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 microM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 microM) significantly depressed the maximum response. 4. 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. 5. Furthermore, after preincubation with 10 microM 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 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 microM), 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.
Centemeri
C
C
Institute of Pharmacological Sciences, University of Milan, Italy.
Bolego
C
C
Abbracchio
M P
MP
Cattabeni
F
F
Puglisi
L
L
Burnstock
G
G
Nicosia
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004963:Estrenes / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology; D010738:Type C Phospholipases / Q000502:physiology; D014544:Uridine Triphosphate / Q000494:pharmacology
D004963:Estrenes; D011760:Pyrrolidinones; D018048:Receptors, Purinergic P2; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D002118:Calcium; D014544:Uridine Triphosphate