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, 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, 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 P2X₄ allosteric effector ivermectin (IVM). The agonist ADPβS 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- or extracellular Ca²⁺. Taken together, the P2Y₁-mediated Ca²⁺ sustain was at least in part via P2X receptors activated by the P2Y₁-induced ATP release, and PKC played a pivotal role in desensitization of P2Y₁ receptors in RBA-2 astrocytes.