This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca 2ϩ influx and activated phospholipase D (PLD). The ATP-stimulated Ca 2ϩ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate (BzATP) stimulated the largest increases in intracellular Ca 2ϩ concentrations ([Ca 2ϩ ] i ); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATP␥S were much less effective, whereas UTP, ADP, ␣,␤-methylene-ATP, and ␤,␥-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg 2ϩ enhanced the ATP-and BzATP-stimulated increases in [Ca 2ϩ ] i . BzATP stimulated PLD in a concentration-and time-dependent manner that could be abolished by removal of extracellular Ca 2ϩ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca 2ϩ mobilization agent, ionomycin, in an extracellular Ca 2ϩ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X 7 receptors induces Ca 2ϩ influx and stimulates a Ca 2ϩ -dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD. Key Words: ATP-2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate -Ca 2ϩ influx-Phospholipase D -P2X 7 receptor-Type-2 astrocytes.

We examined the changes in phospholipid metabolisms in sodium butyrate-treated C6 glioma cells. Treatment of 2.5 mM sodium butyrate for 24 h induced an increase in the activity of glutamine synthetase, suggesting that these cells were under differentiation. Similar treatment was associated with (i) increased arachidonic acid incorporation into phosphatidylcholine, and (ii) decreased arachidonic acid incorporation into phosphatidylinositol and (iii) phosphatidylethanolamine. These effects were subsequently investigated by examining the acylation process, de novo biosynthesis, and the agonist-stimulated phosphoinositides hydrolysis in these cells. Our results indicated that sodium butyrate stimulated the acylation of arachidonic acid into lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol. The glycerol incorporation into these lipids was not affected, but the inositol incorporation into total chloroform extracts and Pl and phosphatidylinositol 4-phosphate was decreased in the sodium butyrate-treated cells. Moreover, the accumulation of the rapid histamine-stimulated phosphoinositide metabolites, i.e., inositol monophosphate, inositol diphosphate, and inositol triphosphate (IP3) was decreased in these cells. To elucidate whether the decreased inositol phosphates were due to a decrease in the phosphoinositides hydrolysis, we measured the transient IP3 production directly by a receptor-binding assay. Our results indicated that histamine-stimulated transient IP3 formations were decreased. Taken together, these results indicated that multiple changes by multiple mechanisms of phospholipid metabolisms were found in sodium butyrate-treated C6 glioma cells. The decreased IP3 formation and its subsequent action, i.e., Ca2+ mobilization, may play an early but pivotal role by which sodium butyrate induces C6 glioma cell differentiation.