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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<jats:sup>2+<\/jats:sup> handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca<jats:sup>2+<\/jats:sup> signalling in HCN\u20102 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST\u20101). Cytosolic Ca<jats:sup>2+<\/jats:sup> concentrations [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub> was measured by the Ca<jats:sup>2+<\/jats:sup>\u2010sensitive fluorescent dye fura\u20102. In HCN\u20102 cells, hypaconitine (10\u201350\u00a0\u03bcmol\/L) induced cytotoxicity and [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub> rises in a concentration\u2010dependent manner. Removal of extracellular Ca<jats:sup>2+<\/jats:sup> partially reduced the hypaconitine's effect on [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub> rises. Furthermore, chelation of cytosolic Ca<jats:sup>2+<\/jats:sup> with BAPTA\u2010AM reduced hypaconitine's cytotoxicity. In Ca<jats:sup>2+<\/jats:sup>\u2010containing medium, hypaconitine\u2010induced Ca<jats:sup>2+<\/jats:sup> entry was inhibited by modulators (2\u2010APB and SKF96365) of store\u2010operated Ca<jats:sup>2+<\/jats:sup> channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn<jats:sup>2+<\/jats:sup> influx indirectly suggesting that hypaconitine evoked Ca<jats:sup>2+<\/jats:sup> entry. In Ca<jats:sup>2+<\/jats:sup>\u2010free medium, treatment with the endoplasmic reticulum Ca<jats:sup>2+<\/jats:sup> pump inhibitor thapsigargin abolished hypaconitine\u2010induced [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub> rises. Conversely, treatment with hypaconitine inhibited thapsigargin\u2010induced [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub> rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine\u2010induced [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub> rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub> rises by Ca<jats:sup>2+<\/jats:sup> influx via store\u2010operated Ca<jats:sup>2+<\/jats:sup> entry involved PKC regulation and evoking PLC\u2010independent Ca<jats:sup>2+<\/jats:sup> release from the endoplasmic reticulum. Because BAPTA\u2010AM loading only partially reversed hypaconitine\u2010induced cell death, it suggests that hypaconitine induced a second Ca<jats:sup>2+<\/jats:sup>\u2010independent cytotoxicity in HCN\u20102 cells.<\/jats:p>","DOI":"10.1111\/1440-1681.13482","type":"journal-article","created":{"date-parts":[[2021,2,20]],"date-time":"2021-02-20T23:02:21Z","timestamp":1613862141000},"page":"801-810","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Mechanism of action of a diterpene alkaloid hypaconitine on cytotoxicity and inhibitory effect of BAPTA\u2010AM in HCN\u20102 neuronal cells"],"prefix":"10.1111","volume":"48","author":[{"given":"Shu\u2010Shong","family":"Hsu","sequence":"first","affiliation":[{"name":"Department of Neurosurgery Kaohsiung Veterans General Hospital Kaohsiung Taiwan"},{"name":"Department of Neurosurgery National Defense Medical Center Taipei Taiwan"},{"name":"College of Health and Nursing 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