Calcified Tissue International

, Volume 51, Issue 4, pp 285–290

Characterization of gap junctions between osteoblast-like cells in culture

  • Karin Schirrmacher
  • Inge Schmitz
  • Elke Winterhager
  • Otto Traub
  • Franz Brümmer
  • David Jones
  • Dieter Bingmann
Laboratory Investigations

Summary

The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4–5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.

Key words

Osteoblast-like cells Gap junctions Connexin43 Electrical and dye coupling 

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References

  1. 1.
    Holtrop ME, Weinger MJ (1971) Proc 4th Parathyroid Conf, Int Congress Series No 243. In: Fawcett DW (ed) Bloom and Fawcett: A textbook of histology. Excerpta Medica. AmsterdamGoogle Scholar
  2. 2.
    Doty SB (1981) Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33:509–512Google Scholar
  3. 3.
    Bennett MVL, Goodenough DA (1978) Gap junctions, electronic coupling, and intercellular communication. Neurosci Res Prog Bull 16:373–485Google Scholar
  4. 4.
    Edelman A, Fritsch J, Balsan S (1986) Short-term effects of PTH on cultured rat osteoblasts: changes in membrane potential. Am J Physiol 251:C483-C490Google Scholar
  5. 5.
    Hohman EL, Elde RP, Rysavy JA, Einzig S, Gebhard RL (1986) Innervation of periosteum and bone by sympathetic vasoactive intestine peptide-containing fibres. Science 232:868–871Google Scholar
  6. 6.
    Ferrier J, Ward-Kesthely AW, Homble F, Ross S (1987) Further analysis of spontaneous membrane potential activity and the hyperpolarizing response to parathyroid hormone in osteblast-like cells. J Cell Physiol 130:344–351Google Scholar
  7. 7.
    Chesnoy-Marchais D, Fritsch J (1988) Voltage-gated sodium and calcium currents in rat osteoblasts. J Physiol 398:291–311Google Scholar
  8. 8.
    Bingmann D, Tetsch P, Massass R (1988) Membraneigen-schaften von Zellen aus Knochenexplantaten. Z Zahnärztl Implantol IV:277–281Google Scholar
  9. 9.
    Bingmann D, Tetsch P, Massass R (1988) Membrane properties of bone cells derived from calvaria of newborn rats (tissue culture). Pflügers Arch S412:R14Google Scholar
  10. 10.
    Massass R, Bingmann D, Korenstein R, Tetsch P (1990) Membrane potential of rat calvaria bone cells: dependence on temperature. J Cell Physiol 144:1–11Google Scholar
  11. 11.
    Rubin CT, Lanyon LE (1985) Regulation of bone mass by mechanical strain magnitude. Calcif Tissue Int 37:411–417Google Scholar
  12. 12.
    Lanyon LE (1984) Functional strain as a determinant for bone remodelling. Calcif Tissue Int 36:556–561Google Scholar
  13. 13.
    Jones DB, Scholübbers J-G (1988) Mechanical stress stimulates phospholipase C and proteinkinase C in periost-derived, but not in haversian-derived osteoblast-like cells in vitro. In: Heuck FHW, Keck E (eds) Fortschritte der Osteologie. Springer Verlag, Berlin-Heidelberg, pp 313–326Google Scholar
  14. 14.
    Jones DB (1989) Vasoactive intestinal peptide stimulates PI-PLC and a rapid raise in intercellular calcium in bone surface cells. Calcif Tissue Int 44 (suppl) S41:G20Google Scholar
  15. 15.
    Jones DB, Nolte H, Scholübbers J-G, Turner E, Veltel D (1991) Biochemical signal transduction of mechanical strain in osteoblast-like cells. Biomaterials 12:101–110Google Scholar
  16. 16.
    Spray DC, Bennett MVL (1985) Physiology and pharmacology of gap junctions. Ann Rev Physiol 47:281–302Google Scholar
  17. 17.
    Hertzberg EL, Johnson RG (1988) Gap junctions. In: Modern cell biology, vol. 7, Liss, New YorkGoogle Scholar
  18. 18.
    Beyer EC (1990) Molecular cloning and developmental expression of two chick embryo gap junction protiens. J Biol Chem 265:14439–14443Google Scholar
  19. 19.
    Barckhaus RH, Bingmann D, Wittkowski W, Tetsch P (1989) Mineralisation in Calvaria-Kulturen: Eine elektronenmikroskopische und röntgenstrahlmikroanalytische Untersuchung. Beitr Elektronenmikroskop Direktabb Oberfl 22:371–380Google Scholar
  20. 20.
    Dixon SJ, Aubin JE, Dainty J (1984) Electrophysiology of a clonal osteoblast-like cell line: evidence for the existence of a Ca2+-activated K+ conductance. J Membr Biol 80:49–58Google Scholar
  21. 21.
    Jeansonne BG, Feagin FF, McMinn RW, Shoemaker RL, Rehm WS (1979) Cell-to-cell communication of osteoblasts. J Dent Res 58(4):1415–1423Google Scholar
  22. 22.
    Jones DB, Schlübbers J-G, Althoff J, Becker M, Ryaby JT (1987) The effect of bone morphogenic protein and pulsed electromagnetic fields on bovine osteoblast-like cells. Trans ORS J Bone Joint Surg 33:00Google Scholar
  23. 23.
    Dalton AJ (1955) A chrome-osmium fixative for electron microscopy. Anat Rec 121:281Google Scholar
  24. 24.
    Traub O, Look J, Dermietzel R, Brümmer F, Hülser D, Willeke K (1989) Comparative characterization of the 21kDa and 26kDa gap junction proteins in murine liver and cultured hepatocytes. J Cell Biol 108:1039–1051Google Scholar
  25. 25.
    Lowry O, Rosebrough N, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 266–275Google Scholar
  26. 26.
    Traub O, Look J, Paul D, Willeke K (1987) Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26kD gap junction protein in cultured mouse hepatocytes. Eur J Cell Biol 43:48–54Google Scholar
  27. 27.
    Stewart WW (1978) Functional connections between cells as revealed by dye-coupling with a high fluorescent naphthalimid tracer. Cell 14:741–759Google Scholar
  28. 28.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–688Google Scholar
  29. 29.
    Yamazaki K (1989) Cell-mediated calcification in collagen gel cultures of fetal rat calvaria cells. Loss of gap junctions precedes calcification. J Bone Miner Metab 7(3):6–17Google Scholar
  30. 30.
    Kuffler SW, Nicholls JG, Martin AR (1984) From neuron to brain. Sinauer Assoc Inc, Sunderland, MassachusettsGoogle Scholar
  31. 31.
    Haas HG, Meyer R, Einwächter HM, Stockem W (1983) Intercellular coupling in frog heart muscle. Electrophysiological and morphological aspects. Pflügers Arch 399(4):321–335Google Scholar
  32. 32.
    Beyer EC, Paul DL, Goodenough DA (1987) Cx43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 1055:2621–2629Google Scholar
  33. 33.
    Yancey SB, John SA, Lal R, Austin BJ, Revel J-P (1989) The 43-kD polypeptide of heart gap junctions: immunolocalization, topology and functional domains. J Cell Biol 108:2241–2254Google Scholar
  34. 34.
    Yamamoto T, Ochalski A, Hertzberg EL, Nagy JI (1990) LM and EM immunolocalization of the gap junctional protein cx 43 in rat brain. Brain Res 508:313–319Google Scholar
  35. 35.
    Dermietzel R, Hertzberg EL, Kessler JA, Spray DC (1991) Gap junctions between cultured astrocytes: immunocytochemical, molecular, and electrophysiological analysis. J Neurosci 11(5):1421–1432Google Scholar
  36. 36.
    Nicholson J, Dermietzel R, Teplow D, Traub O, Willeke K, Revel J-P (1987) Two homologous protein components of hepatic gap junctions. Nature (London) 329:732–774Google Scholar
  37. 37.
    Dermietzel R, Traub O, Hwang TK, Beyer E, Bennett MVL, Spray DC, Willeke K (1989) Differential expression of three gap junction proteins in developing and mature brain tissue. Proc Natl Acad Sci USA 66:10148–10152Google Scholar
  38. 38.
    Crow DS, Beyer EC, Paul DL, Kobe SS, Lau AF (1990) Phosphorylation of cx 43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol Cell Biol 10:1754–1763Google Scholar
  39. 39.
    White RL, Doeller JE, Verselis K, Wittenberg BA (1990) Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca2+. J Gen Physiol 95:1061–1075Google Scholar
  40. 40.
    Rook MB, Jongsma HJ, van Ginneken AC (1988) Properties of single gap junctional channels between isolated neonatal rat heart cells. Am J Physiol 255:H770-H782Google Scholar
  41. 41.
    Maurer P, Weingart R (1987) Cell pairs isolated from adult guinea pig and rat hearts: effects of [Ca2+]i on nexal membrane resistance. Pflügers Arch 409:394–402Google Scholar
  42. 42.
    Weingart R (1986) Electrical properties of the nexal membrane studied in rat ventricular cell pairs. J Physiol 370:267–284Google Scholar
  43. 43.
    Sugiura H, Toyama J, Tsuboi N, Kamiya K, Kodama I (1990) ATP directly affects junctional conductance between paired ventricular myocytes isolated from guinea pig heart. Circ Res 66:1095–1102Google Scholar
  44. 44.
    Saez JC, Connor JA, Spray DC, Bennett MVL (1989) Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-triphosphate, and to calcium ions. Proc Natl Acad Sci USA 86:2708–2712Google Scholar
  45. 45.
    Ferrier J, Illeman A, Zakshek E (1985) Transient and sustained effects of hormones and calcium on membrane potential in a bone cell clone. J Cell Physiol 122:53–58Google Scholar

Copyright information

© Springer-Verlag New York Inc 1992

Authors and Affiliations

  • Karin Schirrmacher
    • 1
  • Inge Schmitz
    • 3
  • Elke Winterhager
    • 2
  • Otto Traub
    • 4
  • Franz Brümmer
    • 5
  • David Jones
    • 6
  • Dieter Bingmann
    • 1
  1. 1.Institute für PhysiologieUniversität-GHSEssenFRG
  2. 2.Institute für AnatomieUniversität-GHSEssenFRG
  3. 3.Institut für Pathologie, BergmannsheilBochumFRG
  4. 4.Institut für GenetikUniversitätBonnFRG
  5. 5.Biologisches InstitutStuttgart 80FRG
  6. 6.Institut für ZellbíologìeOrthopädische KlinikMünsterFRG

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