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     

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

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(-12)M-10(-7)M) 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.     

Stimulation of undermineralized matrix formation by 1,25 dihydroxyvitamin D3 in long bones of rats

Summary We previously reported that pharmacologic doses of 1,25 dihydroxyvitamin D₃ (1,25-(OH)₂D₃) given for 2–3 days, inhibited osteoblastic collagen synthesis in young rats. In this study, we tested the effects of 5, 25, and 125 ng of 1,25(OH)₂D₃ injected subcutaneously into 6-week-old rats for 12 or 18 days. In rats given 125 ng, cortical bone of distal half femurs exhibited decreased calcium (Ca) content but dry weight and hydroxyproline (Hyp) content were no different from control. Trabecular bone Ca was not different from control but dry weight and Hyp were increased. When cortical and trabecular bone were combined, there was a decrease in Ca, an increase in Hyp, and a 50% decrease in Ca:Hyp. Fluorescent labels given after 8 days of treatment were either diffuse or absent in calcified sections from rats given 125 ng, indicating impaired mineralization. The 25 and 125 ng doses produced hypercalcemia with normal serum phosphate. There was a dose-related increase in serum immunoreactive bone gla protein (BGP) and serum 1,25(OH)₂D₃ and a decrease in serum 25 (OH)D₃. At the 5 ng dose, no adverse effects were seen on body growth. With 25 ng and 125 ng, growth was inhibited. Increased serum urea nitrogen and histologic evidence of nephrocalcinosis occurred at the 125 ng dose. When 125 ng was given for 12 days and then withdrawn for 6 days, systemic toxicity decreased and bone Hyp and Ca increased so that Ca:Hyp remained low and comparable to that of rats treated with 1,25(OH)₂D₃ continuously We conclude that pharmacologic doses of 1,25(OH)₂D₃ stimulate trabecular bone matrix formation but produce impairment of mineralization, despite a high Ca×Pi product.     

Human parathyroid hormone (1–34) and salmon calcitonin do not reverse impaired mineralization produced by high doses of 1,25 dihydroxyvitamin D3

Summary We have reported recently that pharmacologic doses of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulated bone matrix formation but impaired mineralization. The objective of this study was to determine if parathyroid hormone (hPTH 1-34) or calcitonin (sCT) would mineralize the osteoid induced by 1,25(OH)₂D₃ in rat long bones. In one experiment, male Sprague-Dawley rats were given daily subcutaneous injections of vehicle: 8 μg hPTH(1-34); 125 ng 1,25(OH)₂D₃; or both 8 μg hPTH and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 12 days. In a second experiment, rats received daily injections of vehicle: 2 U sCT; 125 ng 1,25(OH)₂D₃; or both 2 U sCT and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 18 days. Calcium (Ca), hydroxyproline (Hyp), and dry weight (DW) of the distal femur and serum calcium, phosphate, and serum bone Gla protein (BGP) were measured. In rats given both 1,25(OH)₂D₃ and hPTH, total bone DW and Hyp increased (P<.01) without a corresponding increase in bone Ca so that Ca/Hyp decreased 47% (P<.01) from control and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. In rats treated with both 1,25(OH)₂D₃ and sCT, total bone DW and Hyp increased while Ca decreased so that Ca/Hyp decreased 38% from control (P<.05), and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. These results indicate that hPTH or sCT, given by intermittent injection to rats for 12 or 18 days respectively, failed to mineralize the osteoid induced by high doses of 1,25(OH)₂D₃.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of increased doses of 1,25 dihydroxyvitamin D3 on matrix and DNA synthesis in condylar cartilage of suckling mice

Summary Thein vivo effects of high doses of 1,25(OH)₂D₃ were studied in condylar cartilage of suckling mice. Seven-day-old animals were treated with 20 ng of the hormone for 7 consecutive days. Biochemical assays on collagen content and synthesis were complemented by structural studies using light and electron microscopy. Indirect immunofluorescent methods were used for the localization of type I and II collagens and for fibronectin. This study revealed that the protein content of the condyle decreased substantially following the administration of the hormone. Protein synthesis increased in hormone-treated animals during the first 4 days but was significantly inhibited theeafter. Collagen synthesis, however, was inhibited instantaneously, followed by a decrease in the percentage of cold hydroxyproline of the total protein. Hormone-treated condyles showed a marked decrease in the distribution of type I collagen, no apparent change in the distribution of type II collagen, but an enhanced reactivity for fibronectin especially around hypertrophic chondrocytes. SDS-gel electrophoresis of collagen chains suggested that the hormone did not induce a significant change in the ratios of type I and II collagen chains, yet additional peaks became evident in 1,25(OH)₂D₃-treated specimens. The decrease in collagen synthesis was accompanied by ultrastructural changes in the appearance of the extracellular collagen bundles. They later appeared as a dense meshwork of collagen fibrils, a feature that was lacking in control tissues. The changes in collagen fibrillogenesis could be explained by ourin vitro studies indicating a marked depression of³⁵S-sulfate incorporation secondary to treatment with 1,25(OH)₂D₃. The hormone was also found to suppress the incorporation of³H-thymidine, hence it may be concluded that 1,25(OH)₂D₃, when used in high concentrations, possesses an inhibitory effect upon both the proliferative activity of the cartilage progenitor cells as well as upon the metabolic activity of the condylar cells as related to collagen and glycosaminoglycans synthesis.     

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.