Cell density and growth-dependent down-regulation of both intracellular calcium responses to agonist stimuli and expression of smooth-surfaced endoplasmic reticulum in MC3T3-E1 osteoblast-like cells.
A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.
Koizumi
Toshiyuki
T
Department of Oral and Maxillofacial Surgery, Faculty of Medicine, and Health Service Centre, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan.
Hikiji
Hisako
H
Shin
Wee Soo
WS
Takato
Tsuyoshi
T
Fukuda
Satoru
S
Abe
Takahiro
T
Koshikiya
Noboru
N
Iwasawa
Kuniaki
K
Toyo-oka
Teruhiko
T
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation; D002455:Cell Division; D018871:Endoplasmic Reticulum, Smooth / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007700:Kinetics; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology
D002110:Caffeine; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium