title: Isolated primary osteocytes express functional gap junctions in vitro.
abstract: The osteocyte is the most abundant cell type in bone and is embedded in mineralized bone matrix. Osteocytes are still poorly characterized because of their location and the lack of primary osteocyte isolation methods. Data on the cell biology of osteocytes is especially limited. We have isolated primary osteocytes from rat cortical bone by applying repeated enzymatic digestion and decalcification. The isolated osteocytes expressed typical osteocytic morphology with cell-cell contacts via long protrusions after a 1-day culture. These cells were negative or faintly positive for alkaline phosphatase but expressed high levels of osteocalcin, PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome), and DMP1 (dentin matrix protein 1). These cells also revealed patchy membrane staining for connexin43. For studying the function of gap junctions in isolated osteocytes, we microinjected rhodamine-labeled dextran (MW: 10,000) and Lucifer yellow (MW: 457) and found that Lucifer yellow was rapidly transmitted to several surrounding cells, whereas dextran remained in the injected cells. Heptanol and 18alpha-glycyrrhetinic acid inhibited the transfer of Lucifer yellow. This clearly showed the existence of functional gap junctions in cultured osteocytes. Enveloped viruses, such as vesicular stomatitis virus and influenza A virus, were used for studying cell polarity. We were unable to demonstrate plasma membrane polarization with enveloped viruses in isolated primary osteocytes in culture. Our results suggest that osteocytes do not possess apical and basolateral plasma membrane domains as do osteoblasts, which are their precursors.
authors.affiliation: Department of Anatomy, Institute of Biomedicine, University of Turku, Turku 20520, Finland.
authors.forename: Guoliang
authors.initials: G
authors.lastname: Gu
authors.forename: Martin
authors.initials: M
authors.lastname: Nars
authors.forename: Teuvo A
authors.initials: TA
authors.lastname: Hentunen
authors.forename: Kalervo
authors.initials: K
authors.lastname: Metsikkö
authors.forename: H Kalervo
authors.initials: HK
authors.lastname: Väänänen
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D002469:Cell Separation; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D019850:Heptanol / Q000494:pharmacology; D007546:Isoquinolines / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D010011:Osteocytes / Q000166:cytology* / Q000378:metabolism* / Q000821:virology; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D014760:Viral Fusion Proteins / Q000378:metabolism
chemical_list: D018031:Connexin 43; C081635:Dmp1 protein, rat; D016326:Extracellular Matrix Proteins; D007546:Isoquinolines; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D014760:Viral Fusion Proteins; D015675:Osteocalcin; D019850:Heptanol; C017475:lucifer yellow; D000469:Alkaline Phosphatase; D008666:Metalloendopeptidases; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; C506229:Phex protein, rat; D006034:Glycyrrhetinic Acid