The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5-50 microM range (EC50=4.3 microM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 microM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[gamma-S]=2-MeSATP>/=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'-triphosphate (alpha,beta-MeATP), beta, gamma methylene adenosine-5'-triphosphate (beta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the 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 ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 microM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 microM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor.

We have previously investigated the effects of extracellular ATP on the concentration of free cytosolic calcium ([Ca2+]i) from rat cultured neurohypophysial astrocytes (pituicytes). We demonstrated that ATP acts via a P2Y receptor to increase [Ca2+]i. In the present study, we examine the effect of ATP on K+ efflux using 86Rb+ as an isotopic tracer, in order to characterize the possible presence of a Ca2+-activated K+ conductance and to establish the implications of pituicytes in the regulation of stimulus-secretion coupling. ATP evoked an increase in 86Rb+ efflux from cultured pituicytes. This effect was Ca2+ dependent, as indicated by the unresponsiveness of cells loaded with BAPTA/AM (20 microM). Furthermore, the effect of ATP was mimicked by 2-methylthio-adenosine-5'-triphosphate (2MeSATP), a P2 purinoceptor agonist, and abolished by Reactive Blue 2 (RB-2), a selective P2Y antagonist, implying a role for the P2Y purinoreceptor. A pharmacological study revealed that Ba2+ and tetraethylammonium (TEA), two inhibitors of K+ channels, both strongly reduced the ATP-stimulated 86Rb+ efflux. In addition, the effect of ATP was modulated by different peptidic toxins. Apamin (100 nM), an inhibitor of the small-conductance Ca2+-activated K+ channels, partly blocked ATP-induced 86Rb+ efflux. Leiurus quinquestriatus hebraeus (LQH) scorpion venom (20 microg/ml) and Buthus tamulus (BT) scorpion venom (20-200 microg/ml) inhibited ATP-induced 86Rb+ efflux. The specificity of the effects of the crude venoms was checked using charybdotoxin (100 nM) and iberiotoxin (1 pM), which are the active toxins extracted from the LQH and BT venoms, respectively. These data indicate the involvement of several types of Ca2+-activated K+ channels in the ATP-dependent K+ efflux, and lead to the proposal that, in the neurohypophysis, extracellular ATP released by nerve terminals may act directly on the pituicytes and induce a K+ efflux via a P2Y purinoreceptor.