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Journal of Electron Microscopy Advance Access published online on December 21, 2006
Journal of Electron Microscopy, doi:10.1093/jmicro/dfl033
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© The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received April 11, 2006
Accepted November 3, 2006

Full-length: Biological

Mechanism of Ca2+ increase in myoblasts derived from chicken embryos

Shoji Tabata 1 *, Yoh Takemura 1, Masaaki Kobayashi 1, Masachika Ikeda 1, Shotaro Nishimura 1, Yusuke Sato 2, Ryuichi Tatsumi 2, Yoshihide Ikeuchi 2, and Hisao Iwamoto 1

1 Laboratory of Functional Anatomy, Division of Animal Science, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan
2 Laboratory of Chemistry and Technology of Animal Products, Division of Applied Biological Chemistry, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan

* To whom correspondence should be addressed.
Shoji Tabata, E-mail: stabata{at}agr.kyushu-u.ac.jp


  Abstract

The mechanism of intracellular calcium ions (Ca2+) increase in chicken myoblasts was studied using histological, immunohistochemical, immunoblotting and Ca2+ imaging techniques. Mononuclear myoblasts at embryonic day 12 (E12) contained myofibrils in the peripheral cytoplasm, and the sarcoplasmic reticulum was observed in the cytoplasm. Several Ca2+-related receptors, namely acetylcholine (ACh) receptors, dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), were detected in the tissue as early as E12. Western blotting analyses detected one band corresponding to RyR subtype 3 (RyR3) at E12 and two bands corresponding to RyR1 and RyR3 after E13. Ca2+ imaging of mononuclear myoblasts in vitro revealed an intense Ca2+-increase response to ACh stimulation, and this effect was abolished after EGTA addition to the culture medium. Nifedipine treatment also led to a lack of Ca2+ increase in response to ACh stimulation, while ryanodine treatment led to a weak Ca2+-increase response. On the other hand, multinuclear myoblasts showed a Ca2+-increase response to ACh stimulation in the presence of not only EGTA but also nifedipine, although ryanodine treatment led to a lack of Ca2+ increase. These results suggest that the mechanism of Ca2+ increase in mononuclear myoblasts involves extracellular Ca2+ entry through DHPRs, which is amplified by Ca2+ release from the cytoplasmic Ca2+ store, while multinuclear myoblasts mainly depend on Ca2+ release from the cytoplasmic Ca2+ store.

Keywords: Ca2+ imaging; chicken; immunohistochemistry; myoblast; transmission electron microscopy; western blotting.
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