bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of serum parathyroid hormone and ionized calcium concentrations during reproduction in rats fed a low calcium diet
Authors: Garner Sanford C. , Peng Tai-Chan , Toverud Svein U.
Published: February 2010

Moderate dietary restriction of calcium (0.1% Ca) was used to accentuate the changes in serum immunoreactive parathyroid hormone (iPTH) that had been reported earlier in lactating rats fed 0.4% Ca diet. In addition, the effects of this low-Ca diet on serum total and ionized Ca and iPTH during pregnancy, extended lactation, and weaning were examined. The positive correlation between serum total and ionized Ca was highly significant (r = 0.88, p < 0.001, n = 120). Serum iPTH was significantly higher (36%) in pregnant rats on the day of parturition compared to nonmated controls, and there was a concomitant decrease in both total and ionized serum Ca. Within 1 day after parturition, however, serum Ca had risen to the control level. Serum iPTH remained significantly elevated during the first 2 weeks of lactation, and increased further during the third week of lactation to a level more than twice that of nonlactating controls. Serum Ca fell gradually during the second week of lactation. The high serum iPTH levels were maintained for another 2 weeks when lactation was extended with foster litters. Within 6 hr of removal of the suckling pups on day 16 of lactation, maternal serum ionized and total Ca had risen and serum iPTH had fallen; all three parameters were at levels similar to those of nonmated controls by 24–48 hr after weaning. The data suggest that serum ionized Ca is a major factor contributing to the hyperparathyroid state during lactation in rats fed a low-Ca diet.
Adenine, guanine, and inosine nucleotides of chick growth cartilage: Relationship between energy status and the mineralization process
Authors: Matsumoto Hidehiko , Debolt Kristine , Dr. Shapiro Irving M.
Published: February 2010

The major aim of this investigation was to measure the nucleotide content of the developing chick epiphysis and to relate changes in nucleotide levels to chondrocyte maturation and the development of mineralization. Using a cryostat, sections of cartilage were isolated from the proximal head of the tibial growth cartilage, care being taken to preserve the metabolic integrity of the tissue. Sections were identified microscopically, pooled, and the nucleotide and nucleoside content of each sample determined by HPLC. Procedures used for the study were shown to minimize degradation of nucleotides. Their effectiveness was assessed through an evaluation of the rapid freezing technique and by examination of the effects of apatite on the recovery of endogenous and added nucleotides. Analysis of nucleotide levels in the growth cartilage indicated that chondrocytes undergo a profound change in energy metabolism during development and maturation. Thus, in the premineralized resting and proliferative zones, ATP and, to a lesser extent, GTP values were high, suggesting that the chondrocytes obtained metabolic energy through both glycolytic and mitochondrial oxidative processes. In the hypertrophic zone and in calcified cartilage, there was a profound decrease in the ATP concentration and a corresponding fall in the energy charge and the ATP/ADP ratios. The nucleotide levels in this zone indicated that there was increased reliance on nonoxidative metabolism. Measurement of nucleoside levels in premineralized cartilage suggested that there was little resynthesis of nucleotides through the salvage pathway. These observed changes in nucleotide values are consistent with earlier observations concerning chondrocyte redox and the low pO2 tension of the hypertrophic zone. The decrease in the level of ATP at the site of cartilage calcification lends support to the notion that nucleotides may serve as a source of phosphate ions for the initiation of mineralization.
The effects of muscle-building exercise on Vitamin D and mineral metabolism
Authors: Dr. Bell Norman H. , Godsen Richard N. , Henry David P. , Shary Judith , Epstein Sol
Published: February 2010

Exercise and muscle strength are important determinants of bone mass. Studies were carried out in normal young adult white males to determine the effects of exercise on vitamin D and mineral metabolism. Fourteen men who had engaged in regular muscle-building exercises for at least 1 year and 14 age-matched controls (age range, 19–36 year) were hospitalized on a metabolic ward and were given a constant daily diet estimated to contain 400 mg of calcium, 900 mg of phosphorus, 110 mEq of sodium, 65 mEq of potassium, and 18 mEq of magnesium. Body weight averaged 78 ± 3 kg in the exercisers and 72 ± 2 kg in the controls (NS). Serum calcium, ionized calcium, phosphate, magnesium, somatomedin-C, and immunoreactive parathyroid hormone (PTH) were not different in the two groups, whereas serum Gla-protein (39 ± 5 vs. 24 ± 2 ng/ml, p < 0.01), 25-hydroxyvitamin D (23 ± 2 vs. 16 ± 2, p < 0.05) and 1,25-dihydroxyvitamin D [1,25(OH)2D] (40 ± 2 vs. 29 ± 2 pg/ml, p < 0.01) were higher in the exercisers than in the controls. Urinary calcium, phosphorus, sodium, potassium, creatinine clearance, and norepinephrine were not different in the two groups, whereas urinary magnesium (12.6 ± 1.0 vs. 9.4 ± 0.5 mEq/d, p < 0.01) and urinary cyclic adenosine 3′,5′-monophosphate (cyclic AMP) (2.52 ± 0.19 vs. 1.72 ± 0.20 nM/dl glomerular filtrate, p < 0.01) were higher in the exercisers than in the controls. There were positive correlations between serum 1,25(OH)2D and urinary calcium (r = 0.544, p < 0.01) and between serum 1,25(OH)2D and urinary magnesium (r = 0.407, p < 0.05) in all subjects. The results indicate that muscle-building exercise is associated with increases in serum Gla-protein, serum 1,25(OH)2D, and urinary cyclic AMP. The increase in serum Gla-protein suggests that this form of exercise enhances bone formation.
Growth of rat osteoblast-like cells in a lipid-enriched culture medium and regulation of function by parathyroid hormone and 1,25-dihydroxyvitamin D
Authors: Dr. Catherwood Bayard D. , Addison Judy , Chapman Gail , Contreras Sue , Lorang Marge
Published: February 2010

To examine the role of lipid metabolism in the growth and function of osteoblast-like cells, we studied ROS 17/2.8 osteosarcoma cells and primary cultures of rat calvarial osteoblasts during growth in a serum-free medium supplemented by purified human lipoproteins or by liposomes. Increase in ROS cell number was measured in sparse (1–5 × 103/cm2) cultures over 6–8 days. Liposomes (0–300 μg/ml) and high (HDL), low (LDL), and very low density (VLDL) lipoprotein fractions (0–300 μg apoprotein) markedly stimulated cell growth. Cells plated at 5 × 103/cm2 achieved growth rates in the presence of LDL or HDL comparable to 10% fetal bovine serum. Serum-free culture with exogenous lipid maintained the response of cell cyclic AMP accumulation to parathyroid hormone. Cyclic AMP response to parathyroid hormone was enhanced by glucocorticosteroid, and was attenuated by 1,25-dihydroxyvitamin D (1,25(OH)2D) with an EC50 (10−10M) comparable to that previously observed in serum-cultured cells (J. Biol. Chem. 258:736, 1985). 1,25(OH)2D also increased the alkaline phosphatase activity in ROS cells cultured in lipid-supplemented serum-free culture. Lipoproteins or liposomes also markedly enhanced the proliferative response of sparse cultures of normal rat osteoblasts to polypeptide mitogens.
Hepatic handling of vitamin D3 in micronodular cirrhosis: A structure—function study in the rat
Authors: Plourde Victor , Gascon-Barré Marielle , Coulombe Pierre A. , Vallières Sylvie , Huet P.-Michel
Published: February 2010

The response to vitamin D3 (D3) was studied in a model of micronodular cirrhosis induced by CCl4. The uptake and C-25 hydroxylation of D3 were then studied in isolated-perfused liver preparations. CCl4-treated rats had a significantly lower fractional hepatic D3 uptake than controls; they also had lower 25-hydroxyvitamin D3 (25(OH)D3) concentrations in both liver and perfusate following 150 min of perfusion. CCl4 induced a wide spectrum of hepatic morphologic changes ranging from mild to large collagen infiltration, but micronodular cirrhosis was present in more than 90% of the animals. Histomorphometric analysis of the liver indicated an overall highly significant increase in the volume density (Vv) of collagen infiltration, and a reduction in the Vv normal hepatocytes following CCl4. Linear relationships were also observed between the Vv normal hepatocytes and the liver, perfusate, and total 25(OH)D3, while the 25(OH)D3 production decreased in a logarithmic fashion as the collagen infiltration of the liver parenchyma increased. These data show that the overall production of 25(OH)D3 is decreased in micronodular cirrhosis; they also indicate, however, that the D3-25 hydroxylase seems to stay unimpaired in the remaining hepatocytes of the diseased liver, and that the Vv normal hepatocytes constitute one of the major determinants of the 25(OH)D3 production by the cirrhotic rat liver.
Parathyroid hormone control of free cytosolic Ca2+ in the kidney
Authors: Fujii Yoshio , Fukase Masaaki , Tsutsumi Masaharu , Miyauchi Akimitsu , Tsunenari Toru , Fujita Takuo
Published: February 2010

We have established a perifusion system to monitor free cytosolic calcium concentrations ([Ca2+]i) in mouse kidney slices, which presumably reflects in vivo status more accurately than renal cells in culture, by means of the fluorescent calcium indicators quin-2 and fura-2. An increase in the extracellular calcium concentrations from 0 (no added Ca2+) to 3.0 mM resulted in an increase in [Ca2+]i from 52 to 239 nM. Replacement of 118 mM of extracellular Na+ with choline, or the addition of ouabain, an inhibitor of Na+, K+-ATPase, at 10−6 M in the perfusate caused an increase in [Ca2+]i from 161 ± 13 to 873 ± 78 nM (n = 10) and 161 ± 13 to 395 ± 68 nM (n = 4), respectively, suggesting the possible existence of a Na+, Ca2+ exchange mechanism in the kidney slice. We further examined the effects of PTH on [Ca2+]i mobilization in the kidney. Both human PTH-(1–34) and hPTH-(1-84) increased [Ca2+]i within 60 s at physiologic concentrations of 10−11-10−9 M in a dose-dependent manner. On the other hand, an increase in intracellular cAMP in the slice was also detected above 3 × 10−9 M hPTH-(1–34) [base 2.1 ± 0.4 pmol/mg, 3.2 ± 0.6 pmol/mg (p < 0.05 versus control values) 5 minutes after the application of 3 × 10−9 M hPTH-(1–34) and 17.3 ± 4.3 pmol/mg (p < 0.05 versus control values) 3 × 10−8 M hPTH-(1–34), mean ± SEM, n = 7, p < 0.05 versus control values]. We found that the effects of PTH on [Ca2+]i in our system appear to be similar to those found in cells of isolated proximal tubular cells, suggesting the cAMP and Ca2+ dual second-messenger system in PTH action. Thus, this perifusion system may enable us to study intracellular Ca2+ homeostasis in the kidney.
Increased renal calcium reabsorption in an animal model of hypercalcemia of human malignancy
Authors: Shipley Pauline J. , Shevrin Daniel H. , Kukreja Subhash C.
Published: February 2010

The present studies examined renal calcium (Ca) clearance in an animal model of malignancy-associated humoral hypercalcemia (MAHH) (a human squamous cell lung carcinoma carried in athymic mice). Three groups of animals-controls, normocalcemic tumor-bearing animals and hypercalcemic tumor-bearing animals-were studied in the basal state and during Ca infusion. Baseline Ca clearance was slightly but significantly elevated in the tumor-bearing hypercalcemic animals compared with the other two groups of animals. This clearea value was, however, inappropriately low for the serum Ca value. In the control and in the normocalcemic tumor-bearing animals, Ca clearance increased markedly during Ca infusion. This increase in renal Ca clearance was markedly blunted in the hypercalcemic animals compared with both the controls and the normoclacemic tumor-bearing animals. We conclude that increased renal Ca resorption contributes significantly to the pathogenesis of hypercalcemia of malignancy.
Arachidonic acid metabolism by a vitamin D3-differentiated human leukemic cell line
Authors: Stenson William F. , Teitelbaum Steven L. , Bar-Shavit Zvi
Published: February 2010

HL-60 is a human promyelocytic cell line that differentiates along the granulocytic pathway when incubated with dimethylsulfoxide and along the monocytic pathway when incubated with 1,25-(OH)2D3. We compared arachidonic acid metabolism in undifferentiated, DMSO-differentiated, and 1,25-(OH)2D3-differentiated cells. DMSO- and 1,25-(OH)2D3-differentiated cells metabolized exogenous arachidonic acid to both cyclo-oxygenase products (predominantly thromboxane B2 and prostaglandin E2) and 5-lipoxygenase products, including leukotriene B4. Undifferentiated cells produce these metabolites in much smaller amounts. DMSO-differentiated cells released a large percentage of phospholipid-bound arachidonic acid in response to stimulation with the ionophore A23187, zymosan, or formylmethionylleucylphenylalanine (FMLP). DMSO-differentiated cells stimulated with A23187 converted released arachidonate to LTB4 and TxB2. In contrast, 1,25-(OH)2D3-differentiated cells released a smaller percentage of phospholipid-bound arachidonate in response to stimuli, and undifferentiated cells released none at all. The three cell types (undifferentiated, DMSO-differentiated, and 1,25-(OH)2D3-differentiated) were homogenized and the 10,000 × g supernatant incubated with [14C]arachidonic acid. The supernatants from the homogenates of the DMSO- and 1,25-(OH)2D3-differentiated cells metabolized [14C]arachidonic acid to cyclooxygenase and lipoxygenase products, but the supernatant from the homogenate of undifferentiated cells did not. These data indicate that differentiation of HL-60 cells with DMSO or 1,25-(OH)2D3 induces cyclooxygenase and 5-lipoxygenase and induces mechanisms for the release of arachidonate from phospholipids by soluble and particulate stimuli.
Rapid action of 1,25-dihydroxyvitamin D3 on hepatocyte phospholipids
Authors: Baran Daniel T. , Sorensen Ann Marie , Honeyman Thomas W.
Published: February 2010

Recent studies have reported cellular effects of 1,25-dihydroxyvitamin D3 within 15 minutes, a time period too rapid to be mediated by nuclear activation. The vitamin increases hepatocyte cytosolic calcium levels in the absence of extracellular calcium within 5 minutes. Since metabolites of phosphatidylinositol have been implicated as second messengers in the regulation of cytosolic calcium, we examined the effect of 1,25-dihy-droxyvitamin D3 on hepatocyte phosphatidylinositol turnover and compared these effects to those produced by vasopressin. In isolated hepatocytes labeled with [3H]inositol, 1,25-dihydroxyvitamin D3 (4 nM) increased [3H]glycerophosphoryIinositol by 16% (p < 0.01) within 2.5 minutes, by 18% (p < 0.01) after 5 minutes, and by 11% (p < 0.05) after 10 minutes. At a concentration of 20 nM, 1,25-dihydroxyvitamin D3 increased [3H]glycerophosphorylinositol by 27% (p < 0.01) after 5 minutes. Vitamin D did not affect [3H]inositol polyphosphates. Conversely, vasopressin had no effect on [3H]glycerophosphorylinositol but significantly increased [3H]inositol phosphate, [3H]inositol bisphosphate, and [3H]inositol trisphosphate. 1,25-Dihy-droxyvitamin D3 (4 nM) decreased [3H]phosphatidylinositol by 10% (p < 0.05) after 5 minutes and by 16% (p < 0.01) after 10 minutes. At a concentration of 20 nM, 1,25-dihydroxyvitamin D decreased [3H]phosphatidylinositol by 18% (p < 0.01) after 5 minutes. The vitamin did not affect [3H]phosphatidylinositol bis-phosphate or [3H]phosphatidylinositol trisphosphate. 24,25-Dihydroxyvitamin D had no effect on inositol phospholipids. The effects of 1,25-dihydroxyvitamin D3 on inositol phospholipids were blocked by quinacrine. Bromophenacylbromide inhibited the effects of 1,25-dihydroxyvitamin D3 on inositol phospholipids and also blocked the vitamin-induced increments in cytosolic calcium. In isolated hepatocytes labeled with [3H]arachidonic acid, 1,25-dihydroxyvitamin D3 (20 nM) decreased 3H-labeled phosphatidylcholine and phosphatidylethanolamine, as well as phosphatidylinositol. The data indicate that 1,25-dihydroxyvitamin D3 may enhance deacylation of hepatocyte phospholipids, perhaps by activation of phospholipase A activity. The ability of vitamin D to increase cytosolic calcium requires activation of phospholipase A. Although both 1,25-dihydroxyvitamin D and vasopressin rapidly affect phospholipid metabolism, their mechanisms of action differ.
Effect of pertussis toxin on parathyroid hormone-stimulated cyclic AMP production in cultured kidney cells
Authors: Dr. Rizzoli R. , Bonjour J.-P.
Published: February 2010

The effect of pertussis toxin, which inactivates the guanine nucleotide binding regulatory proteins Gi and Go on cAMP production in response to parathyroid hormone PGE2 or forskolin, was examined in confluent opossum kidney (OK) cells. This effect was compared with that caused by dexamethasone. The response to PTH was increased in cells preincubated with either agent. The effect of pertussis toxin was selective for PTH, since cAMP production in response to neither PGE2 nor forskolin was increased. In contrast, the response to forskolin was enhanced in dexamethasone-treated cells. These results indicate that both stimulatory and inhibitory guanine nucleotides binding regulatory proteins modulate PTH-induced cAMP producion in OK cells. Moreover, pertussis toxin and dexamethasone appear to affect different levels of the PTH-receptor-adenylate cyclase complex.
Calcitonin-like effects of forskolin and choleratoxin on surface area and motility of isolated rabbit osteoclasts
Authors: Dr. Ransjö Maria , Lerner Ulf H. , Heersche Johan N. M.
Published: February 2010

We have previously found that the adenylate cyclase stimulators forskolin and choleratoxin increase cyclic AMP and transiently inhibit bone resorption in cultured mouse calvaria, suggesting that the compounds, directly or indirectly, may inhibit osteoclast activity. In the present study, forskolin and choleratoxin were investigated for their direct effects on surface area and motility of isolated rabbit osteoclasts, and the effects were compared to those of calcitonin (CT). Osteoclasts were cultured on coverslips for different times in the absence or presence of the compounds. The effect on osteoclast mean area was quantified on fixed and stained osteoclasts, and in addition effects were recorded with time-lapse cinemicrography. The effects of CT (100 mU/ml) on mean area and motility were seen within minutes and were maximal after 10–60 minutes. Forskolin (10–30 μmol/liter) produced a rapid (15–60 minutes) inhibition of motility and decrease in area (contraction) of osteoclasts. Choleratoxin (1 μg/ml) treatment also resulted in cell contraction and inhibition of motility; however, the response was not seen before 45–60 minutes. The difference in the kinetics of the osteoclast response between forskolin, CT, and choleratoxin is similar to differences in time course for the effect on cyclic AMP in calvarial bones, which we reported earlier. Although cells were incubated continuously with forskolin, choleratoxin, or CT, the effects were transient. Thus, after 7–8 h incubation with CT, 3–4 h treatment with forskolin, or 4–6 h with choleratoxin, the osteoclasts started to recover from contraction and immotility. The effect of forskolin and choleratoxin on the mean surface area of osteoclasts was dose dependent.

The present study shows that forskolin and choleratoxin have a direct inhibitory action on osteoclast activity and thus provide further evidence that cyclic AMP is a mediator of the action of CT on bone resorption.
Tumor necrosis factor α and interleukin-1 stimulate bone resorption in vivo as measured by urinary [3H]tetracycline excretion from prelabeled mice
Authors: König Adrian , Mühlbauer Roman C. , Fleisch Herbert
Published: February 2010

Tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) have been shown to stimulate bone resorption in vitro. We have now investigated whether these cytokines also cause a similar action when administered in vivo. This was made possible by the adaptation of a newly developed technique that enables the continual assessment of bone resorption in vivo in mice by measuring urinary excretion of 3H from [3H]tetracycline-prelabeled animals.

Experiments using maneuvers known to influence bone resorption, such as a change in dietary calcium or administration of parathyroid hormone or dichloromethylenebisphosphonate, indicate that the technique is reliable and sensitive in mice.

Daily intravenous administration of either recombinant human or recombinant murine TNF-α, as well as subcutaneous administration of recombinant human IL-1α, were found to stimulate bone resorption in a dose-dependent manner. The effect was maximal within 2 days. Thus, exogenous TNF-α and IL-1α can stimulate bone resorption in vivo, suggesting that these cytokines may also exert a systemic effect on bone.
A differentiation-inducing factor produced by the osteoblastic cell line MC3T3-E1 stimulates bone resorption by promoting osteoclast formation
Authors: Abe Etsuko , Ishimi Yoshiko , Takahashi Naoyuki , Akatsu Takuhiko , Ozawa Hiroyuki , Yamana Hiromi , Yoshiki Shusaku , Dr. Suda Tatsuo
Published: February 2010

We have reported that the differentiation-inducing factor (DIF) is present in conditioned medium of mouse osteoblast-like cell (MC3T3-E1) cultures (Ref. 12). In the present study, the DIF from conditioned medium of MC3T3-E1 cells was partially purified and its biologic activity was examined. The DIF was purified by monitoring the induction of phagocytic activity of mouse myeloblastic leukemia cells (M1). The DIF induced differentiation of not only M1 cells but also mouse myelomonocytic cells (WEHI-3). Furthermore, the DIF increased the in vitro bone-resorbing activity and the osteoclast number in mouse calvaria. The increases were inhibited by the addition of either salmon calcitonin or indomethacin. When mouse bone marrow cells were cultured with the DIF for 8 days, formation of osteoclast-like multinucleated cells was stimulated dose dependently. The DIF from MC3T3-E1 cells appeared to be different from interleukin-1 (IL-1), tumor necrosis factor (TNF), and transforming growth factor β (TGF-β). These results suggest that the DIF partially purified from osteoblast-like cell cultures stimulates osteoclastic bone resorption by promoting differentiation and fusion of osteoclast progenitors to form multinucleated osteoclasts.
The direct examination of three-dimensional bone architecture in vitro by computed tomography
Authors: Feldkamp Lee A. , Goldstein Steven A. , Parfitt Michael A. , Jesion Gerald , Kleerekoper Michael
Published: February 2010

We describe a new method for the direct examination of three-dimensional bone structure in vitro based on high-resolution computed tomography (CT). Unlike clinical CT, a three-dimensional reconstruction array is created directly, rather than a series of two-dimensional slices. All structural indices commonly determined from two-dimensional histologic sections can be obtained nondestructively from a large number of slices in each of three orthogonal directions. This permits a comprehensive description of structural variation within a specimen and greatly facilitates the study of structural anisotropy. A measure of three-dimensional connectivity (Euler number/tissue volume) has been determined for the first time in human cancellous bone and shown to correlate with several two-dimensional histomorphometric indices. The method has the potential for overcoming many of the limitations of current approaches to the study of bone architecture at the microscopic level.
Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3′,5′-monophosphate in mouse bone marrow cell cultures
Authors: Akatsu Takuhiko , Takahashi Naoyuki , Debari Kazuhiro , Morita Ikuo , Murota Seiitsu , Nagata Naokazu , Takatani Osamu , Suda Tatsuo
Published: February 2010

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days.(10) Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10−8-10−5 M, 6-keto-PGF1α at 10−5 M, and PGF2α at 10−6-10−5 M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2α and 6-keto-PGF1α in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3′,5′-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.
Effects of transforming growth factors β1 and β2 on a mouse clonal, osteoblastlike cell line MC3T3-E1
Authors: Dr. Ibbotson Kenneth J. , Orcutt Christopher M. , Anglin Ann-Marie , D'Souza Sharyn M.
Published: February 2010

Transforming growth factors (TGF-β1 and TGF-β2) are polypeptide growth factors with a wide range of effects on the growth and differentiated function of a variety of cell types. Transforming growth factors of the β class (TGF-β) are found in large quantities in bone matrix and are synthesized by osteoblasts. For these reasons, it has been suggested that TGF-β may play a major role in the regulation of bone cell metabolism. We have studied the effects of porcine TGF-β1 and the recently described porcine TGF-β2 in a mouse clonal, osteoblastlike cell line MC3T3-EI that has previously been shown to have many characteristics of osteoblasts.

In serum-containing medium, TGF-β1 inhibited alkaline phosphatase activity. The inhibition of alkaline phosphatase activity persisted for at least 72 h following a brief (24 h) exposure to TGF-β1, TGF-β1 also caused a marked change in cell morphology. High doses inhibited collagen synthesis; lower concentrations caused a small increase. Under serum-free conditions, TGF-β1 had biphasic effects on alkaline phosphatase activity inhibiting at high but stimulating at low concentrations and had only a slight stimulatory effect on collagen synthesis. Under the experimental conditions used, the effects of TGF-β1 on alkaline phosphatase activity and collagen synthesis were independent of effects on cell proliferation. In serum-containing medium, TGF-β2 inhibited alkaline phosphatase activity, an effect that was independent of changes in cell proliferation and caused shape changes in an identical fashion to that observed with TGF-β1.
Treatment of congenital osteopetrosis in the rabbit with high-dose 1,25-dihydroxyvitamin D
Authors: Popoff Steven N. , McGuire James L. , Zerwekh Joseph E. , Dr. Marks Sandy C.
Published: February 2010

Osteopetrosis is a congenital metabolic bone disease characterized by skeletal sclerosis resulting from defective osteoclast-mediated bone resorption. Osteopetrosis has been decribed in several animal species (mouse, rat, and rabbit) and in children. Bone marrow transplantation, originally shown to reverse the skeletal sclerosis in some animal mutations, has been effective in curing osteopetrosis in some children. Unfortunately, not all children with osteopetrosis are candidates for or respond to bone marrow transplantation. Recent studies have shown that several animal mutations and some children inheriting osteopetrosis have significantly elevated serum levels of 1,25-(OH)2D. Based on the possibility that there may be a resistance to 1,25-(OH)2D, high-dose calcitriol therapy has been used to treat some children and stimulated some parameters of resorption. In this study, we have examined the effects of high-dose calcitriol therapy on various serum and skeletal parameters in the osteopetrotic rabbit. Mutant rabbits and normal littermates were given continuous infusions of calcitriol via subcutaneously implanted osmotic minipumps for 2 weeks at a dose of 0.5, 2.5, or 25 μg/kg/per day. Untreated mutant rabbits are hypocalcemic and hypophosphatemic in the presence of elevated serum 1,25-(OH)2 levels in comparison with their normal littermates. Calcitriol infusions resulted in dose-dependent increases in circulating 1,25-(OH)2D levels in both normal and mutant rabbits. However, evaluation of other serum parameters and the skeletal response demonstrated significant differences between osteopetrotic and normal rabbits. At the highest dose, normal animals rapidly became hypercalcemic and osteoporotic, accompanied by weight loss and a failure to thrive; mutants remained hypocalcemic and osteopetrotic but did not exhibit the deleterious physical effects seen in treated normal littermates. Although the number of osteoclasts increased in both mutants and normals, osteoclast phenotype in the former remained abnormal. These data indicate that although very high levels of circulating 1,25-(OH)2D were achieved in osteopetrotic mutants, activation of osteoclast-mediated bone resorption with subsequent improvement of skeletal sclerosis was not observed.
Histomorphometric effects of calcium or calcium plus 25-hydroxyvitamin D3 therapy in senile osteoporosis
Authors: Orwoll Eric S. , McClung Michael R. , Oviatt Shelia K. , Recker Robert R. , Weigel Ronald M.
Published: February 2010

To evaluate the effects of calcium and 25-OHD in the therapy of senile osteoporosis, we studied a group of 39 women aged 69 + 7 (standard deviation, SD) years with severe osteoporosis. The group was characterized histomorphometrically by depressed bone remodeling rates without hyperosteoidosis. No subject had risk factors for osteopenia other than their age and postmenopausal status, and no subject was receiving therapy for bone disease at the onset of the study. Subjects were followed for 2 years after randomization to receive either 1200 mg/day of calcium (as calcium carbonate) and 40 μg/day of 25-OHD (calcium-25-OHD group), or 1200 mg/day of calcium plus placebo (calcium-placebo group). Calcium-25-OHD resulted in a clear increase in 25-OHD levels (p < 0.001) and an increase in calcium absorption as indicated by urinary calcium excretion. Nevertheless, there was no significant change in fasting serum calcium, phosphorus, alkaline phosphatase, PTH, or 1,25-(OH)2D in either group. Radial and phalangeal bone mineral content and trabecular bone volume in the biopsied patients remained stable in both groups over the 2 year period. Unexpectedly, repeat bone biopsies revealed a clear improvement in the rate of mineralization in both groups, presumably as a result of the calcium supplementation alone. In summary, calcium-placebo and calcium-25-OHD treatment were both associated with stable appendicular bone mineral content in women with senile osteopenia. The finding of an effect of calcium supplementation on the rate of mineralization indicates that relative calcium deficiency may impair the mineralization phase of remodeling. Calcium therapy may thus provide some benefit in the therapy of osteoporosis characterized by prolonged remodeling rates.
Cosecretion of calcitonin and calcitonin gene-related peptide from cultured rat medullary thyroid C cells
Authors: Seitz Patricia K. , Cooper Cary W.
Published: February 2010

Whether C cells cosecrete calcitonin (CT) and CGRP was examined by exposing cultured rat medullary thyroid carcinoma 6–23 cells for 2 h to high medium Ca and to agents with a potential for affecting Ca-dependent secretion. In every experiment exposure of cells to high medium Ca (2.0–2.5 mM) provoked an increased release of both peptides that was highly correlated (r = 0.73). With other test substances, also, changes in both hormones occurred in parallel. The Ca-channel activator, BAY-K-8644 (10 μM) increased secretion, and this was inhibited by the Ca channel blocker, nitrendipine (10 μM). The Ca2+ ionophore, ionomycin (5 μM), increased release, and the calmodulin-Ca channel inhibitor, phenytoin (100 μM), inhibited Ca-induced release. The active 4β isomer of phorbol-12,13-didecanoate (0.1 μM), but not the inactive 4α isomer, increased secretion. The findings suggest that pathways mediating C cell secretion include plasma membrane Ca channels, intracellular [Ca2+], calmodulin, and protein kinase C. The results show that the secretory process in rat C cells involves the release of CGRP as well as CT.
Ultradistal and cortical forearm bone density in the assessment of postmenopausal bone loss and nonaxial fracture risk
Authors: Dr. Price Roger I. , Barnes Marion P. , Gutteridge Donald H. , Baron-Hay Margaret , Prince Richard L. , Retallack Robert W. , Hickling Caroline
Published: February 2010

Forearm bone mineral density (BMD) was measured by single-energy photon absorptiometry in 360 healthy females without known axial fractures, 202 of whom were postmenopausal. The three sites addressed included an ultradistal (U) region containing approximately 60% trabecular bone. The other sites, distal (D) and shaft (S), were progressively more cortical. Reproducibility was 1.7–1.9% CV. The earliest evidence of a significant correlation between BMD and years since menopause was seen in trabecular bone in subjects aged 45–55 years. Fractional decrease in BMD, relative to the premenopausal value, was significantly larger at U than at S for the decades 55–65 years and above. Fractional rates of bone loss at all sites were a maximum in the first postmenopausal decade, the rate at U being 0.035, approximately 1.5 times that at D or S. A total of 33 subjects reported 54 previous minimally traumatic nonaxial (MTNA) fractures. When BMD measurements of the entire study were divided into quintiles, the prevalence of MTNA fracture cases in the lowest quintile was eight times that of each of the upper three quintiles. Prevalence of fracture cases ranked by quintiles of BMD were not different for the three scan sites. Therefore, ultradistal measurements confer no advantages over distal or shaft BMD for discriminating past MTNA fracture cases but do show larger fractional rates of loss during the first postmenopausal decade. When early postmenopausal women (with years since menopause ≤ 10) having BMD at U below the 15th age-corrected percentile, and their high BMD peers above the 75th percentile, were studied prospectively over 9–24 months there were no differences in their mean fractional rates of bone loss. These rates confirmed cross-sectional measurements based on years since menopause ≤ 10 but without selection for BMD.
Thyroid hormone 5′-deiodinase activity, nuclear binding, and effects on mitogenesis in umr-106 osteoblastic osteosarcoma cells
Authors: Lebron Barbara A. , Pekary A. Eugene , Mirell Carol , Hahn Theodore J. , Hershman Jerome M.
Published: February 2010

The hyperthyroid state in vivo is associated with an increase in osteoblast number and activity, suggesting that thyroid hormone may stimulate osteoblast replication and function. We therefore examined the effects of T3 (16–1170 pM) on replication rate as assessed by cell counts in UMR-106 osteoblastic osteosarcoma cells cultured for 5–10 days in medium supplemented with 10% hormone-stripped fetal calf serum (FCS). Despite the virtual absence of thyroid hormone in the control medium (total T3 concentration, 0.02 ng/ml), the addition of T3 in concentrations to 1000 pM did not increase the cell replication rate. At higher T3 concentrations, a slight decrease in growth rate was observed. No significant 5′-monodeiodinase activity was detected in UMR-106 cell homogenates. However, nuclear binding of T3 was demonstrated in intact cells. A high-affinity nuclear binding component was identified with a Ka of 2.6 × 1010 M−1 and a maximum binding capacity of 7.7 pg T3 per mg DNA, equivalent to 51 binding sites per cell nucleus. A lower affinity nuclear T3 binding component with a Ka of 1.8 × 109 M−1 was also identified. Thus, despite the presence of nuclear T3 receptors, UMR-106 cells do not exhibit a mitogenic response to T3.
Bone demineralization, biochemical indices of bone remodeling, and estrogen replacement therapy in adults with turner's syndrome
Authors: Dr. Štěpán Jan J. , Musilová Jana , Pacovský Vladimír
Published: February 2010

The study centered on a controversy about whether long-term estrogen replacement therapy may ameliorate the osteoporosis seen in patients with Turner's syndrome. This study comprised 26 adult patients with Turner's syndrome (9 treated and 17 untreated or insufficiently treated) and 12 adult women with pure gonadal dysgenesis (8 untreated and 4 treated). A low bone density below -2 standard deviations from the age- and sex-matched predicted normal mean was documented by dual-photon absorptiometry of the lumbar spine in all the untreated and insufficiently treated patients, but only in 6 treated patients. The biochemical indices of bone resorption (urinary hydroxyproline excretion and plasma tartrate-resistant acid phosphatase activity), as well as osteoblastic function (serum osteocalcin and bone alkaline phosphatase isoenzyme), were significantly increased in untreated and insufficiently treated patients compared with treated patients. A significant negative correlation was found between biochemically documented osteoresorption and spinal bone mineral density corrected for age of the patients. Significant positive correlations were found between serum osteocalcin and bone alkaline phosphatase isoenzyme and between biochemical indices of bone resorption and formation. Although in the patients there was an evidence of a high bone remodeling rate, the rate of bone mass loss seemed to be low, comparable with that seen in oophorectomized women who had already passed their accelerated phase of bone loss. The results indicate that long-term hormonal replacement therapy is justified in gonadal dysgenesis, regardless of the karyotype of the patient, to prevent further bone mass loss.
Increased incidence of hip fracture in osteoporotic women treated with sodium fluoride
Authors: Hedlund L. Rune , Gallagher J. C.
Published: February 2010

There has been controversy as to whether fluoride therapy increases the risk of fracture in the appendicular skeleton. In the present study we compared the incidence of hip fracture in four groups of osteoporotic women: 22 treated with placebo, 17 with fluoride and calcium, 18 treated with fluoride and calcitriol, and 21 with calcitriol alone. Four hip fractures occurred in 3 patients on fluoride and calcitriol, and two hip fractures occurred in 2 patients on fluoride and calcium. No hip fractures occurred in patients receiving either calcitriol alone or placebo. The difference in fracture rates for fluoride versus nonfluoride treatment is significant (p = 0.006). Moreover, the six hip fractures occurring in patients receiving fluoride during 72.3 patient years of treatment is 10 times higher than would be expected in normal women of the same age. The probability of observing six fractures in 2 years is extremely small (0.0003). In four of the hip fracture cases, the history suggested a spontaneous fracture. These findings suggest that fluoride treatment can increase the risk of hip fracture in osteoporotic women.
Dog and rat kidney proximal tubule cells in culture: Responses to parathyroid hormone
Authors: Dr. Rao Leticia G. , Yau Chi H. , Kovacs Kalman
Published: February 2010

We have described and compared culture systems for proximal tubule cell (PTC) preparations from dog and rat kidney. Cells were prepared from kidney cortex by enzyme digestion and purified on Percoll density gradient. The dog PTC and rat PTC differed in their growth characteristics in culture. Although the dog PTC tended to overgrow the contaminating fibroblastic cells, the rat PTC tended to be overgrown by the latter cells when cultured in medium containing 15% fetal calf serum (FCS). Cultures of rat PTC in serum-free medium or medium containing only 2% FCS yielded only epithelium-like cells exhibiting characteristics of cells that are proximal tubular in origin. These properties include protrusion of microvilli, high alkaline phosphatase activity, ability to transport sugar, and responsiveness to parathyroid hormone (PTH) in terms of cAMP production. The time course and dose-response curves of PTH-stimulated cAMP accumulation were studied in dog and rat PTC. The estimated half-maximal concentrations (Kact) for PTH in dog and rat PTC were 1.2 and 100 nM, respectively. Both values are within the range reported in the literature for the respective renal membrane preparations. In addition to our previously reported data on dog PTC, this study revealed the presence of PTH-inhibitable 25-hydroxyvitamin D3-24-hydroxylase in dog PTC. These two model cell culture systems should prove useful in studying PTH action in renal cells.
In vitro mineralization of fetal rat parietal bones in defined serum-free medium: Effect of β-glycerol phosphate
Authors: Dr. Gronowicz Gloria , Woodiel Florence N. , Mccarthy Mary-Beth , Raisz Lawrence G.
Published: February 2010

We have developed a bone organ culture system that mineralizes in vitro. Fetal rat parietal bones (20 days old) were cultured in a chemically defined serum-free medium containing physiological 3 mM phosphate. During 5 days in culture, calcium content increased from 26 to 55 μg and dry weight increased from 137 to 194 μg. After 2 days in vivo, the calcium content of the parietal bone showed a comparable increase to 49 μg and dry weight increased to 183 μg. During culture, the mineralized bone area in thick sections increased from 11 to 23%, which paralleled the doubling in calcium content. Fluorescent calcein labeling during the 5 day culture period demonstrated that calcification occurs in an ordered pattern. Protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). The percentage collagen synthesis decreased from 17.5% at 0 time to 5.0% at 2 days and then increased to 9.4% at 5 days of culture.

Varying the inorganic phosphate concentration in the medium or adding β-glycerol phosphate was found to affect mineralization. After 5 days in culture, bones treated with 1 mM phosphate exhibited a large region of unmineralized osteoid with only a 23% increase in calcium content compared with 112% in control (3 mM phosphate) bones and a 28% increase in dry weight compared with a 40% increase in control. Treatment for 5 days with 6 mM phosphate or 1, 3, or 10 mM β-glycerol phosphate had no significant effect on dry weight compared to control bones. However, bone calcium content increased significantly from 55 ± 5 μg in control cultures to 105 ± 7 with 6 mM phosphate, 74 ± 6 with 3 mM β-glycerol phosphate, and 75 ± 5 μg with 10 mM β-glycerol phosphate. Calcified area measured by histomorphometry was also significantly greater than in control bones, but this was mainly due to ectopic calcification in the periosteum, representing from 23 to 74% of the total increase in calcified matrix in bones cultured with 6 mM phosphate or 1–10 mM β-glycerol phosphate. Ultrastructural analysis demonstrated that ectopic calcification was associated with cell death and debris. Therefore, calcification with β-glycerol phosphate and high concentrations of inorganic phosphate differed from mineralization in vivo or in bones cultured with a physiologically concentration of phosphate.
Osteoclast formation in vitro from progenitor cells present in the adult mouse circulation
Authors: Dr. Helfrich Miep H. , Mieremet René H. P. , Wil Thesingh C.
Published: February 2010

The development of multinucleated cells with tartrate-resistant acid phosphatase (TRAP) activity was studied in coverslip cultures of murine blood leukocytes and in cocultures of blood leukocytes with murine fetal bone rudiments. Cells with TRAP activity were not present among the leukocytes before culture and were absent in the bone rudiments at the time of explantation. After 14 days, macrophages with only tartrate-sensitive acid phosphatase activity developed in cultures of leukocytes without long bones. Multinucleated cells were not seen. In cocultures of leukocytes with bone rudiments, however, multinucleated cells with a strong TRAP activity had formed after 10–14 days of coculture. These TRAP-positive cells had invaded the bones and resorbed part of the calcified matrix. Electron microscopy revealed ruffled borders on the resorbing cells. In cocultures, TRAP-positive cells also formed from leukocyte fractions depleted of strongly adherent cells. Also on the cellophane supports of the cocultures, mononuclear cells with a stellate appearance and a strong TRAP activity were seen. We suggest that, in the cocultures, osteoclasts developed from a TRAP-negative, circulating progenitor cell. The presence of osteoclast progenitor cells in the circulation is discussed in light of the descent of osteoclasts from hematopoietic stem cells. That appearance of TRAP activity was always seen in resorbing cells and was not acquired in monocytes present in the leukocyte fraction by mere culture means that in the mouse TRAP is a useful marker for osteoclasts.
Cyclosporin a induces in vivo inhibition of resorption and stimulation of formation in rat bone
Authors: Orcel Philippe , Bielakoff Josette , Modrowski Dominique , Miravet Livia , Dr. Christine de Vernejoul Marie
Published: February 2010

We investigated a possible “in vivo” effect of cyclosporin A, an immunosuppressive agent, on normal rat bone remodeling. At an oral daily dose of 7 mg/kg for 14 days, the blood level of cyclosporin A was in the usual effective range and no change in renal function or magnesium metabolism was observed. Treated rats had decreased bone resorption: urinary hydroxyproline, plasma acid phosphatase, and the number of osteoclasts in caudal vertebrae were significantly reduced. By contrast, bone formation assessed by dynamic histomorphometry after double tetracycline labeling was increased. No modification of calciotropic hormones (vitamin D metabolites and parathyroid hormone as assessed by urinary cyclic AMP) was observed at the end of the treatment. These results suggest that in vivo cyclosporin A treatment induces bone remodeling modifications related to either a direct or a lymphokine-mediated effect on bone cells.