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 bladder female transitional cancer (BFTC) cells was examined using fura-2 as a Ca2+ dye. W-7 (10-1000 microM) caused an increase in [Ca2+]i in a concentration-dependent manner with an EC50 of 75 microM. The [Ca2+]i response was composed of an initial rise and a sustained plateau without significant decaying during the measurement of 250 seconds. Extracellular Ca2+ removal dramatically decreased the Ca2+ signals by 50-90%. W-7 (100 microM) failed to induce a [Ca2+]i increase in Ca(2+)-free medium after pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor; conversely, W-7 pretreatment abolished the Ca2+ release induced by thapsigargin. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 100 microM W-7 in Ca(2+)-free medium. W-7-induced Ca2+ release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17b-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (U73122). Together, this study shows that W-7 caused [Ca2+]i increases in human bladder cancer cells by releasing intracellular Ca2+ from the endoplasmic reticulum and also by causing extracellular Ca2+ influx with the later playing a dominant role. The W-7-induced intracellular Ca2+ release was uncoupled to a prior elevation in intracellular levels of inositol 1,4,5-trisphosphate (IP3).