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.     

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.     

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.     

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.     

1,25 Dihydroxyvitamin D3 is required for growth-independent expression of alkaline phosphatase in cultured rat osteoblasts

Summary Osteoblastic cells were isolated from periosteum-stripped parietal bones of neonatal rat calvaria, seeded at low density (5,000 cells/35 mm of Falcon dish), and cultured for 6 days in BGJ medium supplemented with 20% of vitamin D-depleted FCS or vitamin D and calcium-depleted FCS, with daily addition of 1,25 dihydroxyvitamin D₃ (10⁻⁹ M) or 24,25-dihydroxyvitamin D₃ (10⁻⁹ M). Plating efficiency, clonal growth (number and size distribution of the colonies formed), and the alkaline phosphatase phenotype were evaluated on days 2 and 6 of culture. (1) Culture for 6 days in media not supplemented with 1,25(OH)₂D₃ led to a significant (P<0.001) loss of the alkaline phosphatase phenotype of the osteoblastic cells; the loss was greater in proliferating cells than in nonproliferating ones and occurred in both 0.12 mM or 1.1 mM ionized calcium concentrations. (2) Daily addition of 1,25(OH)₂D₃ (10⁻⁹ M) but not 24,25(OH)₂D₃ maintained the basal percentage of Alk Pase positive cell units in nonproliferating cells and significantly reduced the loss of this phenotype in proliferating colonies. (3) This effect did not stem from an action of the hormone on cell growth. 1,25(OH)₂D₃ was also found to enhance the adhesiveness of the seeded osteoblasts, irrespective of the medium calcium concentration.     

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.     

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     

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.     

Effects of vitamin D metabolites and analogs on bone collagen synthesis in vitro

Summary The effects of selected vitamin D₃ metabolites and analogs on bone collagen synthesis in vitro were examined in organ cultures of neonatal mouse calvarial bone. The incorporation of [³H]proline into the collagenase-digestible fraction of newly synthesized protein was progressively inhibited by 1α,25-dihydroxyvitamin D₃ (1α,25-(OH)₂D₃) (10⁻¹² M to 10⁻⁷ M) in 24-h cultures, and incorporation into noncollagen protein was also blunted at the higher doses employed. The synthetic analog 1α-hydroxyvitamin D₃ (1α-OHD₃) was almost 300-fold less potent an inhibitor of collagen synthesis than was 1α,25(OH)₂D₃, and the natural metabolites 25-hydroxyvitamin D₃ (25OHD₃) and 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃), 1000-fold less potent, although the dose-response curve for each of these compounds was not parallel with that for 1α,25(OH)₂D₃. The 24S,25(OH)₂D₃ enantiomer was four-fold less potent than 24R,25-(OH)₂D₃ or 25OHD₃, and vitamin D₃ showed less than 2% the activity of 25OHD₃. The responses were unaffected by the substitution of 0.4% bovine albumin for 5% horse serum in the medium, and no stimulation of collagen synthesis was observed in response to 25-hydroxylated metabolites between 2×10⁻¹⁴ and 2×10⁻⁶ M or in cultures treated for up to 96 h with 24R,25(OH)₂D₃ (2×10⁻¹⁰M). The overall results emphasize the similarity of the structural requirements for the inhibition of matrix synthesis and the stimulation of resorption by active vitamin D metabolites in bone. In addition, these studies support the importance of the 1-hydroxyl function to the biologic activity of vitamin D in the skeleton.     

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.     

Effect of Vitamin D Metabolites and Analogs on Renal and Intestinal Calbindin-D in the Rat

The effects on renal and intestinal calbindin-D of vitamin D₃ metabolites and synthetic 20-epi-vitamin D₃ analogs with different calcemic actions were examined in Wistar rats. The compounds were administered intraperitoneally once daily for 5 days. The dosages of the metabolites were 1,25-(OH)₂D₃ 0.01, 0.05, 0.1, and 0.4 μg/kg × d, 24,25-(OH)₂D₃ 0.1, 1 and 10 μg/kg × d, and 25-(OH)D₃ 10 and 400 μg/kg × d. The dosage of the synthetic analogs were MC903 0.1, 10, and 100 μg/kg × d, EB1213 0.1 and 10 μg/kg × d, KH1060 0.1 and 0.4 μg/kg × d, and GS1725 0.01 and 0.1 μg/kg × d. Two control groups had either vehicle alone or no treatment. N= 8 in each group. 1,25-(OH)₂D₃ increased renal and intestinal calbindin-D levels, induced hypercalcemia, and suppressed plasma PTH and magnesium concentrations. 24,25-(OH)₂D₃ increased intestinal calbindin-D9k and plasma calcium, but had no effect on renal calbindin-D28k, plasma PTH, and magnesium. The dosage of 24,25-(OH)₂D₃ that was required to increase plasma calcium was larger than the dosage required to increase intestinal calbindin-D9k. 25-(OH)D₃ did not change the calcium metabolic parameters. MC903, a low calcemic analog with a relative high affinity for the vitamin D receptor and a short half-life, increased renal calbindin-D28k without increasing ionized calcium or intestinal calbindin-D9k. EB1213, an analog with a reduced calcemic action and short half-life, increased renal calbindin-D28k and ionized calcium without increasing intestinal calbindin-D9k. The effect of the high calcemic vitamin D analogs KH1060 and GS1725 on calbindin-D was directly related to their calcemic activity. In conclusion, these results demonstrate that 24,25-(OH)₂D₃ increases intestinal calbindin-D9k, but has no effect on renal calbindin-D28k, that low calcemic analogs may increase renal calbindin-D28k without increasing intestinal calbindin-D9k, and that the effect of high calcemic analogs on calbindin-D is directly related to their calcemic activity. Received: 26 May 1995 / Accepted: 29 February 1996     

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.     

Calcium regulating activity of 26,27-dimethyl analog of 24R,25-dihydroxyvitamin D3

To determine the possibility that methyl substitution in 26- and 27-positions of 24R,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] alters activities of the original compound, the effects of 24,25(OH)₂D₃ on calcium (Ca) regulating activity were compared with those of its methyl analog [24,25(OH)₂(CH₃)₂D₃] in addition to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. 24,25(OH)₂D₃ at 10^-6 M and 24,25(OH)₂(CH₃)₂D₃ at 10^-7 M and above significantly stimulated both bone resorption in neonatal mouse calvaria cultures and formation of osteoclast-like multinucleated cells (MNC) in mouse bone marrow cultures. A stimulative effect of 1,25(OH)₂D₃ on bone resorption and MNC formation was recognized in very low concentrations (10^-11 M and above). Although a potency of 24,25(OH)₂(CH₃)₂D₃ in stimulating bone calcium (Ca) mobilization and intestinal Ca transport was higher than that of 24,25(OH)₂D₃, the potencies of both compounds were similar to that of 1,25(OH)₂D₃ unlike in vitro experiments. As 1,24R,25-trihydroxy-26,27-dimethylvitamin D₃ showed almost the same effect as 24,25(OH)₂(CH₃)₂D₃, the dihydroxy form is suggested to be hydroxylated at 1 position and converted to trihydroxy form in vitamin D-deficient rats. From these results, methyl substitution in 26- and 27-position of 24,25(OH)₂D₃ was found to elevate Ca regulating activity of the original compound. In addition, it is suggested that the basis for a similarity in potency between 1,25(OH)₂D₃ and 24,25(OH)₂D₃ or its dimethyl analog in vitamin D-deficient rats is likely the result of 1 -hydroxylation.     

Comparison of the mechanisms of bone resorption induced by 1α,25-dihydroxyvitamin D3 and lipopolysaccharides

Summary The mechanisms of increase in bone resorption induced by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] and bacterial lipopolysaccharides (LPS) were compared in anin vitro dead bone assay and a living bone assay. 1α,25(OH)₂D₃ at concentrations of 0.05–5 ng/ml dose-dependently enhanced the ability of alveolar macrophages to release⁴⁵Ca from prelabeled dead bone particles (dead bone assay). In addition, the vitamin promoted fusion of the macrophages to form multinucleated cells and also enhanced glucose consumption, a marker of activation of macrophages. LPS at 0.05–5 μg/ml similarly enhanced the release of⁴⁵Ca from the dead bone particles and glucose consumption by alveolar macrophages, but it did not induce fusion of the cells at any concentration. Both 1α,25(OH)₂D₃ and LPS dose-dependently stimulated the release of⁴⁵Ca from fetal mouse calvaria prelabeled with⁴⁵Ca (living bone assay). Compared to control bone, there were several times as many osteoclasts per given length of trabecular bone surface in calvaria treated for 5 days with either 5 ng/ml of 1α,25(OH)₂D₃ or 5 μg/ml of LPS. Indomethacin (10⁻⁵ M) completely inhibited the LPS-induced increase of osteoclasts, but not the 1α,25(OH)₂D₃-induced increase. These results suggest that 1α,25(OH)₂D₃ and LPS similarly stimulate bone resorption by activating macrophages as well as by promoting fusion of precursor cells to form multinucleated cells. 1α,25(OH)₂D₃ induced formation of multinucleated cells with bone-resorbing activity directly, whereas LPS appeared to induce multinucleated cells through prostaglandin synthesis by some other types of cells present in living bone tissues.     

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.     

Extrarenal metabolism of 25-hydroxycholecalciferol in the rat: Regulation by 1,25-dihydroxycholecalciferol

Summary To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD₃, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] on³H-25OHD₃ were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of³H-25OHD₃, extracts of serum and pooled small intestinal mucosa were fractionated by Sephadex LH-20 column chromatography followed by high performance liquid chromatography. In intact rats, 1,25(OH)₂D₃ (50 ng/day i.p. for 7 days) increased mean serum³H-24,25-dihydroxycholecalciferol [³H-24,25(OH)₂D₃] from 2±2–210±80 fmol/ml (mean±1 SD), increased mean serum³H-25,26-dihydroxycholecalciferol [³H-25,26(OH)₂D₃] from 2±2–12±6 fmol/ml and lowered mean serum³H-1,25(OH)₂D₃ from 210±40–4±4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)₂D₃ increased mean serum³H-24,25-(OH)₂D₃ from 6±11–115±30 fmol/ml and increased mean serum³H-25,26(OH)₂D₃ from 3±3–26 ± 10 fmol/ml. Nephrectomy increased serum³H-25(OH)D₃ in untreated (from 1450±225–2675±225 fmol/ml serum) and 1,25(OH)₂D₃ treated rats (from 1600±175–3075±100 fmol/ml).³H-1,25(OH)₂D₃ averaged 74±16% of total radioactivity in intestinal mucosa of untreated intact rats and was not detected in either the serum or intestinal mucosa of nephrectomized animals. The results suggest that in intact animals, extrarenal synthesis can account for substantial 24,25(OH)₂D₃ production and for most 25,26(OH)₂D₃ production. Further, the observed stimulation of production of 24,25(OH)₂D₃ and 25,26(OH)₂D₃ by 1,25(OH)₂D₃ in anephric — D rats providesin vivo evidence for regulation of extrarenal 25OHD₃: 24- and 26-hydroxylases.     

Increase in femoral bone mass by ipriflavone alone and in combination with 1α-hydroxyvitamin D3 in growing rats with skeletal unloading

We assessed the possibility that ipriflavone treatment might result in bone restoration in immobilized rats. We also investigated the effect of combined treatment with ipriflavone and vitamin D₃ on the bone. Male Sprague-Dawley rats, 6 weeks of age, were subjected to unilateral sciatic neurectomy. Three weeks after the operation, ipriflavone (100 mg/kg), 1α-hydroxyvitamin D₃ [1α(OH)D₃, 25 ng/kg], or both ipriflavone and 1α(OH)D₃ were orally administered every day for 12 or 24 weeks. After 12 weeks of treatment, only the group receiving combined treatment with ipriflavone and 1α(OH)D₃ showed increases in total femur calcium content (+16.4%, compared with the control). After 24 weeks, both animals treated with ipriflavone alone and those that had received the combination of ipriflavone and 1α(OH)D₃ showed significant increases in femur calcium content (+18.0% and +23.8%, respectively). In these treatment groups, X-ray analysis revealed an increase in bone mineral density over the entire length of the femur, and an increase in cortical diameter at the midshaft without affecting medullary width. Administration of 1α(OH)D₃ (25 ng/kg) alone had no effect. Body weight, femur length, and serum markers of calcium and bone metabolism were not affected in any group. We evaluated the relationship between ipriflavone and vitamin D₃ in bone cells in a culture system using rat bone marrow stromal cells in which the cells subsequently form mineralized bone-like tissue. Continuous treatment with ipriflavone (10⁻⁵ M) for 21 days resulted in an increase in osteocalcin secretion, and enhanced its response to 1α,25-dihydroxyvitamin D₃ (10⁻¹¹ M-10⁻⁸ M). These findings indicate that ipriflavone treatment increases the femoral bone mass in immobilized rats. In addition, a low dose of 1α(OH)D₃, which did not induce hypercalcemia, in combination with ipriflavone, augmented the stimulatory effect of ipriflavone alone on the bone mass, possibly due to a direct effect of each agent on osteoblastic cells.     

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.