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T R Arnett

Publications and source records attributed to T R Arnett.

At least 19 recordsLinked to original sources
PubMed2001

Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y(1) receptor on bone cells.

There is increasing evidence that extracellular nucleotides act on bone cells via P2 receptors. This study investigated the action of ADP and 2-methylthioADP, a potent ADP analog with selectivity for the P2Y(1) receptor, on osteoclasts, the bone-resorbing multinuclear cells. Using three different assays, we show that ADP and 2-methylthioADP at nanomolar to submicromolar levels caused up to fourfold to sixfold increases in osteoclastic bone resorption. On mature rat osteoclasts, cultured for 1 day on polished dentine disks, peak effects on resorption pit formation were observed between 20 nM and 2 microM of ADP. The same concentrations of ADP also stimulated osteoclast and resorption pit formation in 10-day mouse marrow cultures on dentine disks. In 3-day explant cultures of mouse calvarial bones, the stimulatory effect of ADP on osteoclast-mediated Ca(2+) release was greatest at 5-50 microM and equivalent to the maximal effects of prostaglandin E(2). The ADP effects were blocked in a nontoxic manner by MRS 2179, a P2Y(1) receptor antagonist. Using in situ hybridization and immunocytochemistry, we found evidence for P2Y(1) receptor expression on both osteoclasts and osteoblasts; thus, ADP could exert its actions both directly on osteoclasts and indirectly via P2Y(1) receptors on osteoblasts. As a major ATP degradation product, ADP is a novel stimulator of bone resorption that could help mediate inflammatory bone loss in vivo.

Adenosine Diphosphate↗
PubMed2001

pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis.

We examined the effects of HCO(3)(-) and CO(2) acidosis on osteoclast-mediated Ca(2+) release from 3-day cultures of neonatal mouse calvaria. Ca(2+) release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H(+) to culture medium (HCO(3)(-) acidosis). For example, addition of 4 meq/l H(+) reduced pH from 7.12 to 7.03 and increased Ca(2+) release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H(+), were comparable to the maximal Ca(2+) release elicited by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3); 10 nM], parathyroid hormone (10 nM), or prostaglandin E(2) (1 microM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from approximately 7.1 to approximately 7.3. CO(2) acidosis was a less effective stimulator of Ca(2+) release than HCO(3)(-) acidosis over a similar pH range. Ca(2+) release stimulated by HCO(3)(-) acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, approximately 400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO(3)(-) acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO(3)(-) acidosis, however, reduced numbers of osteoclast-like cells by approximately 50%, whereas 1,25(OH)(2)D(3) treatment caused increases of approximately 75%. The results suggest that HCO(3)(-) acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.

Acid-Base Equilibrium↗
PubMed2000

Expression of P2 receptors in bone and cultured bone cells.

Extracellular nucleotides acting through P2 receptors elicit a wide range of responses in many cell types. There is increasing evidence that adenosine triphosphate (ATP) may function as an important local messenger in bone and cartilage. In this study, we used immunocytochemistry, employing novel polyclonal antibodies against P2X(1-7) receptors, and in situ hybridization, using oligonucleotide probes corresponding to P2X(2,4) and P2Y(2,4) messenger RNAs (mRNAs), to localize P2 receptors on undecalcified bone sections and on cultured osteoblasts and osteoclasts. We provide the first direct evidence that the P2X(2) receptor subtype is expressed on osteoclasts, osteoblasts, and chondrocytes. We also obtained evidence for the expression of P2X(5) and P2Y(2) receptors on osteoblasts and chondrocytes, and for P2X(4) and P2X(7) receptors on osteoclasts. Our results confirm earlier reports of P2Y(2) and P2X(4) expression in human osteoclastoma and rabbit osteoclasts, respectively, and are consistent with ATP responses observed on bone cells using electrophysiological techniques. Our novel finding that P2X(2) is expressed by osteoclasts is of particular interest. P2X(2) is the only P2 receptor subtype that requires extracellular acidification to show its full sensitivity to ATP, and our recent functional studies have shown that the stimulatory action of ATP on resorption pit formation by mature osteoclasts is amplified greatly at low pH. These findings point to fundamental new mechanisms for the local modulation of bone resorption.

Adenosine Triphosphate↗
PubMed2000

Bisphosphonates induce apoptosis in human breast cancer cell lines.

Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50 values of 15, 20 and 3 microM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 microM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells.

Antineoplastic Agents↗
PubMed1998

ATP is a potent stimulator of the activation and formation of rodent osteoclasts.

1. There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non-selective cation channels and Ca2+-dependent K+ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation. 2. We found that ATP stimulated resorption pit formation up to 5.6-fold when osteoclast-containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0.2-2 microM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6.9-7.0). Pit formation by acid-activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto-ATPase and by suramin, an antagonist of P2 receptors. 3. Over the same concentration range at which rat osteoclast activation occurred (0.2-2 microM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3.3-fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20-200 microM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation. 4. These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts.

Adenosine Triphosphate↗
PubMed1998

The Escherichia coli chaperonin 60 (groEL) is a potent stimulator of osteoclast formation.

Chaperonins (cpns) are intracellular oligomeric protein complexes that fold and refold proteins in a catalytic manner and aid in the transmembrane transport of cellular proteins. We reported previously that the lipopolysaccharide-free recombinant cpn60 of Escherichia coli (groEL) is able to stimulate the breakdown of murine calvarial bone in culture and showed that such resorption is potently inhibited by an inhibitor of the enzyme cyclo-oxygenase and to a lesser extent by inhibitors of 5-lipoxygenase. In this study, we have investigated the effects of groEL on the resorptive activity and formation of osteoclasts in culture. In low density, osteoclast-containing cultures from neonatal rats incubated for 24 or 96 h on dentine discs, groEL (1-1000 ng/ml) stimulated resorption pit formation up to 4-fold, but this effect was essentially dependent on cell number. Using 12-day cultures of mouse bone marrow to assess osteoclast recruitment, groEL (1-1000 ng/ml) caused a dramatic dose-dependent stimulation of the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and the resorption of the dentine on which bone marrow cells were cultured. Osteoclast formation elicited by groEL was almost completely abolished by indomethacin, an inhibitor of cyclo-oxygenase, but was unaffected by inhibitors of 5-lipoxygenase, suggesting that prostaglandins but not leukotrienes may mediate the action of groEL on osteoclastogenesis. It is possible that bacterial cpn60s such as groEL may play a role in the osteolysis associated with bone infections. Whether endogenous ("self") chaperonins have a role in other bone loss disorders, such as osteoporosis, is an intriguing possibility.

Acid Phosphatase↗
PubMed1996

Modulation of the resorptive activity of rat osteoclasts by small changes in extracellular pH near the physiological range.

We investigated the effect of small shifts in extracellular pH on the resorptive activity of rat osteoclasts in vitro. Osteoclast-containing mixed bone cell populations disaggregated from neonatal rat long bones were cultured for 26 h at low density on bovine bone disks in HCO-3/CO2-buffered medium modified by the addition of small amounts of protons as HCl (2.5-15.0 mEq/L) or hydroxyl ions as NaOH (2.5-10.0 mEq/L). Little or no bone resorption occurred when ambient pH was above about 7.30; at pH 7.00, osteoclasts exhibited near-maximal activity. The greatest changes were associated with a pH difference of as little as 0.10 unit, between pH 7.25 and pH 7.15. Over this narrow range, the average number of resorption pits formed on each bone wafer increased sixfold. The steep sigmoidal pH response curve indicates that very slight alterations in ambient hydrogen ion concentration can effectively "switch on" or "switch off" rat osteoclasts in vitro.

Analysis of Variance↗
PubMed1996

Expression of insulin-like growth factor I (IGF-I) mRNA variants in rat bone.

The rat insulin-like growth factor I (IGF-I) gene is characterized by the presence of multiple mRNA transcripts, which differ in the 5'- and/or 3'-untranslated regions (UTRs). Transcript initiation occurs in either exon 1 or exon 2, giving rise to mRNA species which differ in the length and sequence of the 5'-UTR, while further variation is due to multiple transcription start sites and differential splicing within exon 1. This heterogeneity is indicative of multifaceted regulation of gene expression, and it is likely that differences in the nature of transcript expression reflect cell-specific regulation. As IGF-I is an important factor in skeletal growth and development, the aim of this study was to determine the pattern of transcript expression in rat whole bone and osteoblast-enriched cultures isolated from long bones. The relative proportions of transcripts differing in the 5'-UTR were determined by RNase protection assays and compared to expression in rat liver. These studies revealed a significantly lower expression of exon 2-derived transcripts in bone cells compared to liver (approximately 10% compared to 40% of total transcripts). There were also important differences in start site usage in exon 1 in bone cells. In osteoblastic cells, transcripts initiated at start site 3 were the predominant species (50% +/- 12% of total exon 1-derived mRNAs; Mean +/- SD) whereas the alternately spliced transcripts represented only 20% +/- 3%. This was in contrast to the profile in liver in which 47% +/- 9% of total exon 1-derived transcripts were the alternately spliced mRNAs, but start site 3-initiated transcripts represented only 11% +/- 3%. In addition, the proportion of transcripts initiated at start site 4 was about twofold greater in liver than in bone cells (32% +/- 7% compared to 16% +/- 8%). However, expression of full-length transcripts was similar in both tissues. The distribution in osteoblastic cells reflected that in whole bone. These results demonstrate that the IGF-I transcript profile in bone cells differs to that in liver cells. Since the mRNA variants exhibit different properties, including half-life and translatability, such cell-specific variation in their relative expression is likely to reflect differential regulation of IGF-I in these tissues.

Animals↗
PubMed1996

Selective toxic effects of tamoxifen on osteoclasts: comparison with the effects of oestrogen.

We investigated the actions of the trans- and cis-isomers of tamoxifen on the function of neonatal rat osteoclasts in vitro. Both compounds inhibited resorption pit formation by osteoclast-containing mixed bone cell cultures incubated for 24 h on cortical bone slices. Cell counts revealed that the inhibition was closely related to a cytotoxic effect, to which osteoclasts appeared particularly sensitive. Partial inhibition of resorption was seen in the presence of 2 microM trans-tamoxifen, whereas complete abolition of resorption and osteoclast viability occurred with 10 microM trans-tamoxifen; survival of mononuclear cells was unimpaired at either concentration. Cis-tamoxifen appeared to be slightly more toxic, with significant inhibitions of osteoclast viability and thus resorption pit formation at a concentration of 2 microM, and also of mononuclear cell numbers at 10 microM. Time-lapse video observations indicated that osteoclast death occurred rapidly (within 2-3 h) following exposure to 10 microM of either trans-tamoxifen or cis-tamoxifen. The morphological appearance of the dying cells was consistent with apoptosis. These results may help to explain the anti-resorptive action of tamoxifen seen in vivo in rats and humans. In contrast, oestradiol-17 beta consistently exerted no significant effects on resorption pit formation by rat osteoclasts over 24 h, even at grossly supraphysiological concentrations (up to 10 microM).

Animals↗
PubMed1994

Effects of medium acidification by alteration of carbon dioxide or bicarbonate concentrations on the resorptive activity of rat osteoclasts.

Little is known about the extracellular conditions or factors that stimulate mature osteoclasts to resorb mineralized tissues. Isolated mammalian osteoclasts are strongly stimulated by protons in HEPES-buffered culture media in the absence of CO2 and HCO3-, but it has been reported that cell-mediated Ca2+ release from bone organ cultures is increased only when media are acidified by reduction of HCO3- concentrations, and not by increasing PCO2 (considered models of metabolic and respiratory acidosis, respectively). We investigated this question using disaggregated rat osteoclasts cultured on dentin slices for 24 h. The number of pits resorbed per osteoclast was stimulated in media acidified by manipulation of either HCO3- or CO2 concentrations. In experiments in which incubator CO2 was varied, resorption was almost abolished in the presence of 2.5% CO2 at pH 7.61 but increased in a stepwise manner up to 1.3 pits per osteoclast when dentin slices were cultured with 10% CO2 at pH 6.97. The depths and widths of pits, measured using a confocal laser reflection microscope, also tended to increase with increasing CO2 and decreasing pH. However, in experiments where pH was lowered by reducing medium HCO3-, pit size decreased, partially offsetting the increased number of pits resorbed per osteoclast. These findings suggest that rat osteoclasts may be more sensitive to stimulation by CO2 acidosis than by HCO3- acidosis, at least in the short term, and may possibly reflect local regulatory processes in bone.

Acid Phosphatase↗
PubMed1993

Differences in proximal femur bone density over two centuries.

The incidence of osteoporotic hip fractures in northern Europe has been increasing over the past few decades faster than the rate adjusted for increased life expectancy. One important factor that determines osteoporotic fracture risk is bone density. The restoration of a London church, during which skeletal material dating from 1729 to 1852 was recovered, gave us the opportunity to compare the rate of bone loss in the femora of these samples with that of present-day women. The rate of bone loss, as judged by dual energy X-ray absorptiometry, was significantly greater in modern-day women than in the women from two centuries ago, both pre-menopausally (p < 0.05) and post-menopausally (p < 0.01). The difference in bone loss in the Ward's triangle region between the Spitalfields samples and present-day women remained even when the assumed age at menopause was increased to 48 years or decreased to 42 years. The results suggest that differences in rates of bone loss over two centuries may partly account for the increasing incidence of hip fracture in modern-day women. Reasons for these differences are unclear, but one factor may be a lower degree of physical activity in present-day women.

Adolescent↗
PubMed1992

Interaction of androgen and 1,25-dihydroxyvitamin D3: effects on normal rat bone cells.

We studied the actions of testosterone (T) and 5 alpha-dihydrotestosterone (DHT) in combination with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on primary rat bone cells. The actions of androgens were generally anabolic, although response patterns varied considerably between cultures. For example, DHT caused striking dose- and time-dependent increases in [3H]thymidine incorporation into calvarial cells over the range 1-100 nM, with maximal stimulation of 2.5-fold after 9 days in culture. Testosterone (50 nM) also stimulated [3H]thymidine incorporation into long bone-derived cells. 1,25-(OH)2D3 generally blunted or abolished the proliferative action of androgens but was not itself always inhibitory; in some experiments, stimulation of [3H]thymidine incorporation occurred. Collagen production, as assessed by [3H]proline incorporation into pepsin-resistant protein secreted by calvarial cells, was also stimulated by DHT. In some cultures, androgen responses were absent, although striking inhibitory responses to 1,25-(OH)2D3 were observed. These results illustrate complex patterns of responses to androgens and 1,25-(OH)2D3 in cells derived from rat bone.

Alkaline Phosphatase↗
PubMed1992

Rapid neural growth: calcitonin gene-related peptide and substance P-containing nerves attain exceptional growth rates in regenerating deer antler.

Deer antler is a unique mineralized tissue which can produce very high growth rates of > 1 cm/day in large species. On completion of antler growth, the dermal tissues which cover the antler are shed and the underlying calcified tissue dies. After several months the old antler is discarded and growth of a new one begins. It is known that deer antlers are sensitive to touch and are innervated. The major aims of this study were to identify and localize by immunohistochemical techniques the type of innervation present, and to find out whether nerve fibres could exhibit growth rates comparable to those of antler. We have taken tissue sections from the tip and shaft of growing Red deer (Cervus elaphus) antlers at three stages of development; shortly after the initiation of regrowth, the rapid growth phase, and near the end of growth. Incubation of tissue sections with antisera to protein gene product 9.5 (a neural cytoplasmic protein), neurofilament triplet proteins (a neural cytoskeletal protein), substance P and calcitonin gene-related peptide (both of which are present in and synthesized by sensory neurons) showed the presence of immunoreactive nerve fibres in dermal, deep connective and perichondrial/periosteal tissues at all stages of antler growth. The sparse distribution of vasoactive intestinal polypeptide-like immunoreactivity was found in dermal tissue only at the earliest stage of antler development. Nerve fibres immunoreactive to neuropeptide Y, C-flanking peptide of neuropeptide Y and tyrosine hydroxylase, all present in postganglionic sympathetic nerves, were not observed at any stage of antler growth. Nerves expressing immunoreactivity for any of the neural markers or peptides employed could not be found in cartilage, osteoid or bone. These results show that antlers are innervated mainly by sensory nerves and that nerves can attain the exceptionally high growth rates found in regenerating antler.

Animals↗
PubMed1992

Effect of retinoic acid on the resorptive activity of chick osteoclasts in vitro.

Mixed cell suspensions mechanically isolated from the long-bones of day 20 prehatch chicks were cultured for 24 h on bone and sperm whale dentine slices in the presence of 0, 10, 100, and 1000 nM retinoic acid (RA). Significant inhibitions in the numbers of discrete lacunae resorbed per dentine slice, and in the ratio of lacunae per tartrate-resistant acid phosphatase-positive multinuclear cell were observed with all concentrations of RA studied. Semi-automated, 3-dimensional analysis of 733 pits was performed on one series of experiments using a tandem scanning (confocal) microscope, interfaced to an image analyzing computer. The majority of lacunae were small and unilocular; the plan areas of 90% of control pits were below 500 microns 2. Small but statistically significant increases in lacunar areas, but not mean or maximum depths or volumes, were observed in the presence of 10 and 100 nM RA; however, these changes were much smaller than the magnitude of the decrease in pit numbers. Thus, the overall effect of RA in this system was inhibitory. Our findings contrast with the well known stimulatory action of retinoids on bone resorption both in vivo and in organ culture, but may parallel the inhibitory effects of prostaglandins observed in disaggregated osteoclast systems.

Animals↗
PubMed1991

Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osteoclasts.

Studies with a range of monoclonal and polyclonal antisera to components of the human, rat, and chick vitronectin receptor, alpha V beta 3, and the VLA beta 1 chain show that chick and rat osteoclasts express similar integrin receptors to those described in man. Biochemical analysis with monoclonal antibody 23C6 confirmed the presence on chick osteoclasts of a vitronectin receptor heterodimer of similar size (110/95 kDa reduced) to that immunoprecipitated from human osteoclastoma giant cells. The synthetic peptide GRGDSP, corresponding to the cell adhesion sequence in fibronectin, but not GRGESP peptide, induced significant (P less than 0.005) osteoclast retraction in chick and rat osteoclasts at IC50s (+/- SEM) of 210.0 +/- 14.4 and 191.4 +/- 13.7 microM, respectively; monoclonal anti-vitronectin receptor alpha V beta 3 complex antibody, 23C6, produced similar changes in chick osteoclasts (IC50 = 1.45 +/- 0.22 microM). Antibody 23C6 inhibited the number of pits resorbed in dentine by chick osteoclasts over a concentration range of 4.4 to 88 micrograms/ml; a significant 76% reduction (P = 0.03) was observed at a final concentration of 88 micrograms/ml (6 microM). The effect of peptides upon dentine resorption was less dramatic. No consistent inhibition was seen using chick osteoclasts. Inhibitory effects on resorption by rat osteoclasts were, however, observed; significant reduction in resorption occurred with both GRGDSP (78%; P less than 0.01) and GRGESP (67%; P = 0.02) peptides at 400 microM peptide concentration. These data demonstrate that osteoclast function can be disrupted by low concentrations of the anti-vitronectin receptor antibody, 23C6. The inhibitory effects of the peptides used in this study produced effects on dentine resorption which were generally weaker and variable, although osteoclast cell adhesion was consistently inhibited in an Arg-Gly-Asp (RGD)-dependent manner. We conclude that the vitronectin receptor may play an important role in effecting resorption of mineralized tissues by osteoclasts.

Amino Acid Sequence↗