Korean J Physiol Pharmacol.  2014 Aug;18(4):297-305. 10.4196/kjpp.2014.18.4.297.

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

Affiliations
  • 1Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea. s-hyoon@catholic.ac.kr
  • 2Catholic Agro-Medical Center, The Catholic University of Korea, Seoul 137-701, Korea.
  • 3Department of Functional Crop, National Institute of Crop Science, Rural Development Administration, Miryang 627-803, Korea.

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 (100microM) for 90 sec induced [Ca2+]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1micro g/ml to 100microg/ml) for 30 min inhibited the ATP-induced [Ca2+]i increases in a concentration-dependent manner (IC50=15.3microg/ml). Pretreatment with cyanidin-3-glucoside (15microg/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 omega-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 omega-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.

Keyword

ATP; Calcium; Cyanidin-3-glucoside; Mitochondrial membrane potential; Reactive oxygen species
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