Ca²⁺ mobilization induced by W-7 in MG63 human osteosarcoma cells

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a widely used calmodulin inhibitor, on intracellular free Ca²⁺ levels ([Ca²⁺](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca²⁺ probe. W-7 (20-1000 μM) induced an increase in [Ca²⁺](i) in a dose-dependent manner, with an EC₅₀ of 100 μM. The [Ca²⁺](i) signal comprised an initial rise and a sustained plateau without significant decay within 5 min. External Ca²⁺ removal decreased the Ca²⁺ signals by reducing the peak and sustained phase, indicating W-7-activated intracellular Ca²⁺ release and extracellular Ca²⁺ influx. W-7 (500 μM) failed to induce a [Ca²⁺](i) increase in a Ca²⁺-free medium after pre-treatment with thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor. Conversely, W-7 pre-treatment abolished the Ca²⁺ release induced by thapsigargin. This suggests that W-7 (500 μM) released internal Ca²⁺ mainly from the endoplasmic reticulum. The addition of 3 mM Ca²⁺ increased [Ca²⁺](i) dose-dependently after preincubation with 20-1000 μM W-7 in a Ca²⁺-free medium, implying that W-7 induced capacitative Ca²⁺ entry. W-7-induced Ca²⁺ release was not altered by inhibiting phospholipase C with 2 μM 1-(6-((17 β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (U73122). Tryphan blue assay demonstrated that W-7 (200 μM) caused gradual cell death within 30 min of the initial drug exposure. Together, it was found that W-7 induced [Ca²⁺](i) increases in human osteosarcoma cells by releasing internal Ca²⁺ from the endoplasmic reticulum, and also by triggering Ca²⁺ influx. W-7 may be cytotoxic to osteosarcoma cells. (C) 2000 Academic Press.