Effect of calmidazolium on [Ca²⁺]_i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca2+ concentrations ([Ca2+](i)) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca2+](i) and caused cell death in HA59T cells. [Ca2+](i) and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations a parts per thousand yen1 mu M increased [Ca2+](i) in a concentration-dependent manner with an EC50 value of 1.5 mu M. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Calmidazolium induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to L-type Ca2+ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 mu M thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), calmidazolium-induced [Ca2+](i) rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca2+](i) rises. Inhibition of phospholipase C with 2 mu M U73122 did not change calmidazolium-induced [Ca2+](i) rises. At concentrations between 1 and 15 mu M, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca2+](i) rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via protein kinase C-regulated Ca2+ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.