D3 levels in the cauda region

Normal male rats received six subcutaneous injections of 8.0 pmoles of tritiated 25-hydroxy vitamin D3 ([3H]25(OH)D3) or one intrajugular injection of 8.0 pmoles of high specific radioactivity [3H]-25(OH)D3. Lipid extracts of several tissues including the reproductive organs were subjected to sephadex LH-20 chromatography to determine the tissue distribution of the injected material and of the in vivo produced dihydroxylated cholecalciferol metabolites. The nature of the putative 25(OH)D3 and the 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) from epididymis tissue was confirmed by high performance liquid chromatography (HPLC). The epididymis levels of 24,25(OH)2D3 were considerably higher in the cauda epididymis compared to kidney and caput epididymis levels. The other metabolites levels in this tissue were similar to those determined in the kidneys. The amounts of the three metabolites found in all other tissues were well below the cauda epididymis or kidney levels. The findings suggest a possible physiological role for 24,25(OH)2D3 in the epididymis, and are also consistent with data of others which indicated a possible action of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) in rat reproductive tissues.     

Regulation of 24-hydroxylase activity in mouse skin fibroblast by cholecalciferol derivatives, triamcinolone acetonide and a calcium modulating agent, nicardipine

Mouse skin fibroblasts in culture were used to study the regulation of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) induced 24 hydroxylase (24-OH-ase) under the influence of 3 agents: (1) 24,25-Dihydroxycholecalciferol (24,25(OH)2D3), 62.5 10(-9) M, which led to a significant decrease in the 1,25(OH)2D3-induced 24-OH-ase, probably acted through a nuclear effect mediated by the 1,25(OH)2D3 receptor protein. (2) Triamcinolone acetonide (10(-6)M) which was found to increase the 24-OH-ase enhancement induced by 1.25 and 6.25 nM 1,25(OH)2D3 whereas it did not alter the effect of 31.2 nM 1,25(OH)2D3. (3) A factor which is likely to induce changes in the cell calcium transport or in the Ca pool sizes, i.e. a calcium channel blocker, nicardipine. The effect of 1.25 nM 1,25(OH)2D3 on 24-OH-ase activity was increased by nicardipine (20 microM) which was found to reduce the effect of 6.25 nM 1,25(OH)2D3. The rate of DNA synthesis (measured by [3H]thymidine incorporation) was increased after incubation of fibroblasts with 1,25(OH)2D3 (1.25 nM) plus triamcinolone acetonide (10(-6) M), although it was reduced by nicardipine in comparison with 1,25(OH)2D3 alone. So the effects of these agents on the 1,25(OH)2D3 induced 24-hydroxylase were shown to be independent of the rate of DNA synthesis.     

Specific binding of 24R,25-dihydroxyvitamin D3 to chick intestinal mucosa: 24R,25-dihydroxyvitamin D3 is an allosteric effector of 1,25-dihydroxyvitamin D3 binding

We have previously described a significant decrease in the positive cooperativity level and affinity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] binding to its chick intestinal chromatin receptor induced in vitro by a physiological 10-fold molar excess of (24R)-25-dihydroxyvitamin D3 [24R,25(OH)2D3] [F. Wilhelm and A. W. Norman (1985) Biochem. Biophys. Res. Commun. 126, 496-501]. In this report, we have initiated a comparative study of the binding of 24R,25(OH)2[3H]D3 and 1,25(OH)2[3H]D3 to the the intestinal chromatin fraction obtained from vitamin D-replete birds. 24R,25(OH)2[3H]D3 specific binding to this chromatin fraction was characterized by a dissociation constant (Kd) of 34.0 +/- 6.4 nM, a positive cooperativity level (nH) of 1.40 +/- 0.13, and a capacity (Bmax) of 47 +/- 8 fmol/mg protein. The very low relative competitive index (RCI) of 24R,25(OH)2D3 (0.11 +/- 0.03%) for the 1,25(OH)2D3 binding site/receptor, as well as the inability of 1,25(OH)2D3 to displace 24R,25(OH)2D3 from its binding site at a physiological molar ratio of 1:10, strongly suggest the independence of 24R,25(OH)2D3 and 1,25(OH)2D3 binding sites. Stereospecificity of the 24R,25(OH)2D3 binding sites was attested by the displacement of only 45 +/- 6% of 24R,25(OH)2D3 specific binding by equimolar concentrations of 24S,25(OH)2D3. Collectively these results suggest the existence of a binding domain/receptor for 24,25(OH)2D3 in the chick intestine which is independent of the 1,25(OH)2D3 receptor.     

Pharmacokinetics of 24,25-dihydroxyvitamin D3 in humans

Pharmacokinetic properties of pharmacological doses of 24,25-dihydroxyvitamin-D3 [24,25(OH)2D3] were determined in healthy volunteers. Four male subjects received 25 micrograms of 24,25(OH)2D3 as an intravenous bolus injection. Plasma concentrations of 24,25(OH)2D3, 25-hydroxyvitamin D and 1,25-dihydroxy-vitamin D were monitored during 14 days. In addition, serum ionized calcium, total calcium, inorganic phosphate, albumin, creatinine and intact hPTH(1-84) were measured during 14 days. The concentration-time curve of 24,25(OH)2D3 could be described by a two-exponential curve with half-lives of 3.0 +/- 0.9 hrs and 8.2 +/- 2.9 days (mean +/- SD). The volume of distribution was 0.19 +/- 0.02 liters/kg. None of the mentioned biochemical parameters, except serum 24,25(OH)2D3, changed markedly. In 18 subjects suffering from primary hyperparathyroidism, taking 25 micrograms of 24,25(OH)2D3 daily during three months, an average plateau level of 39 +/- 12 nmol/l of serum was observed. Bioavailability as estimated from this plateau level was approximately 70%.     

Regulation of rat yolk sac 25-hydroxy- and 1,25-dihydroxyvitamin D3 24-hydroxylase activities

The yolk sac of the pregnant rat which functions as a true placenta is a target organ for vitamin D. This tissue can hydroxylate in position 24 both 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). The present report describes an in vitro model for the study of 1,25-(OH)2D3 action on the further metabolism of 25-OH[3H]D3 and 1,25-(OH)2[3H]D3 by yolk sac. The tissue explants were preincubated with 1,25-(OH)2D3 for 18 h in a serum-free culture medium. Physiological concentrations of 1,25-(OH)2D3 were the most effective in stimulating (7.5-fold) the 1,25-(OH)2D3 24-hydroxylase, while the 25-OHD3 24-hydroxylase stimulation (4-fold) required a 1,25-(OH)2D3 concentration of 10(-7) M. The stimulating effect of 1,25-(OH)2D3 on the 1,25-(OH)2D3 24-hydroxylase was temperature-dependent, and, since its was inhibited by actinomycin D and cycloheximide, required de novo protein synthesis. 1,24,25-(OH)3D3, 25-OHD3, and 24,25-(OH)2D3 were 10- to 1000-fold less potent than 1,25-(OH)2D3 in inducing the 1,25-(OH)2D3 hydroxylase. Our results strongly suggest that 1,25-(OH)2D3 regulated the 1,25-(OH)2D3 24-hydroxylase by a receptor-mediated process. Furthermore, 1,25-(OH)2D3 at 10(-9) M induced within 4 h an increase of its own degradation and the formation of an as yet unidentified major 1,25-(OH)2[3H]D3 metabolite. We conclude that the yolk sac can participate in the regulation of 1,25-(OH)2D3 concentration in the fetoplacental unit.     

Single cell analysis of changes in cytosolic calcium induced by vitamin D3 metabolites in cultured rat mesangial cells.

The acute effects of 1,25-Dihydroxy-vitamin D3 [1,25(OH)2D3] on the concentration of cytoplasmic ionized calcium [Ca2+] of cultured rat mesangial cells were studied at the single cell level by microspectrofluorometry of fura-2-loaded cells. Addition of 1,25(OH)2D3 produced an immediate increase of [Ca2]+. This rise in [Ca2+] was sustained and similar to that caused by the Ca2+ channel agonist BAY K 8644. Comparable changes were also observed in cultured human mesangial cells. The effects of the hormone (10 (-10)-10(-7) M) were dose-dependent (62% and 285%). Only 30-40% of the cells responded to stimulation with 1,25(OH)2D3. 25OHD3 also increased Ca2+ whereas 24,25(OH)2D3 and 1aOHD3 were inactive. Addition of 1 mM CoCl2 or 2-5 microM nifedipine largely blocked the effects of 1,25(OH)2D3 suggesting the involvement of Ca2+ channel activation in the rapid 1,25(OH)2D3-induced increase in mesangial cell [Ca2+]. 45Ca uptake studies are consistent with This interpretation.     

The calcification of cartilage matrix in chondrocyte culture: studies of the C-propeptide of type II collagen (chondrocalcin)

We have shown that when chondrocytes are isolated by collagenase digestion of hyaline cartilage from growth plate, nasal, and epiphyseal cartilages of bovine fetuses they rapidly elaborate an extracellular matrix in culture. Only growth plate chondrocytes can calcify this matrix as ascertained by incorporation of 45Ca2+, detection of mineral with von Kossa's stain and electron microscopy. There is an extremely close direct correlation between 45Ca2+ incorporation in the first 24 h of culture and the content of the C-propeptide of type II collagen, measured by radioimmunoassay, at the time of isolation and during culture. Moreover, growth plate cells have an increased intracellular content of the C-propeptide per deoxyribonucleic acid and, during culture, per hydroxyproline (as a measure of helical collagen) compared with nasal and epiphyseal chondrocytes. In growth plate chondrocytes 24,25-dihydroxycholecalciferol (24,25-[OH]2D3), but not 1,25-dihydroxycholecalciferol alone, stimulates the net synthesis of the C-propeptide and calcification; proteoglycan net synthesis is unaffected. Together, these metabolites of vitamin D further stimulate C-propeptide net synthesis but do not further increase calcification stimulated by 24,25-(OH)2D3. These observations further demonstrate the close correlation between the C-propeptide of type II collagen and the calcification of cartilage matrix.