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

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

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     

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.     

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.     

The dichotomy in the effects of 1,25 dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on bone γ-carboxyglutamic acid-containing protein in serum and bone in vitamin D-deficient rats

Summary Vitamin D-deficient, second generation, rachitic rats showed significant decrease in bone Gla protein (BGP) levels in circulation and in the skeleton. 1,25 dehydroxyvitamin D₃ (1,25 (OH)₂D₃) exhibited the most potent influence on serum BGP levels in a dose-dependent manner. At a dose 25 ng/100 g body weight 1,25 (OH)₂D₃ showed a cumulative effect, i.e., the longer the treatment, the more circulating BGP was detected 24,25 dehydroxyvitamin D₃ (24,25(OH)₂D₃) at the same doses did not show similar effect on the serum BGP levels, regardless of the serum calcium levels. Bone BGP levels assayed at various sites representing endochondral and intramenbranous ossification demonstrated an opposite pattern. 1,25(OH)₂D₃ administration was not sufficient to restore bone BGP levels to normalcy, whereas in animals treated with 24,25(OH)₂D₃ bone BGP and calcium levels were significantly higher than control (Vitamin D₃-repleted) levels. The present results can be explained by the dual action of 1,25 (OH)₂D₃ on both synthesis and release of BGP by bone turnover, whereas 24,25 (OH)₂D₃ stimulates synthesis and accumulation of BGP in bone. These observations imply that caution is required in the interpretation of clinical data based solely on serum BGP determination.     

24,25(OH)2D3, bone formation,and bone resorption in vitamin D-deficient,azotemic rats

Summary Bone formation, mineralization, and resorption were measured in vitamin D-deficient, azotemic rats given two different dosages of 24,25(OH)₂D₃ daily and in vehicle-treated controls (C). The intraperitoneal administration of 65 pmol over a 10 day period corrected the hypocalcemia observed in C, whereas 130 pmol produced mild hypercalcemia. Both dosages reduced osteoid width, osteoid area, and mineralization front width form control values. The rates of bone and matrix formation were unaffected by treatment. In C, matrix formation exceeded bone formation and resulted in osteoid accumulation; both dosages of 24,25(OH)₂D₃ reversed this relationship such that bone formation exceeded matrix formation in each treatment group. The rates of osteoid maturation and initial mineralization increased during repletion with 24,25(OH)₂D₃ at both dosage levels. However, the serum calcium concentration was correlated with both osteoid maturation rate (r=0.68,P<0.01) and initial mineralization rate (r=0.63,P<0.01) when all three experimental groups were considered. Bone resorption was unchanged from control values during treatment with 24,25(OH)₂D₃. The results suggest that 24,25(OH)₂D₃ promotes the maturation and mineralization of osteoid, and that this metabolite differs in its effects on bone formation and resorption. It is not clear, however, that the changes in bone dynamics observed are independent of the calcemic response induced by metabolite repletion under the conditions of this experiment.     

Long-term administration of vitamin D3 metabolites alters PTH-responsive osteoblastic adenylate cyclase in rats

Summary We have examined the effect of induced hyper D₃ vitaminosis on bone-related variables in the rat with special reference to the parathyroid (PTH)-sensitive adenylate cyclase (AC) in rat calvariae. Subcutaneous injections three times a week of doses theoretically corresponding to about 10 times the average physiological serum levels of either 25 hydroxyvitamin D₃ (25OHD₃), 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), or 24,25 dihydroxyvitamin D₃ (24,25(OH)₂D₃) for 12 weeks gave the following results: At 12 weeks of treatment, 24,25(OH)₂D₃ levels in the groups receiving 25OHD₃ or 24,25(OH)₂D₃ increased significantly, whereas 1,25(OH)₂D₃ levels remained unaffected. Correspondingly, PTH-sensitive AC activities in crude calvarial membrane fractions from 25OHD₃-and 24,25(OH)₂D₃-treated animals were obliterated. This effect was apparent after 4 weeks of treatment. In the group receiving 25OHD₃, both basal, plus Gpp(NH)p-, and forskolin-sensitive AC activities were significantly reduced after 4 weeks of treatment. Similar effects in crude kidney membrane fractions were, however, not observed. Liver membranes from 25OHD₃- or 24,25(OH)₂D₃-treated animals showed insignificant changes in the isoprenalin-, PGE₁-, Gpp(NH)p-, or forskolin-sensitive AC activities. Finally, the significance of reduced PTH-sensitive bone AC activity has been assessed. 25OHD₃ treatment yielded normocalcemic and hypercalciuric rats, whereas 1,25(OH)₂D₃ enhanced both serum and urine Ca²⁺ levels. 24,25(OH)₂D₃-treated and control animals were undiscernible in this respect. However, the 24,25-(OH)₂D₃ treatment caused reductions in both serum alkaline phosphatase levels and urinary hydroxyproline/creatinine ratio. These results indicate that administration of vitamin D₃ metabolites which increase serum 24,25(OH)₂D₃ levels without affecting renal handling of Ca²⁺, obliterates the PTH-sensitive AC in bone, thereby altering bone turnover.     

Interactions between aluminum and the actions and metabolism of vitamin D3 in the chick

Summary The effects of intraperitoneal injections of aluminum chloride were tested on the intestinal calcium absorption and bone calcium mobilization responses to vitamin D₃ and 1,25(OH)₂D₃, as measured by bioassay in chicks. Aluminum at 5 mg/kg given 5 days before the bioassay in vitamin D— deficient chicks, partially blocked the intestinal calcium absorption response to low (0.65 and 3.2 nmol), but not to higher (32 nmol) doses of vitamin D₃. The responses to all doses (0.32–2.1 nmol) of 1,25(OH)₂D₃ were partially blocked by aluminum treatment. Serum calcium values were elevated in vitamin D—deficient chicks by aluminum administration, but no consistent effects of the treatment on bone calcium mobilization in response to vitamin D₃ or 1,25(OH)₂D₃ were noted. Aluminum treatmentin vivo led to decreased 25-OH-D₃-1-hydroxylase activity subsequently measured in renal homogenates; under a variety of conditions, no direct effect of aluminum on 25-OH-D₃ metabolism by primary cultures of chick kidney cells was observed. The results suggest that the ability of the intestine to respond normally to 1,25(OH)₂D₃ may be compromised by exposure to high levels of aluminum and that the effect of this element on 25-OH-D₃ metabolism observedin vivo may not be exerted by direct action on the renal cell.     

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.     

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 vitamin D and parathyroid hormone on cyclic AMP production by bone cells isolated from rat calvariae

Summary Studies presented here were designed to investigate further the basis for an impaired cAMP response to parathyroid hormone (PTH) in osteoblastlike calvarial bone cells isolated from vitamin D-deficient rat pups. The goal was to perturb Ca, PTH, and vitamin Din vivo in order to see which factors might be responsible for the impairedin vitro bone cell cAMP response. Pups either were parathyroidectomized (PTX) 3–5 days, implanted with osmotic minipumps delivering high doses of PTH, given repeated, high doses of 1,25(OH)₂D₃, or were D-deficient (-D, i.e., born and suckled by D-deficient mothers). Osteoblastlike bone cells, isolated by sequential enzyme digestion and centrifugation, were exposed to PTH for 5 min in the presence of a phosphodiesterase inhibitor. In bone cells isolated from -D rat pups, both basal and PTH-induced cAMP accumulation were significantly lower than in +D bone cells. Earlier, we had shown that two daily injections of -D pups with 50 ng 1,25(OH)₂D₃ restores this reduced bone cAMP response of -D pups toward normal. In the present study, neither basal nor PTH-induced bone cell cAMP accumulation was affected by subjecting D-replete pups to PTX, PTH infusion, or repeated high doses of 1,25(OH)₂D₃ despite the fact that each treatment markedly changed serum Ca or serum immunoreactive PTH. The results indicate that the impaired bone cell cAMP response seen in -D pups is not a direct result of chronic hypocalcemia and that the “heterologous desensitization” seenin vitro with added 1,25(OH)₂D₃ could not be duplicated byin vivo treatment of +D pups with supraphysiologic doses of 1,25(OH)₂D₃. Finally the lack of alteration in the bone cell cAMP response to PTHin vitro after chronic PTH infusionin vivo fails to support the notion that the impaired response in -D bone cells can be explained entirely by “homologous desensitization” induced by high circulating levels of PTH in the hypocalcemic, -D rat pup.     

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.     

Vitamin K2 promotes 1α,25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts

The effect of vitamin K on mineralization by human periosteal osteoblasts was investigated in the absence and presence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). Vitamin K₁ and K₂, but not vitamin K₃, at 2.5 M enhanced in vitro mineralization when cells were cultured with vitamin K for 20 days after reaching confluence in vitro. Vitamin K₂ (2-methyl-3-all-trans-tetraphenyl-1,4-naphthoquinone: menatetrenone) was the most potent of these vitamin K analogs; it slightly inhibited alkaline phosphatase (ALP) activity. Human osteoblasts were mineralized and showed the enhanced ALP activity on treatment with 10^-9 M of 1,25(OH)₂D₃ for 20 or 25 days after confluence. Vitamin K₂ promoted the 1,25(OH)₂D₃-induced mineralization, but slightly inhibited the 1,25(OH)₂D₃-induced ALP activity. Moreover, vitamin K₂ enhanced the 1,25(OH)₂D₃-induced osteocalcin accumulation in the cells and the extracellular matrix (cell layer), but inhibited the osteocalcin content in the medium produced by the 1,25(OH)₂D₃ treatment. However, vitamin K₂ alone did not induce osteocalcin production in the human osteoblasts. On Northern blot analysis, osteocalcin mRNA expression on 1,25(OH)₂D₃-treated cells was enhanced by vitamin K₂ treatment, but vitamin K₂ alone did not induce osteocalcin mRNA expression. Warfarin blocked both the 1,25(OH)₂D₃-induced osteocalcin production and the accumulation in the cell layer, and also blocked the 1,25(OH)₂D₃ plus vitamin K₂-induced osteocalcin production and the accumulation in the cell layer. The 1,25(OH)₂D₃-induced mineralization promoted by vitamin K₂ was probably due to the enhanced accumulation of osteocalcin induced by vitamin K₂ in the cell layer. However, we concluded that the mineralization induced by vitamin K₂ alone was due to the accumulation of osteocalcin in bovine serum on the cell layer, since osteocalcin extracted from the cell layer was not identified by specific antiserum against human osteocalcin, which does not cross-react with bovine osteocalcin. These results suggest that the mechanism underlying the mineralization induced by vitamin K₂ in the presence of 1,25(OH)₂D₃ was different from that of vitamin K₂ alone, and that osteocalcin plays an important role in mineralization by osteoblasts in vitro.     

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

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

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.     

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.     

Vitamin D3 analogs and salmon calcitonin partially reverse the development of renal osteodystrophy in rats

We have previously established an uremic rat model which is suitable for investigating the effect of various treatment modalities on the progression of renal osteodystrophy [1]. Four months subsequent to 5/6 nephrectomy, animals were treated three times a week for 3 months with either vehicle, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 1,25(OH)₂D₃+24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], 1,25(OH)₂D₃+calcitonin (CT), or 1,25(OH)₂D₃+ 24,25(OH)₂D₃+CT. At termination of the study, clinical chemistry, chemical composition, and mechanical properties of femurs, calvarial parathyroid hormone (PTH)-elicited adenylate cyclase (AC), and phospholipase C (PL-C) activities, femoral cross-sectional area, and bone histomorphometry were analyzed. The main findings were that 1,25(OH)₂D₃±24,25(OH)₂D₃ treatment enhanced elasticity as well as time to fracture at the femoral metaphysis. CT potentiated the increase in elasticity obtained by 1,25(OH)₂D₃±24,25(OH)₂D₃ treatment. Only 24,25(OH)₂D₃ administration rectified the supernormal PTH-stimulated uremic bone AC, and only 1,25(OH)₂D₃ medication normalized the diminished CT-elicited AC. The obliterated uremic bone PTH-sensitive PL-C was fully normalized by all drug regimens. Femoral shaft inner zone diameter was enhanced by uremia, however, all drug treatments normalized it. Ditto effect was registered with either drug treatment on the subnormal outer and inner zone widths. Histomorphometrical analyses showed that 1,25(OH)₂D₃ administration reduced both eroded and osteoid surfaces. Most prominently, adjuvant 24,25(OH)₂D₃ or CT administration potentiated the beneficial effect of 1,25(OH)₂D₃ on fibrosis and osteomalacia. We assert that vitamin D₃ treatment markedly reverses the development of renal osteodystrophy, and CT potentiates the effect of vitamin D₃.     

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

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

1,25 Dihydroxyvitamin D3 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.     

Osteoinduction by implants of demineralized allogeneic bone matrix is diminished in vitamin D-deficient rats

Summary Experimental heterotopic bone formation was produced by subcutaneous implants of demineralized allogeneic bone matrix (DABM) in vitamin D-deficient (−D) animals that were either not treated or given vitamin D₃ (+D) or 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) to determine the role of vitamin D and its most active metabolite in osteoinduction and implant remodeling. Histologically, implants in both +D and −D groups caused a similar acute inflammatory response, formation of a fibrous capsule, and chondrogenesis by 1 to 2 weeks after implantation. However, by 3 weeks after implantation implants in the −D animals had formed less bone matrix, had developed a defect in matrix mineralization, had reduced bone forming and bone resorbing surfaces, and had altered bone architecture resulting from defective bone remodeling. The altered histology in −D animals was not corrected by 10 weeks after implantation. Treatment of vitamin D-deficient rats with 1,25(OH)₂D₃, 65 pmol/day for 3 weeks, corrected both the defect in mineralization and the abnormal histology. The results indicate that (1) vitamin D deficiency does not alter either the timing or the sequence of histologic events associated with osteoinduction but dramatically reduces the magnitude of the response, (2) vitamin D deficiency not only impairs mineralization but also reduces bone formation and resorption, and (3) 1,25(OH)₂D₃ mimics all of the actions of vitamin D with regard to correcting the abnormal osteoinductive response and bone histomorphometry.