Activation of purinergic P2X receptors inhibits P2Y-mediated Ca2+ influx in human microglia

Abstract

Purinoceptor (P2X and P2Y) mediated Ca2+ signaling in cultured human microglia was studied using Ca2+ sensitive fluorescence microscopy. ATP (at 100 μM) induced a transient increase in [Ca2+]i in both normal and Ca2+-free solution suggesting a primary contribution by release from intracellular stores. This conclusion was further supported by the failure of ATP to cause a divalent cationic influx in Mn2+ quenching experiments. However, when fluorescence quenching was repeated after removal of extracellular Na+, ATP induced a large influx of Mn2+, indicating that inward Na+ current through a non-selective P2X-coupled channel may normally suppress divalent cation influx. Inhibition of Mn2+ entry was also found when microglia were depolarized using elevated external K+ in Na+-free solutions. The possibility of P2X inhibition of Ca2+ influx was then investigated by minimizing P2X contributions to purinergic responses using either the specific P2Y agonist, ADP-β-S in the absence of ATP or using ATP combined with PPADS, a specific inhibitor of P2X receptors. In quenching studies both procedures resulted in large increases in Mn2+ influx in contrast to the lack of effect observed with ATP. In addition, perfusion of either ATP plus PPADS or ADP-β-S alone caused a significantly enhanced duration (about 200%) of the [Ca2+]i response relative to that induced by ATP. These results show that depolarization induced by P2X-mediated Na+ influx inhibits store-operated Ca2+ entry resulting from P2Y activation, thereby modulating purinergic signaling in human microglia.