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

Abstract

A two-dimensional intracellular Ca2+ ([Ca2+]i) imaging system was used to examine the relationship between [Ca2+]i handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca2+]i level in densely cultured cells was 1.5 times higher than the [Ca2+]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 [Ca2+]i level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 μM), caffeine (10 mN), thapsigargin (1 μM), or ionomycin (10 μM), and the effect on the [Ca2+]i level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca2+]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 Ca2+ response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca2+]i channels. All Ca2+ 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 Ca2+ response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca2+ 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 [Ca2+]i regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.