ATP acting on P-2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes)

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concen tration ([Ca2+](i)) of cultured neurohypophysial astrocytes (pituicytes) wa s studied by fluorescence videomicroscopy. ATP evoked a [Ca2+](i) increase, which was dose dependent in the 2.5-50 mu M range (EC50=4.3 mu M). The ATP -evoked [Ca2+](i) rise was not modified during the first minute following t he removal of external Ca2+. Application of 500 nM thapsigargin inhibited t he ATP-dependent [Ca2+](i) increase. Caffeine (10 mM) and ryanodine (1 mu M ) did not affect the ATP-induced [Ca2+](i) rise. The pituicytes responded t o various P-2 purinoceptor agonists with the following order of potency: AT P=ATP[gamma-S]=2-MeSATP greater than or equal to ADP, where ATP[gamma-S] is adenosine 5'-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine -5'-triphosphate. Adenosine, AMP, alpha,beta-methylene adenosine-5'-triphos phate (alpha,beta-MeATP), beta,gamma methylene adenosine-5'-triphosphate (b eta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P- 2 purinoceptor antagonists blocked the ATP-evoked [Ca2+](i) increase with t he following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. The A TP-evoked [Ca2+](i) increase was substantially blocked by pertussis toxin t reatment, suggesting that it might be mediated by a pertussis-toxin-sensiti ve G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 mu M) abolis hed the ATP-evoked [Ca2+](i) rise, whereas its inactive stereoisomer U-7334 3 (0.5 mu M) remained ineffective. Our results indicate that, in rat cultur ed pituicytes, ATP stimulation induces an increase in [Ca2+](i) due to PLC- mediated release from intracellular stores through activation of a pertussi s-toxin-sensitive, G-protein-linked P-2Y receptor.