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.     

Alkaline phosphatase inhibition by levamisole prevents 1,25-dihydroxyvitamin D3-stimulated bone mineralization in the mouse

Summary To determine the relationship between alkaline phosphatase (AP), 1,25(OD)₂D₃ and bone formationin vivo, we have examined the effects of levamisole, a stereospecific inhibitor of AP on bone formation and on 1,25(OH)₂D₃-stimulated bone mineralization in the mouse. Normal mice were injected daily with levamisole at doses of 40 and 80 mg/kg/b.w. The compound was given alone or in combination with 1,25(OH)₂D₃ infusion (0.05 μg/kg/d) for 7 days. Treatment with levamisole alone inhibited the serum AP activity (mainly of skeletal origin in mice) by 18.4 and 61.3% for the low and high dose respectively. No deleterious effect on body growth, tibia length, and bone cells population was detected. The moderate inhibition of AP activity produced by the lower dose of levamisole alone (18.4%) or in combination with 1,25(OH)₂D₃ (37.9%) was associated with a reduced endosteal matrix apposition rate (MaAR) determined by double³H-proline labeling method. This effect was related to a levamisole-induced fall in serum phosphate. Despite the moderate inhibition of AP activity, the mineral apposition rate (MiAR) determined by the double tetracycline labeling method remained normal. Moreover, 1,25(OH)₂D₃ infusion still resulted in increased MiAR which was stimulated to the same extent as in the absence of levamisole. By contrast, the more severe inhibition of AP activity induced by 80 mg/kg of levamisole alone (61.3%) or in combination with 1,25(OH)₂D₃ (45.8%) inhibited both the MaAR and the MiAR and prevented the stimulatory effect of 1,25(OH)₂D₃ on bone mineralization. The data show that AP activity affects the bone matrix and mineral apposition ratesin vivo and that severe inhibition of AP activity inhibits the 1,25(OH)₂D₃-induced stimulation of bone mineralization in the mouse.     

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.     

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.     

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.     

Idiopathic juvenile osteoporosis: Evidence of normal osteoblast function by 1,25-dihydroxyvitamin D3 stimulation test

Summary Idiopathic juvenile osteoporosis (IJO) is a rare form of bone demineralization that occurs during childhood. The mechanism of bone loss is unclear. Some bone hystomorphometric studies have found osteoblast failure and decreased bone formation in the affected patients whereas others have reported increased bone resorption. To elucidate this issue, we studied osteoblast function in six patients with IJO (five males, one female; aged 2.3–14.6 years) and five healthy sex- and age-matched subjects (four males, one female; aged 2.0–15.1 years) measuring serum values of osteocalcin under basal condition and during an osteoblast stimulation test performed by oral 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] administration (1.8 g/1.73 m²/daily). After a baseline day (day 0), all the subjects (patients and controls) received 1,25(OH)₂D₃ in four divided doses for 6 days (days 1–6). Fasting blood samples were obtained every morning (0800 h) for the determination of serum osteocalcin. Baseline osteocalcin levels were not significantly different between IJO and controls (13.58±6.05 ng/ml versus 16.04±5.09 ng/ml, respectively) even if two patients had low osteocalcin values. During 1,25(OH)₂D₃ administration, serum osteocalcin values significantly increased (P<0.001) from baseline in both children with IJO and controls, reaching peak values not significantly different in the two groups. Our results do not support the hypothesis that defective osteoblast function is the primary factor of bone demineralization in IJO.     

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.     

1,25-Dihydroxyvitamin D3 promotes prostaglandin E1-induced differentiation of HL-60 cells

Human promyelocytic HL-60 cells can be induced by biochemical agents to differentiate in vitro towards divergent types of myelomonocytic cells. It has been reported that prostaglandin E₁ (PGE₁) can induce granulocytic differentiation and that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) can induce monocytic differentiation. We have now examined the effects of these compounds, both alone and in combination, on HL-60 cell differentiation. PGE₁ (1 g/ml) or 1,25(OH)₂D₃ (10 nM) each inhibited cell proliferation over 48–96 hours of treatment, but combined treatment with both agents was necessary to produce a strong inhibition. The percentage of HL-60 cells that can reduce nitroblue tetrazolium (NBT) (a characteristic index of early monocytic or granulocytic differentiation) increased 13-fold within 72 hours of PGE₁ treatment, and 1,25(OH)₂D₃ produced a fivefold stimulation. However, combined treatment (PGE₁ plus 1,25(OH)₂D₃) produced a dramatic 35-fold increase. HL-60 cells did not produce significant levels of nitric oxide (NO) before 48 hours in culture, and treatment with PGE₁ or 1,25(OH)₂D₃ did not significantly increase cellular NO elaboration over control levels. However, combined treatment produced a striking 12-fold increase over control levels. Similarly, combined treatment was necessary to obtain the maximal time-dependent stimulation of cellular lactate dehydrogenase (LDH) activity (a marker of granulocytic differentiation) as well as acid phosphatase (ACP) activity. During this same period of time, PGE₁, but not 1,25(OH)₂D₃, markedly stimulated cellular claboration of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-, and 1,25(OH)₂D₃ cotreatment strongly augmented these effects. Thus, combined treatment with 1,25(OH)₂D₃ plus PGE₁ generally augmented the apparent conversion of these cells, producing synergistic (multiplicative) or additive effects. Furthermore, PGE₁ induced within 48 hours the more general phenotypic changes classically associated with the differentiation of these cells: increased expression of chloroacetate esterase (ChAE) (a granulocytic marker), decreases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in morphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)₂D₃ had little effect itself on most of these parameters, but augmented the morphological changes induced by PGE₁ treatment. Within 48 hours, the ability of these cells to reduce the tetrazolium salt WST-1, a general measure of cellular metabolic activity, was increased by PGE₁, but not by 1,25(OH)₂D₃; however, the combination of 1,25(OH)₂D₃ and PGE₁ again produced the strongest stimulation. Similarly, only PGE₁ significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. In summary, our findings suggest that PGE₁, not 1,25(OH)₂D₃, is sufficient to promote rapid in vitro differentiation of HL-60 cells along the granulocytic pathway; however, the PGE₁-induced conversion of these cells is markedly augmented by cotreatment with 1,25(OH)₂D₃. In addition, these converted HL-60 cells preferentially utilize the glycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mature granulocytes.     

1,25 Dihydroxyvitamin D3 affects calmodulin distribution among subcellular fractions of skeletal muscle

Summary 1,25 Dihydroxyvitamin D₃ has been shown to stimulate calcium fluxes across skeletal muscle membranes. The involvement of calmodulin in the effects of the metabolite was investigated. Primary cultures of chick embryo skeletal muscle myoblasts and soleus muscles from vitamin D-deficient or 1,25 (OH)₂D₃-treated chicks were used. Culture of myoblasts and vitamin D-deficient soleus with 1,25 (OH)₂D₃ (0.05 ng/ml) for 24 and 1 hour, respectively, significantly increased⁴⁵Ca uptake by the preparations. In the presence of the calmodulin antagonists flufenazine or compound 48/80 in the uptake medium, no differences between control and treated cultures were observed. The calmodulin content of myoblasts and soleus homogenates and subcellular fractions derived therefrom was estimated by measuring their capacity to stimulate calmodulin-depleted cAMP phosphodiesterase. No changes in total calmodulin cellular content could be detected in response to 1,25(OH)₂D₃. However, the sterol produced an increase in calmodulin levels of microsomes, mitochondria, and crude myofibrillar fraction and a proportional decrease in cytosolic calmodulin concentration. The 1,25(OH)₂D₃-dependent changes in calmodulin distribution among subcellular fractions of soleus muscle were observed eitherin vivo orin vitro. The effectsin vitro were already detectable after 5 minutes of treatment with the sterol and parallel 1,25(OH)₂D₃-dependent changes in tissue Ca uptake. The results suggest that changes in calmodulin intracellular distribution may underly part of the mechanism by which 1,25(OH)₂D₃ affects muscle calcium transport.     

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.     

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.     

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.     

1,25-dihydroxyvitamin D3 causes an increase in the number of osteoclastlike cells in cat bone marrow cultures

Summary The direct effect of 1,25(OH)₂D₃ upon osteoclast formation from precursor cells is still unknown. In the present experiments we have tested the effects of 1,25(OH)₂D₃ on the generation of osteoclastlike cells in cat bone marrow cultures. These cultures contain proliferating nonattached mononuclear cells and precursor cells that subsequently attach to the culture flask surface and then fuse to form multinucleated osteoclastlike cells. After 7 days of culture we separated the nonattached precursor cells from the attached cells and studied the effects of 1,25(OH)₂D₃ (10⁻¹⁰ M–10⁻⁸ M) on multinucleated cell formation in these two cell populations. In cultures derived from the non-attached precursor cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) resulted in a 180% increase in the number of attached mononuclear cells and a 90% increase in the number of nuclei contained within multinucleated cells. These effects were dose-dependent. 1,25(OH)₂D₃ did not have a consistent effect on the number of nonattached precursor cells. In cultures derived from attached cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) induced a 50% increase in the number of mononuclear attached cells and a 40% increase in the number of nuclei within polykaryons. The most likely explanation for these results is that 1,25(OH)₂D₃ promotes the differentiation and subsequent adhesion of nonattached precursor cells, stimulates proliferation of attached mononuclear precursor cells, and possibly stimulates fusion of these attached precursor cells.     

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.     

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.     

Enhanced sensitivity of young suckling rats to the toxic effects of 1,25-dihydroxyvitamin D3

Summary The responses of suckling rat pups of different ages to high doses of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) were determined. Four daily oral doses of 1,25(OH)₂D₃ (2 ng/g body wt) given to 9–13-day-old pups produced severe hypercalcemia 24 h after the last dose (15.52 ± 0.14 mg/dl vs. 10.94 ± 0.15 mg/dl in controls of the same age) and a 9-fold increase in kidney Ca content; the same doses given to 16–20-day-old pups produced only modest hypercalcemia (12.34 ± 0.22 mg/dl vs. 10.57 ± 0.22 mg/dl in controls of the same age) and a 4-fold increase in kidney Ca content. There was no change in serum phosphorus (P) at either age. Six-week-old weaned rats, given the same doses of 1,25(OH)₂D₃, showed neither hypercalcemia nor kidney calcification and thus were protected against the toxic effects of the treatment. The difference in responses of the twoages of suckling pups was also observed with lower doses. Removal of the solid food from the diet of the 16–20-day pups showed that the consumption of solid food, in addition to milk, in this age group was not the cause of the lower serum Ca response. The changes in both serum and kidney Ca after intraperitoneal (i.p.) injections of 1,25(OH)₂D₃ at the same dose in each age group were similar to those observed with oral administration. The time course of the rise in serum Ca following a single dose of 1,25(OH)₂D₃, given either orally or i.p., showed that the hypercalcemia was more pronounced and lasted longer in the 9–13-day pups than in the 16–20-day pups. The results suggest that weaned rats are relatively well protected against hypervitaminosis D and that younger pups gradually develop such protection during the suckling period.     

Induction of macrophagic and granulocytic differentiation of murine bone marrow progenitor cells by 1,25-dihydroxyvitamin D3

Summary 1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) was recently shown to promote maturation of 5-fluorouracil (5FU)-treated bone marrow cells by up-regulating macrophage-colony stimulating factor (M-CSF) receptors in the presence of interleukin la (IL-1). In order to reveal how 1,25(OH)₂D₃ interacts with colony-stimulating factors and regulates the differentiation of bone marrow progenitor cell populations, in the present study, natural bone marrow cells were isolated from untreated mice and used in a-minimum essential medium supplemented with 20% heat-inactivated horse serum without added appropriate cytokines. Under the conditions, cells spontaneously differentiated gradually with days of culture, as assessed by expression of macrophage differentiation antigens such as Mac-1 (CD11b) and F4/80. Both M-CSF and granulocyte macrophage-colony stimulating factor (GM-CSF) induced only Mac-1 antigen expression. Simultaneous treatment with M-CSF and 1,25(OH)₂D₃ enhanced the M-CSF's effect on expression of both antigens, although (1,25(OH)₂D₃) per se has no effect on the expression for up to 11 days. In addition, successive treatment with 1,25(OH)₂D₃ and M-CSF or GM-CSF dramatically enhanced expression of both antigens or Mac-1 antigen, respectively. Similarly, both simultaneous and successive treatment with 1,25(OH)₂D₃ and M-CSF significantly enhanced phagocytic activity and H₂O₂ production, whereas successive treatment with (1,25(OH)₂D₃) and GM-CSF significantly enhanced only phagocytic activity. Enzymehistochemical study demonstrated that cells treated simultaneously or successively with 1,25(OH)₂D₃ and M-CSF were strongly positive for nonspecific esterase (NSE), a macrophage-specific marker, and that simultaneous or successive treatment with 1,25(OH)₂D₃ and GM-CSF yielded cells strongly positive for NSE or for chloroacetate esterase (ChAE), a granulocyte-specific marker, respectively. These findings suggest that 1,25(OH)₂D₃ primes bone marrow progenitor cell populations not only to M-CSF but also to GM-CSF and thereby accelerates the CSFs-dependent differentiation of the cells to the macrophage or granulocyte.     

Monolayer cultures of normal human bone cells contain multiple subpopulations of alkaline phosphatase positive cells

Summary Cytochemical staining of normal human bone cells in monolayer cultures for alkaline phosphatase (ALP) indicated that the cultures contained mixed-cell populations. Time course evaluations of the cytochemical staining revealed, in addition to the ALP-negative cell population, at least two subpopulations of ALP-positive human bone cells with different levels of ALP. A cytochemical method has been developed which separates the ALP-positive cells into high and intermediate ALP subpopulations. In this method, human bone cells were stained for ALP using an azo-dye method and incubating at 4°C for 10 and 30 minutes, respectively. We defined the cell population that stained positively for ALP at 10 minutes as strong ALP-positive cells, and both strong and intermediate cells were stained at 30 minutes. The intermediate cells were determined from the difference between the values at the two time points. The intra- and interassay variations of the assay, with the same investigator in blinded investigations, were both less than 10% and the interobserver variation was approximately 25%. Analysis of the distribution of ALP levels in cells with a laser densitometer confirmed the presence of at least three cell subpopulations. 1,25(OH)₂D₃ treatment increased the proportions of both ALP-positive cell populations, whereas TGF-beta treatment increased only the intermediate ALP-positive cell population. On the contrary, fluoride increased the proportion of the strong ALP cells, and IGF-1 had no effect on the proportions of either ALP-positive subpopulation. When the ALP-specific activity was compared with the percentage of each ALP-positive subpopulations for the cells treated with effectors, the ALP-specific activity correlated with the total ALP-positive and with the strong ALP-positive populations but not with the intermediate ALP-positive subpopulation. In summary, this study represents the first evidence that normal human bone cells in monolayer cultures contained at least two subpopulations of ALP-positive cells, and that bone cell effectors could have differential effects on each cell population.