17-β雌二醇对去卵巢大鼠小肠粘膜钙结合蛋白基因表达的影响

用硫氰酸胍一步法提取小肠、肾脏和骨组织总ＲＮＡ，用ＰｓｔＩ和ＳｍａＩ双酶切携带钙结合蛋白（ＣａＢＰ）９Ｋ基因的ｐＵＣ１２质粒，获取ＣａＢＰ９ＫｍＲＮＡ在各组雌性大鼠中的变化。结果：卵巢切除组，小肠粘膜ＣａＢＰ９ＫｍＲＮＡ明显低于对照组；而卵巢切除后注射１７－β雌二醇治疗组（０．５μｇ／１００ｇ体重）未出现降低。１７－β雌二醇可影响小肠粘膜ＣａＢＰ９Ｋ的基因表达。本结果提示，雌激素可能通过钙结合蛋白参与钙在大鼠体内的代谢。     

Effect of Vitamin D Metabolites and Analogs on Renal and Intestinal Calbindin-D in the Rat

The effects on renal and intestinal calbindin-D of vitamin D₃ metabolites and synthetic 20-epi-vitamin D₃ analogs with different calcemic actions were examined in Wistar rats. The compounds were administered intraperitoneally once daily for 5 days. The dosages of the metabolites were 1,25-(OH)₂D₃ 0.01, 0.05, 0.1, and 0.4 μg/kg × d, 24,25-(OH)₂D₃ 0.1, 1 and 10 μg/kg × d, and 25-(OH)D₃ 10 and 400 μg/kg × d. The dosage of the synthetic analogs were MC903 0.1, 10, and 100 μg/kg × d, EB1213 0.1 and 10 μg/kg × d, KH1060 0.1 and 0.4 μg/kg × d, and GS1725 0.01 and 0.1 μg/kg × d. Two control groups had either vehicle alone or no treatment. N= 8 in each group. 1,25-(OH)₂D₃ increased renal and intestinal calbindin-D levels, induced hypercalcemia, and suppressed plasma PTH and magnesium concentrations. 24,25-(OH)₂D₃ increased intestinal calbindin-D9k and plasma calcium, but had no effect on renal calbindin-D28k, plasma PTH, and magnesium. The dosage of 24,25-(OH)₂D₃ that was required to increase plasma calcium was larger than the dosage required to increase intestinal calbindin-D9k. 25-(OH)D₃ did not change the calcium metabolic parameters. MC903, a low calcemic analog with a relative high affinity for the vitamin D receptor and a short half-life, increased renal calbindin-D28k without increasing ionized calcium or intestinal calbindin-D9k. EB1213, an analog with a reduced calcemic action and short half-life, increased renal calbindin-D28k and ionized calcium without increasing intestinal calbindin-D9k. The effect of the high calcemic vitamin D analogs KH1060 and GS1725 on calbindin-D was directly related to their calcemic activity. In conclusion, these results demonstrate that 24,25-(OH)₂D₃ increases intestinal calbindin-D9k, but has no effect on renal calbindin-D28k, that low calcemic analogs may increase renal calbindin-D28k without increasing intestinal calbindin-D9k, and that the effect of high calcemic analogs on calbindin-D is directly related to their calcemic activity. Received: 26 May 1995 / Accepted: 29 February 1996     

In 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     

Avian Osteoclast Cells are Stimulated to Resorb Calcified Matrices by and Possess Receptors for Leukotriene B4

Leukotriene B₄ (LTB₄) is elevated in inflammatory conditions and appears to be a potential mediator of inflammation. We have recently shown that this 5-lipoxygenase metabolite of arachidonic acid stimulates bone resorption in vitro and in vivo. In order to determine the mechanism whereby LTB₄ causes bone resorption, avian osteoclasts were examined for the effects of LTB₄ and for the presence of LTB₄ receptors. Isolated avian osteoclast mononuclear precursor cells, which fuse in culture to form multinucleated cells, were chosen for receptor binding studies because this population is a morphologically similar source of osteoclasts, and large numbers of these cells can be obtained from egg-laying hens. Binding of LTB₄ and activation would support the hypothesis of a direct effect of this compound on osteoclasts. LTB₄ stimulated isolated avian osteoclasts to form resorption lacunae on calcified matrices and to increase their content of tartrate-resistant acid phosphatase (TRAP), a marker of activated osteoclasts. Receptor binding studies were performed at day 1, when the cells were mononuclear, at day 4, when mononuclear precursors were actively fusing, and at day 7, when fusion has slowed. Scatchard analysis of saturation binding data showed two classes of binding sites, a high- and low-affinity binding site with dissociation constants (K_D) of 0.2–0.4 nM and 5.6–24 nM. Association studies showed rapid binding of LTB₄ to the cells within 10 minutes. These data show that LTB₄ accelerates fusion and activates highly enriched populations of avian osteoclasts and that LTB₄ receptors are present in this cell population. Received: 30 November 1997 / Accepted: 12 May 1998     

Calcium Absorption and Bone Loss in Ovariectomized Rats Fed Varying Levels of Dietary Calcium

The following studies were undertaken to examine whether estrogen deficiency impairs calcium absorption in aged rats, and to determine whether impaired calcium absorption and the level of dietary calcium are related to the degree of bone loss due to estrogen deficiency. Sixty rats were sham operated (Sham) or ovariectomized (Ovx) to make them estrogen deficient and divided into three dietary groups of 10 rats per group: Group 1 (Sham) and Group 2 (Ovx) were maintained on a diet containing 0.5% calcium; Group 3 (Sham) and Group 4 (Ovx) were maintained on a diet containing 0.1% calcium; Group 5 (Sham) and Group 6 (Ovx) were maintained on a diet containing 0.02% calcium. Calcium absorption was measured in all animals at the beginning of the study and 2 weeks, 1 month, 2 months, and 3 months following surgery, then the animals were sacrificed. In Ovx rats fed 0.5% Ca diet, calcium absorption decreased progressively and the decrease became statistically significant 8 and 12 weeks following ovariectomy (P < 0.05). A similar ovariectomy-related impairment of calcium absorption was not observed in animals fed diets with lower calcium content, making the Ovx rat a tenuous model of intestinal calcium malabsorption. Low dietary calcium decreased cancellous bone mineral content and density at the proximal tibial metaphysis and the decrease was augmented by ovariectomy. The degree of osteopenia due to ovariectomy was not related to the level of dietary calcium or the efficiency of calcium absorption. Received: 7 July 1998 / Accepted: 23 December 1998     

Hormonal Regulation of the Osteoblastic Phenotype Expression in Neonatal Murine Calvarial Cells

Osteoblastic cell cultures from fetal rat calvariae have provided a popular model for studying the effects of dexamethasone (DEX) and 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] on gene expression but data from murine calvarial cells are scarce. Species-specific responses of rat and mouse osteoblastic cells to these hormones have been reported previously. In the present study, we investigated the effects of DEX and 1,25(OH)₂D₃ on expression of the osteoblastic phenotype by mouse calvarial cells. These murine osteoblast-like (MOB) cells expressed alkaline phosphatase (ALP) activity and osteocalcin and formed calcified nodules. Unlike the rat calvarial cells, ALP activities and nodule formation in MOB were inhibited by DEX. 1,25(OH)₂D₃ enhanced and DEX lowered the amount of osteocalcin synthesized by MOB. 1,25(OH)₂D₃ did not affect the number of nodules, but increased their sizes. Treating the cells for 2 days with only DEX at the beginning of the culture enhanced the effect of 1,25(OH)₂D₃ on ALP. We found that in murine calvarial cells, DEX inhibits and 1,25(OH)₂D₃ enhances ALP activity, osteocalcin synthesis, and calcified nodule formation. This is in contrast to previous reports of rat calvarial cells where DEX is a positive and 1,25(OH)₂D₃ can be a negative regulator of the osteoblastic phenotype. These results suggest that profound species-specific differences exist between mice and rats in the regulation of the osteoblastic phenotype. Received: 15 October 1997 / Accepted: 16 June 1998     

Differences in vitamin D status and calcium intake: Possible explanations for the regional variations in the prevalence of hypercalcemia in tuberculosis

The prevalence of hypercalcemia in patients with untreated tuberculosis (TB) varies widely between countries. Since the vitamin D status and calcium intake are important determinants of hypercalcemia in TB, these two factors were compared among four populations (U.K., Hong Kong, Malaysia, Thailand) with a low prevalence (<3%) and two populations (Sweden, Australia) with a high prevalence (>25%). In the three Asian countries, the circulating vitamin D levels are abundant, but the calcium intakes are low. Subjects from the U.K. have the lowest circulating vitamin D level of all, although their calcium intake is high. In Sweden and Australia, both the circulating vitamin D levels and calcium intakes are high. Since serum 1,25(OH)₂D concentration will only be raised if its substance for extrarenal conversion, 25(OH)D, is plentiful and the effect of a given serum 1,25 (OH)₂D concentration on serum calcium is determined by the calcium intake, it is postulated that the regional variation in the prevalence of hypercalcemia in TB may be due to differences in the circulating vitamin D levels and calcium intakes in these populations. Received: 18 March 1996 / Accepted: 17 June 1996     

Oral 1,25-dihydroxyvitamin D3 loading test in normal subjects and patients with chronic renal failure under haemodialysis

Summary: The present study was designed to determine the criterion for 1,25-dihydroxyvitamin D₃ (1,25 (OH)₂D₃) loading test in normal subjects and haemodialysis patients. Fourteen normal subjects were administered 1.0 μg of 1,25(OH)₂D₃ per os and serum 1,25(OH)₂D was monitored every hour up to 6 h afterwards under conditions of overnight fasting, and six haemodialysis patients were administered 2.0 μg of 1,25(OH)₂D₃ per os and serum 1,25(OH)₂D was monitored every 2 h up to 12 h afterwards. Peak time of serum 1,25 (OH)₂D varied between 2 and 5 h after administration in normal subjects. However, there was a good correlation between the maximum increment of 1,25(OH)₂D (maxΔ1,25(OH)₂D) and the increment at 4 h after administration (Δ1,25(OH)₂D(4 h)). the peak time of Δ1,25(OH)₂D in six haemodialysis patients was also at 4 h after administration. From these observations, Δ1,25(OH)₂D(4 h) was evaluated in subsequent studies. Twenty-six normal subjects and 24 haemodialysis patients were administered 0.5–2.0 μg of 1,25(OH)₂D₃ per os, according to their bodyweights, under conditions of overnight fasting. Blood samples were drawn for measuring 1,25(OH)₂D prior to and 4 h after administration. Δ1,25(OH)₂D(4 h) showed good correlation with the dose of 1, 25 (OH)2D3 adjusted by bodyweight (ng/kg bodyweight). the ratio of Δ1,25(OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ was more than 2.0 in all normal subjects (range: 1.97?2.89, mean ± SD: 2.38 ± 0.287). Moreover, the ratio of Δ1,25(OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ showed a good reproducibility (CV%= 5.7 Δ 0.32, n=5), and did not depend on the administered dose of 1,25(OH)₂D₃, suggesting that this ratio is a good parameter for the intestinal absorption of 1,25(OH)₂D₃. In haemodialysis patients, the mean ratio of Δ1,25 (OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ was 2.14 Δ 0.489, which was not significantly different from the ratio in normal subjects, suggesting that, fundamentally, there was no impairment of intestinal absorption of 1,25(OH)₂D₃ in these patients. However, low ratios of Δ(4 h) and the dose of 1,25(OH)₂D₃ with low basal levels of 1,25(OH)₂D were observed in some patients (less than 1.5 in four patients), suggesting that there exist haemodialysis patients with malabsorption of 1,25(OH)₂D₃. From these results, the criterion for normal response in 1,25(OH)₂D loading test was proposed, namely, that the ratio of Δ1,25(OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ be more than 2.0.     

Parathyroid Hormone,Prostaglandin E2, and 1,25-Dihydroxyvitamin D3 Decrease the Level of Na+-Ca2+ Exchange Protein in Osteoblastic Cells

We previously described Na⁺-Ca²⁺ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na⁺-dependent Ca²⁺ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E₂ (PGE₂), or 1,25(OH)₂D₃. To determine whether this inhibition of Na⁺-Ca²⁺ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE₂, or 1,25(OH)₂D₃. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na⁺-Ca²⁺ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10⁻⁷ M PTH, and required 16–24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10⁻⁶ M PGE₂ or 10⁻⁸ M 1,25(OH)₂D₃. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na⁺-Ca²⁺ exchanger. Thus, the previously reported inhibition of Na⁺-Ca²⁺ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells. Received: 5 February 1996 / Accepted: 18 October 1996     

1,25 Dihydroxyvitamin-D3 Attenuates the Confluence-Dependent Differences in the Osteoblast Characteristic Proteins Alkaline Phosphatase, Procollagen I Peptide, and Osteocalcin

In the present study a cell culture model of primary human osteoblasts based on degrees of confluence was investigated by measuring basal and 1,25(OH)₂D₃stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as well as the corresponding gene expression. Primary osteoblast-like cell cultures from seven donors were treated in the second passage with 1,25(OH)₂D₃ (5 × 10⁻⁸ M for 48 hours) and investigated at four stages of confluence (stage I 50%, stage II 75%, stage III 100%, and stage IV 7 days postconfluence). In untreated cultures passing through the different stages of confluence, we saw a 1.8-fold increase of AP activity, a 2.3-fold increase of OC secretion, but a decrease of PICP levels to 0.36-fold. Gene expression showed only minor variation between the different confluence stages. 1,25(OH)₂D₃ did not significantly affect PICP production. Alkaline phosphatase protein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differentiation-stage dependent changes of basal levels of osteoblast-specific proteins. However, 1,25(OH)₂D₃ stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)₂D₃ stimulation might improve the reproducibility of results obtained at different confluence stages from cultures of clinical samples. Received: 25 November 1997 / Accepted: 2 September 1998     

Up-Regulation of Inducible Nitric Oxide (NO) Synthase and NO Production in HL-60 Cells Stimulated to Differentiate by Phorbol 12-Myristate 13-Acetate Plus 1,25-Dihydroxyvitamin D3 Is Not Obtained With Dimethylsulfoxide Plus 1,25-Dihydroxyvitamin D3

In previous studies we found that the calciotropic hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] augments the action of either prostaglandin E₁ (PGE₁) or NaF to induce differentiation of human promyelocytic HL-60 cells, a process that features increased generation of nitric oxide (NO) via up-regulation of inducible nitric oxide synthase (iNOS). We have now examined the short-term interaction of 1,25(OH)₂D₃ with phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) in these cells. PMA (100 nM) alone generally up-regulated several classical indices of macrophagic differentiation and stimulated cellular production of interleukin (IL)-1α, IL-6, tumor-necrosis factor (TNF)-α, PGE₂, and NO. Increased generation of NO primarily resulted from increased expression of cellular iNOS. When 1,25(OH)₂D₃ (10 nM) was added to PMA treatments, most PMA-induced changes, particularly its effects to up-regulate iNOS-dependent NO production and change cell morphology, were multiplicatively augmented. In contrast, DMSO (1.3%) alone, an inducer of granulocytic differentiation, increased cytokine production, but failed to stimulate NO production or induce iNOS. In contrast to its striking interaction with PMA, 1,25(OH)₂D₃ could not augment DMSO's differentiative effects. Changes in cellular cytokine production were eliminated as the driving force in HL-60 differentiation when specific neutralizing antibodies failed to produce any attenuation of iNOS up-regulation or of the shifts in cell morphology. However, indomethacin (30 μM) blocked the synergistic interaction between 1,25(OH)₂D₃+ PMA to shift cell morphology and stimulate NO production. Subsequently adding PGE₂ (1 ng/ml) to indomethacin-treated cells restored the ability of 1,25(OH)₂D₃+ PMA to interactively increase cellular NO production, but failed to fully replicate the strong shift in cell morphology typical of PMA + 1,25(OH)₂D₃ treatments. Our findings suggest that interaction between 1,25(OH)₂D₃ and PMA to induce macrophagic differentiation increases iNOS-dependent NO production by a mechanism involving a cyclooxygenase product(s), possibly PGE₂. Received: 13 March 1997 / Accepted: 14 November 1997     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

Increased Serum 1,25-Dihydroxyvitamin D after Growth Hormone Administration is not Parathyroid Hormone-Mediated

To assess the effects of growth hormone (GH) on serum 1,25-dihydroxyvitamin D [1,25(OH)₂D], we performed the following prospective crossover study in six healthy, young, adult, white men. During each of two admissions for 2? days to a general clinical research center, subjects were placed on a daily dietary calcium intake of 400 mg. Serum calcium, phosphorus, 1,25(OH)₂D, immunoreactive intact parathyroid hormone (PTH), insulin-like growth factor I (IGF-I), IGF binding protein 3 (IGFBP3), tubular reabsorption of phosphate (TRP), and maximum tubular reabsorption of phosphate (TMP/GFR) were measured. Recombinant human GH (rhGH, Humatrope) (25 μg/kg/day subcutaneously for 1 week) was administered prior to and during one of the admissions. Results are expressed as mean ± SEM. Whereas serum 1,25(OH)₂D (58.9 ± 7.7 versus 51.6 ± 7.4 pg/ml, P < 0.01), serum phosphorus (4.5 ± 0.1 versus 3.7 ± 0.1 mg/dl, P < 0.01), TRP (92.0 ± 0.5 versus 87.8 ± 0.7 mg/dl, P < 0.005), TMP/GFR (4.6 ± 0.1 versus 3.5 ± 0.2, P < 0.005), and urinary calcium (602 ± 49 versus 346 ± 25 mg/day, P < 0.001) increased significantly, serum PTH decreased significantly (19.9 ± 1.9 versus 26.8 ± 4.0 pg/ml, P < 0.05) and serum calcium did not change when subjects received rhGH. These findings indicate that in humans, GH affects serum 1,25(OH)₂D independently of circulating PTH and that this effect is mediated by IGF-I. We propose, therefore, that one potential mechanism by which GH stimulates increases in bone mass is via modest increases in serum 1,25(OH)₂D. Received: 2 May 1996 / Accepted: 18 October 1996     

Regulation of Osteogenic Differentiation of Human Bone Marrow Stromal Cells: Interaction Between Transforming Growth Factor-β and 1,25(OH)2 Vitamin D3 In Vitro

Bone marrow stromal cells are believed to play a major role in bone formation as a major source of osteoprogenitor cells, however, very little is known about how the osteogenic differentiation of these cells is regulated by systemic hormones and local growth factors. We examined the effects of TGF-β and its interaction with 1,25(OH)₂ Vitamin D₃ [1,25(OH)₂D₃] on the differentiation and proliferation of human bone marrow stromal cells (hBMSC) in secondary cultures. Alkaline phosphatase (ALP) activity was inhibited by TGF-β (0.1–10 ng/ml) and increased by 1,25(OH)₂D₃ (50 nM), however, co-treatment of TGF-β and 1,25(OH)₂D₃ synergistically enhanced ALP activity with maximal stimulation occurring at about 8 days after treatment. This synergistic effect was independent of proliferation because, in contrast to TGF-β alone, combined treatment with TGF-β and 1,25(OH)₂D₃ had no effect on hBMSC proliferation. As no synergistic effect was seen with combinations of 1,25(OH)₂D₃ and other osteotrophic growth factors, including BMP-2, IGF-I, and basic fibroblast growth factor (bFGF), it would seem likely that the synergistic interaction is specific for TGF-β. The increased ALP activity was due to an enhancement of 1,25(OH)₂D₃-induced ALP activity by TGF-β, rather than vice versa. In contrast, TGF-β inhibited 1,25(OH)₂D₃-induced osteocalcin production. Taken together, these results indicate that TGF-β and 1,25(OH)₂D₃ act synergistically to stimulate the recruitment of BMSC to the osteoblast lineage. This interaction may play an important role in bone remodeling. Received: 24 March 1998 / Accepted: 1 February 1999     

Calcium and Vitamin D Therapy in Corticosteroid-Induced Bone Loss: What is the Evidence?

Corticosteroid-induced osteoporosis (CIO) is a serious disorder that results in significant long-term morbidity. Increased bone resorption is caused by decreased Ca absorption and increased urinary Ca excretion leading to secondary hyperparathyroidsim. Calcium prophylaxis alone, when patients start corticosteroids, is associated with rapid rates of spinal bone loss and offers only partial protection from corticosteroid-induced spinal bone loss. Though calcium supplementation may have some benefit, it clearly cannot completely prevent corticosteroid-induced bone loss. At most, Ca therapy should only be considered adjunctive therapy in the treatment or prevention of corticosteroid-induced bone loss and should be administered in combination with other treatments. Earlier work demonstrated increases in forearm bone mineral density (BMD) with the use of Ca and vitamin D in patients with established CIO. However, caution should be taken when interpreting these results, since bone loss generally tapers or plateaus after the first 12 months of corticosteroid treatment; as such, any therapy might show benefit. In addition, bone density was only taken at the radius and not the spine where most of the bone loss takes place. Nonetheless, in recent trials of at least 2-year duration in which calcium and vitamin D therapy served as placebos, the result indicated that bone mass was maintained at the spine and hip throughout treatment in patients who had used chronic corticosteroids. In primary prevention trials, the amount of bone loss observed in the spine after therapy with Ca and vitamin D combinations is similar to that observed in other prevention studies in the Ca alone-treated control groups. Furthermore, Ca and vitamin D therapy appears to be less effective than other agents in the prevention of corticosteroid-induce bone loss. Although several studies do not report side effects that may be associated with Ca and vitamin D therapy, the few that do frequently report hypercalciuria. In the absence of other studies that support the use of Ca and vitamin D in the prevention of CIO, the data are too limited to generally recommend them alone as a preventative therapy. Activated vitamin D may be of greater benefit.     

Effect of Vitamin D Receptor Genotypes on Calcium Absorption,Duodenal Vitamin D Receptor Concentration,and Serum 1,25 Dihydroxyvitamin D Levels in Normal Women

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established. The VDR gene codes for the vitamin D receptor protein which regulates intestinal calcium absorption. In order to assess the biochemical basis we studied the effect of common allelic variation of the VDR gene on intestinal VDR protein concentration, calcium absorption, and serum 1,25 dihydroxyvitamin D (1,25(OH)₂D). Ninety-two Caucasian women were genotyped for Bsm I and Taq I polymorphism at the VDR gene locus. From these we compared 49 young women aged 25–35 years and 43 elderly women aged 65–83 years, who had all three measurements performed. There were no significant differences in intestinal VDR protein concentration, serum 1,25(OH)₂D, or radioactive calcium absorption among VDR genotype groups. Therefore, the small intestine does not seem to be a target for VDR gene polymorphism. Received: 12 August 1996 / Accepted: 3 January 1997     

Voltage-Gated calcium channels and nonvoltage-gated calcium uptake pathways in the rat incisor odontoblast plasma membrane

Odontoblasts participate actively in the transport and accumulation of Ca²⁺ ions to the mineralization front during dentinogenesis. These cells are known to carry membrane-bound ATP-driven pumps and Na⁺/Ca²⁺ antiports for Ca²⁺ extrusion, but little is known about Ca²⁺ influx mechanisms into these cells. It has been shown that the administration of Ca²⁺ channel blockers in vivo strongly impairs Ca²⁺ uptake in the mineral phase during dentinogenesis in the rat; the present in vitro study is aimed at further elucidating odontoblast Ca²⁺ uptake mechanisms. Dissected rat incisor odontoblasts exhibited a pronounced fluorescence when incubated with a fluorescently-labeled (STBodipy) dihydropyridine, which is specific for voltage-gated Ca²⁺ channels of the L-type, and this binding was competitively abolished by nifedipine. As assayed by fluorescence spectrometry, odontoblast Ca²⁺ uptake was enhanced by the agonistic dihydropyridine BAYK-8644 (5 μM) as well as by plasma membrane depolarization in a high K⁺ (120 mM) medium. The Ca²⁺ uptake after depolarization was impaired by nifedipine (5 μM). When treated with the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), a nonvoltage-gated uptake of ⁴⁵Ca²⁺ was identified. This uptake was not influenced by nifedipine (20 μM) but was impaired by lanthanum ions (200 μM). A nonvoltage-gated uptake of Mn²⁺ into CPA-treated cells could be traced using the fura-2 quenching technique. This CPA-induced Ca²⁺ flux was not caused by an alteration of the plasma membrane potential, as assayed with di-8-ANEPPS. The results demonstrate that Ca²⁺ flux into dentinogenically active odontoblasts occurs through voltage-gated Ca²⁺ channels of the L-type and by nonvoltage-gated, agonist-sensitive Ca²⁺ uptake pathways. Received: 6 November 1995 / Accepted: 21 February 1996     

Calcidiol and PTH Levels in Women Attending an Osteoporosis Program

We performed a retrospective study of 237 patients attending a specialty osteoporosis practice. Secondary causes for reduced bone mineral density (BMD) were evaluated in 196 postmenopausal women and 41 premenopausal women; mean age was 56 ± 13.8 years (mean ± SD). BMD was measured by dual-energy X-ray absorptiometry (DXA) (QDR 1000W/2000 Hologic). Levels of intact parathyroid hormone (iPTH), calcidiol [25(OH)D], thyroid-stimulating hormone, and 24-hour urinary calcium were measured, and serum and urine protein (SPEP and UPEP) electrophoresis were performed. Overall, 16% of our patients had 25(OH)D levels <15 ng/ml, the lowest acceptable vitamin D level without a concomitant rise in iPTH levels. Among the osteoporotic patients (T score <−2.5 SD), 17% had 25(OH)D levels <15 ng/ml and 7% <10 ng/ml. Among the osteopenic patients (−2.5 < T < −1.0 SD), 11% had 25(OH)D levels <15 ng/ml. Seventeen percent of patients with Z score ≤−1.0 SD (low range normal value) had 25(OH)D levels <15 ng/ml. Low 25(OH)D levels were inversely related to high iPTH values (r = 0.30, P < 0.0001). Hypercalciuria was present in 15% of our patients, elevations of PTH levels (>65 pg/ml, upper normal limit of assay) were present in 11.5%, and hyperthyroidism in 4%. A 25(OH)D level of <25 ng/ml in women (n = 86) with no known secondary causes of low BMD was associated with an iPTH level above 49 pg/ml. The measurement of 25(OH)D levels is recommended in the evaluation of secondary causes for reduced BMD. Supplementation with vitamin D appears needed to keep 25(OH)D above 25 ng/ml, the level required to prevent increments in iPTH levels. Received: 9 February 1998 / Accepted: 1 October 1998     

The effect of estrogen deficiency on bone mineral density,renal calcium and phosphorus handling and calcitropic hormones in the rat

The oophorectomized (OOX) rat has been proposed as a good model of postmenopausal osteroporosis in women. The aim of this study was to compare the effect of OOX in 6-month-old rats to the effects of menopause in women with respect to bone mass, the renal handling of calcium and phosphorus, and calcitropic hormones. To more closely replicate the human situation the rats were pair fed a 0.1% calcium diet. Thirty four, 6-month-old rats were randomized to sham operation or OOX. Whole body and regional bone density was performed at baseline and 6 weeks postoperation. Blood and 24-hour urine samples were obtained at baseline, 1, 3, and 6 weeks and assayed for various biochemical variables, parathyroid hormone (PTH), and calcitriol. The OOX rats lost significantly more bone than the sham-operated rats (change in global bone mineral density, sham −1.7 ± 2.0%, OOX −3.9 ± 2.6%, P < 0.001). In the OOX animals, an increase in the 24-hour urine calcium was observed at 1 and 3 weeks, which had returned to sham-operated levels by 6 weeks. In the whole group, the increase in urine calcium at 1 week was negatively correlated with the change in bone mass at 6 weeks (r =−0.39, P= 0.029). OOX resulted in an increased filtered load of calcium and phosphorus. There was an increase in the maximal renal tubular reabsorption of phosphorus (TmP-GFR) but no clear change in renal calcium handling. Neither calcitriol nor parathyroid hormone showed a significant change as a result of OOX. As in postmenopausal women, following oophorectomy in the rat, there was significant generalized bone loss and a negative calcium balance. This was associated with an initial rise in urine calcium due to a rise in the filtered calcium load; plasma phosphorus and TmP-GFR also rose. The rat model may differ from postmenopausal bone loss in that the initial rise in urine calcium was not present at later time points as occurs in natural menopause in women. Calcitropic hormone levels did not change. This study has shown that the 6-month-old OOX rat fed a 0.1% calcium diet has many similarities of calcium and phosphorus homeostasis to that seen at menopause in women. Received: 14 August, 1995 / Accepted: 8 March 1996