Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: The effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids

Summary Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)₂D₃ are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13–13nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60–70% increment) with similar sensitivity to dexamethasone (ED₅₀ ∼ 1.0 nm). On the other hand, 1,25(OH)₂D₃ decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)₂D₃ treatment. Introduction of physiological concentrations of 1,25(OH)₂D₃ (0.013–1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)₂D₃ and a complex interplay among PTH, dexamethasone, and 1,25(OH)₂D₃.     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

Summary We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)₂D₃ and for glucocorticoids, and have examined the modulation of the 1,25(OH)₂D₃ receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)₂D₃ receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)₂D₃; this phenomenon occurred in a dosedependent fashion for triamcinolone (10⁻⁹ to 10⁻⁷ M) with a maximum increase of 1,25(OH)₂D₃ receptor concentration of ⋍twofold. During the culture period, the 1,25(OH)₂D₃ receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)₂D₃ receptor concentration was higher in the triamcinolone-treated cells.     

1,25 dihydroxyvitamin D3 alone or in combination with parathyroid hormone does not increase bone mass in young rats

Summary Parathyroid hormone (PTH) alone is known to increase bone mass, but clinical studies of osteoporotic men suggest that when 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) is given in combination with PTH, the effect on bone growth is enhanced. To determine if 1,25(OH)₂D₃ alone would stimulate bone growth, young male rats were given daily subcutaneous injections of either vehicle or 2.5, 5, 10, or 20 ng 1,25(OH)₂D₃ per 100 g body weight for 30 days. To determine if 1,25(OH)₂D₃ would augment the PTH anabolic response, rats were given daily subcutaneous injections of either vehicle for 12 days; or 4 μg/100 g hPTH alone or in combination with 5 ng/100 g 1,25(OH)₂D₃; or 8 μg/100 g hPTH alone or in combination with 5 ng/100 g 1,25(OH)₂D₃. Calcium (Ca), dry weight (DW), and hydroxyproline (Hyp) of the distal femur; the rate of mineralization in the metaphysis of the proximal tibia; and serum calcium and phosphate were measured. Low normocalcemic doses of 1,25(OH)₂D₃ did not significantly stimulate bone growth. 1,25(OH)₂D₃ did not augment the PTH-stimulated anabolic effect in young male rats. Low doses (2.5 and 5 ng) of 1,25(OH)₂D₃ were not hypercalcemic, and there was no increase in total bone calcium or dry weight although the 5 ng dose increased trabecular bone calcium. 1,25(OH)₂D₃ at 10 and 20 ng increased trabecular bone DW and Hyp, but mineralization was impaired and rats were hypercalcemic. 1,25(OH)₂D₃ in combination with PTH did not augment the PTH stimulation of bone growth as trabecular and cortical bone Ca, DW, and HYP were not increased in rats given both hPTH and 1,25(OH)₂D₃ compared with values for rats treated with hPTH alone.     

1,25(OH)2D3 inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells

Summary Increasing the extracellular Ca²⁺ concentration from 0.5 to 3.0 mM induced marked increments in cytoplasmic Ca²⁺ concentration (Ca²⁺ _i) and inhibition of parathyroid hormone (PTH) release of freshly isolated bovine parathyroid cells. 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃; 0.1–100 ng/ml) did not affect (Ca²⁺ _i) and was also without acute effect on the secretion. During 4 days of monolayer culture, the parathyroid cells underwent significant increases in both number and size, and presence of 10–100 ng/ml 1,25(OH)₂D₃ almost completely inhibited the cell proliferation, whereas the hypertrophy was unaffected. One day of culture with 0.1–100 ng/ml 1,25(OH)₂D₃ was without effect on PTH release but after 4 days there was a dose-related reduction of recretion. At this time point and irrespective of the culture condition, PTH release was no longer suppressed by high extracellular Ca²⁺. Furthermore, Ca²⁺ _i increased little upon increments in the extracellular Ca²⁺ concentration as compared with freshly isolated cells. It is concluded that after prolonged exposure to 1,25(OH)₂D₃, PTH release is inhibited and, at high concentrations, the parathyroid cells cease to proliferate. However, 1,25(OH)₂D₃ does not affect the development of functional dedifferentiation of parathyroid cells during monolayer culture.     

Evidence forin vivo upregulation of 1,25(OH)2 vitamin D3 receptor in human monocytes

Summary Mammalian cells increase net expression of 1,25(OH)₂D₃ receptors after exposure to physiological concentrations of 1,25(OH)₂D₃ in vitro. we examined specific binding of 1,25(OH)₂D₃ by human monocytes before and after daily administration of 1.5–2 ug 1,25(OH)₂D₃ p.o. for 3 days in 5 healthy normal D-replete probands. Median specific binding (N_max) at baseline was 793 molecules/cell and 2052 or 2828 at 24h and 72h of 1,25(OH)₂D₃ treatment respectively. The results suggest (a) up-regulation of 1,25(OH)₂D₃ receptors occurs in man and (b) monocyte preparations can be used to assess receptor regulationin vivo.     

Biochemical characterization of 1,25(OH)2D3 receptors in chick embryonal duodenal cytosol

Summary This study presents measurements of serum vitamin D metabolites, calcium and phosphorus as well as measurements of the equilibrium dissociation constant for duodenal 1,25(OH)₂D₃ receptor in 15-, 18-, 19-, and 20-day chick embryos in comparison to that in 1- and 118-day-old chicks and to vitamin D-deficient chicks. The present results showed that: (a) serum 1,25(OH)₂D and 24,25(OH)₂D levels rise from 15 and 18 to days 19 and 20 of embryonic development while serum phosphate levels are stable; (b) serum calcium levels rise at hatching to adult levels; (c) the duodenal 1,25(OH)₂D₃ receptor is detectable in 15-day-old embryo and has a Kd similar to that of 118-day-old vitamin D-replete chicks; and (d) the activity of 1,25(OH)₂D₃ receptor in chick duodenal cytosol is maximal at hatching.     

Enhanced suppression of 1,25(OH)2D3 and intact parathyroid hormone in Graves' disease as compared to toxic nodular goiter

Summary 1,25(OH)₂D₃, 25OHD₃, and intact parathyroid hormone, as well as various parameters of calcium-phosphorus metabolism were measured in 38 patients with Graves' disease (GD) and in 24 patients with toxic nodular goiter (TNG). Plasma 1,25(OH)₂D₃ levels were lower in GD patients (82 ±29 pmol/liter) than in those with TNG (155±32 pmol/liter) (P<0.0005). The mean value of 1,25(OH)₂D₃ in 45 controls was intermediate between the two groups of patients (140±41) and the difference was statistically significant. GD patients before and after treatment had higher alkaline phosphatase (P<0.05), lower intact parathyroid hormone (PTH) (P<0.05), and lower 1,25(OH)₂D₃ levels (P<0.0005 in the hyperthyroid andP<0.01 in the euthyroid state) than TNG patients. We conclude that increased skeletal calcium resorption is due to elevated levels of T₃ causing suppression of 1,25(OH)₂D₃ production and of PTH levels in both groups of patients albeit of different degrees. Furthermore, we postulate that the profound suppression of 1,25(OH)₂D₃ in GD is secondary to an immune-mediated phenomenon.     

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

Summary Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)₂D₃ receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [³H]-1,25(OH)₂D₃ is noted in both the logarithmic growth phase and at confluence (N_max 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. K_D was 10⁻¹¹ M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)₂D₃ receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest N_max in the proliferating cartilage. 1,25(OH)₂D₃ in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10⁻¹² M and dose-dependent inhibition at 10⁻¹⁰ M and below. Inhibition was specific and not seen with 24,25(OH)₂D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and effects of 1,25(OH)₂D₃ on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.     

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cellsIn vitro

Summary Binding of [³H] 1,25 (OH)₂D₃ and effects of 1,25 (OH)₂D₃ on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [³H] 1,25 (OH)₂D₃ by a 3.5 S macromolecule with DNA binding, K_D 6.2×10⁻¹⁰M and N_max 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10⁻⁸ M 1,25 (OH)₂D₃, but not 25 (OH)D₃, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.     

1,25(OH)2D3 and dihydrotestosterone interact to regulate proliferation and differentiation of epiphyseal chondrocytes

Growth plate chondrocytes are affected by 1,25(OH)₂D₃ and androgens, which may critically interact to regulate proliferation and differentiation during the male pubertal growth spurt. We investigated possible interactions of 1,25(OH)₂D₃ and the non-aromatizable androgen dihydrotestosterone (DHT) in primary chondrocyte cultures from young male rats. DHT and 1,25(OH)₂D₃ independently stimulated DNA synthesis and cell proliferation in a dose-dependent manner with maximally effective doses of [10^-8 M] and [10^-12 M], respectively. Both DHT and 1,25(OH)₂D₃ stimulated the expression and release of IGF-I, and the proliferative effects of each hormone were prevented by an IGF-I antibody. DHT and 1,25(OH)₂D₃ increased messenger RNAs (mRNAs) of their cognate receptors and of IGF-I receptor mRNA (IGF-I-R). 1,25(OH)₂D₃ also stimulated mRNA of the androgen receptor (AR), whereas DHT did not affect mRNA of the vitamin-D receptor (VDR). Coincubation with both steroid hormones did not stimulate receptor mRNAs more than either hormone alone. The proliferative effects of DHT and 1,25(OH)₂D₃ were completely inhibited by simultaneous incubation with both hormones, despite potentiation of IGF-I synthesis. In contrast, both hormones synergistically stimulated cell differentiation as judged by alkaline phosphatase activity, collagen X mRNA, and matrix calcification in long-term experiments. We conclude that DHT and 1,25(OH)₂D₃ interact with respect to chondrocyte proliferation and cell differentiation. The proliferative effects of both hormones are mediated by local IGF-I synthesis. Simultaneous coincubation with both hormones blunts the proliferative effect exerted by either hormone alone, in favor of a more marked stimulation of cell differentiation.     

The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells

Summary Controversy exists over a direct effect of 1,25(OH)₂D₃ on PTH secretion. To investigate the possibility that the suppressive effect of 1,25(OH)₂D₃ on PTH secretion may be demonstrable in 1,25(OH)₂D₃-depleted tissue and/or after prolonged periods of exposure to 1,25(OH)₂D₃, primary monolayer cultures of bovine parathyroid cells were established in 1∶1 DMEM/Ham's F-12 media supplemented with 2% calf serum but not 1,25(OH)₂D₃. Ionized calcium was maintained at 1.0 mM. Experiments were performed on 4-day-old culture cells. PTH concentration was measured using both a mid-region/carboxyl and an amino-terminal PTH antisera. 1,25(OH)₂D₃ at a concentration of 0.1 ng/ml suppressed PTH secretion by 32±7% after 48 hours. High calcium concentration (2.0 mM) suppressed PTH secretion by 37±10% and this effect was not additive over that of 1,25(OH)₂D₃. PTH secretion rate recovered fully 48 hours after normalization of the external calcium concentration but not after the removal of 1,25(OH)₂D₃. It is concluded that 1,25(OH)₂D₃ directly suppresses PTH secretion by monolayer culture of bovine parathyroid cells.     

Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse

Summary The purpose of this study was to evaluate whether the 1,25(OH)₂D₃-induced increased bone mineralization in the mouse occurs in response to stimulation of bone resorption. In order to inhibit bone resorption, 35-day-old mice were given 16 μmol/kg/day of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) for 10 days, the first injection occurring 3 days prior to the continuous infusion of 0.06, 0.13, or 0.20 μg/kg/day of 1,25(OH)₂D₃ for 7 days. Two groups of mice were treated with AHPrBP or 1,25(OH)₂D₃ alone. The skeletal changes were assessed by histomorphometric study of caudal vertebrae after double³H-proline and double tetracycline labelings for evaluation of the matrix apposition rate (MaAR) and mineral apposition rate (MiAR), respectively. Treatment with AHPrBP alone or combined to 1,25(OH)₂D₃ decreased the number of acid phosphatase-stained osteoclasts and reduced the endosteal MaAR and MiAR and the amount of osteoid. When given alone, 1,25(OH)₂D₃ increased serum calcium above normal, enhanced the number of histochemically active osteoclasts, and stimulated the endosteal MiAR. Pretreatment with AHPrBP blocked both the increase in serum calcium and the stimulation of the MiAR induced by 1,25(OH)₂D₃ infusion though serum 1,25(OH)₂D₃ levels rose according to the dose given. The results show that 1) the serum calcium and the bone resorbing responses to 1,25(OH)₂D₃ infusion are prevented by pretreatment with AHPrBP, and 2) the stimulatory effect of 1,25(OH)₂D₃ on the mineralization rate is blocked when bone resorption is inhibited. The data indicate that 1,25(OH)₂D₃ promotes bone mineralization in the mouse mainly in response to stimulation of bone resorption.     

Interaction between 24R,25-dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol on45Ca release from bone in vitro

Summary Interaction among vitamin D₃ metabolites on bone receptor sites is not known. Therefore, interaction between the most potent vitamin D₃ metabolite, 1,25(OH)₂D₃, and the most abundant dihydroxymetabolite, 24R,25(OH)₂D₃, was studied on isolated rat fetal bone by measuring⁴⁵Ca release from prelabeled bones. 24R,25(OH)₂D₃ at concentrations of 10–50 ng/ml caused marked inhibition of the bone-resorbing activity of 1,25 (OH)₂D₃ at concentrations of 10–50 pg/ml. 24S,25-(OH)₂ (unnatural enantiometer), on the other hand, at a concentration of 100 ng/ml did not inhibit the bone-resorbing effect of 10 pg/ml 1,25(OH)₂D₃. 24R,25(OH)₂D₃ at a concentration of 20 ng/ml did not inhibit the⁴⁵Ca-releasing effect of a submaximal concentration of PTH (500 ng/ml). Therefore, the inhibitory effect of 24R,25(OH)₂D₃ on the bone response to 1,25(OH)₂D₃ appeared to be specific and probably due to a competitive inhibitory effect. In addition, the inhibitory effect of 24R,25(OH)₂D₃ was weak, since it could be partially overcome by increasing the concentration of 1,25 (OH)₂D₃.     

Histological observations on the failure of rachitic rat bones to respond to 1,25/OH)2D3

Summary Rachitic rats, maintained on diets with low or normal P contents, were given daily intraperitoneal doses of 1,25(OH)₂D₃ or 25OHD₃ at levels of 100 or 200 ng. Plasma chemistry was measured and the ash content and histological appearance of the bones investigated. Using labeled material it was shown that the dosing levels of 1,25(OH)₂D₃ employed ensured a higher than normal plasma concentration of that metabolite over the period between doses. 1,25(OH)₂D₃ was not as effective as 25OHD₃ in raising bone ash or reducing the amount of osteoid. The difference between the effects of the metabolites was evident at both dietary P levels, but more marked at the higher P level. In contrast, the metabolites reduced the width of the epiphyseal plate to an approximately similar degree, and this is possibly the reason why there are discrepancies between previous reports of the effectiveness of 1,25(OH)₂D₃ compared with 25OHD₃ or vitamin D₃. Dosing with 1,25(OH)₂D₃ failed to maintain a constant plasma P_i value over the period between doses in animals fed the low P diet.     

Effects of 1,25(OH)2D3 on bone tissue in the rabbit: Studies on fracture healing,disuse osteoporosis,and prednisone osteoporosis

Summary A closed tibial fracture, which was controlled by an intramedullary stainless steel pin, was created in 16 rabbits. Eight rabbits were treated with 75 ng of 1,25(OH)₂D₃ daily as subcutaneous (s.c.) injections. After three weeks, the fractured tibia resisted a force of 101,7±21.0 Newtons in the control group and 57.3±8.0 Newtons in animals given 1,25(OH)₂D₃ (m±SE,P<0.05). In another group of eight rabbits, the left hindleg was immobilized in a plastic splint. Four rabbits were given 75 ng of 1,25(OH)₂D₃/day s.c. and the effect of immobilization was studied on the calcaneus. Bone ash/cm³ of the calcaneus on the immobilized side was decreased by 11±2% in control rabbits and by 20±2% in the group treated with 1,25(OH)₂D₃ indicating a more advanced immobilization osteoporosis (m±SE,P<0.05), which was also demonstrated by studies of bone density. Eighteen rabbits were used in a study of the effects of 1,25(OH)₂D₃ on the development of prednisolone osteoporosis. The dose of prednisolone was 2.5 mg per day, given by the oral route. After four months, the density of the femur was 1.53±0.02 g/cm² in control rabbits and 1.42±0.01 in prednisolonetreated animals (P<0.01). In rabbits additionally given 1,25(OH)₂D₃, the mean value for bone density was further lowered (n.s.). It appears that 1,25(OH)₂D₃ exaggerates disuse osteoporosis and prednisolone osteoporosis and impairs fracture healing in rabbits. These results differ from what has been shown earlier with 1,25(OH)₂D₃ treatment in the rat.     

1,25 dihydroxyvitamin D3 modifies cyclosporine-induced bone loss

Summary We have previously shown that cyclosporin A (CsA) produces high bone remodeling with resorption exceeding formation and loss of bone volume in the rat. This may have important clinical implications where CsA is widely used in organ transplantation. 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a bone mineralizing hormone which also has immune modifying properties. Consequently, we studied the effect of combined CsA and 1,25(OH)₂D₃ administration over 28 days in four groups of rats. Group A received vehicle (n=10), group B CsA (15 mg/kg) (n=10) alone, group C 1,25(OH)₂D₃ plus CsA (n=15), and group D 1,25(OH)₂D₃ alone (20 ng/100 g) (n=15). Rats were bled periodically at day 0, 7, 14, and 28 and Ca, parathyroid hormone (PTH), 1,25(OH)₂D, osteocalcin (bone Gla-protein, BGP), BUN, and creatinine were measured. Rats were sacrificed on day 28 and bones were examined histomorphometrically. Compared to controls, CsA resulted in significant elevation of BGP and a transient increase in 1,25(OH)₂D with excess bone remodeling and loss of bone volume. 1,25(OH)₂D₃ administration produced hypercalcemia, a significant rise in BGP, with suppression of PTH and increased osteoid volume. Combined therapy prevented the loss of bone volume probably due to increased osteoid tissue and enhanced osteoblast activity. Renal dysfunction, a side-affect of CsA, was not a factor. In conclusion, 1,25(OH)₂D₃ combined with CsA restores bone volume which is accompanied by increases in serum calcium and BGP.     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Lack of In vitro evidence for storage of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 1,25(OH)2D3 binding protein in skeletal matrix

A few studies have reported on the measurement of 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) in bone, using chloroform/methanol extraction and radioreceptor assay. As the significance of bone 1,25(OH)₂D₃ content was not defined in any of these reports, the objective of the current investigation was to determine whether 1,25(OH)₂D₃ may be stored in skeletal matrix. Bone powder samples from the iliac crest were extracted in ethylacetate/cyclohexane and 1,25(OH)₂D₃ isolated from the extract by means of Sephadex LH-20 and high pressure liquid chromatographic separation and subsequently measured by radioimmunoassay (RIA). Within the detection range of the RIA, no 1,25(OH)₂D₃ could be measured, suggesting that 1,25(OH)₂D₃ is not stored in skeletal matrix. Vitamin D bone concentrations previously measured may therefore have reflected plasma contamination. Consistent with this hypothesis, only traces of skeletal 1,25(OH)₂D₃ binding protein were measured when compared with serum values. Although 1,25(OH)₂D₃ may act as a potential local determinant of bone remodeling, there is no evidence supporting a delayed paracrine function by matrix-derived 1,25(OH)₂D₃.     

Effects of phosphate and 1,25(OH)2D3 on in vitro bone collagen synthesis in the hypophosphatemic mouse

Summary Calvarial bones from hypophosphatemic (Hyp) mice and normal littermates were cultured in a chemically defined medium to determine: (a) the effect of medium phosphate (Pi) concentration (1, 2, and 3 mM) on collagen synthesis; (b) the effect of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] (10⁻¹²M–10⁻⁷M) on collagen synthesis; and (c) whether bone responsiveness to 1,25(OH)₂D₃ was affected by changes in medium Pi concentration. Bone collagen synthesis was evaluated by measuring [³H]hydroxyproline formation. The distribution of labeled hydroxyproline between bone explant and culture medium (total and dialyzable fraction) was studied. These experiments confirm that 1,25(OH)₂D₃ inhibits specifically bone collagen synthesis in vitro. We did not detect any effect of medium Pi concentration on basal collagen synthesis but were able to demonstrate that lowering medium Pi concentration increased the 1,25(OH)₂D₃-induced inhibition of collagen synthesis. Bones from both genotypes responded to 1,25(OH)₂D₃, but modulation of this response by changes in Pi concentration was altered in Hyp bone as, in contrast to normal bone, its response to 1,25(OH)₂D₃ was unaffected when medium Pi concentration was decreased from 3 to 2 mM. These findings support the hypothesis of an altered response of bone to 1,25(OH)₂D₃ in the Hyp mouse.     

Studies on the mode of action of vitamin D XXXVII 1α-hydroxyvitamin D3: A long-acting 1,25-dihydroxyvitamin D3 analog

Summary This study was undertaken to determine whether 1α-hydroxyvitamin D₃ [1α(OH)D₃] administration to chicks in vivo results in 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] intestinal receptor occupancy and to compare the temporal characteristics of the physiological effects of 1α(OH)D₃ and 1,25(OH)₂D₃ for several days after a single dose of either steroid. Occupied 1,25(OH)₂D₃ receptors of the chick duodenal mucosa were measured by the recently developed exchange assay procedure [J Biol Chem (1980) 255:9534–9537]. Within 2 h after 1α(OH)D₃ injection in rachitic chicks, there was a significant elevation of 1,25(OH)₂D₃ receptor occupancy in the intestinal mucosa. This observation represents the first direct confirmation that this synthetic analog exerts biological effects through occupancy of 1,25(OH)₂D₃ receptors. Serum 1,25(OH)₂D₃ levels reached a 3-fold higher peak after 1,25(OH)₂D₃ injection (3.25 nmol) than after 1α(OH)D₃ injection (6.5 nmol); further, after 1α(OH)D₃ injection the peak was delayed by 2–4 h. However, serum 1,25(OH)₂D₃ levels remained elevated for only 3–6 h after 1,25(OH)₂D₃, compared to 48 h after 1α(OH)D₃ injection. Occupied 1,25(OH)₂D₃ receptor levels paralleled serum 1,25(OH)₂D₃ levels at all times after administration of either steroid. At 24 h, duodenal vitamin D-dependent calcium binding protein (CaBP) levels were similarly elevated in both treatment groups, but by 48 and 72 h after 1α(OH)D₃ administration CaBP and serum Ca²⁺, respectively, were more significantly elevated. These data confirm that 1α(OH)D₃ induces its major biological effects via intracellular 1,25(OH)₂D₃ receptors and reinforce the concept that 25-hydroxylation is a prerequisite for these effects. These results also suggest that 1α(OH)D₃ may become useful in the therapy for sustained treatment of vitamin D deficiency diseases.