Clomiphene, an ovulation-inducing agent, causes [Ca ²⁺ ] _i increases in human osteoblast-like cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca ²⁺ levels ([Ca ²⁺ ] _i ) in MG63 human osteosarcoma cells was explored by using fura-2 as a Ca ²⁺ indicator. Clomiphene at concentrations between 5-75 μM increased [Ca ²⁺ ] _i in a concentration-dependent manner with an EC ₅₀ of 50 μM. The [Ca ²⁺ ] _i signal consisted of an initial rise and a sustained phase. Ca ²⁺ removal reduced the Ca ²⁺ signal by 40±10%. The [Ca ²⁺ ] _i increase induced by 50 μM clomiphene was inhibited by 80±5% by 10 μM nifedipine, but was insensitive to 50 μM La ³⁺ or 10 μM verapamil. In Ca ²⁺ -free medium, pretreatment with 50 μM brefeldin A (to disrupt the Golgi complex Ca ²⁺ store), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca ²⁺ pump), and carbonylcyanide m-chlorophenylhydrazone (CCCP; to uncouple mitochondria) inhibited 51±3% of 50 μM clomiphene-induced Ca ²⁺ release; conversely, pretreatment with 50 μM clomiphene abolished the [Ca ²⁺ ] _i increase induced by thapsigargin, CCCP, and brefeldin A. The Ca ²⁺ release-induced by 50 μM clomiphene was unchanged by inhibition of phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Collectively, the results suggest that clomiphene increased [Ca ²⁺ ] _i in osteoblast-like cells, by releasing intracellular Ca ²⁺ in a phospholipase C-independent manner and by causing nifedipine-sensitive Ca ²⁺ influx.