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 mu M) induced an increase in [Ca2+](i) in a dose-dependent manner, with an EC50 of 100 mu M. 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 mu M) failed to induce a [Ca2+](i) increase in a Ca2+-free medium after pre-treatment with thapsigarin (1 mu M), an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, W-7 pre-treatment abolished the Ca2+ release induced by thapsigargin. This suggests that W-7 (500 mu M) released internal Ca2+ mainly from the endoplasmic reticulum. The addition of 3 mM Ca2+ increased [Ca2+](i) dose-dependently after preincubation with 20-1000 mu M 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 mu M 1-(6-((17 beta-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 mu M) 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.