Department of Neurosurgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Department of Neurosurgery, National Defense Medical Center, Taipei, Taiwan.
Clin Exp Pharmacol Physiol. 2021 May;48(5):801-810. doi: 10.1111/1440-1681.13482. Epub 2021 Feb 20.
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 handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca signalling in HCN-2 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST-1). Cytosolic Ca concentrations [Ca ] was measured by the Ca -sensitive fluorescent dye fura-2. In HCN-2 cells, hypaconitine (10-50 μmol/L) induced cytotoxicity and [Ca ] rises in a concentration-dependent manner. Removal of extracellular Ca partially reduced the hypaconitine's effect on [Ca ] rises. Furthermore, chelation of cytosolic Ca with BAPTA-AM reduced hypaconitine's cytotoxicity. In Ca -containing medium, hypaconitine-induced Ca entry was inhibited by modulators (2-APB and SKF96365) of store-operated Ca channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn influx indirectly suggesting that hypaconitine evoked Ca entry. In Ca -free medium, treatment with the endoplasmic reticulum Ca pump inhibitor thapsigargin abolished hypaconitine-induced [Ca ] rises. Conversely, treatment with hypaconitine inhibited thapsigargin-induced [Ca ] rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine-induced [Ca ] rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca ] rises by Ca influx via store-operated Ca entry involved PKC regulation and evoking PLC-independent Ca release from the endoplasmic reticulum. Because BAPTA-AM loading only partially reversed hypaconitine-induced cell death, it suggests that hypaconitine induced a second Ca -independent cytotoxicity in HCN-2 cells.
乌头碱是一种作用于神经肌肉的阻滞剂，属于二萜生物碱，存在于乌头属植物的根中。虽然乌头碱已被证明会影响神经模型中的各种生理反应，但它对皮质神经元细胞活力的影响及其钙处理机制仍不清楚。本研究旨在探讨乌头碱是否会改变 HCN-2 神经元细胞系的活力和钙信号。通过细胞增殖试剂（WST-1）测量细胞活力。通过钙敏感荧光染料 fura-2 测量胞质 Ca 浓度 [Ca ]。在 HCN-2 细胞中，乌头碱（10-50 μmol/L）以浓度依赖的方式诱导细胞毒性和 [Ca ]升高。去除细胞外 Ca 部分减少了乌头碱对 [Ca ]升高的作用。此外，用 BAPTA-AM 螯合胞质 Ca 可降低乌头碱的细胞毒性。在含有 Ca 的培养基中，乌头碱诱导的 Ca 内流被储存操作 Ca 通道调节剂（2-APB 和 SKF96365）和蛋白激酶 C（PKC）抑制剂（GF109203X）抑制。乌头碱诱导 Mn 内流间接表明乌头碱引起了 Ca 内流。在无 Ca 培养基中，内质网 Ca 泵抑制剂 thapsigargin 的处理消除了乌头碱诱导的 [Ca ]升高。相反，乌头碱处理抑制了 thapsigargin 诱导的 [Ca ]升高。然而，用 U73122 抑制 PLC 并不抑制乌头碱诱导的 [Ca ]升高。总之，乌头碱引起的细胞毒性与通过储存操作 Ca 内流引起的先前的 [Ca ]升高有关，涉及 PKC 调节和内质网中 PLC 非依赖性 Ca 释放。由于 BAPTA-AM 加载仅部分逆转了乌头碱诱导的细胞死亡，因此表明乌头碱在 HCN-2 细胞中诱导了第二种与 Ca 无关的细胞毒性。
