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.     

Oral 1,25(OH)2D3 pulse therapy

Conclusion In our experience, after a few months of therapy, every patient showed a marked improvement in both X-ray abnormalities derived from osteitis fibrosa and symptoms of renal osteodystrophy, especially bone pain, unless the serum phosphorus level was very high. The effectiveness of this therapy on the suppression of PTH secretion apparently depends on the initial PTH level, and also on the size of the gland itself. One of the major current difficulties in this therapy is the prevention of hypercalcemia when calcium carbonate is used. The calcium concentration of the dialysate must be reduced to 2.5 mEq/l not only for pulse therapy, but also for conventional therapy by vitamin D with calcium carbonate. Parathyroidectomy should be indicated only for the patient who does not respond to pulse therapy.     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

1,25(OH)2D and 24,25(OH)2D production in the developing kidney

To clarify the state of vitamin D production by the developing kidney, firstly, we measured serum levels of 1,25(OH)₂D and 24,25(OH)₂D in humans of different ages (pregnant and nonpregnant women, adult males, children and newborn infants) and secondly, we measured 1- and 24-hydroxylase activity in the kidney mitochrondria of rats at different ages. The mean serum levels of 1,25(OH)₂D in pregnant women, cord blood and newborns were significantly higher than those in children and non-pregnant women and adult males. In newborns, the level increased with gestational age. Synthesis of 1,25(OH)₂D was, at least in part, under the control of the fetus and newborn, rather then being solely a reflection of the conditions prevailing in the mother. The 1-hydroxylase activity in mitochondria was highest in the 1- to 2-month-old rats, and it decreased gradually thereafter. The change in 1-hydroxylase activity with age was due to a change in the V_max of the system.     

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

Summary Calvarial bones from hypophosphatemic (Hyp) mice and normal littermates were cultured in a chemically defined medium to determine: (a) the effect of medium phosphate (Pi) concentration (1, 2, and 3 mM) on collagen synthesis; (b) the effect of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] (10⁻¹²M–10⁻⁷M) on collagen synthesis; and (c) whether bone responsiveness to 1,25(OH)₂D₃ was affected by changes in medium Pi concentration. Bone collagen synthesis was evaluated by measuring [³H]hydroxyproline formation. The distribution of labeled hydroxyproline between bone explant and culture medium (total and dialyzable fraction) was studied. These experiments confirm that 1,25(OH)₂D₃ inhibits specifically bone collagen synthesis in vitro. We did not detect any effect of medium Pi concentration on basal collagen synthesis but were able to demonstrate that lowering medium Pi concentration increased the 1,25(OH)₂D₃-induced inhibition of collagen synthesis. Bones from both genotypes responded to 1,25(OH)₂D₃, but modulation of this response by changes in Pi concentration was altered in Hyp bone as, in contrast to normal bone, its response to 1,25(OH)₂D₃ was unaffected when medium Pi concentration was decreased from 3 to 2 mM. These findings support the hypothesis of an altered response of bone to 1,25(OH)₂D₃ in the Hyp mouse.     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

Summary We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)₂D₃ and for glucocorticoids, and have examined the modulation of the 1,25(OH)₂D₃ receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)₂D₃ receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)₂D₃; this phenomenon occurred in a dosedependent fashion for triamcinolone (10⁻⁹ to 10⁻⁷ M) with a maximum increase of 1,25(OH)₂D₃ receptor concentration of ⋍twofold. During the culture period, the 1,25(OH)₂D₃ receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)₂D₃ receptor concentration was higher in the triamcinolone-treated cells.     

Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse

Summary The purpose of this study was to evaluate whether the 1,25(OH)₂D₃-induced increased bone mineralization in the mouse occurs in response to stimulation of bone resorption. In order to inhibit bone resorption, 35-day-old mice were given 16 μmol/kg/day of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) for 10 days, the first injection occurring 3 days prior to the continuous infusion of 0.06, 0.13, or 0.20 μg/kg/day of 1,25(OH)₂D₃ for 7 days. Two groups of mice were treated with AHPrBP or 1,25(OH)₂D₃ alone. The skeletal changes were assessed by histomorphometric study of caudal vertebrae after double³H-proline and double tetracycline labelings for evaluation of the matrix apposition rate (MaAR) and mineral apposition rate (MiAR), respectively. Treatment with AHPrBP alone or combined to 1,25(OH)₂D₃ decreased the number of acid phosphatase-stained osteoclasts and reduced the endosteal MaAR and MiAR and the amount of osteoid. When given alone, 1,25(OH)₂D₃ increased serum calcium above normal, enhanced the number of histochemically active osteoclasts, and stimulated the endosteal MiAR. Pretreatment with AHPrBP blocked both the increase in serum calcium and the stimulation of the MiAR induced by 1,25(OH)₂D₃ infusion though serum 1,25(OH)₂D₃ levels rose according to the dose given. The results show that 1) the serum calcium and the bone resorbing responses to 1,25(OH)₂D₃ infusion are prevented by pretreatment with AHPrBP, and 2) the stimulatory effect of 1,25(OH)₂D₃ on the mineralization rate is blocked when bone resorption is inhibited. The data indicate that 1,25(OH)₂D₃ promotes bone mineralization in the mouse mainly in response to stimulation of bone resorption.     

Modulation by epidermal growth factor of the basal 1,25(OH)2D3 receptor level and the heterologous up-regulation of the 1,25(OH)2D3 receptor in clonal osteoblast-like cells

Summary The effects of epidermal growth factor (EGF) on basal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) receptor level and on parathyroid hormone (PTH)-induced 1,25-(OH)₂D₃ (OH)₂D₃ receptor up-regulation were studied in the phenotypically osteoblastic cell line UMR 106. EGF in concentrations exceeding 0.1 ng/ml reduced the number of 1,25(OH)₂D₃ binding sites without changing the binding affinity. Maximal reduction was 30% at about 1 ng/ml. This reduction was independent of a change in cAMP content. EGF dose-dependently attenuated both PTH-induced 1,25(OH)₂D₃ receptor up-regulation and PTH-stimulated cAMP production without and effect on the ED₅₀ of the PTH effects. For both PTH responses the IC₅₀ and the maximal effective dose were similar, 0.1 ng/ml an 1 ng/ml EGF, respectively. Reduction was first seen at 0.01 ng/ml EGF. At this concentration. EGF reduced PTH-stimulated 1,25-(OH)₂D₃ receptor binding without an inhibition of the cAMP response. Time-course studies with 1 ng/ml EGF revealed that at 2 h preincubation EGF reduced the heterologous up regulation by PTH, and maximal inhibition was seen after 4 h. In contrast, PTH-stimulated cAMP production was just significantly inhibited only after 6 h, with 60% inhibition after 24 h preincubation. The effects of prostaglandin E₂ and forskolin on both 1,25(OH)₂D₃ binding and cAMP production were inhibited in a similar fashion. On the other hand, dibutyryl cAMP- and 3-isobutyl-1-methylxanthinestimulated 1,25(OH)₂D₃ binding were not affected by EGF. Taken together, our results demonstrate that EGF reduces both the basal number of 1,25(OH)₂D₃ binding sites and the heterologous up-regulation of the 1,25(OH)₂D₃ receptor. The current data suggest that EGF reduces heterologous upregulation of the 1,25(OH)₂D₃ receptor independent of as well as dependent on the cAMP messenger system. The EGF effect is not primarily located at the PTH receptor, at cAMP phosphodiesterase, or at protein kinase A level.     

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: The effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids

Summary Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)₂D₃ are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13–13nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60–70% increment) with similar sensitivity to dexamethasone (ED₅₀ ∼ 1.0 nm). On the other hand, 1,25(OH)₂D₃ decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)₂D₃ treatment. Introduction of physiological concentrations of 1,25(OH)₂D₃ (0.013–1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)₂D₃ and a complex interplay among PTH, dexamethasone, and 1,25(OH)₂D₃.     

Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption

The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by ⁴⁵Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.     

Metabolic effects of thiazide and 1,25-(OH)2 vitamin D in postmenopausal osteoporosis

It was previously shown that the stimulation of intestinal calcium absorption by exogenous 25-hydroxyvitamin D (25-OHD) in postmenopausal women with osteoporosis was attenuated when thiazide was added, probably due to the suppression of endogenous synthesis of 1,25-(OH)₂ vitamin D (1,25-(OH)₂D). To test whether the above attenuation could be averted if exogenous 1,25-(OH)₂D replaced 25-OHD, 10 women with postmenopausal osteoporosis participated in a three-phase study comprising control (pretreatment), treatment with 1,25-(OH)₂D 0.5 µg/day for 4 weeks, and combined 1,25-(OH)₂D and trichlormethiazide (TZ) 2 mg/day for 4 weeks. The 1,25-(OH)₂D treatment significantly increased serum 1,25-(OH)₂D from 60±7.2 (SD) to 154±48 pmol/1, fractional intestinal calcium absorption () from 0.386 ±0.055 to 0.613±0.081, and urinary calcium from 3.7±0.8 to 6.6±1.9 mmol/day. Addition of TZ significantly reduced urinary calcium from 6.6±1.9 to 4.8±1.3 mmol/day, without changing (0.613±0.081 to 0.584±0.070), serum calcium or 1,25-(OH)₂D (154±48 to 154±38 pmol/1). Thus, estimated calcium balance (absorbed minus urinary calcium, increased marginally to +5.6 mmol/day on 1,25-(OH)₂D alone (p=0.028) and significantly to +6.8 mmol/day on 1,25-(OH)₂D+TZ, from the control value of +4.0 mmol/day. Seven patients who were treated long-term with combined 1,25-(OH)₂D and TZ for 11–29 months maintained their a (0.593±0.099) and a marginally more positive estimated calcium balance (+6.4 mmol/day,p=0.025 from the control phase). Moreover, there was a stability of bone density of radial shaft, femoral neck, and lumbar spine. In conclusion, when exogenous 1,25-(OH)₂D is provided, TZ does not lower serum 1,25-(OH)₂D and in patients with postmenopausal osteoporosis. Thus, the hypocalciuric action of TZ may lead to improved calcium balance and may potentially attenuate further bone loss.     

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.     

Enhanced suppression of 1,25(OH)2D3 and intact parathyroid hormone in Graves' disease as compared to toxic nodular goiter

Summary 1,25(OH)₂D₃, 25OHD₃, and intact parathyroid hormone, as well as various parameters of calcium-phosphorus metabolism were measured in 38 patients with Graves' disease (GD) and in 24 patients with toxic nodular goiter (TNG). Plasma 1,25(OH)₂D₃ levels were lower in GD patients (82 ±29 pmol/liter) than in those with TNG (155±32 pmol/liter) (P<0.0005). The mean value of 1,25(OH)₂D₃ in 45 controls was intermediate between the two groups of patients (140±41) and the difference was statistically significant. GD patients before and after treatment had higher alkaline phosphatase (P<0.05), lower intact parathyroid hormone (PTH) (P<0.05), and lower 1,25(OH)₂D₃ levels (P<0.0005 in the hyperthyroid andP<0.01 in the euthyroid state) than TNG patients. We conclude that increased skeletal calcium resorption is due to elevated levels of T₃ causing suppression of 1,25(OH)₂D₃ production and of PTH levels in both groups of patients albeit of different degrees. Furthermore, we postulate that the profound suppression of 1,25(OH)₂D₃ in GD is secondary to an immune-mediated phenomenon.     

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.     

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.     

1,25(OH)2D3体外抑制小鼠Th17细胞分化的用研究

目的 探讨1,25(OH)2D3对Th17细胞分化的体外抑制作用.方法 通过对分离出的脾淋巴细胞进行CD4+T细胞分选,将不同浓度的1,25(OH)2D3加入到CD4+T细胞中并在诱导因子的作用下诱导其分化.通过ELISA检测培养上清中IL-17A和IL-22的浓度;流式细胞仪分析Th17细胞的比例及RT-PCR检测Th17细胞分化的转录因子的表达.结果 培养上清ELISA检测显示:与对照组相比,1,25(OH)2D3组的IL-17A和IL-22浓度随着加入1,25(OH)2D3浓度的增大而呈现逐渐降低的现象,差异且具有统计学意义(P＜0.01);流式结果显示:诱导分化后对照组Th17细胞比例最多,1,25(OH)2D3组处理后Th17细胞比例降低,且呈剂量依赖性;RT-PCR检测显示:与对照组相比,1,25(OH)2D3组IL-17A、IL-22,RORγt和RORα 表达量随着剂量的增加而逐渐降低,并具有统计学意义(P＜ 0.01).结论 体外实验表明,1,25(OH)2D3具有抑制Th17细胞分化的作用,表现为Th17细胞比例、分泌的细胞因子及特异的转录因子表达均减少.     

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⁺.     

Simulated acidosis does not impair 1,25-dihydroxyvitamin D3 production by cultured kidney cells

Summary Cultured mouse kidney cells grown in serum-free medium were used to assess the metabolism of 25-hydroxyvitamin D₃ in the presence of simulated metabolic acidosis. Kidney epithelial cells isolated from 4–6 week old mice were grown to confluence in a defined serum-free medium at pH 7.4. The confluent monolayers were incubated with tritiated 25-hydroxyvitamin D₃ for 6 hours, the samples were extracted, and vitamin D metabolites were separated and quantitated by high pressure liquid chromatography (HPLC). The pH of the incubation medium was set at 6.9, 7.1, 7.4, or 7.7 by adjusting the bicarbonate concentration, using chloride as the balancing anion at constant Pco₂. When pH was altered at the beginning of the 6 hour assay, production of 1,25-dihydroxyvitamin D₃ was the same at each pH. More prolonged pH perturbation for a total of 30 hours likewise had no influence on 1,25-dihydroxyvitamin D₃ production. These results confirm that intact mammalian kidney cells in serum-free culture possess an active 25-hydroxyvitamin D₃-1-hydroxylase and that the activity of the enzyme is unaffected by pH over the range 6.8–7.7. In experiments where acidosis has been shown to alter 1,25-dihydroxyvitamin D₃ production, the mechanism was probably indirect.     

Influence of 1,25-dihydroxyvitamin D3 on cell proliferation during odontogenesis of the mouse embryonic molarsIn vitro

Summary Mandibular first molars from 17-day-old mouse embryos were cultured for 2 and 4 days in alpha-MEM supplemented with 10% newborn bovine serum containing 0.02% ethanol or 0.1, 1.0 or 10 ng/ml 1,25-(OH)₂D₃. After embedding, every 6th section was stained and mitotic features of inner dental epithelium (IDE) and dental papilla cells (DP) were counted under a light microscope. On the 2nd day, no significant differences were observed in the IDE and DP mitotic indexes among the control, ethanol, and the three vitamin D groups. On the 4th day, the indexes for all groups decreased. But the IDE indexes for all vitamin D groups were significantly different from those for the control (P<0.01) and ethanol (P<0.05) groups, whereas the DP index was significant (P<0.05) only in the 10 ng/ml vitamin D group. The present results suggest that vitamin D₃ may have an influence on cell proliferation of IDE and DP in mouse embryonic molarsin vitro.