Effect of calmidazolium on Ca²⁺ movement and proliferation in human osteosarcoma cells

1. In human MG63 osteosarcoma cells, the effect of calmidazolium on [Ca²⁺]_i and proliferation was explored using fura-2 and ELISA, respectively. 2. Calmidazolium, at concentrations greater than 0.1 μmol/L, caused a rapid increase in [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ = 0.5 μmol/L). The calmidazolium-induced [Ca²⁺]_i increase was reduced by 66% by removal of extracellular Ca²⁺. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, caused a monophasic increase in [Ca²⁺]_i, after which the effect of calmidazolium to increase [Ca²⁺]_i was completely inhibited. U73122, an inhibitor of phospholipase C (PLC), abolished histamine (but not calmidazolium)-induced increases in [Ca²⁺]_i. Pretreatment with phorbol 12-myristate 13-acetate to activate protein kinase C inhibited the calmidazolium-induced increase in [Ca²⁺]_i in Ca²⁺-containing medium by 47%. 3. Separately, it was found that overnight treatment with 2-10 μmol/L calmidazolium inhibited cell proliferation in a concentration-dependent manner. 4. These results suggest that calmidazolium increases [Ca²⁺]_i by stimulating extracellular Ca²⁺ influx and also by causing release of intracellular Ca²⁺ from the endoplasmic reticulum in a PLC-independent manner. Calmidazolium may be cytotoxic to osteosarcoma cells.