Purinergic P2Y receptors on astrocytes are directly coupled to phospholipase A2

ATP stimulates arachidonic acid mobilization and eicosanoid production in cultured astrocytes via P2Y-purinergic receptors. To assist in determining the mechanism of phospholipase A2 activation and the role of calcium in eicosanoid production, cultures were pretreated with pertussis toxin (PTx). ATP-evoked eicosanoid release was inhibited by PTx in a concentration-dependent fashion. Inositol phospholipid hydrolysis was partially attenuated by PTx, but the concentrations required were approximately 50 times greater than those for inhibition of eicosanoid production, suggesting that phospholipase C activation is not necessary for eicosanoid synthesis. Stimulation of eicosanoid release by other P2Y-purinergic receptor agonists was also inhibited by PTx; however, PTx had no effect on eicosanoid release evoked by ionomycin or thapsigargin, nor did it affect ATP-stimulated calcium influx or mobilization from intracellular stores. Increases in intracellular free calcium concentration alone were insufficient to stimulate eicosanoid production, but maximal production was dependent upon the concentration of extracellular calcium. These results suggest that the P2Y-purinergic receptor is coupled to phospholipase A2 via a guanine nucleotide-binding protein, and that extracellular calcium may also be involved in the synthesis of eicosanoids by astrocytes.