Effect of calmidazolium on [Ca 2+ ] i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca 2+ concentrations ([Ca 2+ ] i ) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca 2+ ] i and caused cell death in HA59T cells. [Ca 2+ ] i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca 2+ ] i in a concentration-dependent manner with an EC 50 value of 1.5 μM. The Ca 2+ signal was reduced partly by removing extracellular Ca 2+ . Calmidazolium induced Mn 2+ quench of fura-2 fluorescence implicating Ca 2+ influx. The Ca 2+ influx was insensitive to L-type Ca 2+ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca 2+ -free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor), calmidazolium-induced [Ca 2+ ] i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca 2+ ] i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca 2+ ] i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca 2+ ] i rises by causing Ca 2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca 2+ influx via protein kinase C-regulated Ca 2+ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.