Effect of vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion

Summary The active vitamin D metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] causes marked suppression of both pre-proparathyroid hormone messenger RNA (pre-proPTH mRNA) and parathyroid hormone (PTH) secretion. These effects are dose dependent and reversible when tested in anin vitro primary tissue culture cell system using normal bovine parathyroid cells. In the current studies, the precursors of 1,25(OH)₂D₃ and the related metabolite 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], were used in the same culture system to test for possible regulatory effects. The results were compared with identically prepared cells exposed to 1,25(OH)₂D₃. In short-term studies (30–120 minutes), none of the vitamin D-related compounds produced any effect on PTH secretion. In long-term studies (24–48 hours, using primary tissue culture in the presence of test agents), neither vitamin D₃ nor 25(OH)D₃ affected PTH secretion or pre-proPTH mRNA over the concentration range 10⁻¹¹–10⁻⁷M. On the other hand, 24,25(OH)₂D₃ produced significant suppression of both pre-proPTH mRNA (77% of control,P<.01) and PTH secretion (75% of control,P<.005) at 10⁻⁷ M. By comparison, 10⁻¹¹ M 1,25(OH)₂D₃ produced levels of suppression (25–30%) of both pre-proPTH mRNA and PTH secretion comparable to 10⁻⁷ M 24,25(OH)₂D₃, while even greater suppression (40–50%) occurred at 10⁻⁹-10⁻⁷ M 1,25(OH)₂D₃. From these studies, we conclude that vitamin D₃ and 25(OH)D₃ do not have significant effects on PTH synthesis and secretion over the range of doses tested. Compared with 1,25(OH)₂D₃, 24,25(OH)₂D₃ exhibits mild suppression at pharmacologic concentrations. The effect of 24,25(OH)₂D₃ prabably occurs through weak interaction of 24,25(OH)₂D₃ with the 1,25(OH)₂D₃ receptor.     

In vitro effects of vitamin D3 metabolites on rat calvaria cAMP content

Summary Results from in vitro works suggest that 1,25- and 24,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃ and 24,25-(OH)₂D₃) act on bone via different mechanisms. The present investigation was performed to study the effect of these two metabolites and of their precursor 25-hyxdroxyvitamin D₃ (25-(OH)D₃) on bone cAMP content in vitro. Rats' paired half calvaria were incubated under sterile conditions with one vitamin D₃ derivative (10⁻¹³ to 10⁻⁹ M) or with ethanol (0.005 ml for 15 min to 24 h in 1 ml medium containing 0, 0.2, 1, 2, or 3 mM calcium. In some experiments: (a) cycloheximide (10⁻⁵M) was added simultaneously with the vitamin D₃ metabolites; (b) 1–84 bPTH (5 × 10⁻⁸ M) was added for 5 or 15 min at the end of the 24 h incubation. Calvaria were immersed in 1 ml TCA 5% 4°C and homogenized. The cAMP was extracted with diethylether and measured by a competitive protein binding assay. Results bring further evidence for a particular effect of low doses of 24,25-(OH)₂D₃ (10⁻⁹ to 10⁻¹²M) and of 25-(OH)D₃ (10⁻⁹ to 10⁻¹¹M) on bone, different from that of 1,25-(OH)₂D₃: cAMP content was higher in 24,25-(OH)₂D₃- or 25-(OH)D₃-treated and lower in 1,25-(OH)₂D₃-treated calvaria than in ethanol-treated ones with 1 mM calcium. The 1,25-(OH)₂D₃ effect persisted in calcium-free medium whereas 25-(OH)D₃ and 24,25-(OH)₂D₃ effects could not be observed with 0 mM nor with 3 mM calcium. The required duration of the preincubation (over 1 h) as well as the inhibitory action of cycloheximide may suggest an involvement of protein synthesis in the vitamin D₃ metabolites effects. Neither 1,25-(OH)₂D₃ nor 24,25-(OH)₂D₃ affected the PTH-induced increase in bone cAMP content.     

In vitro effects of aluminum on bone phosphatases: A possible interaction with bPTH and vitamin D3 metabolites

Summary The present study was undertaken to test the in vitro action of aluminum on bone phosphatase activities and the possible interaction of this metal with parathyroid hormone (bPTH) or vitamin D₃ dihydroxymetabolites [1,25- and 24,25(OH)₂D₃). Three-day-old rat calvaria were incubated for 24 h with one of the following: bPTH at 5×10⁻⁸M, 1,25-or 24,25(OH)₂D₃ at 2.5×10⁻⁹M, Al at concentrations ranging from 3×10⁻¹¹M to 6×10⁻⁶M, or their corresponding solvents. Al effects were also investigated when the medium phosphate or calcium concentrations were modified. In some experiments, Al was added simultaneously with bPTH or one of the vitamin D₃ metabolites at the beginning of the 24 h incubation. At the end of all incubations, acid and alkaline phosphatase activities were measured in bone cytoplasmic extract. The results show that: (a) When compared to the value found in half calvaria incubated in a control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with Al (3×10⁻¹¹M to 1.5×10⁻⁶M) as well as with bPTH, 1,25-, or 24,25(OH)₂D₃ and sharply decreased with higher Al concentrations (6×10⁻⁶M). (b) The Al effect on phosphatase activities is modified in a free phosphate or a free calcium medium. (c) The presence of Al at 1.5×10⁻⁶M or 6×10⁻⁶M significantly decreases the bPTH or 1,25(OH)₂D₃-induced stimulation of bone phosphatase activities. (d) A similar interaction could not be found between Al and 24,25-(OH)₂D₃.     

1,25-dihydroxyvitamin D3 causes an increase in the number of osteoclastlike cells in cat bone marrow cultures

Summary The direct effect of 1,25(OH)₂D₃ upon osteoclast formation from precursor cells is still unknown. In the present experiments we have tested the effects of 1,25(OH)₂D₃ on the generation of osteoclastlike cells in cat bone marrow cultures. These cultures contain proliferating nonattached mononuclear cells and precursor cells that subsequently attach to the culture flask surface and then fuse to form multinucleated osteoclastlike cells. After 7 days of culture we separated the nonattached precursor cells from the attached cells and studied the effects of 1,25(OH)₂D₃ (10⁻¹⁰ M–10⁻⁸ M) on multinucleated cell formation in these two cell populations. In cultures derived from the non-attached precursor cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) resulted in a 180% increase in the number of attached mononuclear cells and a 90% increase in the number of nuclei contained within multinucleated cells. These effects were dose-dependent. 1,25(OH)₂D₃ did not have a consistent effect on the number of nonattached precursor cells. In cultures derived from attached cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) induced a 50% increase in the number of mononuclear attached cells and a 40% increase in the number of nuclei within polykaryons. The most likely explanation for these results is that 1,25(OH)₂D₃ promotes the differentiation and subsequent adhesion of nonattached precursor cells, stimulates proliferation of attached mononuclear precursor cells, and possibly stimulates fusion of these attached precursor cells.     

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.     

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.     

Avian medullary bone in organ culture: Effects of vitamin D metabolites on collagen synthesis

Summary A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of³H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10–15% of the total protein labeled. The addition of 1,25-dihydroxycholecalciferol (1,25 (OH)₂D₃) resulted in a dose-dependent inhibition of³H-proline incorporation into CDP at doses from 10⁻¹⁰M to 10⁻⁷M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since³H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)₂D₃-induced decrease in the³H-hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)₂D₃ is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaiored incorporation of collagen into bone matrix, or bone resorption Medullary bone collagen synthesis was not affected by 24,25(OH)₂D₃, either alone or in combination with 1,25(OH)₂D₃. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)₂D₃ and 1,24,25(OH)₃D₃ were the most potent metabolites tested, followed by 1-alpha(OH)D₃. 25(OH)D₃ and 24,25(OH)₂D₃ had no effect at concentrations as high as 10⁻⁷M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as anin vitro model for the study of bone formation     

Inhibition by 1,25 dihydroxycholecalciferol of hormonal secretion of rat parathyroid gland in organ culture

Summary The effect of vitamin D metabolites on parathyroid hormone secretion was studied using rat parathyroid gland cultured in basal medium Eagle containing 5% serum obtained from thyroparathyroidectomized rat, 1 mM magnesium, and calcium concentration varying from 0.75–2.25 mM, and radioimmunoassay for rat parathyroid hormone (rPTH). 1,25 dihydroxycholecalciferol (1,25(OH)₂D₃), 5×10⁻¹⁰−2.5×10⁻⁸M, consistently decreased rPTH secretion in dose-related manner; the effect reached steady state after 24 hin vitro addition of 1,25(OH)₂D₃ and was also observed at different medium calcium concentrations (0.75, 1.25, 1.75 mM). Comparison of dose-responses for inhibitory activity of some vitamin D metabolites on rPTH secretion showed: 1,25(OH)₂D₃=1,24,25(OH)₃D₃>1α OHD₃>25 OHD₃. Cholecalciferol (10⁻⁵M), 24,25-dihydroxycholecalciferol (10⁻⁸−10⁻⁶M) and 25,26-dihydroxycholecalciferol (5×10⁻⁹−5×10⁻⁷M) did not inhibit rPTH secretion. Analysis of structural activity relation of vitamin D metabolites studied indicated that 1α or pseudo-1α hydroxylated metabolites or analogs were active in inhibiting rPTH secretion, while, non-1α hydroxylated metabolites were without or were weakly inhibitory only at very high concentrations. This study provides further evidence for a direct role of 1,25(OH)₂D₃ on a negative feedback loop for regulation of parathyroid gland function.     

Interaction of 24,25-Dihydroxyvitamin D3 and parathyroid hormone on bone enzymes in vitro

Summary The in vitro effects of vitamin D₃ metabolites, parathyroid extract (PTE), purified parathyroid hormone (bPTH), vitamin A, and heparin on acid and alkaline phosphatases in rat or mouse calvaria in culture were investigated. Results show that: (a) when compared to values found in half calvaria incubated for 24 h in control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with PTE (1 USP/ml), bPTH (4×10⁻⁸M), heparin (5 USP/ml), vitamin A (23 USP/ml), 25-(OH)D₃ (2.5×10⁻¹¹ to 2.5×10⁻⁸M), 24,25-(OH)₂D₃, and 1,25(OH)₂D₃ (2.5×10⁻¹² to 2.5×10⁻⁷M); (b) the presence of 24,25-(OH)₂D₃ at low concentrations in the incubation medium decreases significantly the PTE, bPTH, vitamin A, or heparin induced stimulation of the phosphatase activities. This interaction is also observed when measuringβ glucuronidase and glucose-6-phosphatase activities and⁴⁵Ca release from previously labeled mouse calvaria; (c) a similar activity could not be found with 1,25-(OH)₂D₃ suggesting that 24,25-(OH)₂D₃ may have a specific role in bone metabolism.     

Somatostatin inhibits duodenal calcium transport mediated by 1,25-dihydroxyvitamin D3 in perfused duodena from normal chicks

We have reported that physiological dose (30pM-650pM) of 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] increased the unidirectional movement of⁴⁵Ca²⁺ from the lumen to the venous effluent within a few minutes in perfused duodena from normal chicks, and hypercalcemia inhibited this rapid stimulatory effect on calcium transport mediated by 1,25(OH)₂ D₃. The purpose of the present study was to determine the effect of somatostatin on calcium transport in chicks. The basal Ca²⁺ transport, in the absence of 1,25(OH)₂ D₃, did not change when 10⁻⁸M to 10⁻⁶M of somatostatin was added to the perfusate. The effect of 1,25(OH)₂D₃ on calcium transport, however, was completely abolished on addtion of 10⁻⁶M somatostatin in the perfusate, and partially blocked on addition of 10⁻⁷M somatostatin and 10⁻⁸M somatostatin had no effect on 1,25(OH)₂ D₃ mediated calcium transport. These results suggest that somatostatin may decrease intestinal calcium transport mediated by the rapid direct action of 1,25(OH)₂ D₃.     

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(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.     

Comparison of 1,25-, 25-, and 24,25-hydroxylated vitamin D3 binding in fetal rat calvariae and osteogenic sarcoma cells

Summary The hormonal metabolite of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], exerts its biological effects by binding to a cytosolic receptor protein. Such a protein has been demonstrated in vitamin D₃ target organs including fetal rat calvariae and more recently in rat osteogenic sarcoma cells. In this study we have compared the binding of 25-hydroxyvitamin D₃ [25(OH)D₃] and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] to that of 1,25-(OH)₂D₃ in fetal rat calvariae and osteogenic sarcoma (OS) cells. Sucrose density sedimentation, DNA-cellulose chromatography, and intracellular uptake studies have been employed to evaluate these interactions. In cytosol preparations from calvariae, [³H]-1,25(OH)₂D₃ bound to a 3.3S macromolecule and to a much greater extent to a 5.8S macromolecule while both [³H]25(OH)D₃ and [³H]24,25(OH)₂D₃ bound to the 5.8S macromolecule. By incubating intact calvariae and OS cells with labeled metabolites and thus establishing binding intracellularly prior to cell disruption, we have found that the 3.3S protein which has high specificity for 1,25(OH)₂D₃ occurs inside the cells; the 5.8S protein, however, does not occur inside the cells but is generated after cell disruption. The [³H]-1,25(OH)₂D₃-receptor complex adsorbed to DNA-cellulose and was eluted from this affinity resin at 0.28M KCl. In contrast, [³H]25(OH)D₃ and [³H]-24,25(OH)₂D₃ binding activity did not adsorb to DNA-cellulose. We conclude that, in contrast to the 3.3S protein, the 5.8S macromolecule does not fulfill receptor criteria but is rather generated by the experimental manipulation of the bone cells. Our data suggest that the vitamin D₃ actions on bone are mediated only via the 3.3S receptor, and hence quantitative but not qualitative differences of the effects of the various metabolites are feasible. With technical assistance by M. Larsen, D. Meler, and M. LaFrance.     

Effects of vitamin D-binding protein on bone resorption stimulated by 1,25 dihydroxyvitamin D3

Summary Vitamin D and its metabolites are tightly bound to the serum vitamin D-binding protein (DBP) and only the free hormone is considered to be physiologically active. On the other hand, DBP could interact with cell membranes and even favor its intracellular entry. The present study was undertaken to examine the effects of DBP on bone resorption stimulated by 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Forelimb bones from 19-day-old fetal rats were cultured for 5 days in the presence of purified human or rat serum albumin (hSAP or rSAP) and 1,25(OH)₂D₃, with or without human or rat DBP (hDBP or rDBP). Bone resorption was assessed by measuring the release of previously incorporated⁴⁵Ca. We found that the resorptive response to 1,25(OH)₂D₃ was minimally altered by hDBP (5 μM). The minimal effects of hDBP on 1,25(OH)₂D₃ activity on rat bones might be explained by a 6-fold lower affinity of hDBP (1.1×10⁷ M⁻¹) than rDBP (5.9×10⁷ M⁻¹) for 1,25(OH)₂D₃ or by species differences in cellular recognition of DBP. In a homologous rat system, however, rDBP at low (0.5 μM) or physiological (5 μM) concentration significantly decreased 1,25(OH)₂D₃-induced bone resorption. These data therefore support the hypothesis that free rather than DBP-bound 1,25(OH)₂D₃ is physiologically important.     

Vitamin D3 and avian bonein vitro: Specificity of effect on Japanese quail calvaria

Summary Calcitriol (1,25(OH)₂D₃) has been shown, under certain conditions, to elicit anin vitro response in adult avian calvarium which may be interpreted as calcium uptake by the bone. The present investigation was undertaken to study the specificity of this response. Calvaria were removed from 6-week-old female Japanese quail and cultured for periods of up to 96 hours at 37°C in 5% CO₂/95% air. 1,25(OH)₂D₃ induced a fall in the medium total and ionic calcium concentrations at both 48 hours and 96 hours of incubation; these responses were not blocked by the presence of 10⁻⁴ M acetazolamide. Bovine parathyroid hormone (bPTH (1–34)) at 10⁻⁷ M, and dibutyryl cyclic AMP (DBcAMP) at 10⁻⁴M, had no effect on the medium calcium. In contrast, forskolin at 10⁻⁴ M induced a marked fall in medium calcium concentrations, particularly at 48 hours. The specificity was also studied with respect to vitamin D₃ and its two major metabolites. 1,25(OH)₂D₃ exhibited a bellshaped dose-response relationship with the maximal effect at 10⁻⁷ M. In contrast, the other two compounds elicited no effects at 10⁻⁷ M or 10⁻⁶ M; significant responses were observed at 10⁻⁵ M with both agents. In general, 25-dihydroxyvitamin D₃ (25OHD₃) was more potent than vitamin D₃. These findings suggest that the medium calcium response to 1,25(OH)₂D₃, interpreted as calcium uptake by the cultured adult avian bone, is relatively specific among calcemic agents; the response was elicited by forskolin but not by bPTH(1–34) or DBcAMP. The potency ratio exhibited by the vitamin D₃ analogs (1,25(OH)₂D₃>25OHD₃>vitamin D₃) reinforces the specificity claim.     

Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro

Summary The present investigation was undertaken to study the role of carbonic anhydrase in 1,25 dihydroxyvitamin D₃-induced bone resorption. Calvaria were removed from 5- to 6-day-old mice and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium (high glucose, 4,500 mg/dl) supplemented with antibiotics and either heat-inactivated horse and fetal calf sera or bovine serum albumin. The experimental cultures contained 1×10⁻⁸ M 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). All cultures were incubated at 37°C in 5% CO(in2)/95% air. Bone resorption was assessed by release of stable calcium into the medium. Bone enzymes (acid and alkaline phosphatases and carbonic anhydrase) were determined following homogenization in 0.25 M sucrose. The effects of 1,25(OH)₂D₃ were studied in the presence and absence of the carbonic anhydrase inhibitor acetazolamide and its analogue (CL 13,850), which lacks inhibitory activity. Acetazolamide inhibited 1,25(OH)₂D₃-induced calcium release in a dose-dependent fashion from 10⁻⁵–10⁻⁴ M. When added to the cultures at a concentration of 1×10⁻⁴ M, acetazolamide completely blocked the 1,25(OH)₂D₃-induced calcium release, a phenomenon not seen with an equimolar concentration of CL 13,850. The most significant finding was that 1,25(OH)₂D₃-induced calcium release was accompanied by a significant increase in the carbonic anhydrase activity of bone at both 48 (treated/control ratio=2.05) and 96 (treated/control ratio=2.59) hours. Bone alkaline phosphatase activity decreased and acid phosphatase activity increased in response to 1,25(OH)₂D₃. These findings support the concept that carbonic anhydrase is involved in bone resorption inducedin vitro by certain calcemic hormones and related compounds.     

Vitamin D Metabolites Regulate Matrix Vesicle Metalloproteinase Content in a Cell Maturation-Dependent Manner

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcificationin vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-\. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1,25(OH)₂D₃ and 24,25 (OH)₂D₃. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10¹⁰-10^-7 M24,25(OH)₂D₃ or growth zone chondrocytes incubated with 10^-11-l0^-8 M 1,25(OH)₂D₃, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)₂D₃ increased alkaline phosphatase by 35–60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)₂D₃ increased alkaline phosphatase by 35–60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)₂D₃ regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A₂ specific activities as well as metalloprotemases which degrade proteoglycans.     

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.     

The effects of parathyroid hormone or 1,25-dihydroxyvitamin D3 on monocyte-osteoclast fusion

Summary ³H-thymidine-labeled blood monocytes were cultured with osteoclasts in the presence or absence of parathyroid hormone or 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in order to evaluate (1) the percentage of monocytes capable of fusing with osteoclasts, (2) if parathyroid hormone or 1,25(OH)₂D₃ influences the contribution of blood monocytes to osteoclast nuclear turnover. We found that within 24 hours of culture, about 8% of blood monocytes fuse with osteoclasts regardless of the presence of parathyroid hormone (PTH) or 1,25(OH)₂D₃. On the other hand, formation of nonosteoclastic giant cells by fusion of monocytes is enhanced by 5×10⁻⁹ M 1,25(OH)₂D₃ but only in the presence of the bone resorptive cells.     

The influence of dihydroxylated vitamin D metabolites on bone formation in the chick

Summary The ability of 1,25(OH)₂D₃ and of 24,25(OH)₂D₃ to prevent or to heal rickets in chicks was evaluated by studies of plasma biochemistry, growth plate histology, bone morphometry and microradiography, and bone mineralization. 1,25(OH)₂D₃ at a dose of 100 ng/day produced fewest abnormalities compared with vitamin D₃-treated control chicks. Bone growth was slightly greater than vitamin D₃-treated controls in chicks given a lower dose of this metabolite; the reverse was observed in chicks given a higher dose. 24,25(OH)₂D₃ was less effective than 1,25(OH)₂D₃ in preventing rickets even at doses as high as 400 ng/day. Treatment of rachitic chicks with doses of 24,25(OH)₂D₃ up to 300 ng/day produced no healing effect on the bone lesions, in marked contrast to the beneficial effects observed with 1,25(OH)₂D₃.