The effects of stable strontium on calcium metabolism.: II. Effects of 1α-hydroxyvitamin D3 in strontium-fed rats,and inhibitory effect of strontium on bone resorptionin vitro

It has been postulated that the effect of strontium on bone metabolism due to the reduced plasma 1,25-dihydroxyvitamin D₃ level following the inhibition of 1α-hydroxylation by strontium. The effects of strontium were examined on intestinal calcium absorption when rats were received synthetic 1α-hydroxyvitamin D₃. Four groups of rats at the age of 36 days were fed a semi-synthetic vitamin D-deficient diet for 4 weeks containing 1% strontium and vitamin D₃ (Sr-D group), 1% strontium and 1α-hydroxyvitamin D₃ (Sr-α group), vitamin D₃ (Co-D group), or 1α-hydroxyvitamin D₃ (Co- α group), respectively. At the age of 60 days, calcium and strontium balance studies were conducted to determine intestinal calcium absorption over a 3-day period, and 1,25-dihydroxyvitamin D level was then measured. Serum 1,25-dihydroxyvitamin D in Sr-D group was undetectable, and intestinal calcium absorption significantly decreased. Replacement of vitamin D₃ with 1α-hydroxyvitamin D₃ recovered serum 1,25-dihydroxyvitamin D to the level in Co-D group. However, this substitution in Sr-α group failed to increase intestinal calcium absorption. We also examined the direct of strontium on bone resorption using⁴⁵Ca pre-labeled mouse calvaria. Strontium was injected every day until sacrifice, and percent⁴⁵Ca release from cultured calvariae was measured. Bone resorption was inhibited by strontium dose-dependently in groups which had and had not received parathyroid hormone in culture. These results suggest that strontium inhibits intestinal calcium absorption and has a direct inhibitory effect on bone resorption.     

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.     

Bioavailability of calcium from oyster shell electrolysate and DL-Calcium lactate in vitamin D-replete or vitamin D-deficient rats

Bioavailability of calcium (Ca) from oyster shell electrolysate (Active Absorbable Ca: AACa) and DL-Ca lactate in rats was investigated. Plasma Ca metabolism, bone mineral density and vitamin D-deficient rats were measured and compared each other. Vitamin D-deficient rats were fedad libitum a vitamin D-free diet containing 0.44 % Ca or 1.20 % Ca adjusted with either AACa or DL-Ca lactate with or without oral supplementation of physiological dose (10 IU/rat/day) of vitamin D₃ for 28 days. During and after feeding, concentrations of Ca, phosphorus, parathyroid hormone (PTH), 25-hydroxyvitamin D₃ (25-OH-D₃), 1α, 25-dihydroxyvitamin D₃ [1, 25(OH)₂D₃] and alkaline phosphatase (Alp) activity in plasma were measured. Furthermore, femoral Ca and phosphorus contents, mineral density and mechanical strength were also measured. Despite of the differences of contents and chemical forms of Ca in diets, all the groups showed prompt and complete recoveries in both Ca and vitamin D malnutritions under vitamin D-replete condition and there was no significant difference among all the groups in increases of body weight gains and plasma Ca levels, and in decreases of plasma PTH level and Alp activity. In contrast, the groups fed the same diets under vitamin D-deficient condition showed no significant recoveries in both plasma Ca metabolism and bone mineralization although Ca bioavailability of AACa appeared to be slightly better than that of DL-Ca lactate. These results suggest that bioavailability of Ca from AACa and DL-Ca lactate are substantially equal in increasing plasma Ca levels, bone mineral density and mechanical strength in both vitamin D-replete and vitamin D-deficient rats.     

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.     

Cyclic AMP-dependent membrane protein phosphorylation and calmodulin binding are involved in the rapid stimulation of muscle calcium uptake by 1,25-dihydroxyvitamin D3

Summary Rapidin vivo effects of 1,25-dihydroxyvitamin D₃ on muscle calcium metabolism have been reported.In vitro studies have shown that exposure of vitamin D-deficient chick soleus muscles to the sterol for 1–10 minutes causes a significant stimulation of tissue⁴⁵Ca uptake which can be suppressed by Ca channel blockers. A parallel increase in muscle membrane calmodulin content that could be mimicked by forskolin was observed. Experiments were carried out to obtain information about the mechanism underlying the fast action of 1,25-dihydroxyvitamin D₃. Like the sterol, forskolin (10 μm) rapidly increased (+48% at 5 min) soleus muscle⁴⁵Ca uptake and its effect could be reversed by nifedipine (50 μm). In agreement with these observations, 1,25-dihydroxyvitamin D₃ markedly elevated tissue cAMP levels within 45 seconds to 5 minutes of treatment in a dose-dependent manner (10⁻¹¹–10⁻⁷M). Moreover, incubation of isolated muscle microsomes with 1,25-dihydroxyvitamin D₃ increased adenylate cyclase activity and caused a similar profile of stimulation of protein phosphorylation with [γ-³²P]-ATP as forskolin. Major changes were detected in proteins whose calmodulin binding ability has been previously shown to be increased by 1,25-dihydroxyvitamin D₃. In addition, the calmodulin antagonists fluphenazine and compound 48/80 abolished the increase in muscle Ca uptake and membrane calmodulin content produced by the sterol. The results suggest that 1,25-dihydroxyvitamin D₃ activates muscle Ca channels through a direct membrane action which involves cAMP-dependent protein phosphorylation and calmodulin binding.     

Effects of dietary supplementation of calcium and vitamin don bone growth in growing male rats

Effects of dietary supplementation of calcium (Ca) and vitamin D(D) on bone growth in growing male rats were investigated. We performed this study using D-deficient rats of 3-month-old. In the experiment 1, the D-deficient rats were fed either low-Ca (0.22% Ca) or high-Ca (1.20% Ca) diets with oral supplementation of different amounts of D₃ (0, 0.7, 7 or 70 IU/week) for 28 days. In the dxperiment 2, the D-deficient rats were fed diets containing different concentrations of Ca (0.22, 0.44, 0.88 or 1.20%) with oral D₃ supplementation of either low-dose (0.7 IU/week) or relatively high-dose (70 IU/week) for 28 days. After the feeding period, plasma levels of Ca, 1α, 25 (OH)₂D₃, PTH, bone Gla protein were measured. Bone ash weight, bone mineral density, mechanical bone strength were also measured. In the both experiments, the plasma levels of PTH decreased to the normal levels in response to the increased amounts of dietary Ca intakes as well as D supplementation. In contrast, the bone markers increased to the respective normal levels in response to the increased amounts of dietary Ca intakes as well as D supplementation. In the experiments 1 and 2, a high correlation between the plasma levels of PTH and the bone markers was observed. These results suggest that both dietary Ca and D supplementation may affect bone growth in growing rats by controlling PTH secretion.     

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.     

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.     

Effects of vitamin D deficiency in the chicken embryo

Summary Vitamin D-deficient chicken embryos were obtained by feeding laying hens a diet in which 5 μg 1,25(OH)₂D₃/kg feed were substituted for the vitamin D₃ supplement in the control diet. Hatchability, total Ca and inorganic P concentration in blood, and tibial ash/dry weight ratio were determined in the vitamin D-deficient embryos and in embryos obtained from hens fed the control diet supplemented with 1100 IU vitamin D₃/kg feed. After 5 weeks on the substituted diet the hens laid eggs that showed decreased hatchability in spite of excellent shell quality. All determinations in blood and bones were made on embryos of eggs laid after 6–12 weeks on the diets. On the 17th day of incubation the embryos derived from hens fed the substituted diet showed significant hypocalcemia and hyperphosphatemia and a low tibial ash/dry weight ratio. Injection of 1,25(OH)₂D₃ 3 days before killing corrected the hypocalcemia of the deficient embryos. Those chicks that managed to hatch had normal levels of calcium and inorganic phosphate 1 day after hatching. These findings support previous suggestions by us and other authors that vitamin D metabolites are required by the embryo in order to mobilize calcium from the shell, and decreased hatchability in vitamin D-deficient embryos is related to a defect in calcium mobilization from the shell. While in previous studies a decrease in hatchability was the only parameter used to judge D deficiency of the embryos in our present studies, the deficiency is confirmed by demonstrating a deficit in mineral metabolism which is a more specific sign of D deficiency.     

Role of parathyroid hormone and 1,25-dihydroxyvitamin D3 in the development of osteopenia in oophorectomized rats

Summary The effect of ovarian insufficiency on calcium metabolism has been thought to involve an increased bone resorptive effect of parathyroid hormone and possible impaired synthesis of 1,25-dihydroxyvitamin D₃. In the present study a rat model allowing for controlled serum levels of parathyroid hormone and 1,25-dihydroxyvitamin D₃ was used. Oophorectomy in this species is associated with increased serum levels of 1,25-dihydroxyvitamin D₃ and decreased bone mass. Although thyroparathyroidectomy increased bone mass, an increased sensitivity of bone to parathyroid hormone in oophorectomized rats was not observed. Thus the development of the osteopenia did not seem to be related to increased parathyroid hormone sensitivity or to reduced levels of 1,25-dihydroxyvitamin D₃. Exogenous 1,25-dihydroxyvitamin D₃ increased bone mass in oophorectomized as well as intact rats. Intestinal calcium transport was increased by moderate doses of 1,25-dihydroxyvitamin D₃. Intestinal calcium transport was also reduced by thyroparathyroidectomy and increased by the administration of parathyroid extract. A tendency for increased accumulation of 1,25-dihydroxyvitamin D₃ in blood in oophorectomized rats has been noted. It is suggested that the tendency to hypercalcemia in ovarian-insufficient females given 1-hydroxylated vitamin D compounds may be related to a diminished metabolism of 1,25-dihydroxyvitamin D₃.     

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.     

Chlorpromazine alters bone metabolism of ratsin vivo

Summary The acute effect of chloropromazine (CPZ) on metabolic changes in rat was investigated. CPZ was found to markedly suppress⁴⁵Ca incorporation into the calvarium and ileumin vitro. According to the serum and/or urinary levels of certain markers for bone metabolism, the increases of Ca and P in the serum and Ca, P, and γ-carboxyglutamate (Gla) in the urine were observed in rats given 10 mg CPZ/kg of their body weight, whereas the amount of alkaline phosphatase (ALP) activity, ionized Ca, calcitonin, 25-hydroxyvitamin D₃ (25(OH)D₃), 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) as clearly or slightly reduced, suggesting the inhibitory effect of their bone formation. This was well supported since only bone type ALP was detected in the urine and loss of the vertebral bone density from rats given daily CPZ administration for a week. Moreover, in this case, there is little if any difference in the levels of mid-molecule parathyroid hormone (mid-PTH), osteocalcin, and urinary cAMP for the nontreated and CPZ-treated animals, resulting in the fact that CPZ may mainly inhibit the hydroxylase for active vitamin D₃.     

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.     

Effects of prostaglandin E2 and indomethacin on 25-hydroxyvitamin D3-1α-hydroxylase activity in isolated kidney cells of normal and streptozotocin-induced diabetic rats

Summary The effects of prostaglandin E₂ (PGE₂) (1 μM) and indomethacin (IN) (20 μM) on 1,25 dihydroxyvitamin D₃ production were studied in renal cell suspensions isolated from control, stroptozotocin-induced diabetic, and insulin-treated diabetic rats. Renal cortex cells were isolated by enzymatic digestion, and preincubated for 30 min at 37°C with the appropriate additive(s) followed by a 1 h incubation with 8 nM 25-hydroxyvitamin D₃ in serumfree medium. Radioactivity incorporated into that fraction of the cell suspension extract co-eluting with synthetic 1,25-dihydroxyvitamin D₃ on high pressure liquid chromatography was determined All animals were raised for 5 weeks on a vitamin D-deficient diet. Isolated kidney cells from vitamin D-deficient rats showed dose-dependent response of 1,25 dihydroxyvitamin D₃ production to PGE₂. Cells from control animals demonstrated a stimulatory effect of PGE₂ (P<0.05) and a suppressive effect of IN (P<0.01) on 1,24 dihydroxyvitamin D₃ production. In contrast, cells from diabetic rat kidneys failed to respond to these agents, alterations which were reversed by insulin treatment. The accumulated data suggest that depressed synthesis of 1,24 dihydroxyvitamin D₃ previously observed in the experimental diabetic rat is due, at least in part, to an impaired production and response to PGE₂-like prostaglandins.     

Calcium regulating activity of 26,27-dialkyl analogs of 1α,25-dihydroxyvitamin D3

Summary A series of analogs of 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] with alkyl substitutions in 26- and 27-positions were tested for calcium (Ca) regulating activity. The potencies of dialkyl analogs in stimulating bone resorption in neonatal mouse calvaria cultures were the highest in 1,25-dihydroxy-26,27-dimethylvitamin D₃[1,25(OH)₂-(Me)₂D₃], followed by 1,25(OH)₂D₃, 1,25-dihydroxy-26,27-diethylvitamin D₃[1,25(OH)₂(Et)₂D₃], and 1,25-dihydroxy-26,27-dipropylvitamin D₃[1,25(OH)₂(Pr)₂D₃] in that order. A similar order of potential regarding formation of osteoclast-like cells in mouse bone marrow cell cultures and on bone Ca mobilization with long-term vitamin D-deficient rats was observed in the same series. The relative potencies of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 1,25(OH)₂(Et)₂D₃, and 1,25(OH)₂(Pr)₂D₃ in competing with 1,25(OH)₂D₃ for binding to chick intestinal cytosol receptors were 1:1:0.16:0.036. A similar order of potential in case of intestinal Ca transport in situ was observed in the same series. The potencies of dialkyl analogs in competing with 25-hydroxy-vitamin D₃ for binding to rat serum vitamin D binding protein were much lower than that of 1,25(OH)₂D₃. Effect of 1,25(OH)₂(Me)₂D₃ on osteopenia in rats induced by ovariectomy and right sciatic neurotomy was higher than that of 1,25(OH)₂D₃. From these results, the lengthening by one carbon at 26- and 27-positions was shown to maintain the Ca regulatory activity of 1,25(OH)₂D₃.     

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.     

Studies on the role of 24-hydroxylation of vitamin D in the mineralization of cartilage and bone of vitamin D-deficient rats

Summary To test the importance of 24-hydroxylation of vitamin D₃ on bone mineralization, rat pups born to vitamin D-deficient females were given either 25-hydroxyvitamin D₃ or 24,24-difluoro-25-hydroxyvitamin D₃ for 16 days beginning at the time of weaning. Following such treatment analysis of blood samples revealed no detectable 24R,25-(OH)₂D₃ and 1,25-(OH)₂D₃ in the rats given the difluoro compound while revealing the expected 24,24-difluoro-25-hydroxyvitamin D₃ and 24,24-difluoro-1,25-dihydroxyvitamin D₃. The rats given 25-hydroxyvitamin D₃ had the expected levels of 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and 1,25-dihydroxyvitamin D₃. Following sacrifice at day 17, postweaning bone mineralization and modeling were studied in long bones using histological methods. Bones taken from vitamin D-deficient rats at the beginning and end of the experimental period had lesions typical of rickets. These included wide growth plates, excessive amounts of osteoid, and metaphyseal fibrosis. Following treatment with either 25-hydroxyvitamin D₃ or 24,24-difluoro-25-hydroxyvitamin D₃, bone mineralization returned to normal. Growth plate widths and the amount of osteoid on bone surfaces were both substantially reduced and to a similar degree in both treatment groups. Normal cartilage core formation and trabecularization of the metaphyseal primary spongiosa were also restored to a similar degree in both groups. In effect, no difference was observed in any bone parameter studied between the 25-hydroxyvitamin D₃- and the 24,24-difluoro-25-hydroxyvitamin D₃-treated animals. These results provide strong evidence that 24-hydroxylation of the vitamin D molecule plays little or no role in the modeling and mineralization of bone.     

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     

In vitro calcium transport properties of skeletal muscle mitochondria from vitamin D-deficient and 1,25-dihydroxy-vitamin D3-treated chicks

Summary Previous work has shown that vitamin D₃ or 1,25-dihydroxy-vitamin D₃ affect calcium content and fluxes in mitochondria of chick skeletal musclein situ. Studies were performed to investigate whether these effects are related to variations in the Ca²⁺ transport properties of mitochondrial membranes. Mitochondria isolated from skeletal muscle of vitamin D-deficient chicks and chicks dosed with 1,25(OH)₂D₃ for 3 or 7 days (50 ng/day) were employed. No changes in the rate and affinity for calcium of the Ruthenium Red-sensitive Ca²⁺ uptake system were detected after treatment with 1,25(OH)₂D₃. The metabolite did not cause either modifications in Ca²⁺ efflux from mitochondria preloaded with the cation induced by Na⁺ or blockage of mitochondria energy supply. Prior treatment of animals with vitamin D₃ was also without effects. However, a significant stimulation of Ca²⁺ uptake by intact muscle preparations from the same experimental animals was observed in response to treatment with 1,25(OH)₂D₃ in vivo (50 ng/day, 3 days) orin vitro (10⁻¹⁰ M, 60 minutes). In addition, the Ca content of muscle mitochondria was markedly diminished in chicks treated with the sterol. It is suggested that the effects of 1,25(OH)₂D₃ on muscle mitochondrial Ca metabolism may be secondary to changes in cytoplasmic Ca²⁺.