Exposure of rat cortical astrocytes to αβmethyleneATP results in features of reactive astrogliosis via activation of a novel P2Y receptor linked to cyclooxygenase-2 (COX-2) upregulation [Brambilla et al., J Neurochem 2002, 83:1285-1296]. Here, we have investigated the role of extracellular signal-regulated kinases (ERK1/2) in αβmethyleneATP-induced gliosis. Challenge of cultures with αβpmethyleneATP resulted in early (5-10 min) and marked time-dependent ERK1/2 activation. Pertussis toxin completely abolished this effect. ERK1/2 activation was also completely prevented by the selective ERK1/2 inhibitor PD 098059 and by the P2 antagonist pyridoxalphosphate-6-azophenyl-2′-4′-disulphonic acid. Because αβpmethyleneATP induced no changes of intracellular calcium concentrations [Brambilla et al., 2002], a role for phosphoinositide-specific phospholipase C was ruled out. Conversely, D609, an inhibitor of phosphatidylcholine-PLC (PC-PLC) and PLD, fully abolished both αβmethyleneATP-induced ERK1/2 activation and the associated gliosis. The Ras inhibitor FTI-277 could also partially abolish ERK1/2 activation. Exposure of cells to αβmethyleneATP resulted in a time-dependent increase in the DNA binding activity of AP-1 and NF-κB, two transcription factors known to be phosphorylated by ERKs and involved in COX-2 gene transcription. We conclude that stimulation of a gliotic P2Y receptor leads to ERK1/2 activation via multiple parallel signaling pathways involving Gi/o-dependent and calcium-independent stimulation of PC-PLC and/or PLD, and consequent activation of the Ras/Raf systems. We are currently trying to clone this receptor from these cells. Its molecular identification may lead to the development of a new class of antineurodegenerative agents, which, by counteracting excessive gliosis and COX-2 upregulation, may prove useful in neurological disorders characterized by astrogliosis and inflammation. © 2003 Wiley-Liss, Inc.