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

Abstract

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 Ca 2 þ 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 Ca 2 þ 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 0 -3 0 -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 P2X 7 and/or other purinebinding 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 Ca 2 þ 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 Ca 2 þ fluxes are independent of P2X 7 -associated pore formation.

Intracellular Signaling Pathways Integrating the Pore Associated with P2X7R Receptor with Other Large Pores

“…We have shown in 2005 that intracellular Ca 2+ acts as a second messenger in the large conductance channel opening in peritoneal macrophages and 2BH4 cells (Faria et al, 2005). Similar data were found by other groups (Cankurtaran-Sayar et al, 2009;Roger et al, 2008Roger et al, , 2010Schachter et al, 2008), but others did not observe this effect (da Cruz et al, 2006;Iglesias et al, 2008;Schachter et al, 2008;Virginio et al, 1999). We continued to investigate the Ca 2+ participation in more detail, and we described a major Ca 2+ dependence in the P2X7R pore formation, but we also observed Ca 2+ independent events in the same cell types (Faria et al, 2010).…”

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

“…Whether this second permeability state is attributed to intrinsic channel dilation [3], the pannexin-1 channel [4] or an alternate but unknown uptake pathway [5][6][7] remains controversial. Moreover, our understanding of this permeability state is further complicated with some [8][9][10] but not other [11][12][13] studies showing that P2X7-induced dye uptake involves the p38 mitogen-activated protein kinase. Regardless of the true identity of the P2X7 pore and the mechanism by which it opens, P2X7 activation stimulates several intracellular signalling pathways to induce various cellular events including inflammatory mediator release, reactive oxygen and nitrogen species formation, and cell proliferation or death [14,15].…”

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

“…Peritoneal macrophages typically express mRNA for P2Y1, P2Y2, P2Y6 and possibly P2Y4 as well as P2X1, P2X4 and P2X7 receptors (120)(121)(122). P2X7 activation is associated with Interleukin-1 secretion and killing of Mycobacteria, Chlamydia and fungi in monocyte-macrophages (52,(123)(124)(125)(126)(127)(128).…”