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.