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transport mechanism of anionic dyes displayed by macrophages was also able to support dye efflux and, once activated at 37°C, it remained active at 4°C, whereas uptake of cationic dyes was temperature-dependent and unidirectional. Our results indicate that the mechanism of ATP e -induced dye uptake, usually called a 'permeabilization phenomenon' and associated with a 'permeabilization pore' can be ascribed to at least two distinct mechanisms in macrophages: a diffusional pathway, possibly associated with the 440 pS Z pores, and a cation uptake mechanism that is not diffusional and should be ascribed to an, as yet, unidentified transport mechanism.Key words: ATP, P2 receptor, P2X 7 , Permeabilization, Macrophage, Cation, Anion, Pore Journal of Cell Science 3262 extracellular concentration of the dye (Steinberg et al., 1987a), it has been proposed that the mechanism underlying the dye uptake phenomena is free diffusion through a permeabilization pore, and it has been assumed that cations and anions uses the same pathway. However, evidence from several different sources indicate the involvement of distinct mechanisms (North, 2002;Egan et al., 2006). Whole-cell patch-clamp recordings performed on cells transfected with P2X 7 receptors indicate a permeability shift from low to high M r molecules in the first seconds after stimulation by ATP e , while keeping the selectivity for cations (Virginio et al., 1999). In addition, although ATP e induces the uptake of both cationic and anionic dyes in macrophages (Steinberg et al., 1987a), the uptake of anions has not been reported either in HEK-293 cells or astrocytomas transfected with P2X 7 , neither has it been reported in lymphocytes (Wiley et al., 1993;Surprenant et al., 1996;Rassendren et al., 1997;Chessell et al., 1998;Ferrari et al., 2000;Paukert et al., 2002;Duan et al., 2003;Suadicani et al., 2006), suggesting that in these experimental situations, ATP e might not induce membrane permeabilization to anions.Based on cell-attached patch-clamping experiments demonstrating the opening of large cation-and anion-permeant channels (Z pores) induced by ATP e in macrophages, we have hypothesized that the permeabilization pore of macrophages could be distinct from the receptor itself, but coupled to P2X 7 through an unidentified signaling mechanism (Coutinho-Silva and Persechini, 1997;Persechini et al., 1998). This possibility has recently been corroborated by data showing that pannexin-1, a protein that forms large non-selective transmembrane channels (Barbe et al., 2006), is involved in the phenomenon of P2X 7 -associated, ATP e -induced membrane permeabilization (Pelegrin and Surprenant, 2006;Locovei et al., 2007). However, there have been no reports of any large and non-selective unitary channels similar to the Z pores in cells transfected with P2X 7 receptors under conditions of ATP einduced dye uptake. Moreover, pannexin-1 channels are expected to be permeable to both cations and anions (Bao et al., 2004;Locovei et al., 2006;Locovei et al., 2007), whereas there are no ...

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.

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