Activation of ERK1/2 by extracellular nucleotides in macrophages is mediated by multiple P2 receptors independently of P2X7-associated pore or channel formation

1 Macrophages express several P2X and P2Y nucleotide receptors and display the phenomenon of ATP-induced P2X(7)-dependent membrane permeabilization, which occurs through a poorly understood mechanism. Several P2 receptors are known to be coupled to the activation of mitogen-activated protein kinases (MAPKs) and Ca2+ signaling. 2 Here, we use macrophages to investigate the phosphorylation of extracellular signal-regulated kinases 1 and 2( ERK1/2) by nucleotides and the involvement of MAPKs and intracellular Ca2+ concentration in ATP-induced membrane permeabilization. 3 Short-term (5 min) pre-exposure to oxidized ATP (oATP), a P2X(7) antagonist that does not inhibit P2X(7)-associated inward currents or membrane permeabilization, inhibits the activation of ERK1/2 by ATP, ADP, the P2X(7) agonist 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP), but not by UTP and UDP. We conclude that macrophages display several P2Y receptors coupled to the ERK1/2 pathway and that oATP antagonizes the action of purine nucleotides, possibly binding to P2X7 and/or other purine-binding P2Y receptors. 4 We also show that BzATP and ATP activate ERK1/2 by two different pathways since ERK1/2 activation by BzATP, but not by ATP, is blocked by the tryrosine kinase inhibitor, genistein, and the Src protein kinase inhibitor, tyrphostin. However, the activation of ERK1/2 by ATP is blocked by the protein kinase C (PKC) inhibitor, chelerythrine chloride. Under the same conditions, membrane permeabilization is not blocked by genistein, tyrphostin, or chelerythrine chloride, indicating that tyrosine kinase, Src protein kinase, and PKC are not required for pore opening. 5 Membrane permeabilization is independent of ERK1/2 activation since chelerythrine, or short-term exposure to oATP or PD98059, efficiently block ERK1/2 activation without inhibiting membrane permeabilization. In addition, membrane permeabilization is not inhibited by SB203580 and SB202190, two inhibitors of p38 MAPK, nor by intracellular BAPTA, which blocks ATP-induced Ca2+ signals. 6 These results suggest that multiple P2 receptors lead to ERK1/2 activation, that ligation of the same receptors by agonists with different affinities can lead to differential stimulation of separate pathways, and that MAPKs and intracellular Ca2+ fluxes are independent of P2X(7)-associated pore formation.