Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca²⁺Concentration, ROS Formation and Mitochondrial Depolarization in PC12 Cells

Abstract

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 Ca2+ concentration ([Ca2+]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP (100µM) for 90 sec induced [Ca2+]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1µ g/ml to 100µg/ml) for 30 min inhibited the ATP-induced [Ca2+]i increases in a concentration-dependent manner (IC50=15.3µg/ml). Pretreatment with cyanidin-3-glucoside (15µg/ml) for 30 min significantly inhibited the ATP-induced [Ca2+]i responses following removal of extracellular Ca2+ or depletion of intracellular [Ca2+]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [Ca2+]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [Ca2+]i responses in the presence of nimodipine and ω-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [Ca2+]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular Ca2+ chelator BAPTA-AM or the mitochondrial Ca2+ 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 Ca2+ through the nimodipine and ω-conotoxin-sensitive and -insensitive pathways and the release of Ca2+ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting Ca2+-induced mitochondrial depolarization.

Knockdown of CLIC4 enhances ATP-induced HN4 cell apoptosis through mitochondrial and endoplasmic reticulum pathways

“…The reduction of the mitochondrial membrane potential or enhanced mitochondrial membrane depolarization is an early sign of cell apoptosis ahead of DNA breakage and activation of caspase 3 [ 19 ]. ATP can increase [Ca 2+ ] i and induce mitochondrial membrane depolarization [ 20 ]. Therefore, we assessed whether knockdown of CLIC4 affected ATP-induced mitochondrial membrane depolarization.…”

“…Previous studies have suggested that the depletion of ER Ca 2+ stores leads to cell apoptosis and growth arrest [ 21 , 22 ]. The Ca 2+ stores can be depleted by both ATP and thapsigargin (TG), an ER Ca 2+ -ATPase inhibitor [ 20 , 23 ]. Therefore, we examined the effect of CLIC4 on the ATP- and TG-induced Ca 2+ release from ER Ca 2+ stores.…”

“…The mitochondrial membrane potential, which is formed by the normal function of proton pumps plays key roles in maintaining oxidative phosphorylation and ATP production [ 29 ]. Enhanced mitochondrial membrane depolarization is a well-recognized sign of apoptosis in the early stage [ 20 ]. Our data show that the knockdown of CLIC4 significantly enhanced ATP-induced mitochondrial membrane depolarization in HN4 cells, further proving that the knockdown of CLIC4 exerts a pro-apoptotic effect through the mitochondrial apoptosis pathway and strengthened ATP-induced apoptosis in HN4 cells.…”

Nutritional intervention with cyanidin hinders the progression of muscular dystrophy

“…Several pieces of evidence showed that oxidative stress and accumulation of reactive oxygen species (ROS) strongly contribute to aggravate the dystrophic pathology 6,7 . One of the considered strategies is to use antioxidant molecules to counteract the oxidative stress generated by muscle contraction and degeneration [6][7][8][9][10][11][12][13] .…”

Effects of Cyanidin-3-O-glucoside on Synthetic and Metabolic Activity of Ethanol Stimulated Human Pancreatic Stellate Cells

“…reduced oxygen consumption and ATP synthesis in the ethanol activated hPSC. Perveen et al () has demonstrated that in rat pheochromocytoma (PC‐12) cells, ATP induced increase in intracellular calcium concentration and consequent ROS formation led to mitochondrial injury, whereas C‐3‐O‐G protected mitochondria from ATP‐induced intracellular calcium increase (Perveen et al, ).…”