The influence of dihydroxylated vitamin D metabolites on bone formation in the chick

Summary The ability of 1,25(OH)₂D₃ and of 24,25(OH)₂D₃ to prevent or to heal rickets in chicks was evaluated by studies of plasma biochemistry, growth plate histology, bone morphometry and microradiography, and bone mineralization. 1,25(OH)₂D₃ at a dose of 100 ng/day produced fewest abnormalities compared with vitamin D₃-treated control chicks. Bone growth was slightly greater than vitamin D₃-treated controls in chicks given a lower dose of this metabolite; the reverse was observed in chicks given a higher dose. 24,25(OH)₂D₃ was less effective than 1,25(OH)₂D₃ in preventing rickets even at doses as high as 400 ng/day. Treatment of rachitic chicks with doses of 24,25(OH)₂D₃ up to 300 ng/day produced no healing effect on the bone lesions, in marked contrast to the beneficial effects observed with 1,25(OH)₂D₃.     

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.     

Vitamin D Metabolites Regulate Matrix Vesicle Metalloproteinase Content in a Cell Maturation-Dependent Manner

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcificationin vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-\. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1,25(OH)₂D₃ and 24,25 (OH)₂D₃. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10¹⁰-10^-7 M24,25(OH)₂D₃ or growth zone chondrocytes incubated with 10^-11-l0^-8 M 1,25(OH)₂D₃, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)₂D₃ increased alkaline phosphatase by 35–60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)₂D₃ increased alkaline phosphatase by 35–60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)₂D₃ regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A₂ specific activities as well as metalloprotemases which degrade proteoglycans.     

Hypercalcemia associated with increased circulating 1,25 dihydroxyvitamin D in a patient with pulmonary tuberculosis

Summary Studies are described in a 53-year-old man with far-advanced pulmonary tuberculosis who developed transient increases in circulating 1,25 dihydroxyvitamin D (1,25(OH)₂D) and hypercalcemia while on antituberculous treatment. Serial dilution of an extract of the patient's serum obtained while he was hypercalcemic displaced [³H]-1,25(OH)₂D₃ from chick intestinal receptor in a manner identical to authentic 1,25(OH)₂D₃. Serum 25-hydroxyvitamin D (25OHD) was suppressed during the abnormal elevation of serum 1,25(OH)₂D. It is concluded that tuberculosis is another chronic granulomatous disease in which hypercalcemia may result from abnormal metabolism of vitamin D.     

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.     

Lack of influence of 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion from normal or hyperplastic glands

Summary The role of 24,25(OH)₂D₃ on parathyroid gland function remains controversial. The present studies were performedin vitro using (a) dispersed normal bovine parathyroid cells (bPTC) and (b) dispersed canine PTC (cPTC) prepared from glands of normal dogs, dogs with chronic renal failure (CRF), and dogs with CRF treated with 24,25(OH)₂D₃, 2.5 μg orally every day for more than 6 months. Bovine parathyroid cells were incubated for up to 180 min at 0.5, 1.0, and 3.0 mM external calcium in the presence or absence of 24,25(OH)₂D₃ (100 or 1000 nM). Similar experiments were conducted with cells incubated for 24 h in the presence of either the ethanol vehicle or 24,25(OH)₂D₃ (1000 nM). Parathyroid hormone secretion, measured in the supernatant by both C-terminal and N-terminal assays, did not show any differences between control and experimental groups at any time interval. Canine parathyroid cells obtained from uremic animals showed an average threefold increase in the total amount of PTH secreted, on a per cell basis over 180 min at 0.5 mM Ca²⁺, when compared with normal controls. However, there was no significant difference in PTH secretion at any level of calcium concentration between the cells obtained from parathyroid glands of CRF dogs and 24,25(OH)₂D₃-treated CRF dogs. Acute exposure to 24,25(OH)₂D₃ (1000 nM)in vitro of the cells obtained from the glands of CRF dogs also had no effect on PTH secretion. We conclude that 24,25(OH)₂D₃ has no direct effect on PTH secretion from dispersed parathyroid cells of either normal or uremic animals.     

The effect of endogenous estrogen fluctuation on metabolism of 25-hydroxyvitamin D

Summary To test the hypothesis that estrogen modulates the metabolism of 25-hydroxyvitamin D (25(OH)D) to 1,25-dihydroxyvitamin D (1,25(OH)₂D) and 24, 25-dihydroxyvitamin D (24, 25(OH)₂D), we studied 20 normal premenopausal women at four consecutive weekly intervals during one menstrual cycle. Estrogen stimulation was semiquantitatively defined into baseline, lowgrade, or medium-grade categories, based on endogenous estrone and estradiol concentrations. 1,25(OH)₂D increased incrementally from baseline levels of 34±3(SE) pg/ml to 39±3 pg/ml (P=0.2) with low-grade estrogen stimulation and to 43±3 pg/ml (P<0.05) with medium-grade estrogen stimulation, while 25(OH)D, 24,25(OH)₂D, vitamin D binding protein, parathyroid hormone, calcium, and phosphate did not change. 24,25(OH)₂D was correlated to 25(OH)D at baseline (r=0.65,P<0.01) and with low-grade estrogen stimulation (r=0.062,P<0.01), but not with medium-grade stimulation (r=0.13); these relationships are consistent with the concepts that 25(OH)D is metabolized predominantly to 24,25(OH)₂D at low estrogen levels, but not at higher estrogen levels. We conclude that endogenous estrogen elevation promotes formation of 1,25(OH)₂D from 25(OH)D, and that it may reciprocally inhibit synthesis of 24,25(OH)₂D.     

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.     

Efficacy of antisense monocyte chemoattractant protein-1 (MCP-1) in a rat model of mesangial proliferative glomerulonephritis

Abstract

Objective: The effects of inhibition of monocyte chemoattractant protein-1 (MCP-1) on a rat model of mesangial proliferative glomerulonephritis (MsPGN) were evaluated. Methods: The anti-Thy-1 MsPGN model was developed by intravenously injecting anti-Thy-1 monoclonal antibodies into rats, followed by an injection of mesangial cells transfected with antisense MCP-1 into the renal artery. Exogenous cells were detected by in situ hybridization. Rats (40 total) were randomly divided into five groups: SO (sham operation), TG (Thy-1 glomerulonephritis model), MC (non-transfected normal rat mesangial cell), BC (pLXSN empty vector or blank control), and AM (antisense MCP-1 transfection) groups. Effects of exogenous MCP-1 on urinary protein excretion rate, biochemical parameters, and pathological changes were evaluated. Expression of MCP-1 and transforming growth factor-β₁ (TGF-β₁) were detected by immunohistochemistry. mRNA expression of MCP-1, TGF-β₁, and CC chemokine receptor 2 (CCR2) were detected by RT-PCR. Results: Exogenous MCP-1 cDNA was successfully transfected into mesangial cells. Exogenous mesangial cells were detected in glomeruli by in situ hybridization. Glomerular mesangial cell proliferation, 24-h urinary protein excretion rate, mRNA expression of MCP-1, TGF-β₁, and CCR2, and protein expression of MCP-1 all decreased in the AM group as compared to the control group (p?<?0.05), but there was no significant difference in the expression level of TGF-β₁ protein. Conclusions: (1) Mesangial cells can be used as a vector to transfect exogenous genes into kidneys; (2) antisense MCP-1 decreases mesangial cell proliferation and pathological injury in MsPGN model rats by decreasing expression of MCP-1 and CCR2; and (3) antisense MCP-1 suppressed mesangial cell proliferation and matrix accumulation in anti-Thy-1 MsPGN model rats, which did not entirely depend on TGF-β_1.     

Serum calcium regulating hormones in the perinatal period

Summary To clarify perinatal vitamin D metabolism, we measured 25-hydroxyvitamin D (25OHD), 24,25-dihydroxyvitamin D [24,25(OH)₂D], 1,25-dihydroxyvitamin D [1,25(OH)₂D], calcium (Ca), phosphorus (P), parathyroid hormone (PTH), and human calcitonin (CT) in paired maternal, cord, and infant serum. Cord serum 25OHD was significantly lower than the maternal level, and cord serum 24,25(OH)₂D was also significantly below the maternal concentrations. Maternal, cord, and infant serum 1,25(OH)₂D, on the other hand, was significantly higher than the normal adult level. The serum PTH was low, but the CT concentration was high in the cord. Cord serum Ca and P levels were significantly higher than maternal. The reason for the elevated circulating 1,25(OH)₂D level in the perinatal period is uncertain, and we speculate that the possible factors are gonadal steroids, placental lactogen, prolactin, and CT. In addition, serum 24,25(OH)₂D and 1,25(OH)₂D concentrations are under some control by the fetus.     

Interaction between 24R,25-dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol on45Ca release from bone in vitro

Summary Interaction among vitamin D₃ metabolites on bone receptor sites is not known. Therefore, interaction between the most potent vitamin D₃ metabolite, 1,25(OH)₂D₃, and the most abundant dihydroxymetabolite, 24R,25(OH)₂D₃, was studied on isolated rat fetal bone by measuring⁴⁵Ca release from prelabeled bones. 24R,25(OH)₂D₃ at concentrations of 10–50 ng/ml caused marked inhibition of the bone-resorbing activity of 1,25 (OH)₂D₃ at concentrations of 10–50 pg/ml. 24S,25-(OH)₂ (unnatural enantiometer), on the other hand, at a concentration of 100 ng/ml did not inhibit the bone-resorbing effect of 10 pg/ml 1,25(OH)₂D₃. 24R,25(OH)₂D₃ at a concentration of 20 ng/ml did not inhibit the⁴⁵Ca-releasing effect of a submaximal concentration of PTH (500 ng/ml). Therefore, the inhibitory effect of 24R,25(OH)₂D₃ on the bone response to 1,25(OH)₂D₃ appeared to be specific and probably due to a competitive inhibitory effect. In addition, the inhibitory effect of 24R,25(OH)₂D₃ was weak, since it could be partially overcome by increasing the concentration of 1,25 (OH)₂D₃.     

The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells

Summary Controversy exists over a direct effect of 1,25(OH)₂D₃ on PTH secretion. To investigate the possibility that the suppressive effect of 1,25(OH)₂D₃ on PTH secretion may be demonstrable in 1,25(OH)₂D₃-depleted tissue and/or after prolonged periods of exposure to 1,25(OH)₂D₃, primary monolayer cultures of bovine parathyroid cells were established in 1∶1 DMEM/Ham's F-12 media supplemented with 2% calf serum but not 1,25(OH)₂D₃. Ionized calcium was maintained at 1.0 mM. Experiments were performed on 4-day-old culture cells. PTH concentration was measured using both a mid-region/carboxyl and an amino-terminal PTH antisera. 1,25(OH)₂D₃ at a concentration of 0.1 ng/ml suppressed PTH secretion by 32±7% after 48 hours. High calcium concentration (2.0 mM) suppressed PTH secretion by 37±10% and this effect was not additive over that of 1,25(OH)₂D₃. PTH secretion rate recovered fully 48 hours after normalization of the external calcium concentration but not after the removal of 1,25(OH)₂D₃. It is concluded that 1,25(OH)₂D₃ directly suppresses PTH secretion by monolayer culture of bovine parathyroid cells.     

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.     

The immunosuppressive effects of a leukotriene B4 receptor antagonist on liver allotransplantation in rats

The immunosuppressive effects of a leukotriene B₄ (LTB₄) receptor antagonist, ONO4057, on liver allotransplantation in rats were evaluated, and the levels of prostaglandin E₂ (PGE₂) in the liver tissue during rejection of the allografts examined. The rats were divided into four groups: group 1: Lewis rats (LEW) given a sham operation with dimethyl sulfoxide (DMSO); group 2: LEW given syngenic orthotopic liver transplantation (OLT) from LEW, with DMSO; group 3: LEW given allogenic OLT from ACI rats (ACI), with DMSO; and group 4: LEW given allogenic OLT from ACI, with ONO4057 as 10, 30, or 100 mg/kg per day dissolved in DMSO to subgroups 4a, 4b, and 4c, respectively. Histological examinations were performed, survival times monitored, and liver tissue PGE₂ levels 3, 5, 7, and 14 days after transplantation measured. The mean graft survival times in groups 4a, b, and c, at 37.5±10.4, 52.2±24.4, and 34.0±4.9 days (mean±SEM), respectively, were significantly longer than that in group 3 (at 13.0±3.2 days). Moreover, the levels of tissue PGE₂ in the liver allografts in group 4a were significantly higher than those in group 3 on days 5 and 7. These results suggest that ONO4057 has an immunosuppressive effect on liver allotransplantation since it reduces the activities of LTB₄ which augments immune responses, and also because it indirectly increases the PGE₂ level.     

Treatment of postherpetic neuralgia with prostaglandin E1 ointment a preliminary study

Journal of Anesthesia -     

Comparison of the effects of 1,25-dihydroxycholecalciferol,prostaglandin E2, and osteoclast-activating factor with parathyroid hormone on the ultrastructure of osteoclasts in cultured long bones of fetal rats

Summary The effects of 1,25-dihydroxy vitamin D₃ [1,25(OH)₂D₃], prostaglandin (PGE₂), and osteoclast-activating factor (OAF) on the size of osteoclasts, nuclei, ruffled borders, and clear zones in cultured long bones of fetal rats were quantitated. In addition, the number of osteoclasts in the bones was counted and the release of calcium from the bone into the culture medium was determined. These data were compared with the corresponding effects of parathyroid hormone (PTH). All agents tested increased the size of the ruffled borders significantly after 3 h, the size of the clear zones after 12 h, and the size of the cells after 12–24 h. No important differences in sizes were noticed between the agents tested or between the agents and PTH. The number of osteoclasts was increased after 24 h of treatment with PTH, but not after the other agents. Calcium release was significantly increased for all agents between 12 and 24 h. It is concluded that bone resorption by 1,25(OH)₂D₃, OAF, and PGE₂ is mediated primarily by increased activity of existing osteoclasts similar to PTH activation.     

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.     

Treatment of renal osteodystrophy in children with 1αOHD3 and 24, 25(OH)2D3

Three pediatric patients with renal osteodystrophy were treated with 1αOHD₃ and 24, 25(OH)₂D₃. While serum calcium level significantly decreased, no significant effects were found on serum phosphorus, alkaline phosphatase, parathyroid hormone and urinary excretion of calcium. These results suggest that 24, 25(OH)₂D₃ may play some roles in bone and mineral metabolism.     

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.     

Correlation between 1.25 dihydroxyvitamin D serum levels and fractional rate of intestinal calcium absorption in hypercalciuric nephrolithiasis. role of phosphate

Summary Different mechanisms could explain the elevated calcium elimination, the main cause of calcium oxalate renal stones. Our results suggest that phosphate levels are decreased in patients with absorptive hypercalciuric nephrolithiasis and elevated serum dihydroxyvitamin D. This could be the reason why in this group of patients oral phosphate treatment prevented hypercalciuria and renal lithiasis.This work was supported by FlSss 89/0799