Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

ABSTRACT

Phenolic compounds affect intracellular free Ca2+ concentration ([Ca2+]i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca2+ signaling in PC12 cells using fura-2-based digital Ca2+ imaging and whole-cell patch clamping. Treatment with ATP (100 micrometer) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 micrometer) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84 micrometer). Treatment with octyl gallate (3 micrometer) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca2+ with nominally Ca2+-free HEPES HBSS or depletion of intracellular Ca2+ stores with thapsigargin (1 micrometer). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1 micrometer) significantly inhibited the ATP-induced [Ca2+]i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca2+]i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 micrometer) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca2+]i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca2+ from extracellular space and P2Y receptor-induced release of Ca2+ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca2+ responses by inhibiting the secondary activation of voltage-gated Ca2+ channels.