Journal of Electron Microscopy Advance Access originally published online on December 21, 2006
Journal of Electron Microscopy 2006 55(5):265-271; doi:10.1093/jmicro/dfl033
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Mechanism of Ca2+ increase in myoblasts derived from chicken embryos
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. E-mail: stabata{at}agr.kyushu-u.ac.jp
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
Received 11 April 2006, accepted 3 November 2006