Abstract
Objective: To examine the intracellular calcium response to basic calcium phosphate (BCP) crystals in fibroblasts.
Design: In this study, intracellular calcium [Ca2+]ilevels 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+]iover the baseline level in human fibroblasts. This peak was derived mostly from extracelluar calcium and was not seen when BCP crystals in calcium-free media were added to fibroblasts. The [Ca2+]iconcentration returned to the baseline level within 8 min. A second rise of [Ca2+]istarted 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 mmammonium chloride.
Conclusion: The initial transient [Ca2+]iincrease probably serves as a second messenger leading to activation of early cellular responses such as c-fosexpression which is important in BCP crystal-induced mitogenesis. The second, slower and more sustained rise of [Ca2+]iprobably initiated other cellular processes needed for fibroblast mitogenesis.
