Intracellular calcium responses to basic calcium phosphate crystals in fibroblasts.

OBJECTIVE

To examine the intracellular calcium response to basic calcium phosphate (BCP) crystals in fibroblasts.

DESIGN

In this study, intracellular calcium [Ca2+]i levels in fibroblasts were determined using the photoactive dye, fura-2. Interruption of these responses was accomplished by either removal of Ca2+ from the extracellular medium or addition of ammonium chloride that inhibits intracellular dissolution of BCP crystals by alkalinizing phagolysosomes. The effects of such interruptions on BCP induction expression of proto-oncogenes were demonstrated by the Northern blot analysis.

RESULTS

Addition of media containing BCP crystals yielded an immediate 10-fold rise of [Ca2+]i over the baseline level in human fibroblasts. This peak was derived mostly from extracellular calcium and was not seen when BCP crystals in calcium-free media were added to fibroblasts. The [Ca2+]i concentration returned to the baseline level within 8 min. A second rise of [Ca2+]i started at 60 min and continued to increase up to at least 3 h. This peak was derived from intracellular dissolution of phagocytosed crystals and almost completely inhibited by 10 mM ammonium chloride.

CONCLUSION

The initial transient [Ca2+]i increase probably serves as a second messenger leading to activation of early cellular responses such as c-fos expression which is important in BCP crystal-induced mitogenesis. The second, slower and more sustained rise of [Ca2+]i probably initiates other cellular processes needed for fibroblast mitogenesis.