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 Ca²⁺ handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca²⁺ signalling in HCN-2 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST-1). Cytosolic Ca²⁺ concentrations [Ca²⁺ ]_i was measured by the Ca²⁺ -sensitive fluorescent dye fura-2. In HCN-2 cells, hypaconitine (10-50 μmol/L) induced cytotoxicity and [Ca²⁺ ]_i rises in a concentration-dependent manner. Removal of extracellular Ca²⁺ partially reduced the hypaconitine's effect on [Ca²⁺ ]_i rises. Furthermore, chelation of cytosolic Ca²⁺ with BAPTA-AM reduced hypaconitine's cytotoxicity. In Ca²⁺ -containing medium, hypaconitine-induced Ca²⁺ entry was inhibited by modulators (2-APB and SKF96365) of store-operated Ca²⁺ channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn²⁺ influx indirectly suggesting that hypaconitine evoked Ca²⁺ entry. In Ca²⁺ -free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished hypaconitine-induced [Ca²⁺ ]_i rises. Conversely, treatment with hypaconitine inhibited thapsigargin-induced [Ca²⁺ ]_i rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine-induced [Ca²⁺ ]_i rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca²⁺ ]_i rises by Ca²⁺ influx via store-operated Ca²⁺ entry involved PKC regulation and evoking PLC-independent Ca²⁺ release from the endoplasmic reticulum. Because BAPTA-AM loading only partially reversed hypaconitine-induced cell death, it suggests that hypaconitine induced a second Ca²⁺ -independent cytotoxicity in HCN-2 cells.