Spatial and temporal aspects of Ca²⁺ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes

ATP produces a variety of Ca²⁺ responses in astrocytes. To address the complex spatio-temporal Ca²⁺ signals, we analyzed the ATP-evoked increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca²⁺ imaging techniques. ATP at less than 10 nM produced elementary Ca²⁺ release event "puffs" in a manner independent of extracellular Ca²⁺. Stimulation with higher ATP concentrations (3 or 10 μM) resulted in global Ca²⁺ responses such as intercellular Ca²⁺ wave. These Ca²⁺ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca²⁺ release by inositol 1,4,5-trisphosphate (InsP₃). When cells were stimulated with ATP much longer, the transient [Ca²⁺]i elevation was followed by sustained Ca²⁺ entry from the extracellular space. This sustained rise in [Ca²⁺]i was inhibited by Zn²⁺ (<10 μM), an inhibitor of capacitative Ca²⁺ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca²⁺ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca²⁺ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca²⁺ release by InsP₃, and CCE in response to depletion of InsP₃-sensitive Ca²⁺ stores. It should be noted that these Ca²⁺ signals and their modulation by Zn²⁺ could occur in the hippocampus in situ since both ATP and Zn²⁺ are rich in the hippocampus and could be released by excitatory stimulation.