Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes:: Elucidate the roles of ATP release and protein kinase C

A physiological concentration of extracellular ATP stimulated biphasic Ca2+ signal, and the Ca2+ transient was decreased and the Ca2+ sustain was eliminated immediately after removal of ATP and Ca2+ in RBA-2 astrocytes. Reintroduction of Ca2+ induced Ca2+ sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca2+ signaling and the Ca2+ sustains were eliminated using Ca2+-free buffer. The 2MeSADP-mediated biphasic Ca2+ Signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca2+ entry (CCE) decreased the Ca2+-induced Ca2+ entry; nevertheless, ATP further enhanced the Ca2+-induced Ca2+ entry in the intracellular Ca2+ store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca2+ entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca2+ sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPPS stimulated a lesser P2Y(1)-mediated Ca2+ signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH3 and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- orextracellularCa(2+). Taken together, the P2Y(1)-mediated Ca2+ sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes.