Physiological activity of osteoblast including bone formation is known to be closely related to the increase of intracellular Ca2+ activity ([Ca2+]i) in osteoblast. Ca2+ is an important intracellular
messenger
in diverse cellular functions, and regulation of its level is mediated by the transmembrance Ca2+ movement via Ca2+ channels, Na+-Ca2+ exchange, and by intracellular Ca2+ movement through the
intracellular stroes. The purpose of this study is to investigate how the intracellular Ca2+ is regulated in osteoblast-like cells(OLCs) by measuring Ca2+ activity with cell imaging technique. OLCs were isolated from femur
and
tibia of neonatal rats, and cultured for 7 days. Cultured OLCs were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored with a cooled CCD camera. The images were processed and analyzed with an
image
analyzing software. The results were as follows. (1) [Ca2+]i of OLC decreased as the Ca2+ concentration in the superfusing Tyrode solution was lowered. When Na+ concentration in the superfusing solution
was
decreased, [Ca2+]i increased.. These suggest that Ca2+ flux occurs via the Na+-Ca2+ exchange mechanism. (2) When Na+ in the superfusing solution was removed, a transient
Ca2+
increase(Ca2+ spike) was occasionally observed. However, Ca2+ spike was not observed after adding 1 μM thapsigargin. This implies that the generation of Ca2+ spike is mediated by the release of Ca2+
from
endoplasmic reticulum(ER). (3) As the Ca2+ concentration in the superfusing solution was raised, the frequency of 0mM Na+-induced Ca2+ spike increased, suggesting that Ca2+-induced Ca2+
release(CICR) mechanism exists. (4) After [Ca2+]i was decreased with the supefusion of Ca2+-free solution containing thapsigargin, the recovery of [Ca2+]i with reperfusion of 2.5mM
Ca2+
solution transiently exceeded the control level, suggesting that the reperfusion of 2.5mM Ca2+ solution transiently exceeded the control level, suggesting that the depletion of Ca2+ in ER indeces Ca2+ influx from
extracellular medium via store-operated Ca2+ influx(SOCI) mechanism. (5) [Ca2+]i was not affected by the superfusion of 25mM K+ Tyrode solution. These results suggest that intracellular Ca2+
activity in osteoblast is regulated by transmembrance Ca2+ flux via Na+-Ca2+ exchange, Ca2+ release from the internal store (ER) via Ca2+-induced Ca2+ release, and stroe-operated
Ca2+ influx acorss the cell membrane.
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