| Research Abstract |
It is generally accepted that cytosolic free Ca^<2+> ([Ca^<2+>]i) is one of second messengers as is cAMP. We had investigated the effect of parathyroid hormone (PTH) on alkaline phosphatase (ALP) activity and cAMP production in the presence or absence of extracellular Ca^<2+> at various concentrations in rat dental pulp cells (DP cells), including odontblasts. To compare this responses of DP cells with that of bone-forming cells (BF cells, i.e. osteoblast-like cells) in primary culture, we examined the effects of various reagents on [Ca^<2+>]i in BF cells.
In the first year, we established the isolation technique of BF cells from newborn (or neonatal) mouse calvaria using sequential enzyme digestion. Furthermore, we studied the basic technique of subculture with maintaining the differentiated functions of the cells. On the other hand, to master the method of [Ca^<2+>]i measurement using a Nikon microspectrofluorometry system, we first examined the effect of NaF on [Ca^<2+>]i in single L
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-929 mouse fibroblasts cultured on coverslips (the single cell method). F- induced a transient increase in [Ca^<2+>]i.
In BF cells in premary culture, we demonstrated that [Ca^<2+>]i rapidly elevated in response to phosphatidic acid (PA). In primary cultures, however, the single cells method is unsuitable to [Ca^<2+>]i measurement, since it is impossible to avoid contamination of many cell types. such as fibroblasts. To resolve this problem, we tried to establish an osteoblast-like cell line.
In the final year, we established a clonal osteoblast-like cell line (MOB 3-4) derived from neonatal mouse calvaria. In the cells, an increase in the culture density enhanced ALP activity and induced the response to PTH. The pattern of the NaF-increased [Ca^<2+>]i was similar to that of the PTH action in the cells in a dense culture. In addition, the cells display many osteoblastic characteristics including high bone-specific ALP activity, production of I collagen, and the responses to PTH, PGE2, and 1,25(OH)2D3 resulting in increased ALP activity and [Ca^<2+>]i.
In conclusion, it is suggested that cytosolic free Ca^<2+> may modulate the regulation of ALP activity in a clonal osteoblast-like cell line, MOB 3-4, and that this modulation by Ca^<2+> may be applied to odontblasts. Less
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