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 cytosoli c free calcium ion, [Ca2+](i), in a population of rat striatal astrocy tes loaded with the fluorescent Ca2+ probe Fura2, by means of fluoresc ence spectrometry. 2 ATP triggered a fast and transient elevation of [ Ca2+](i) in a concentration-dependent manner. The responses of the pur ine analogues 2-methylthio-ATP (2-meSATP), adenosine-5'-O-(2-thiodipho sphate) (ADP beta S), as well as uridine-5'-triphosphate (UTP) resembl ed 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, where as pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 m u M) significantly depressed the maximum response. 4 Extracellular Ca2 + did not contribute to the observed [Ca2+]i1 rise: removing calcium f rom the extracellular medium (with 1 mM EGTA) or blocking its influx b y means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleo tide responses. 5 Furthermore, after preincubation with 10 mu M thapsi gargin, the nucleotide-evoked [Ca2+](i) increments were completely abo lished. In contrast, 10 mM caffeine did not affect the responses, sugg esting 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 per tussis toxin (PTx) (350 mg ml(-1)) partially inhibited the nucleotide- mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73 122, but not its inactive stereoisomer U-73343 (5 mu M), significantly reduced the ATP-evoked [Ca2+](i) rise. 7 In conclusion, our results s uggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca 2+](i) is due solely to release from intracellular stores and is media ted by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.