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 mumol/L, caused a rapid increase in [Ca2+](i) in a concentration-dependent manner (EC50=0.5 mumol/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 mumol/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.