Biomedical subjects

B Burt-Pichat

Publications and source records attributed to B Burt-Pichat.

9 recordsLinked to original sources

PubMed2006

Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells.

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.

Animals↗

PubMed2005

Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A.

Animals↗

PubMed2005

Relationship between compressive properties of human os calcis cancellous bone and microarchitecture assessed from 2D and 3D synchrotron microtomography.

The aim of this study was to determine the contribution of 2D and 3D microarchitectural characteristics in the assessment of the mechanical strength of os calcis cancellous bone. A sample of cancellous bone was removed in a medio-lateral direction from the posterior body of calcaneus, taken at autopsy in 17 subjects aged 61-91 years. The sample was first used for the assessment of morphological parameters from 2D morphometry and 3D synchrotron microtomography (microCT) (spatial resolution=10 microm). The 2D morphometry was obtained from three slices extracted from the 3D microCT images. Very good concordance was shown between 3D microCT slices and the corresponding physical histologic slices. In 2D, the standard histomorphometric parameters, fractal dimension, mean intercept length, and connectivity were computed. In 3D, histomorphometric parameters were computed using both the 3D mean intercept length method and model-independent techniques. The 3D fractal dimension and the 3D connectivity, assessed by Euler density, were also evaluated. The cubic samples were subjected to elastic compressive tests in three orthogonal directions (X, Y, Z) close to the main natural trabecular network directions. A test was performed until collapse of trabecular network in the main direction (Z). The mechanical properties were significantly correlated to most morphological parameters resulting from 2D and 3D analysis. In 2D, the correlation between the mechanical strength and bone volume/tissue volume was not significantly improved by adding structural parameters or connectivity parameter (nodes number/tissue volume). In 3D, one architectural parameter (the trabecular thickness, Tb.Th) permitted to improve the estimation of the compressive strength from the bone volume/tissue volume alone. However, this improvement was minor since the correlation with the BV/TV alone was high (r=0.96). In conclusion, which is in agreement with the statistic's rules, we found, in this study, that the determination of the os calcis bone compressive strength using the 3D bone volume fraction cannot be improved by adding 3D architectural parameters.

Aged↗

PubMed2004

Dramatic decrease of innervation density in bone after ovariectomy.

Recent studies have demonstrated that bone is highly innervated and contains neuromediators that have functional receptors on bone cells. However, no data exist concerning the quantitative changes of innervation during bone loss associated with estrogen withdrawal. To study the involvement of nerve fibers in the regulation of bone remodeling, we have evaluated the modifications of innervation in a classical in vivo model of osteopenia in rats, ovariectomy (OVX). Skeletal innervation was studied by immunocytochemistry using antibodies directed against specific neuronal markers, neurofilament 200 and synaptophysin, and the neuromediator glutamate. Sciatic neurectomy, another model of bone loss due to limb denervation and paralysis, was used to validate our quantitative image analysis technique of immunostaining for nerve markers. Female Wistar rats at 12 wk of age were sham-operated (SHAM) or ovariectomized (OVX). Bone mineral density measurement and bone histomorphometry analysis of tibiae 14 d after surgery demonstrated a significant bone loss in OVX compared with SHAM. We observed an important reduction of nerve profile density in tibiae of OVX animals compared with SHAM animals, whereas innervation density in skin and muscles was similar for OVX and control rats. Quantitative image analysis of immunostainings demonstrated a significant decrease of the percentage of immunolabeling per total bone volume of neurofilament 200, synaptophysin, and glutamate in both the primary and secondary spongiosa of OVX rats compared with SHAM. These data indicate for the first time that OVX-induced bone loss in rat tibiae is associated with a reduction in nerve profile density, suggesting a functional link between the nervous system and the bone loss after ovariectomy.

Animals↗

PubMed1999

High-resolution computed tomography for architectural characterization of human lumbar cancellous bone: relationships with histomorphometry and biomechanics.

The aim of the present study on human vertebral cancellous bone was to validate structural parameters measured with high-resolution (150 microm) computed tomography (HRCT) by referring to histomorphometry and to try to predict mechanical properties of bone using HRCT. Two adjacent vertical cores were removed from the central part of human L2 vertebral body taken after necropsy in 22 subjects aged 47-95 years (10 women, 12 men; mean age 79 +/- 14 years). The right core was used for structural analysis performed by both HRCT and histomorphometry. Two cancellous bone specimens were extracted from the left core: a cube for HRCT and a compression test, and a cylinder for a shear test. Significant correlations were found between HRCT and histomorphometric measurements (BV/TV, trabecular thickness, separation and number, and node-strut analysis), but with higher values for most of the tomographic parameters (BV/TV and trabecular thickness determined by HRCT were overestimated by a factor 3.5 and 2.5 respectively, as compared with histomorphometry). The maximum compressive strength and Young's modulus were highly correlated (rho = 0.99, p<0.0005). Significant correlation was obtained between bone mineral density (determined using dual-energy X-ray absorptiometry) and the maximum compressive strength (rho = 0. 64, p = 0.002). In addition the maximum compressive strength and architectural parameters determined by HRCT or histomorphometry showed significant correlations (e.g., for HRCT, BV/TV: rho = 0.88, p<0.0005, N.Nd/TV: rho = 0.73, p<0.001). The shear strength was significantly correlated with BV/TV (rho = 0.62, p = 0.002), Tb.Sp (rho = -0.58, p = 0.004) and TSL (rho = 0.55, p = 0.006) measured by HRCT. In conclusion, an HRCT system with 150 microm resolution is not sufficient to predict the true values of the structural parameters measured by histomorphometry, although high correlations were found between the two methods. However, we showed that a resolution of 150 microm allowed us to predict the mechanical properties of human cancellous bone. In vivo peripheral systems with such a resolution should be of interest and would deliver an acceptable radiation dose to the patient.

Aged↗

PubMed1998

Glutamate receptors are expressed by bone cells and are involved in bone resorption.

The excitatory amino acid glutamate (Glu) is a potent neurotransmitter in the central nervous system and exerts its action via a variety of glutamate receptors (GluRs). Because we had previously shown that a poly-glutamate (poly-Glu) peptide stimulates bone resorption in vitro, an effect specific to Glu (Raynal, C., Delmas, P. D., and Chenu, C. Bone sialoprotein stimulates in vitro bone resorption. Endocrinology 137:2347-2354; 1996), we investigated the possibility that bone cells express GluRs, and whether they may be important for osteoclast activity. Using immunocytochemistry on rat bone sections, we have shown that all mature bone cells (osteoblasts and osteoclasts) express GluRs, and that the ionotropic N-methyl-D-aspartate (NMDA) receptor 1 subunit (NMDAR1) appears most highly expressed. Osteoclasts isolated from rabbit long bones also possessed NMDAR1 GluRs. Bone resorption in vitro by isolated osteoclasts was inhibited by a monoclonal antibody directed against NMDAR1, as well as by two pharmacological antagonists of this receptor (D-AP5 and MK 801), which also antagonized poly-Glu stimulated bone resorption. These results suggest a possible new mechanism for regulating osteoclast activity and indicate that excitatory amino acids such as glutamate may be important local regulators of bone cell functions.

2-Amino-5-phosphonovalerate↗

PubMed1996

Variations of muscle mitochondrial creatine kinase activity in mitochondrial diseases.

Mitochondrial creatine kinase (mtCK) activity has been measured in the mitochondria isolated from the muscle of 69 patients suspected of mitochondrial diseases. The isolated mitochondria did not contain significant amounts of the muscle isoform of creatine kinase, as checked by an immunoassay performed after electrophoretic separation of the various isoforms. Hence, the enzyme assay reliably represented the mtCK activity. Therefore, a simple measurement of CK activity in isolated mitochondria permitted the measurement of mtCK activity. An absence of mtCK activity in muscle was never observed. The lowest activities were not associated to defined mitochondrial diseases linked to defects of respiratory chain complexes or to defects of citric cycle enzymes. On the contrary, mtCK activity was significantly increased in the muscle of patients exhibiting ragged red fibers. This increase was generally associated to an increase of citrate synthase activity. Since ragged-red fibers and elevated mtCK activities were generally not found in children younger than 3 years, even in cases of characteristic oxidative phosphorylation deficiency, it is suggested that the increase in mtCK activity as well as the appearance of ragged-red fibers are not the first events which occur during the evolution of mitochondrial diseases but would rather be long-term secondary processes which slowly develop in deficient mitochondria.

Adolescent↗

PubMed1996

Molecular histology of mitochondrial and nuclear transcripts in the muscle of patients harbouring a single mitochondrial DNA deletion.

The distribution of transcripts of mitochondrial and nuclear genes involved in oxidative phosphorylation and of the mitochondrial creatine kinase nuclear gene was examined, using in situ hybridisation, in the skeletal muscle of 11 patients harbouring a heteroplasmic mitochondrial DNA (mtDNA) single deletion. Levels of mRNAs transcribed from genes located within the deletions were not decreased, suggesting that the remaining wild-type mtDNA was still transcribed. Those muscle fibres with characteristic abnormal mitochondrial proliferation always showed overexpression of mRNAs and rRNAs transcribed from mitochondrial genes located outside the deletions. Interestingly, they also showed overexpression of the nuclear-encoded ATP synthase beta subunit mRNA, but not of mitochondrial creatine kinase mRNA. These observations lead to three proposals: (1) overexpression of mitochondrial transcripts within fibres harbouring mitochondrial proliferation, together with the apparently normal expression of the remaining wild-type mtDNA, is not related to decreased mitochondrial translation; (2) it is more probably related to an up-regulation mechanism which co-ordinates both mitochondrial and nuclear expression; and (3) this mechanism is restricted to transcripts directly involved in oxidative phosphorylation and to fibres with mitochondrial accumulation.

Adolescent↗

PubMedDate not supplied

Molecular identification of NMDA glutamate receptors expressed in bone cells.

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor has recently been identified in bone, but the molecular composition of this receptor expressed by bone cells is unknown. NMDA receptor (NMDAR) is a hetero-oligomeric protein composed of two classes of subunits, the essential subunit NR1 and NR2A to D subunits that do not by themselves produce functional channels but potentiate NR1 activity and confer functional variability to the receptor. These subunits coassemble in different combinations to form functionally distinct NMDAR. In this study, we have investigated the molecular composition of NMDAR expressed by osteoblasts and osteoclasts in culture, using RT-PCR analysis, in situ hybridization and immunocytochemistry. Specific probes were designed for the different subunits of the NMDAR, and we showed by RT-PCR analysis that mammalian osteoclasts expressed NR2B and NR2D subunits mRNAs but not NR2A and NR2C mRNAs. Rat calvaria and MG63 osteoblastic cells also expressed several NR2 subunits mRNAs, namely NR2A, NR2B, and NR2D. In situ hybridization on isolated rabbit osteoclasts and MG63 cells has confirmed the localization of NR1, NR2B, and NR2D transcripts in osteoclasts and NR1, NR2A, NR2B, and NR2D transcripts in MG63 cells. The expression of NR2D protein by bone cells was shown by immunofluorescence. These results demonstrate for the first time that osteoblasts and osteoclasts express several NR2 subunits, suggesting a molecular diversity of NMDAR channels similar to what was shown for brain. The presence of distinct functional NMDAR on bone cells may be associated with various states of bone cell differentiation and function.

Animals↗