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Primary Cultures of Chick Osteocytes Retain Functional Gap Junctions between Osteocytes and between Osteocytes and Osteoblasts

Published online by Cambridge University Press:  15 February 2007

Hiroshi Kamioka ,
Yoshihito Ishihara ,
Hans Ris ,
Sakhr A. Murshid ,
Yasuyo Sugawara ,
Teruko Takano-Yamamoto  and
Soo-Siang Lim
Hiroshi Kamioka
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Yoshihito Ishihara
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Hans Ris
Affiliation:
Department of Zoology and Integrated Microscopy Resource, University of Wisconsin, Madison, WI 53706-1794, USA
Sakhr A. Murshid
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Yasuyo Sugawara
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Teruko Takano-Yamamoto
Affiliation:
Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Soo-Siang Lim
Affiliation:
National Science Foundation, Arlington, VA 22230, USA
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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 (18α-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.

Keywords

osteocytesosteoblastsgap junctionsintercellular communicationdye couplingFRAP
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 13 , Issue 2 , April 2007 , pp. 108 - 117
Copyright
© 2007 Microscopy Society of America

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