The renal metabolism of 25-hydroxyvitamin D3 in the rat: Regulation by 1,25-dihydroxyvitamin D3

Summary To determine the effects of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on the renal metabolism of 25-hydroxyvitamin D₃ [25(OH)D₃], the influence of 1,25(OH)₂D₃ and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] was compared in vitamin D-deficient rats. Serum calcium (Ca), serum immunoreactive parathyroid hormone (iPTH) and the specific activities (SA) of renal 25(OH)D₃: 24-hydroxylase (24-hydroxylase) and 25(OH)D₃: 1α-hydroxylase (1-hydroxylase) were measured. In vitamin D-deficient rats, mean serum Ca was low, serum iPTH was increased, renal 1-hydroxylase was increased, and renal 24-hydroxylase was below the limit of detection. Treatment with either 1,25(OH)₂D₃ or 24,25(OH)₂D₃, 50 ng/d for 2 days, significantly increased mean serum Ca but did not change serum iPTH, renal 1-hydroxylase SA, or renal 24-hydroxylase SA. 1,25(OH)₂D₃, 50 ng/d for 7 days, returned serum Ca and iPTH to normal, lowered renal 1-hydroxylase SA, and induced renal 24-hydroxylase activity. In contrast, 24,25(OH)₂D₃, 50 ng/d for 7 days, similarly lowered renal 1-hydroxylase SA but did not induce renal 24-hydroxylase activity. Thyroparathyroidectomy of vitamin D-deficient rats resulted in a rapid decline in 1-hydroxylase SA. The results indicate that in vitamin D-deficient rats a) 1,25(OH)₂D₃ reduces renal 1-hydroxylase SA and increases renal 24-hydroxylase SA and b) 24,25(OH)₂D₃ reduces renal 1-hydroxylase SA and does not alter renal 24-hydroxylase SA. Inhibition of renal 1-hydroxylase by the two metabolites is apparently mediated through changes in serum Ca and circulating iPTH, whereas stimulation by 1,25(OH)₂D₃ of renal 24-hydroxylase activity is direct.     

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.     

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.     

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⁺.     

Effect of vitamin D on growth cartilage cell proliferation in vitro

Disturbed calcification of the growth plate and stunting is a frequent finding in vitamin D-deficiency rickets, vitamin D-dependency rickets and renal osteodystrophy, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of Ic, 25-dihydroxy vitamin D₃ [1,25(OH)₂D₃] receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In tibial growth plates and in primary cultures of tibial growth cartilage of male Sprague-Dawley rats (80 g) specific binding of [³H]-1,25(OH)₂D₃ was noted. Scatchard analysis revealed the presence of a single class of non-interacting binding sites.K _d was 10^–11 M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA-cellulose affinity chromatography. By immunohistology, growth cartilage cells (rabbit tibia) were shown to express nuclear 1,25(OH)₂D₃ receptors, most prominently in the proliferative and early hypertrophic zone. This corresponds to binding data which showed highest binding of 1,25(OH)₂D₃ in the logarithmic growth phase (12,780 molecules/cell versus 4,538 molecules/cell in confluent cells) in primary cultures of growth plate chondrocytes. In the presence of delipidated fetal calf serum 1,25(OH)₂D₃ had a biphasic effect on cell proliferation and density, i.e. stimulation at 10^–12 M and dose-dependent inhibition at 10^–10 M and below. Inhibition was specific and not seen with 24 (R), 25-dihydroxyvitamin D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and specific effects of 1,25(OH)₂D₃ on chrondrocyte proliferation in vitro point to a role for vitamin D in the homeostasis of growth cartilage of the rat.     

1,25(OH)2D3 inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells

Summary Increasing the extracellular Ca²⁺ concentration from 0.5 to 3.0 mM induced marked increments in cytoplasmic Ca²⁺ concentration (Ca²⁺ _i) and inhibition of parathyroid hormone (PTH) release of freshly isolated bovine parathyroid cells. 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃; 0.1–100 ng/ml) did not affect (Ca²⁺ _i) and was also without acute effect on the secretion. During 4 days of monolayer culture, the parathyroid cells underwent significant increases in both number and size, and presence of 10–100 ng/ml 1,25(OH)₂D₃ almost completely inhibited the cell proliferation, whereas the hypertrophy was unaffected. One day of culture with 0.1–100 ng/ml 1,25(OH)₂D₃ was without effect on PTH release but after 4 days there was a dose-related reduction of recretion. At this time point and irrespective of the culture condition, PTH release was no longer suppressed by high extracellular Ca²⁺. Furthermore, Ca²⁺ _i increased little upon increments in the extracellular Ca²⁺ concentration as compared with freshly isolated cells. It is concluded that after prolonged exposure to 1,25(OH)₂D₃, PTH release is inhibited and, at high concentrations, the parathyroid cells cease to proliferate. However, 1,25(OH)₂D₃ does not affect the development of functional dedifferentiation of parathyroid cells during monolayer culture.     

In Vivo Treatment with Calcitriol (1,25(OH)2D3) Reverses Age-Dependent Alterations of Intestinal Calcium Uptake in Rat Enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D₃; [1,25(OH)₂D₃] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)₂D₃ stimulation of Ca²⁺ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20–24-month-old rats given 1,25(OH)₂D₃ for 3 days (30 ng/100 g bw/day), calcitriol (10⁻¹⁰ M, 3–5 minutes) stimulated Ca²⁺ uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)₂D₃. When the aged rats were injected with 25(OH)D₃, similar Ca²⁺ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1,25(OH)₂D₃-dependent changes in Ca²⁺ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)₂D₃ stimulation of duodenal cell Ca²⁺ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)₂D₃ or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system. Received: 26 December 1997 / Accepted: 12 May 1998     

Acquired alterations in vitamin D metabolism in the acidotic state

Summary Classic (type I) renal tubular acidosis in children is attended by growth retardation and rickets, abnormalities that can be corrected by alkali therapy alone. We have employed the NH₄Cl-treated rachitic chick as a model to investigate vitamin D metabolism in the acidotic state. NH₄Cl ingestion for 96 h was associated with a rise in serum calcium, a significant decrease in blood pH (7.42+0.08 vs 7.30±0.08,P<0.005), decreased [³H]1,25(OH)₂D₃ following [³H]25OHD D₃ injections, and enhanced metabolic clearance of administered [³H]1,25(OH)₂D₃. The data collectively suggest that metabolic acidosis in the chick alters the production and degradation of 1,25(OH)₂D₃.     

1α,25‐Dihydroxyvitamin D3 and its analogues,EB1089 and CB1093, profoundly inhibit the in vitro proliferation of the human hepatoblastoma cell line HepG2

Background : 1α,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃) has been shown to inhibit the proliferation of various cancer cells including colon, prostate, melanoma, osteosarcoma and breast cancer. Methods : The human hepatoma cell line (HepG2) was cultured with 1,25(OH)₂D₃ or one of two analogues EB1089 or CB1093 for various durations. Cellular proliferation was measured by uptake of [³H]thymidine, and cell numbers were determined by trypan blue exclusion counting. Results : 1,25(OH)₂D₃, EB1089 and CB1093 all inhibited proliferation of HepG2 by up to 90% after 5 days of treatment, compared to the untreated controls. Decreased proliferation was associated with an approximately 50% reduction in cell numbers at concentrations of up to 10^–10 mol/L after 5 days of treatment with 1,25(OH)₂D₃. Cell proliferation rapidly recovered in cultures treated with lower concentrations of 1,25(OH)₂D₃ (10^–10 and 10^–11 mol/L) when 1,25(OH)₂D₃ was removed from the cultures by placing cells in serum containing medium without 1,25(OH)₂D₃. When HepG2 cells were treated with 10^–8 mol/L 1,25(OH)₂D₃ for 5 weeks, there was still significant inhibition of proliferation, although at week 5 there was 66% inhibition compared to 93% at the end of week 1. Conclusions : 1,25(OH)₂D₃, EB1089 and CB1093 all significantly inhibit the proliferation of HepG2 hepatoblastoma cells, with EB1089 being the most potent at lower concentrations. Inhibition can be maintained for at least 4 weeks, but is reversed after removal of vitamin D₃.     

Cell density-dependent vitamin D effects on calcium accumulation in rat osteogenic sarcoma cells (ROS 17/2)

Summary Rat osteogenic sarcoma cells have been used widely as a model system to study actions of 1,25(OH)₂D₃ and other hormones in osteoblastlike cells. However, some of the pleiotypic manifestations of hormones in these cells vary greatly dependent upon the cell population density and other conditions of culture. Therefore, we have studied the effect of cell density on the relationship between 1,25(OH)₂D₃ and the initial⁴⁵Ca accumulation in ROS 17/2 cells in order to establish conditions suitable for studying the effect of 1,25(OH)₂D₃ on calcium fluxes in these cells. Cells were grown in the presence and absence of 1,25(OH)₂D₃ for 48 hours and then incubated for 4 min in the culture medium containing 0.5 μCi/ml of⁴⁵CaCl₂. In high population density cultures, 0.25–1.0 pg/ml of 1,25(OH)₂D₃ stimulated the intracellular accumulation of⁴⁵Ca (cpm/mg protein), whereas 80 pg/ml or higher concentrations inhibited accumulation of⁴⁵Ca. In low density cultures, concentrations less than 80 pg/ml had no effect, 80–120 pg/ml increased the intracellular accumulation, and as much as 200 pg/ml failed to show the inhibitory effect. These results indicate that the ROS 17/2 cell responses to 1,25(OH)₂D₃ are biphasic—low concentrations stimulating and high concentrations inhibiting⁴⁵Ca accumulation. The sensitivity of the cells to 1,25(OH)₂D₃ increases as the cell population density increases. These observations suggest that the culture density and dose-response relationship must be carefully defined inin vitro studies utilizing osteogenic cell culture systems.     

Early manifestations of vitamin D effects in rat osteogenic sarcoma cells

Summary We have recently demonstrated that 48 hour exposure of ROS 17/2 cells to low concentrations of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) (1.0 pg/ml) stimulated the cellular accumulation of⁴⁵Ca, and exposure to high concentrations (160 pg/ml) inhibited such accumulation. In the present study, short-term (15 min) effects of the sterol on⁴⁵Ca accumulation in ROS 17/2 cells were compared with the long-term (48 hours) effects in order to clarify mechanisms responsible for 1,25(OH)₂D₃ control of calcium metabolisms in ROS 17/2 cells. ROS 17/2 cells were grown for 48 hours in the presence and absence of 1,25(OH)₂D₃ and then incubated for an additional 15 min in the presence and absence of 1,25(OH)₂D₃ immediately before measuring⁴⁵Ca accumulation. Cellular⁴⁵Ca was measured after incubating the cells in the medium containing 0.5 μCi/ml of⁴⁵CaCl₂ for 4 min at 25°C. The effect of actinomycin D was determined by preincubating the cells in 0.1 μg/ml of actinomycin D for 45 min at 25°C. Exposure to low concentrations (1.0 pg/ml) of 1,25(OH)₂D₃ for either 48 hours or 15 min increased⁴⁵Ca in the cells by 10–20%. An additional 15 min exposure following 48 hour exposure yielded an increase in the cellular⁴⁵Ca similar to that after 48 hours or 15 min exposure. Exposure to high concentrations (160 pg/ml) for either 48 hour or 15 min decreased cell⁴⁵Ca by approximately 20%.An additional 15 min exposure to the high concentrations did not change the 48 hour effect. Actinomycin D reversed early inhibitory effects of high concentrations, but had no effect on the early stimulatory effects of low concentrations. These results suggest that the mechanisms underlying the 15 min and 48 hour effects of 1,25(OH)₂D₃ are the same. Moreover, the mechanism responsible for the inhibitory effect of high concentrations is dependent onde novo protein synthesis whereas that for the stimulatory effects of low concentrations is not.     

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.     

Vitamin D Therapy of Osteoporosis: Plain Vitamin D Therapy Versus Active Vitamin D Analog (D-Hormone) Therapy

Normal intestinal calcium (Ca) absorption is an essential feature of bone homeostasis. As with many other organ systems, intestinal Ca absorption declines with aging, and this is one pathological factor that has been identified as a cause of senile osteoporosis in the elderly. This abnormality leads to secondary hyperparathyroidism, which is characterized by high serum parathyroid hormone (PTH) and an increase in bone resorption. Secondary hyperparathyroidism due to poor intestinal Ca absorption has been implicated not only in senile osteoporosis but also in age-related bone loss. Accordingly, in population-based studies, there is a gradual increase in serum PTH from about 20 years of age onward, which constitutes a maximum increase at 80 years of age of 50% of the basal value seen at 30 years of age. The cause of the increase in PTH is thought to be partly due to impaired intestinal Ca absorption that is associated with aging, a cause that is not entirely clear but at least in some instances is related to some form of vitamin D deficiency. There are three types of vitamin D deficiency: (1) primary vitamin D deficiency, which is due to a deficiency of vitamin D, the parent compound; (2) a deficiency of 1,25(OH)₂D₃ resulting from decreased renal production of 1,25(OH)₂D₃; and (3) resistance to 1,25(OH)₂D₃ action owing to decreased responsiveness to 1,25(OH)₂D₃ of target tissues. The cause for the resistance to 1,25(OH)₂D₃ could be related to the finding that the vitamin D receptor level in the intestine tends to decrease with age. All three types of deficiencies can occur with aging, and each has been implicated as a potential cause of intestinal Ca malabsorption, secondary hyperparathyroidism, and senile osteoporosis. There are two forms of vitamin D replacement therapies: plain vitamin D therapy and active vitamin D analog (or D-hormone) therapy. Primary vitamin D deficiency can be corrected by vitamin supplements of 1000 U a day of plain vitamin D whereas 1,25(OH)₂D₃ deficiency/resistance requires active vitamin D analog therapy [1,25(OH)₂D₃ or 1α(OH)D₃] to correct the high serum PTH and the Ca malabsorption. In addition, in the elderly, there are patients with decreased intestinal Ca absorption but with apparently normal vitamin D metabolism. Although the cause of poor intestinal Ca absorption in these patients is unclear, these patients, as well as all other patients with secondary hyperparathyroidism (not due to decreased renal function), show a decrease in serum PTH and an increase in Ca absorption in response to therapy with 1,25(OH)₂D₃ or 1α(OH)D₃. In short, it is clear that some form of vitamin D therapy, either plain vitamin D or 1,25(OH)₂D₃ or 1α(OH)D₃, can be used to correct all types of age-dependent impairments in intestinal Ca absorption and secondary hyperparathyroidism during aging. However, from a clinical standpoint, it is important to recognize the type of vitamin D deficiency in patients with senile osteoporosis so that primary vitamin D deficiency can be appropriately treated with plain vitamin D therapy, whereas 1,25(OH)₂D₃ deficiency/resistance will be properly treated with 1,25(OH)₂D₃ or 1α(OH)D₃ therapy. With respect to postmenopausal osteoporosis, there is strong evidence that active vitamin D analogs (but not plain vitamin D) may have bone-sparing actions. However, these effects appear to be results of their pharmacologic actions on bone formation and resorption rather than through replenishing a deficiency.     

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

Summary Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)₂D₃ receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [³H]-1,25(OH)₂D₃ is noted in both the logarithmic growth phase and at confluence (N_max 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. K_D was 10⁻¹¹ M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)₂D₃ receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest N_max in the proliferating cartilage. 1,25(OH)₂D₃ in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10⁻¹² M and dose-dependent inhibition at 10⁻¹⁰ M and below. Inhibition was specific and not seen with 24,25(OH)₂D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and effects of 1,25(OH)₂D₃ on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.     

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.     

Elongation of the side chain of analogs of 1α,25-dihydroxyvitamin D3 prevents osteopenia in a rat model

Five analogs of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] [1], 26,27-dimethyl-1,25-dihydroxyvitamin D₃ [1,25(OH)₂(Me)₂D₃] [2], 26,27-dimethyl-1,25-dihydroxyvitamin D₃ [1,25(OH)₂(Et)₂D₃] [3], 26,27-dipropyl-1, 25-dihydroxyvitamin D₃ [1,25(OH)₂(Pr)₂D₃] [4], 26,27-dimethyl-24, 24-difluoro-1,25-dihydroxyvitamin D₃ [24F₂-1,25(OH)₂(Me)₂D₃, and [5] 24a-homo-24,24-difluoro-1,25-dihydroxyvitamin D₃ [24aF₂-homo-1,25(OH)₂D₃] were investigated to clarify the possibility that prevents osteopenia induced in rats by ovariectomy and sciatic neurotomy. The objective of our studies was to determine whether these analogs may be effective for treatment of subjects with osteoporosis. 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃ prevented decreases in bone mineral density (BMD) of the femur, as measured by dual energy X-ray absorptiometry (DXA). The potency of 1,25(OH)₂(Me)₂D₃ in this test was higher than that of 1,25(OH)₂D₃. The potencies of 24F₂-1,25(OH)₂(Me)₂D₃ and 24aF₂-homo-1,25(OH)₂D₃ were similar to that of 1,25(OH)₂D₃. On the other hand, though 1,25(OH)₂(Et)₂D₃ and 1,25(OH)₂(Pr)₂D₃ had a preventive effect on the decease in BMD, the potency of two analogs was lower than that of 1,25(OH)₂D₃. Decreases in cortical and trabecular bone areas of the femur were prevented by three analogs of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃. Serum calcium (Ca) concentration was elevated at the last administration of three analogs of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃ and 24aF₂-homo-1,25(OH)₂D₃. Decreases in the Ca concentration in untreated rats were noted a few days after the last administration. In light of these positive effects, we are continuing research on 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃ as putative treatment for osteoporosis.     

Effects of vitamin D3 metabolites on calcium fluxes in intact chicken skeletal muscle and myoblasts culturedin vitro

Summary 25-hydroxycholecalciferol (25OHD₃) and 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) at physiological concentrations exerted direct effects on Ca fluxes in cultured vitamin D-deficient chick soleus muscle and myoblasts. Isotopic desaturation curves of soleus muscle prelabeled with⁴⁵Ca indicated that the action of 25OHD₃ is localized in a slow-exchangeable Ca pool where it stimulates net Ca uptake. On the other hand, the predominant effects of 1,25(OH)₂D₃ consist in an increase of the rate constant of Ca efflux of this pool and in an increase of net Ca uptake in a fast-exchangeable pool. 24,25-dihydroxycholecalciferal proved to be inactive on both Ca uptake and efflux. In addition, 1,25(OH)₂D₃ significantly increased⁴⁵Ca labeling of cultured chick myoblasts. These effects were accompanied by changes in the growth and differentiation of the cultures. The results suggest a direct involvementin vivo of 25OHD₃ and 1,25(OH)₂D₃ on muscle cellular calcium.     

The effect of induced metabolic acidosis on vitamin D3 metabolism in rachitic chicks

Summary The metabolism of vitamin D₃ was studied in 3-week-old, vitamin D deficient chicks, fed since hatching with a diet containing 3% ammonium chloride, 1% calcium, and 0.7% phosphorus. When kidney homogenates were incubated in vitro with [³H]25-(OH)D₃, the production of 1,25-(OH)₂D₃ was reduced by 40% in acidotic birds. During in vivo experiments, after injection of [³H]D₃ (1220 pM/bird), the level of 1,25-(OH)₂D₃ was also reduced in blood plasma, intestine, and tibiae in acidotic chicks as compared with the controls. As a large increase in plasma phosphate was found during acidosis, these results are discussed in relation to the possible role of phosphorus in the control of 1,25-(OH)₂D₃ synthesis.     

Coordinate inhibition of alkaline phosphatase and type X collagen syntheses by 1,25-dihydroxyvitamin D3 in primary cultured hypertrophic chondrocytes

Summary The effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) (2.3×10^-12-1.4×10^-6 [M]) on alkaline phosphatase, collagen, and cell proliferation were examined in primary cultured hypertrophic chondrocytes prepared from the distal epiphyseal growth plate of the tibias of 12-day chick embryos. 1,25(OH)₂D₃ showed time- and dose-dependent inhibitory effects on the alkaline phosphatase and collagen levels. The inhibition of alkaline phosphatase activity became detectable at 2×10^-11 [M] and reached 10% of control at 10^-7 [M]. The concentration of 1,25(OH)₂D₃ giving a 50% inhibition of the enzyme level was approximately 3×10^-10 [M]. Of the two extracellular collagen pools, a cell-associated matrix pool showed a more dramatic decrease (to 10% of control) than a culture medium pool (to 50% of control) at increased 1,25(OH)₂D₃ concentrations. The degree of inhibition was different for each type of chondrocyte-specific collagen (types II, IX, X, and XI). Types II and IX were inhibited in a parallel manner to only 60–80% of control. On the other hand, types X and XI were more greatly reduced up to 10% of control, and their dose-dependent inhibitory curves were similar to that of alkaline phosphatase. On cell proliferation, 1,25(OH)₂D₃ had a biphasic effect: stimulation at 10^-10–10^-8 [M] and inhibition at higher levels. The results revealed the significant involvement of 1,25(OH)₂D₃ in the metabolism of two probable calcification-related products, alkaline phosphatase and type X collagen.