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  • 1
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 64 (1997), S. 161-170 
    ISSN: 0730-2312
    Keywords: calcium-regulating hormone ; bone cells ; acridine orange ; signal transduction ; GTP-binding protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The involvement of multiple G-proteins in parathyroid hormone regulation of acid production was demonstrated in a highly enriched osteoclast population. Osteoclasts were isolated from the endosteum of 2.5 to 3-week-old chicken tibia using sequential enzymatic digestion. Single cell analysis of acid production was accomplished using microscope photometry and vital staining with acridine orange, a hydrogen ion concentration sensitive fluorescent dye. Lithium chloride, an uncoupler of G-proteins from their respective receptors, blocked parathyroid hormone stimulated production of acid. Cholera toxin, which permanently activates Gs-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with Gi- and Go-proteins, blocked both 10 8 M and 10 11 M PTH stimulated acid production, suggesting that the pertussis toxin-sensitive G-protein is utilized at both PTH concentrations. Immunoblots of osteoclast plasma membrane proteins, using a panel of antibodies generated against specific G-protein α subunits, revealed a 48 kDa Gsα, a 41 Goα, a 34 kDa Giα-3, and a unique 68 kDa Gα subunit, with the 41 kDa and 34 kDa bands being the most intense. Immunoblots of osteoblast plasma membrane proteins had a substantially different profile with the most intense bands being a Gsα (48 kDa) and a Goα (36 and 38 kDa). The studies suggest the utilization of at least two different G-proteins in the parathyroid hormone regulation of acid formation by osteoclasts, a Gs and a pertussis toxin-sensitive G-protein (Go and/or Giα-3). J. Cell. Biochem. 64:161-170. © 1997 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 65 (1997), S. 565-573 
    ISSN: 0730-2312
    Keywords: calcium-regulating hormones ; bone cells ; acridine orange ; signal transduction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteoclasts, isolated from the endosteum of 2.5- to 3-week-old chickens, were treated with acridine orange, a hydrogen ion concentration-sensitive fluorescent dye, in order to monitor changes in acid production. The adenylate cyclase inhibitor, alloxan, blocked parathyroid hormone (PTH)-stimulated acid production. Dibutyryl cyclic adenosine monophosphate, a membrane-permeant form of cyclic adenosine monophosphate, mimicked the PTH effect. Bisindolylmaleimide, a specific inhibitor of protein kinase C (PKC), blocked the initial stimulation (15, 30, and 60 min) of acid production by PTH but had no effect on long-term stimulation (120 min). Confocal microscopy of osteoclasts stained with fluorescein-conjugated bisindolylmaleimide revealed a shift in location of PKC from the cytoplasm to the plasma membrane region after treatment with parathyroid hormone. The results of these studies support the hypothesis that PTH regulation of acid production in osteoclasts involves both adenylate cyclase and PKC as effectors. J. Cell. Biochem. 65:565-573. © 1997 Wiley-Liss Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 71 (1998), S. 204-215 
    ISSN: 0730-2312
    Keywords: osteoclast ; spectrin ; membrane skeleton ; bone ; bone resorption ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The presence of spectrin was demonstrated in chick osteoclasts by Western blotting and light and electron microscopic immunolocalization. Additionally, screening of a chick osteoclast cDNA library revealed the presence of α-spectrin. Light microscope level immunocytochemical staining of osteoclasts in situ revealed spectrin staining throughout the cytoplasm with heavier staining found at the marrow-facing cell margin and around the nuclei. Confocal microscopy of isolated osteoclasts plated onto a glass substrate showed that spectrin encircled the organelle-rich cell center. Nuclei and cytoplasmic inclusions were also stained and the plasma membrane was stained in a nonuniform, patchy distribution corresponding to regions of apparent membrane ruffling. Ultracytochemical localization showed spectrin to be found at the plasma membrane and distributed throughout the cytoplasm with especially intense staining of the nuclear membrane and filaments within the nuclear compartment. J. Cell. Biochem. 71:204-215, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 372-382 
    ISSN: 0730-2312
    Keywords: bFGF ; extracellular matrix ; in situ hybridization ; RT-PCR ; immunocytochemistry ; cell proliferation ; Western blotting ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Basic fibroblast growth factor (bFGF) is a permissive mitogen for cultured chondrocytes and has been localized in the specific zones of the epiphyseal growth plate. In this study, we demonstrate that bFGF present in cartilage originates from within the cellular constituents of this tissue. Utilizing reverse transcription coupled to the polymerase chain reaction (PCR), bFGF mRNA was found in extracts of cartilage tissue. Immunocytochemical studies revealed that bFGF was present intracellularly in freshly isolated proliferative chondrocytes and in the extracellular matrix (ECM) after 24 h of culture. Western blot analysis of protein extracts from isolated proliferative chondrocytes identified a bFGF immunoreactive species with a molecular weight of approximately 18 kDa. In situ hybridization confirmed the presence of bFGF mRNA in freshly isolated proliferative chondrocytes. The bFGF in the ECM seemed to be sequestered and not available for biological activity, since these cells still required exogenous bFGF for cell proliferation. This sequestered bFGF could be released to stimulate cell proliferation when cultures were treated with plasmin, a proteolytic enzyme. These data support the hypothesis that bFGF is synthesized by chondrocytes and functions as an autocrine/paracrine mitogen via its deposition into the ECM with subsequent release from the ECM of cartilage being a critical step in biological activity. In addition, the study provides further evidence that locally produced bFGF plays an important role in normal growth and development of cartilage tissue. © 1996 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 143 (1990), S. 100-104 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Effects of interstitial fluid flow on osteoblasts were investigated. Intracellular cyclic adenosine monophosphate (cAMP) levels were monitored in cultured osteo-blasts subjected to shear rates ranging from 10 to 3,500 sec-1. Cyclic AMP levels were significantly increased at all shear rates from 1 pmole/mg protein to 10-16 pmole/mg protein. Osteoblasts subjected to a shear rate of 430 sec-1 for 0.5-15 minutes exhibited elevated levels (12-fold) of intracellular cAMP, which were sustained throughout the perfusion period. Osteoblasts were three times more sensitive to flow stimulation than human umbilical vein endothelial cells and baby hamster kidney fibroblasts, which also displayed higher cAMP levels (4-fold) after exposure to flow. To distinguish streaming potential effects from shear stress effects, viscosity was increased 5-fold by addition of neutral dextran to the perfusing medium. Shear stress is a function of viscosity, and streaming potentials are not for a given shear rate. The mechanism of this cellular response to flow was shown to be shear stress dependent. Inhibition of cyclooxygenase by 20 μM ibuprofen completely inhibited the flow-dependent cAMP response, indicating the cAMP response is mediated by prostaglandins. Our results suggest that fluid flow induced by mechanical stress may be an important mediator of bone remodeling.
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