Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

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Abstract
This study was aimed at characterizing ATP‐induced rises in cytosolic free calcium ion, [Ca2+]i, in a population of rat striatal astrocytes loaded with the fluorescent Ca2+ probe Fura2, by means of fluorescence spectrometry. ATP triggered a fast and transient elevation of [Ca2+]i in a concentration‐dependent manner. The responses of the purine analogues 2‐methylthio‐ATP (2‐meSATP), adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), as well as uridine‐5′‐triphosphate (UTP) resembled that of ATP, while α,β‐methylene‐ATP (α,β‐meATP) and β,γ‐methylene‐ATP (β,γ‐meATP) were totally ineffective. Suramin (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response. Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleotide responses. Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide‐evoked [Ca2+]i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin‐, but not caffeine/ryanodine‐sensitive stores are involved. Both application of the G‐protein blocker guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml−1) partially inhibited the nucleotide‐mediated responses. Moreover, the phospholipase C (PLC) inhibitor U‐73122, but not its inactive stereoisomer U‐73343 (5 μM), significantly reduced the ATP‐evoked [Ca2+]i rise. In conclusion, our results suggest that, in rat striatal astrocytes, ATP‐elicited elevation of [Ca2+]i is due solely to release from intracellular stores and is mediated by a G‐protein‐linked P2Y receptor, partially sensitive to PTx and coupled to PLC. British Journal of Pharmacology (1997) 121, 1700–1706; doi:10.1038/sj.bjp.0701293