bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative evaluation of anticalciuretic effects of synthetic parathyroid hormonelike peptides
Authors: Scheinman Steven J. , Mitnick Maryann E. , Stewart Andrew F.
Published: February 2010

The PTH-like peptide (PTHLP) responsible for hypercalcemia in many patients with humoral hypercalcemia of malignancy (HHM) acts on PTH receptors in bone and kidney, and large doses of PTHLPs have been shown to reduce urinary calcium excretion. However, PTHLPs have not been assessed quantitatively for effects on renal calcium excretion at concentrations (5-100 pM) now known to be found in the serum of patients with HHM. We perfused isolated rat kidneys with synthetic [tyr-36] PTHLP-(1-36)amide [PTHLP-(1-36)], PTHLP-(1-74), and synthetic bovine PTH-(1-34). The ratio of calcium to sodium clearances (Cc2/CN2), a measure of distal tubular calcium transport, was reduced to the same extent by PTH, PTHLP-(1-36), and PTHLP-(1-74) (54.3 ± 3.9, 52.9 ± 3.9, and 52.7 ± 1.3% reductions from control), respectively at maximal doses (35-50 pM and higher), with half-maximal effects at 10, 18, and 32 pM, respectively. PTH, PTHLP-(1-36), and PTHLP-(1-74) all increased fractional phosphate excretion over control (p < 0.05 each). All three peptides were natriuretic, at least doubling fractional Na excretion (p < 0.05 or less). Urinary cAMP excretion was increased by all three. None had any effect on GFR or renal vascular resistance.

These results indicate that clinically relevant concentrations of PTHLPs are anticalciuretic and natriuretic, with maximal effects similar to those of PTH. Differences in anticalciuretic potencies are small but may explain differences among patients, depending on the size(s) and concentrations of the native circulating form(s) of the peptide.
Ascertainment of risk factors for osteoporosis: Comparison of interview data with medical record review
Authors: Beard C. Mary , Dr. Melton L. Joseph , Cedel Sandra L. , Richelson L.S. , Riggs B. Lawrence
Published: February 2010

To determine the accuracy of self-reported risk factors for osteoporosis, an age-stratified random sample of Rochester, MN, women was studied. Results from a structured face-to-face interview were compared with information documented in contemporary inpatient and outpatient health care records in the community. Using the × statistic to evaluate concordance of these two data sources, we found substantial agreement for a history of proximal femoral and distal forearm fractures, peptic ulcer disease, estrogen replacement therapy and oral contraceptive use, and cigarette and alcohol exposure. Moderate agreement was seen for histories of other age-related fractures, hysterectomy or oophorectomy, thyroidectomy, and use of thyroid supplements. Poor agreement was found for prior thyroid disease, gastrectomy, and corticosteroid or anticonvulsant use. This study demonstrates a need for greater attention to the quality of self-reported risk factor data in studies of bone loss and fractures.
Measurement of vertebral area on spine X-rays in osteoporosis: Reliability of digitizing techniques
Authors: Nelson Dorothy , Peterson E. , Tilley B. , Otallon W.M. , Chao E. , Riggs B.L. , Kleerekoper M.
Published: February 2010

Much of the clinical research in osteoporosis is directed toward documenting a reduction in vertebral fracture rate, but there is considerable disagreement about defining and quantifying vertebral fractures. We have evaluated the technique of digitizing landmarks identified on lateral radiographs of thoracic and lumbar vertebrae and computing vertebral body area. Reduction in area indicates that fractures occurred. Radiographs from 10 patients with osteoporosis and vertebral fractures were obtained from each of two centers, Henry Ford Hospital (HFH) and Mayo Clinic (MC), and vertebral area for each individual in the complete set of 20 radiographs was calculated at each center.

Measurements at the two centers differed by a multiplicative constant related to the method of recording landmarks on the radiographs that was estimated using 300 x-rays from HFH. After adjusting the MC areas for this multiplicative relationship, the average ratio of the HFH areas to the transformed MC areas of individual vertebrae (T4-L5) ranged from 0.98 to 1.06. The correlation between HFH and transformed MC areas for individual vertebrae averaged 0.85, with slopes between 0.87 and 1.00, intercept average -0.57. Within-patient rank correlation averaged 0.97. We conclude that radiographic digitization is a reliable and reproducible method of determining vertebral body dimensions that is suitable for evaluating radiographs obtained at different clinical sites and for comparison with normal data. This technique should prove useful for documenting the presence of a vertebral fracture that may not be readily apparent on visual inspection of radiographs and for monitoring serial changes in vertebral body dimensions in long-term epidemiologic and therapeutic studies.
Promotion of calvarial cell osteogenesis by endothelial cells
Authors: Villanueva Jorge E. , Nimni Marcel E.
Published: February 2010

Bone development and remodeling are associated with changes in the pattern of vascularization. Here we show that endothelial cells isolated from rat liver or bovine aorta can greatly enhance bone formation when implanted in diffusion chambers with rat fetal calvarial cells. The latter cells are unable to form bone when implanted alone at low initial cell density. The amount of mineralization measured by calcium deposition was 70 times higher in chambers containing calvarial cells mixed with endothelial cells from isologous liver or bovine aorta than in chambers containing endothelial or calvarial cells alone. Alkaline phosphatase activity was increased 20-fold. Calvarial cells in the presence of demineralized bone matrix powder did not form bone when implanted under similar conditions. Endothelial cells implanted alone seemed to enhance neovascularization around the Millipore diffusion chambers.
Release of parathyroid hormonelike peptides by fetal rat long bones in culture
Authors: Bergmann P. , Wolf N. Nijs-De , Pepersack T. , Corvilain J.
Published: February 2010

We observed that culture medium conditioned with fetal rat long bones stimulated cyclic AMP production by canine renal cortical membranes. This cyclase-stimulating activity (CSA) was retained by an ultrafiltration membrane with a molecular weight cutoff of 5000; three biologically active peaks with an approximate molecular weight of 18,000-25,000, 9000-12,000, and 4000-6000 were separated by high-performance liquid chromatography. The biologic activity was destroyed by trypsin digestion. The stimulation of adenylate cyclase by the medium and by the three peaks was inhibited by [N-leu8.18, Tyr34]parathyroid hormone-(3-34)-amide and by [Tyr34]parathyroid hormone-(7-34)amide. Preincubation of the bone culture medium and of the three peaks with an antibody raised against human parathyroid hormone-(l-34) did not decrease the biologic activity more than incubation with nonimmune serum. However, the biologic activity of the three active peaks was significantly suppressed after preincubation with an antiserum directed against the N-terminal region of the parathyroid hormone-related peptide of malignancy. The release of CSA into the bone culture medium was enhanced by parathyroid hormone induction and by 1,25-dihydroxycholecalciferol. It was decreased by calcitonin. We conclude that fetal murine bones in culture release peptides that stimulate the adenylate cyclase of renal cortical membranes. These peptides are antigenically similar to the parathyroid hormone-related peptide of malignancy. Their release from bones is modulated by hormones that control bone resorption.
Bilirubin as an inhibitor of cartilage metabolism: Effect on avian chondrocyte proliferation in cell culture
Authors: Vassilopoulou-Sellin Rena , Rey-Bear Norse , Oyedeji Caroline O.
Published: February 2010

Elevated levels of bilirubin and other tetrapyrroles are common to a number of chronic hematologic and liver diseases that can result in growth failure. This report establishes a cellular model system for the study of these endogenous growth inhibitors. Primary chondrocyte cultures were prepared from embryonic chick cartilage; cells were incubated (0.3 ± 105 cells per plate) in tissue culture medium containing 10% fetal bovine serum (FBS) with or without added bilirubin, 0.01-0.10 mM. Bilirubin caused profound, dose-dependent inhibition of chondrocyte proliferation: after 7 days, control incubations contained 11.45 ± 2.0 ± 105 cells per plate versus 4.92 ± 0.55 ± 105 cells per plate for wells with added 0.01 mM bilirubin and 1.76 ± 0.30 ± 105 for cultures with added 0.05 mM bilirubin. In chondrocytes cultured for 3 days, the addition of 0.05 mM bilirubin was associated with inhibition of [3H]thymidine incorporation into DNA (47 ± 5% of control), [3H]uridine incorporation into RNA (11 ± 0.05% of control), and [14C]leucine incorporation into proteins (16 ± 1% of control). The inhibition of chondrocyte proliferation induced by a range of bilirubin concentrations (0.01-0.10 mM) was in no way attenuated by the addition of physiologic concentrations of albumin (4 g/dl). After 3 days in media containing bilirubin or heme (at equimolar concentrations), chondrocytes were subsequently incubated in FBS alone for an additional 3 days; only partial reversal of the bilirubin (or heme)-induced inhibition was then observed. Our data show that bilirubin (and heme) produces profound, dose-dependent, partially reversible inhibition of chondrocyte proliferation in culture without affecting cell viability, supporting the concept that these endogenous tetrapyrroles are potent growth inhibitors. We also conclude that chick chondrocytes in cell culture are a suitable model for delineating the mechanisms of tetrapyrrole tissue action.
Long-term effects of physiologic concentrations of dexamethasone on human bone-derived cells
Authors: Wong Min-Min , Rao Leticia G. , Ly Hao , Hamilton Lynn , Tong Jeffrey , Sturtridge William , Mcbroom Robert , Aubin Jane E. , Dr. Murray Timothy M.
Published: February 2010

Bone cells derived from human trabecular explants display osteoblastic features. We examined the modulation of alkaline phosphatase activity and cAMP production as the result of exposing trabecular explants to physiologic concentrations of dexamethasone for 4 weeks during cellular outgrowth and subculture. Cells treated with dexamethasone were observed to grow generally more slowly than control cells. Cells appeared larger and more polygonal, and staining for alkaline phosphatase was more intense in the dexamethasone-exposed cultures. There was a progressive increase in cellular PTH responsiveness with increasing duration of exposure of cells to dexamethasone. Cells grown for 6 weeks in 3 x 10−8 M dexamethasone had a 10-fold increase in PTH-stimulated cyclic AMP accumulation. Dexamethasone-treated cells also had a significantly increased alkaline phosphatase activity. 1,25-(OH)2D3-stimulated alkaline phosphatase activity was increased approximately 20-fold. cAMP responses were significantly increased to PTH (21.7-fold), PGE, (2.67-fold), and forskolin (4.81-fold), but not to cholera toxin. Dexamethasone-treated cells also had a mean decrease in 1,25-(OH)2D3-stimulated osteocalcin production to 26.2% of control values (p < 0.001). Hydrocortisone treatment gave rise to similar effects but of smaller magnitude than those of dexamethasone. Testosterone did not have a significant effect on alkaline phosphatase activity or cAMP production. Skin fibroblasts showed a significant enhancement of alkaline phosphatase activity in response to dexamethasone, but of a much smaller magnitude than in bone cells. The phenotypic changes induced by long-term culture in dexamethasone are consistent with the promotion of a more differentiated osteoblastic phenotype.
Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization
Authors: Weinreb Miron , Shinar Doron , Rodan Gideon A.
Published: February 2010

Alkaline phosphatase (AP), osteopontin (OP), and osteocalcin (OC) are expressed during osteoblastic differentiation. However, previous studies suggested differences in the timing and possibly the site of their expression. In this study we used in situ hybridization to follow the distribution of these osteoblastic markers during bone development.

Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S-labeled RNA probes complementary to the AP, OP, and OC mRNAs. Controls included sections hybridized with the sense (mRNA) probes or pretreated with RNase.

Positive cells were identified in all areas of bone formation of the long bones and calvariae. Based on quantitative silver grain distribution and density, high levels of OP expression were present only in osteoblasts in close proximity to bone (one to two cell layers). OC expression, apparently at lower levels than OP, was also localized to osteoblasts in contact with bone. In contrast AP, which was expressed at lower levels than OP, was present in a large number of cells, including preosteoblasts that were many layers removed from the bone-forming surface.

These findings are consistent with the asynchronous expression of phenotypically related genes and suggest that AP is an earlier differentiation marker than OP and OC during the formation of endochondral and membranous bone.
Effect of sodium warfarin on vitamin K-dependent proteins and skeletal development in the rat fetus
Authors: Feteih Rabab , Tassinari Melissa S. , Lian Jane B.
Published: February 2010

Sodium warfarin was administered daily to Sprague-Dawley rats from gestational day 8 to day 22 to examine the effects of this compound on the developing fetal skeleton and on the vitamin K-dependent bone and cartilage proteins. At a dose of 175 μg/kg of sodium warfarin there was a 43% mortality rate among the dams. Maternal prothrombin times and serum osteocalcin levels were slightly elevated but not significantly. In the surviving litters, fetal bone osteocalcin and γ-carboxyglutamic acid were significantly reduced (50 and 57%, respectively, on gestational day 22) when compared to age- and/or weight-matched control pups. The high correlation of osteocalcin content in long bone (R = 0.64) and calvariae (R = 0.77) to fetal body weight observed in control fetuses was not seen in the warfarin-exposed pups. Examination of alizarinstained warfarin-exposed fetal skeletons for ossification centers showed no difference from controls. However, analysis of the tibial growth showed several changes compared to control that included (1) widened hypertrophic zones, (2) increased calcification of the hypertrophic zones, and (3) disorganization of the hypertrophic cells. These results suggest that the growth plate abnormalities seen with prenatal warfarin exposure relate to the inhibition of the vitamin K-dependent proteins of the skeletal system.
Vitamin D metabolism in rats with adjuvant-induced arthritis
Authors: Langman Craig B. , Ford Kathy K. , Pachman Lauren M. , Glorieux Francis
Published: February 2010

Adjuvant-induced arthritis in rats shares many of the features of humans with rheumatoid arthritis, including the development of osteopenia in areas distal to erosive joint disease. We established adjuvant arthritis in male and female Sherman strain rats and then studied external calcium balances and vitamin D metabolism during the period of acute active clinical, serologic, and pathologic arthritis and osteopenia and in the preclinical period. While ingesting a calcium-sufficient vitamin D-replete diet (0.6% calcium, 0.65% phosphorus, and 2.2 IU D3 per g food), female rats with arthritis demonstrated reduced calcium balance (arthritic, 36 + 8 versus control, 169 + 13 mg per 6 days, p < 0.02) because of inefficient gastrointestinal absorption of calcium (arthritic 9.7% versus control 37%). This was associated with calcitriol deficiency (arthritic 52 + 7 versus control 70 + 10 pg/ml) and reduced osteocalcin levels. Male rats with arthritis demonstrated an inability to raise serum calcitriol levels to the same degree as control rats (200 + 30 versus 440 + 70, respectively) while ingesting a calcium-deficient diet (0.002% calcium, 0.34% phosphorus, and 2.2 IU D3 per g food) and also had reduced balance (59 + 7 versus 85 + 10 mg per 6 days, respectively) due in part to decreased efficiency of absorption (55 versus 67%). No abnormalities in calcium balance or in serum calcitriol levels on the sufficient diet were present in the preclinical period. Physiologic calcitriol replacement to arthritic female rats increased osteoid available for mineralization and increased mineral apposition rates. Calcitriol deficiency in adjuvant-induced arthritis is responsible, in part, for reduced calcium balance and likely contributes to the development of osteopenia during the acute stage of the systemic inflammatory disease.
Osteocalcin secretion by the human osteosarcoma cell line MG-63
Authors: Lajeunesse Daniel , Frondoza Carmelita , Schoffield Brian , Sacktor Bertram
Published: February 2010

The human osteosarcoma cell line MG-63 has been used to study the production of the bone-specific protein, osteocalcin. In the absence of any stimuli, MG-63 cells secreted very low levels of osteocalcin. The secretion of osteocalcin started after a lag time of 10-12 h upon 1,25-(OH)2D3 treatment. Osteocalcin secretion was measured at doses as low as 0.03 nM (fourfold increase, p < 0.05), and this activity increased further with higher doses of 1,25-(OH)2D3 to reach a plateau at 50 nM. The secretion increased transiently from very low levels in sparse cell cultures to peak values in subconfluent cultures (+ 40%), two- to threefold above values obtained for confluent cells. Values for confluent cells average 55.9 + 2.0 ng/ml protein per 48 h. A similar behavior is observed for 1,25-(OH)2D3 receptor concentration under similar experimental conditions. Bmax increased transiently from sparse to subconfluent cell cultures (40 -60% confluent) and reached values 50% lower in confluent cells. However, the receptor affinity was not affected by cell density. MG-63 cells also possessed an alkaline phosphatase isoenzyme of the bone-liver-kidney type that was stimulated by 1,25-(OH)2D3 treatment (two- to threefold) and inhibited by parathyroid hormone (40 nM, -25%, p < 0.025). PTH and PGE2 increased cAMP production in a dose-dependent manner, but the cells were irresponsive to salmon calcitonin. Basal and PTH-responsive cyclic AMP production were also modulated by cell density. Dexamethasone pretreatment (100 nM, 48 h) stimulated the PTH-dependent cAMP production but failed to influence the response to PGE2. Vitamin D3-induced osteocalcin secretion was inhibited by 40 nM PTH (-20%, p < 0.01) and 5 nM PGE2 (-36%, p < 0.005), a situation that could be related to the ability of these hormones to stimulate cAMP in these cells. These results show that the MG-63 cell line is a good human osteoblastlike cell model in which bone-specific protein synthesis (osteocalcin and alkaline phosphatase) is modulated in response to 1,25-(OH)2D3 and PTH.
Sequential histomorphometric changes in cancellous bone from ovariohysterectomized dogs
Authors: Boyce R.W. , Franks A.F. , Jankowsky M.L. , Orcutt C.M. , Piacquadio A.M. , White J.M. , Bevan J.A.
Published: February 2010

To evaluate potential pharmacologic agents for the prevention or treatment of the bone loss associated with ovarian insufficiency, a predictable animal model is needed. To assess the potential utility of the ovariohysterectomized dog as a model of this condition, we characterized the sequential histomorphometric changes in canine cancellous bone in response to the loss of ovarian function. A group of 25 adult beagle dogs were ovariohysterectomized and terminated at 1, 3, 6, and 10 months following surgery. Iliac biopsies were performed following double-fluorochrome labeling at the time of surgery and at termination. Static and dynamic histomorphometry was performed on undecalcified sections. By 3 months postovariohysterectomy, there was activation of cancellous bone remodeling as indicated by significant increases in mineralizing surface and bone formation rate. Increases in osteoid surface, mineralizing surface, and bone formation rate were also apparent at 1 month postovariohysterectomy, and although not statistically significant, these trends suggest the skeletal response to acute loss of ovarian function was rapid. This increase in bone remodeling was transient. By 6 months, mineralizing surface and bone formation rate were depressed below presurgical levels. In addition to a reduction in bone formation, a reduction in osteoblast function characterized by reduced labeling of osteoid and a disproportionate increase in eroded surface also occurred. By 10 months postovariohysterectomy, cancellous bone remodeling was not significantly different from presurgical levels. At no time was a significant reduction in bone volume detected. These data suggest that the changes in cancellous bone remodeling in the ovariohysterectomized dog are a series of transient phenomena. The duration and/or magnitude of these changes do not appear to be sufficient to effect a sizable or significant reduction in bone volume. The brief nature of the transients in bone remodeling and the lack of a sizable bone loss in the ovariohysterectomized dog limit the utility of this model for the routine assessment of agents for either the prevention or treatment of bone disease associated with ovarian insufficiency.
Stimulation of collagenase production by rat osteosarcoma cells can occur in a subpopulation of cells
Authors: Strege David W. , Kahn Arnold J. , Jeffrey John J. , Dr. Partridge Nicola C.
Published: February 2010

Recent studies have indicated that neutral collagenase can be produced in bones of rats. In addition, it has been demonstrated by in vitro studies that the enzyme is likely secreted by osteoblasts. Cells of the osteoblastic tumor cell line UMR-106 can be stimulated to produce not only collagenase, but also collagenase inhibitor and plasminogen activator. However, it is conceivable that not all osteoblasts produce all of these proteins. In this study, in which UMR cells were maximally stimulated with PTH, only a subpopulation of cells was observed to produce enhanced levels of collagenase but all cells had the ability to synthesize plasminogen activator.

Cells of the rat osteosarcoma line UMR-106-01 were stained for the presence of collagenase and tissue plasminogen activator using an immunohistochemical procedure. In many cases, the cells were exposed to monensin for the final 3 h of incubation as well as to the inducing agent PTH. Monensin prevented export of the enzymes, enabling them to be visualized within their cell of origin. Maximal stimulation of collagenase was demonstrated to occur 8 h after exposure to 10−8-10−7 M PTH. Under these conditions, 14-17% of the cells appeared to synthesize elevated amounts of collagenase (as determined by intense staining). Without PTH stimulation, there was a low level of collagenase in all cells, but < 1% of the cells stained heavily for the enzyme. In contrast, strong staining for plasminogen activator was observed in all cells with or without PTH treatment. This suggests that the regulation of collagenase production in osteoblasts occurs at the level of the individual cell such that there appear to be subpopulations of cells that respond differently to PTH stimulation.
Human parathyroid hormone-(1-34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats
Authors: Dr. Liu Chung-Ching , Kalu Dike N.
Published: February 2010

A group of 3-month-old Sprague-Dawley rats were sham operated or ovariectomized and given daily injections of human PTH-(1-34) (8 or 16 μg per 100 g body weight) for 5 weeks. At the termination of the study histomorphometric techniques were used to examine changes in cortical and cancellous bone in the diaphysis and proximal metaphysis of the tibia. Ovariectomy resulted in a 50% decrease in cancellous bone that was accompanied by a 41 and 120% increase in osteoclasts and osteoblasts, respectively. In contrast, in the ovariectomized animals treated with PTH, the metaphyseal cancellous bone increased by over 300% to a level in excess of that present in the sham-operated control animals. The increase in cancellous bone induced by PTH was associated with an over 70% increase in osteoblasts and tetracycline-labeled area and an unexpected decrease in trabecular osteoclasts. In the tibial diaphysis PTH also decreased endosteal osteoclasts and at the same time increased osteoblast size and number as well as endosteal and periosteal bone formation; ovariectomy increased only periosteal bone formation. Our findings demonstrate that intermittent administration of PTH prevents ovariectomy-induced bone loss and augments cancellous and cortical bone formation in sexually mature ovariectomized rats. Although the basis of the bone anabolic action of PTH remains elusive, our data indicate that it may involve the uncoupling of bone formation and resorption such that the latter is inhibited as bone formation is enhanced. Our findings are also compatible with the view that intermittent administration of PTH increases bone mass, in part by stimulating the proliferation and differentiation of osteoblast progenitors while inhibiting osteoclast proliferation.
Human PTH-(3–34) inhibited the effects of human parathyroid hormone-related protein on phosphate uptake in a cultured renal cell line (OK cells)
Authors: Dr. Nakai Masamichi , Fukase Masaaki , Yamaguchi Toru , Tsukamoto Tatsuo , Fujii Nobutaka , Fujita Takuo
Published: February 2010

The action mechanism of hPTH and hPTHrP-(1–34) on phosphate uptake in opossum kidney (OK) cells was studied using [Nle8,18Tyr34]hPTH-(3–34)-NH2, a potent competitive inhibitor of adenylate cyclase-coupled PTH receptor. We examined the effects of hPTH-(1–34), hPTHrP-(1–34), and hPTH-(3–34) separately or in combination on the change in renal cyclic AMP production and phosphate uptake in OK cells. Both hPTH-(1–34) and hPTHrP-(1–34) stimulated intracellular cyclic AMP production to the same degree at concentrations between 10−10 and 10−7 M and inhibited phosphate uptake equipotently on a molar basis (27.5 ± 2.0 and 33.2 ± 1.2% inhibition at 10−7 M, respectively). Both exogenous addition of (Bu)2cAMP and endogenous stimulation of cAMP by forskolin inhibited phosphate uptake in a dose-dependent manner. Cyclic AMP production induced by either hPTH-(1–34) or hPTHrP-(1–34) was inhibited by both [Nle8,18Tyr34]-hPTH-(3–34)-NH2 and [Tyr34]-hPTH-(7–34)-NH2. However, [Nle8,18Tyr34]hPTH-(3–34)-NH2 and [Tyr34]-hPTH-(7–34)-NH2 inhibited hPTH-induced cAMP production more strongly. The inhibitory action of phosphate uptake by hPTH-(1–34) and hPTHrP-(1–34) was prevented in the presence of a 100-fold greater concentration of [Nle8,18Tyr34]hPTH-(3–34)-NH2. The antagonistic action of [Nle8,18Tyr34]hPTH-(3–34)-NH2 on the inhibition of phosphate uptake induced by hPTH-(1–34) and hPTHrP-(1–34) became weaker with time (0–120 minutes), and [Nle8,18Tyr34]hPTH-(3–34)-NH2 did not antagonize the inhibition of phosphate uptake induced by hPTHrP-(1–34) at 120 minutes of incubation. Our results indicated that PTHrP inhibits renal phosphate transport, at least in part through an adenylate cyclase-cAMP-coupled PTH receptor, but the response to the competitive inhibition of this peptide-induced cAMP production and inhibition of phosphate uptake with PTH receptor antagonists was different. PTH receptor heterogeneity in OK cells may exist and contribute to this phenomenon.
Characterization of the major parathyroid hormone target cell in the endosteal metaphysis of rat long bones
Authors: Rouleau Marie F. , Mitchell Jane , Dr. Goltzman David
Published: February 2010

The majority of in vivo competitive binding of parathyroid hormone (PTH) in the endosteal metaphysis of rat long bones was recently shown to be localized in the intertrabecular tissue to a cell that is distinct from a differentiated osteoblast. In the present report we have further characterized this cell, termed a parathyroid hormone target (PT) cell, by light and electron microscopy using radioautography and histochemical techniques. These studies demonstrate that the PT cell is a mononuclear cell with a large cell body located at times between clusters of differentiated osteoblasts, as well as in other regions of the intertrabecular tissue. Its long cytoplasmic processes extend from the bone matrix through the intertrabecular region toward vascular structures, interdigitating with various cells of the endosteum. A distinctive tubular structure originating in the Golgi system and often associated with long mitochondria and glycogen particles extends throughout the cytoplasmic processes of the PT cell. Based on its capacity to incorporate [3H]thymidine, the PT cell appears to divide rather slowly. The identification of occasional hybrid cells with ultrastructural features of both the PT cell and the differentiated osteoblast and the presence of histochemical evidence for alkaline phosphatase activity suggest that the PT cell is of the osteoblast lineage. These studies therefore morphologically define a major osseous target cell for PTH that, although of the osteoblast lineage, is not a differentiated osteoblast and provide in vivo evidence that characteristics of the “osteoblast phenotype” are not restricted to a sole osseous cell type.
Ultrastructural localization of a tartrate-resistant acid ATPase in bone
Authors: Dr. Reinholt Finn P. , Widholm Silwa Mengarelli , Ek-Rylander Barbro , Andersson Göran
Published: February 2010

Osteoclasts are effector cells in bone breakdown, and the active bone resorption is confined to the ruffled border zone of these cells. An acid milieu is maintained in this zone which is probably a prerequisite for bone resorption. Tartrate-resistant acid phosphatase (TRAP) activity has been recognized as a characteristic property of osteoclasts and in several studies proposed as a cytochemical marker of osteoclasts. We have previously isolated and characterized a tartrate-resistant and iron-activated acid ATPase (TrATPase) from rat bone, the enzyme being a member of the TRAP family. In the present study the ultrastructural localization of this enzyme was delineated by employing immunogold technique on low temperature-embedded maxillar rat bone. Intensive immunolabeling was seen on the bone surfaces facing the ruffled border zone while lower amounts of marker were seen in adjacent bone areas, that is, on the bone surfaces facing the clear zone and deeper into the bone. Within the osteoclasts gold markers were observed mainly in vesicular structures interpreted as lysosomes. Immunolabeling was also observed in the recently described endocytic cells located near osteoblasts and osteoclasts. Also in these cells, the marker was confined to lysosomelike structures. The amount of label in bone facing osteoblasts was low, as was the amount within osteoblasts. Our observation of extracellular localization, in particular accumulation of TrATPase in bone matrix facing the ruffled border area of the osteoclasts, favors the view that the enzyme is exported to areas of active bone resorption, thereby indicating a potential role for the enzyme in this process.
In vivo effects of human recombinant transforming growth factor β on bone turnover in normal mice
Authors: Marcelli Christian , Yates A. John , Dr. Mundy Gregory R.
Published: February 2010

Reports of the effects of TGF-β on bone cells are conflicting and controversial. Different cell culture and organ culture models for both osteoblasts and osteoclasts have given different responses. In some the effects are dependent on prostaglandin synthesis, and in others they are prostaglandin independent. To determine the effects of TGF-β on osteoblasts and osteoclasts in vivo and the role of prostaglandins in mediating these effects, we injected 2.5–5 μg TGF-β into the subcutaneous tissue overlying the calvariae of normal mice for 2–5 days and compared the morphologic responses in underlying calvarial bone with those in mice injected with vehicle alone. TGF-β treatment had no effect on plasma calcium concentration. However, TGF-β caused a marked increase in periosteal thickness (fivefold) and cellularity, morphologic changes in osteoblasts, and new mineralized bone formation. These effects were localized to the site of injection and were partially inhibited by concomitant indomethacin treatment. There was a parallel increase in osteoclast numbers in adjacent marrow spaces, and the osteoclasts formed were unusually large. In contrast, no increase in the numbers of osteoclasts was seen in indomethacin-treated animals. These data show that TGF-β has powerful effects on local bone cell function in vivo and that these effects may be mediated, in part, by prostaglandin generation.
Synergistic effect of transforming growth factor β and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes
Authors: Crabb Ian D. , O'Keefe Regis J. , Puzas J. Edward , Rosier Randy N.
Published: February 2010

Transforming growth factor β and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-β stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-β and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-β and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-β, corresponded to the time required for [125I]TGF-β receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-β or FGF. Finally, TGF-β and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.
Inhibition of mediator release in systemic mastocytosis is associated with reversal of bone changes
Authors: Graves Leland , Stechschulte Daniel J. , Morris David C. , Lukert Barbara P.
Published: February 2010

A 59-year-old male presented with systemic mastocytosis with extensive skeletal involvement resulting in vertebral compression fractures and bone pain. Histomorphometric analysis of bone revealed increased mast cells, elevated static parameters of bone resorption, and low bone formation. Serum calcium, phosphorus, and alkaline phosphatase were normal; however, serum 1,25-dihydroxyvitamin D3 and osteocalcin levels were low. Histamine levels in plasma and urine were elevated. Following therapy with ketotifen, the patient had resolution of bone pain along with decreased flushing and pruritus. Elevated plasma and urine histamine levels normalized, as did 1,25-dihydroxy vitamin D3 and osteocalcin levels. Indices of low bone formation improved on therapy. Eroded surfaces improved but remained elevated. This case is the first demonstration that bone symptoms and histomorphometric change in systemic mastocytosis are reversed with inhibition of mast cell degranulation. The role of mast cells and their products in bone metabolism is poorly understood, but the therapy of bone disease in systemic mastocytosis should include inhibition of the release of mast cell products along with the use of histamine antagonist.
Corticosteroid effects on proximal femur bone loss
Authors: Dr. Sambrook Philip , Birmingham Joan , Kempler Susan , Kelly Paul , Eberl Stefan , Pocock Nicholas , Yeates Michael , Eisman John
Published: February 2010

Prolonged high-dose corticosteroid therapy is known to result in an increased risk of osteoporotic fracture. Reductions in bone density have been demonstrated at the distal radius and lumbar spine in patients receiving corticosteroids; however there have been few studies of bone density in the hip (the most important site of osteoporotic fracture) in this context. To examine the effect of corticosteroids on the hip we measured bone mineral density (BMD) by dual-photon absorptiometry at three sites in the proximal femur as well as the lumbar spine in 32 patients aged 18–77 years who had been treated with corticosteroids (mean daily prednisone dose 12.7 mg) for up to 23 years. BMD was compared with the expected values using age regressions in normal subjects. BMD was significantly reduced in the femoral neck, Ward's triangle, and the trochanteric region (p < 0.001 all sites). In the lumbar spine BMD was also significantly reduced (p < 0.001). We also measured BMD serially in 29 patients receiving corticosteroids. BMD measurements were made in 12 patients who had already been treated with long-term corticosteroids at the time of first BMD measurement (chronic group) and from the commencement of corticosteroid therapy in 17 patients (acute group). The mean (± SEM) change in BMD (g/cm2 per year) in the lumbar spine and femoral neck were 0.006 ± 0.006 and –0.021 ± 0.007, respectively, for the chronic group and –0.02 ± 0.005 and –0.039 ± 0.006 for the acute group. The rate of bone loss from the lumbar spine was significantly greater in the acute than the chronic group (p < 0.01), and a similar trend was seen in the femoral neck (p = 0.07). These data suggest that bone loss with corticosteroids is most rapid soon after starting treatment and occurs to a similar extent from the proximal femur to the lumbar spine. The bone loss at the femoral neck suggests an increased risk of fracture at that site, particularly in older subjects on long-term corticosteroid therapy.
Treatment of paget's disease with intermittent low-dose infusions of disodium pamidronate (APD)
Authors: Stone Michael D. , Hawthorne A. Barney , Kerr David , Webster Gillian , Hosking David J.
Published: February 2010

Disodium pamidronate (APD) is a potent inhibitor of bone resorption, with less risk of defective mineralization than earlier bisphosphonates. We assessed the response to six spaced low-dose intravenous infusions of APD given at intervals of approximately 6 weeks followed by weekly infusions if bone turnover remained abnormal. Three groups of 10 patients were studied, each group receiving infusions of 15, 30, or 45 mg. Hydroxyproline excretion fell by 62% and alkaline phosphatase was reduced by 72%, with no difference between the dose levels. A total of 21 patients (70%) achieved normal levels of bone turnover, indicating that low-dose infusions of APD are a safe and effective treatment for Paget's disease.
Interleukin-6 does not stimulate bone resorption in neonatal mouse calvariae
Authors: Al-Humidan Abdulkarim , Ralston Stuart H. , Hughes David E. , Chapman Karen , Aarden Lucien , Russell R. Graham G. , Dr. Gowen Maxine
Published: February 2010

Recombinant human interleukin-6 (IL-6) was assessed for its ability to stimulate bone resorption in prelabeled mouse calvariae in vitro. IL-6 had no effect on bone resorption at concentrations ranging from 300 to 10,000 U/ml (3–1000 pg/ml). Neither the presence of indomethacin nor prolonged incubation periods (96 h) affected this result. IL-6 did not affect resorption stimulated by human recombinant IL-1α (rIL-1α) but inhibited resorption stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. rIL-1α, PTH, and 1,25-(OH)2D3 induced IL-6 release by calvariae. We conclude from these studies that IL-6 does not stimulate bone resorption in neonatal mouse calvariae. However, it may act as a locally produced inhibitor and therefore a paracrine regulator of bone resorption induced by osteotropic hormones. IL-6 could also function as a long-range stimulator of systemic reactions and acute-phase responses to local inflammatory and neoplastic lesions in bone.
Insulinlike growth factor II and transforming growth factor β regulate collagen expression in human osteoblastlike cells in vitro
Authors: Dr. Strong Donna D. , Beachler Anthony L. , Wergedal Jon E. , Linkhart Thomas A.
Published: February 2010

Insulinlike growth factor II (IGF-II) and transforming growth factor β (TGF-β) are the most abundant polypeptide growth factors found in human bone matrix and are produced by human bone cells in vitro. IGF-II and TGF-β, increased total protein synthesis, collagenous protein synthesis, and the steady-state level of type I procollagen mRNA in a time-dependent manner in osteoblastlike cells isolated from human bone. Type III procollagen mRNA expression was low in untreated cultures and was not affected by IGF-II or TGF-β. TGF-β, elevated type I procollagen mRNA rapidly, with the maximal observed change at 10 h. In contrast, procollagen mRNA levels increased more slowly in response to IGF-II and reached a lower maximal level than with TGF-β, but the response was sustained through 24 h. Collagenous protein synthesis in IGF-II- and TGF-β-treated cells increased in parallel with increases in procollagen mRNA levels and was higher at 21 h for TGF-β, and at 36 h for IGF-II. The difference in the time course and magnitude of change in type I procollagen mRNA levels in response to IGF-II and TGF-β, suggests that these two growth factors work through distinct mechanisms that provide both a rapid transient response and a later sustained response in bone matrix biosynthetic activity.
In vitro resorptive activity of isolated chick osteoclasts: Effects of carbonic anhydrase inhibition
Authors: Dr. Hunter Susan J. , Rosen Clifford J. , Gay Carol V.
Published: February 2010

A potent inhibitor of carbonic anhydrase, 5-[3-hydroxybenzoyl]thiophene-2-sulfonamide (HTS), was shown to cause a 37% reduction in the area of resorption pits formed by isolated chick osteoclasts when used at a dose of 10−7 M. HTS at doses of 10−9 and 10−7 M was also effective in reducing acid formation by the osteoclasts (14 and 36%, respectively). Additionally, the effect of HTS was found to be readily reversed by removing the agent, showing that it does not exert a toxic effect on the cells. This study indicates that the inhibitory effect of HTS on bone resorption is at the level of the acid-forming mechanism in osteoclasts and supports the view that carbonic anhydrase has a central role in the process.
Bone mass in an urban and a rural population: A comparative, population-based study in Southern Sweden
Authors: Dr. Gärdsell Per , Johnell Olof , Nilsson Bo E. , Sernbo Ingemar
Published: February 2010

Several previous studies have reported regional differences in the incidence of hip fractures. A population-based study was performed in the city of Malmö (urban population) and in the municipality of Sjöbo (rural population), 60 km apart. A total of 961 men and women, randomly selected and of Scandinavian ethnic background, participated in the study. Bone mineral content (BMC) of the forearm was measured with single-photon absorptiometry (SPA).

Women and men in the city had significantly lower BMC compared with the rural population. The differences were even more pronounced when comparing a true urban population (lived their entire life in a city) with a true rural population (never lived in a city). The differences in BMC between Malmö and Sjöbo were more obvious in men.

These data suggest that differences in bone mass between an urban and a rural population could to some extent explain differences in fracture incidence.
Structural requirements for the action of parathyroid hormone-related protein (PTHrP) on bone resorption by isolated osteoclasts
Authors: Evely Roger S. , Bonomo Anthony , Schneider Hans-Gerhard , Moseley Jane M. , Gallagher James , Professor Martin T. John
Published: February 2010

Parathyroid hormone-related protein (PTHrP) plays a major role in the syndrome of humoral hypercalcemia of malignancy (HHM) by its actions on bone and kidney. In this study an isolated osteoclast bone resorption assay was used to investigate the actions of this peptide and the structure-activity relationships for its resorption effect. As with PTH, neither synthetic nor recombinant PTHrP preparations stimulated resorption within highly purified osteoclast populations. Resorption was stimulated only in the presence of contaminating osteoblasts or in cocultures with the osteoblastlike cell line UMR-106. In the presence of osteoblasts PTHrP-(1–34) and PTHrP-(1–84) stimulated bone resorption in a dose-dependent manner with a potency comparable to that of PTH-(1–34) on a molar basis. The biologic activity of the PTHrP was shown to reside in the first 34 amino acids, and within that region the structural requirements for promotion of osteoclastic resorption resembled closely those for promotion of cyclic AMP formation in osteoblastlike cells. Using emulsion autoradiography with iodinated PTHrP-(1–34) and PTHrP-(1–84) on mixed bone cell preparations from neonatal rats, specific binding was demonstrated only to osteoblasts, not to osteoclasts. These results clearly demonstrate that PTHrP is a potent stimulator of bone resorption and that these effects are, like those of PTH, mediated by initial actions upon cells of the osteoblast lineage.
Calcium regulation of parathyroid and C cell function in familial benign hypercalcemia
Authors: Rajala Mary M. , Klee George G. , Heath Hunter
Published: February 2010

The roles of parathyroid hormone (PTH) and calcitonin (CT) in the pathogenesis of familial benign hypercalcemia (FBH, or hypocalciuric hypercalcemia) are uncertain. Thus we performed studies in 26 patients with FBH, 12 patients with primary hyperparathyroidism (HPT), and 20 normal volunteers, to answer these questions: are plasma levels of intact or biologically active PTH frequently elevated in FBH? Is plasma intact PTH nonsuppressible during calcium infusion? Is there blunting of the C cell CT response to calcium infusion as occurs in primary HPT? We used three methods for measurement of PTH: a mid region-specific radioimmunoassay (iPTH, antiserum GP-1M), an extraction-concentration bioassay (bioPTH, stimulation of cAMP generation in osteoblastlike cells), and a two-site immunoradiometric assay (IRMA) for intact PTH. PTH levels were significantly elevated in primary HPT by all three methods, but mean PTH was normal in FBH and 85–92% of values overlapped the normal range. During 5 minute calcium infusions (2 mg Ca2+ per kg) iPTH values fell little, but bioPTH and intact PTH fell sharply in all three groups. Mean calcium-induced decreases of intact and bioPTH were indistinguishable from normal in FBH, but PTH levels generally remained elevated at 5 minutes in primary HPT. In FBH basal and postinfusion CT levels were normal. The data show that, in the majority of patients with FBH, PTH concentrations and bioactivity in blood are within the normal range and are suppressed rapidly to very low levels with further increases of calcium. The data suggest that the abnormality of parathyroid function in FBH differs from that in primary HPT. There was no deficiency of CT or C cell responsiveness in FBH, another difference from primary HPT. Nonetheless, in 8–15% of cases, measurement of PTH could not discriminate FBH from primary HPT.