Hormonal, pH, and calcium regulation of connexin 43-mediated dye transfer in osteocytes in chick calvaria

Yoshihito Ishihara, Hiroshi Kamioka, Tadashi Honjo, Hirotaka Ueda, Teruko Takano-Yamamoto, Takashi Yamashiro

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Gap junctional intercellular communication among osteocytes in chick calvaria, their natural 3D environment, was examined using FRAP analysis. Cell-cell communication among osteocytes in chick calvaria was mediated by Cx43 and was regulated by extracellular pH, extracellular calcium ion concentration, and PTH. Introduction: The intercellular network of communication among osteocytes is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is thought to play an important role in integration and synchronization of bone remodeling. We hypothesized that extracellular pH (pHo) and extracellular calcium ion concentration ([Ca 2+]e), both of which are dynamically altered by osteoclasts during bone remodeling, affect GJIC among osteocytes. Using fluorescence replacement after photobleaching (FRAP) analysis, we examined the effect of changes in pHo and [Ca2+]e and addition of PTH on GJIC in osteocytes in chick calvaria. Additionally, we examined the role of intracellular calcium on the regulation of GJIC among osteocytes. Materials and Methods: Anti-Connexin43 (Cx43) immunolabeling was used to localize gap junctions in chick calvaria. GJIC among osteocytes in chick calvariae was assessed using FRAP. Results: Cx43 immunoreactivity was detected in most of the osteocyte processes. FRAP analysis showed dye-coupling among osteocytes in chick calvariae. In untreated osteocytes, fluorescence intensity recovered 43.7 ± 2.2% within 5 min after photobleaching. Pretreatment of osteocytes with 18 α-GA, a reversible inhibitor of GJIC, significantly decreased fluorescence recovery to 10.7 ± 2.2%. When pHo was decreased from 7.4 to 6.9, fluorescence recovery significantly decreased from 43.3 ± 2.9% to 19.7 ± 2.3%. Conversely, when pHo was increased from 7.4 to 8.0, fluorescence recovery was significantly increased to 61.9 ± 4.5%. When [Ca2+]c was increased from 1 to 25 mM, fluorescence recovery was significantly decreased from 47.0 ± 6.1% to 16.1 ± 2.1%. In bone fragments exposed to 1.0-10 nM rPTH for 3 h, replacement of fluorescence was significantly increased to 60.7 ± 7.2%. Chelating intracellular calcium ions affected GJIC regulation by [Ca 2+]e and PTH. Conclusions: Our study of cell-cell communication between osteocytes in chick calvaria showed for the first time that GJIC among osteocytes is regulated by the extracellular environment and by hormonal stimulation during bone remodeling. This method may be more biologically relevant to living bone than current methods.

Original languageEnglish
Pages (from-to)350-360
Number of pages11
JournalJournal of Bone and Mineral Research
Volume23
Issue number3
DOIs
Publication statusPublished - Mar 2008

Keywords

  • Extracellular calcium
  • Extracellular pH
  • Fluorescence replacement after photobleaching
  • Gap junction
  • Intercellular communication
  • Intracellular calcium
  • Osteocyte
  • PTH

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Orthopedics and Sports Medicine

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