title: Primary cultures of chick osteocytes retain functional gap junctions between osteocytes and between osteocytes and osteoblasts.
abstract: The inaccessibility of osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to osteocyte and between adjacent osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18alpha-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent osteocytes and between osteoblasts and osteocytes. This role is consistent with the ability of osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.
authors: Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan. kamioka@md.okayama-u.ac.jp
authors: Hiroshi
authors: H
authors: Kamioka
authors: Yoshihito
authors: Y
authors: Ishihara
authors: Hans
authors: H
authors: Ris
authors: Sakhr A
authors: SA
authors: Murshid
authors: Yasuyo
authors: Y
authors: Sugawara
authors: Teruko
authors: T
authors: Takano-Yamamoto
authors: Soo-Siang
authors: SS
authors: Lim
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002642:Chick Embryo; D018031:Connexin 43 / Q000502:physiology; D005456:Fluorescent Dyes / Q000737:chemistry; D017629:Gap Junctions / Q000502:physiology*; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007546:Isoquinolines / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D010011:Osteocytes / Q000166:cytology / Q000502:physiology*; D012886:Skull / Q000166:cytology / Q000502:physiology*
chemical_list: D018031:Connexin 43; D005456:Fluorescent Dyes; D007546:Isoquinolines; C119129:18alpha-glycyrrhetinic acid; C017475:lucifer yellow; D006034:Glycyrrhetinic Acid