Mechanism of action of a diterpene alkaloid hypaconitine on cytotoxicity and inhibitory effect of BAPTA‐AM in HCN‐2 neuronal cells

Hypaconitine, a neuromuscular blocker, is a diterpene alkaloid found in the root of Aconitum carmichaelii . Although hypaconitine was shown to affect various physiological responses in neurological models, the effect of hypaconitine on cell viability and the mechanism of its action of Ca 2+ handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca 2+ signalling in HCN‐2 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST‐1). Cytosolic Ca 2+ concentrations [Ca 2+ ] i was measured by the Ca 2+ ‐sensitive fluorescent dye fura‐2. In HCN‐2 cells, hypaconitine (10–50 μmol/L) induced cytotoxicity and [Ca 2+ ] i rises in a concentration‐dependent manner. Removal of extracellular Ca 2+ partially reduced the hypaconitine's effect on [Ca 2+ ] i rises. Furthermore, chelation of cytosolic Ca 2+ with BAPTA‐AM reduced hypaconitine's cytotoxicity. In Ca 2+ ‐containing medium, hypaconitine‐induced Ca 2+ entry was inhibited by modulators (2‐APB and SKF96365) of store‐operated Ca 2+ channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn 2+ influx indirectly suggesting that hypaconitine evoked Ca 2+ entry. In Ca 2+ ‐free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin abolished hypaconitine‐induced [Ca 2+ ] i rises. Conversely, treatment with hypaconitine inhibited thapsigargin‐induced [Ca 2+ ] i rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine‐induced [Ca 2+ ] i rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca 2+ ] i rises by Ca 2+ influx via store‐operated Ca 2+ entry involved PKC regulation and evoking PLC‐independent Ca 2+ release from the endoplasmic reticulum. Because BAPTA‐AM loading only partially reversed hypaconitine‐induced cell death, it suggests that hypaconitine induced a second Ca 2+ ‐independent cytotoxicity in HCN‐2 cells.