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 (100µM) for 90 sec induced [Ca(2+)](i) increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1µ g/ml to 100µg/ml) for 30 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50)=15.3µg/ml). Pretreatment with cyanidin-3-glucoside (15µg/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 ω-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 ω-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.