Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free $Ca^{2+}$ Concentration, ROS Formation and Mitochondrial Depolarization in PC12 Cells

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5'-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free $Ca^{2+}$ concentration ([$Ca^{2+}$]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP ($100{\mu}M$) for 90 sec induced [$Ca^{2+}$]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside ($1{\mu}g/ml$ to $100{\mu}g/ml$) for 30 min inhibited the ATP-induced [$Ca^{2+}$]i increases in a concentration-dependent manner ($IC_{50}=15.3{\mu}g/ml$). Pretreatment with cyanidin-3-glucoside ($15{\mu}g/ml$) for 30 min significantly inhibited the ATP-induced [$Ca^{2+}$]i responses following removal of extracellular $Ca^{2+}$ or depletion of intracellular [$Ca^{2+}$]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [$Ca^{2+}$]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [$Ca^{2+}$]i responses in the presence of nimodipine and ${\omega}$-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [$Ca^{2+}$]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular $Ca^{2+}$ chelator BAPTA-AM or the mitochondrial $Ca^{2+}$ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular $Ca^{2+}$ through the nimodipine and ${\omega}$-conotoxin-sensitive and -insensitive pathways and the release of $Ca^{2+}$ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting $Ca^{2+}$-induced mitochondrial depolarization.