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

1 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. 2 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) (ADP beta S), as well as uridine-5'-triphosphate (UTP) resembled that of ATP, while alpha,beta-methylene-ATP (alpha,beta-meATP) and beta,gamma-methylene-ATP (beta,gamma-meATP) were totally ineffective. 3 Suramin (50 mu M) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 mu M) significantly depressed the maximum response. 4 Extracellular Ca2+ did not contribute to the observed [Ca2+]i1 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. 5 Furthermore, after preincubation with 10 mu M 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. 6 Both application of the G-protein blocker guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) (1 mM) and preincubation with pertussis toxin (PTx) (350 mg ml(-1)) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 mu M), significantly reduced the ATP-evoked [Ca2+](i) rise. 7 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.