Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X7 agonists ATP and 3'-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X7-mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ERK2, p38, and JNK1/JNK2) phosphorylation and that the antioxidants N-acetylcysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.     

Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function

Activation of the P2X(7) receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg(578), Lys(579)) within this motif are essential for LPS binding to P2X(7) in vitro. Because P2X(7) can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X(7) is critical for receptor function and trafficking. In these studies we mutated Arg(578) and Lys(579) of P2X(7), and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X(7)-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25 degrees C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25 degrees C the P2X(7)-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X(7) C terminus controls receptor trafficking and modulates channel activity.     

Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus

The binding of extracellular ATP to the P2X(7) receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1beta secretion and eventual death. The latter three effects are unique to the P2X(7) receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X(7) receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X(7) receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X(7) was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathione S-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X(7). The P2X(7) receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X(7) receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X(7) C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X(7) receptor activation.     

不同诱导因子对人外周血单个核细胞P2X7受体表达的作用

ATP激活P2X7受体可产生一系列的白细胞功能反应,因此P2X7受体的表达调控引起我们的兴趣。然而P2X7受体在正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)、单核细胞中的表达调控机制尚未阐明。本文用半定量RT-PCR方法检测多种细胞因子、细菌抗原、丝裂原对P2X7受体表达的诱导作用,探索P2X7受体的诱导表达模式。结果表明,单个核细胞和单核细胞可检出P2X7受体的表达;白细胞介素2、4、6(interleukin-2、-4、-6,IL-2、IL-4、IL-6)、肿瘤坏死因子仪(tumour necrosis factor-α,TNF-α)等细胞因子和金黄色葡萄球菌CowanⅠ株(Staphylococcus aureus Cowan strainⅠ,SAC)、脂多糖(lipopolysaccharide,LPS)能上调PBMC的P2X7受体表达,而γ干扰素(interferon-γ,IFN-γ)、粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)、巨噬细胞集落刺激因子(macmphage colony-stimulating factor,M-CSF)和植物血凝素(phytohemagglutinin-M,PHA-M)等则没有作用;LPS和M-CSF可以提高单核细胞的P2X7受体表达,IFN-γ、TNF-α、GM-CSF作用较弱,但是这些因子的预处理并不能增强LPS对P2X7受体表达的诱导。炎症因子促进P2X7受体的表达,提示P2X7受体可能在对抗细菌感染的免疫反应中起一定作用,这有待于进一步研究。     

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

The P2X7 receptor is widely recognized to mediate the proinflammatory effects of extracellular ATP. However this receptor in the absence of ATP may have a function unrelated to inflammation. Our data show that P2X7 expressed on the surface of monocyte/macrophages or on epithelial HEK-293 cells greatly augments the engulfment of latex beads and live and heat-killed bacteria by effector phagocyte in the absence of ATP and serum. The expression of P2X7 on the effector also confers the ability to phagocytose apoptotic target cells and an accumulation of P2X7 can be seen at the attachment point to the target. Activation of the P2X7 receptor by ATP causes a slow dissociation (over 10–15 min) of nonmuscle myosin from the P2X7 membrane complex and abolishes further P2X7-mediated phagocytosis of these targets. The recent crystal structure of the homologous zebrafish P2X4 receptor shows an exposed “nose” of the ectodomain (residues 115–162) which contains three of the five disulfide bonds conserved in all P2X receptors. Three short biotin-labeled peptides mimicking sequence of this exposed region bound to apoptotic target cells but not to either viable cells or to other target particles. All three peptides contained one or two cysteine residues and their replacement by alanine abolished peptide binding. These data implicate thiol-disulfide exchange reactions in the initial tethering of apoptotic cells to macrophage and establish P2X7 as one of the scavenger receptors involved in the recognition and removal of apoptotic cells in the absence of extracellular ATP and serum.     

Regulation of arachidonic acid mobilization in lipopolysaccharide-activated P388D(1) macrophages by adenosine triphosphate

Murine P388D(1) macrophages exhibit a delayed prostaglandin biosynthetic response when exposed to bacterial lipopolysaccharide (LPS) for prolonged periods of time that is dependent on induction of the genes coding for Group V secretory phospholipase A(2) and cyclooxygenase-2. We herein report that LPS-induced arachidonic acid (AA) metabolite release in P388D(1) macrophages is strongly attenuated by the P2X(7) purinergic receptor antagonists periodate-oxidized ATP and pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid, and this is accompanied by suppression of the expression of both Group V secretory phospholipase A(2) and cyclooxygenase-2. The effect appears to be specific for LPS, because the P2 purinergic receptor antagonists do not affect P388D(1) cell stimulation by other stimuli such as platelet-activating factor or the Ca(2+) ionophore A23187. Moreover, extracellular nucleotides are found to stimulate macrophage AA mobilization with a pharmacological profile that implicates the participation of the P2X(7) receptor and that is inhibited by periodate-oxidized ATP. Collectively these results demonstrate coupling of the P2X(7) receptor to the AA cascade in P388D(1) macrophages and implicate the participation of this type of receptor in LPS-induced AA mobilization.     

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.     

Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages

Interaction of P2X7 receptor with P2X4 receptor has recently been suggested, but it remains unclear whether P2X4 receptor is involved in P2X7 receptor-mediated events, such as cell death of macrophages induced by high concentrations of extracellular ATP. Here, we present evidence that P2X4 receptor does play a role in P2X7 receptor-dependent cell death. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), and cell death via activation of P2X7 receptor. P2X4-knockdown cells, established by transfecting RAW264.7 cells with two short hairpin RNAs (shRNAs) targeting P2X4 receptor, showed a decrease of the initial peak of intracellular Ca(2+) after treatment with ATP, though pore formation and the P2X7-mediated activation of ERK1/2 and p38 MAPK were not affected. Intriguingly, P2X4 knockdown resulted in significant suppression of cell death induced by ATP or P2X7 agonist BzATP. In conclusion, our results suggest that P2X4 receptor is involved in P2X7 receptor-mediated cell death, but not pore formation or MAPK signaling.     

3-(Naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride attenuates NLRP3 inflammasome-mediated signaling pathway in lipopolysaccharide-stimulated BV2 microglial cells

The nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasome is a multiprotein complex with a role in innate immune responses. NLRP3 inflammasome dysfunction is a common feature of chronic inflammatory diseases. Microglia activation is also associated with neuroinflammatory pathologies. We previously reported that 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) reduced hypoxia-induced toxicity by modulating inflammation. However, no studies have elucidated the precise mechanisms for the anti-inflammatory action of KHG26792, in particular via inflammasome mediation. This study investigated the effects of KHG26792 on the inflammasome-mediated signaling pathway in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. KHG26792 significantly attenuated several inflammatory responses including tumor necrosis factor-α, interleukin-1β, interleukin-6, reactive oxygen species, and mitochondrial potential in these cells. KHG26792 also suppressed LPS-induced increase NLRP3, activated caspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) levels. Furthermore, KHG26792 successfully blocked LPS-activated adenosine triphosphate (ATP) level, likely through the purinergic receptor P2X ligand-gated ion channel 7 (P2X7) receptor. Our results suggest that the anti-inflammatory functions of KHG26792 may be, at least in part, due to regulation of the P2X7R/NLRP3-mediated signaling pathway during microglial activation.     

The first transmembrane domain of the P2X receptor subunit participates in the agonist-induced gating of the channel.

Based on pharmacological properties, the P2X receptor family can be subdivided into those homo-oligomers that are sensitive to the ATP analog alphabeta-methylene ATP(alphabetameATP) (P2X(1) and P2X(3)) and those that are not (P2X(2), P2X(4), P2X(5), P2X(6), and P2X(7)). We exploited this dichotomy through the construction of chimeric receptors and site-directed mutagenesis in order to identify domains responsible for these differences in the abilities of extracellular agonists to gate P2X receptors. Replacement of the extracellular domain of the alphabetameATP-sensitive rat P2X(1) subunit with that of the alphabetameATP-insensitive rat P2X(2) subunit resulted in a receptor that was still alphabetameATP-sensitive, suggesting a non-extracellular domain was responsible for the differential gating of P2X receptors by various agonists. Replacement of the first transmembrane domain of the rat P2X(2) subunit with one from an alphabetameATP-sensitive subunit (either rat P2X(1) or P2X(3) subunit) converted the resulting chimera to alphabetameATP sensitivity. This conversion did not occur when the first transmembrane domain came from a non-alphabetameATP-sensitive subunit. Site-directed mutagenesis indicated that the C-terminal portion of the first transmembrane domain was important in determining the agonist selectivity of channel gating for these chimeras. These results suggest that the first transmembrane domain plays an important role in the agonist operation of the P2X receptor.     

ERK7 is an autoactivated member of the MAPK family

Extracellular signal-regulated kinase 7 (ERK7) shares significant sequence homology with other members of the ERK family of signal transduction proteins, including the signature TEY activation motif. However, ERK7 has several distinguishing characteristics. Unlike other ERKs, ERK7 has been shown to have significant constitutive activity in serum-starved cells, which is not increased further by extracellular stimuli that typically activate other members of the mitogen-activated protein kinase (MAPK) family. On the other hand, ERK7's activation state and kinase activity appear to be regulated by its ability to utilize ATP and the presence of its extended C-terminal region. In this study, we investigated the mechanism of ERK7 activation. The results suggest that 1) MAPK kinase (MEK) inhibitors do not suppress ERK7 kinase activity; 2) intramolecular autophosphorylation is sufficient for activation of ERK7 in the absence of an upstream MEK; and 3) multiple regions of the C-terminal domain of ERK7 regulate its kinase activity. Taken together, these results indicate that autophosphorylation is sufficient for ERK7 activation and that the C-terminal domain regulates its kinase activity through multiple interactions.     

Inflammatory events in hippocampal slice cultures prime neuronal susceptibility to excitotoxic injury: a crucial role of P2X7 receptor-mediated IL-1beta release

We investigated the consequences of transient application of specific stimuli mimicking inflammation to hippocampal tissue on microglia activation and neuronal cell vulnerability to a subsequent excitotoxic insult. Two-week-old organotypic hippocampal slice cultures, from 7-day-old C57BL/6 donor mice, were exposed for 3 h to lipopolysaccharide (LPS; 10 ng/mL) followed by 3 h co-incubation with 1 mM ATP, or 100 microM 2'3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate triethylammonium, a selective P2X(7) receptor agonist. These treatments in combination, but not individually, induced a pronounced activation and apoptotic-like death of macrophage antigen-1 (MAC-1)-positive microglia associated with a massive release of interleukin (IL)-1beta exceeding that induced by LPS alone. Antagonists of P2X(7) receptors prevented these effects. Transient pre-exposure of slice cultures to a combination of LPS and P2X(7) receptor agonists, but not either one or the other alone, significantly exacerbated CA3 pyramidal cell loss induced by subsequent 12 h exposure to 8 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propinate (AMPA). Potentiation of AMPA toxicity was prevented when IL-1beta production or its receptor signaling were blocked by an inhibitor of interleukin-converting-enzyme or IL-1 receptor antagonist during application of LPS + ATP. The same treatments did not prevent microglia apoptosis-like death. These findings show that transient exposure to specific pro-inflammatory stimuli in brain tissue can prime neuronal susceptibility to a subsequent excitotoxic insult. P2X(7) receptor stimulation, and the consequent IL-1beta release, is mandatory for exacerbation of neuronal loss. These mechanisms may contribute to determine cell death/survival in acute and chronic neurodegenerative conditions associated with inflammatory events.     

P2X7 receptor as sensitive flow sensor for ERK activation in osteoblasts

The involvement of the P2 receptor in the activation of ERK induced by a short transient fluid flow stimulation in MC3T3-E1 osteoblasts was examined in the current study. The ERK activation induced by this transient fluid flow stimulation was followed by an increase in c-fos mRNA expression. Suramin, a non-selective P2 receptor antagonist, and two different P2X7 receptor (P2X7R) antagonists, ATP analogue (oxidized ATP) and dye (Brilliant blue G), inhibited fluid flow-induced ERK activation. However, the P2Y receptor pathway inhibitor U73122 did not abolish this ERK activation. The P2X7R agonist 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) significantly increased ERK activation and this activation could be completely inhibited by oxidized ATP and Brilliant blue G. Our results suggest that P2X7R is a highly sensitive P2 receptor for fluid flow-induced ERK activation in osteoblasts.     

N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.     

Phosphorylation of DCC by ERK2 is facilitated by direct docking of the receptor P1 domain to the kinase

Netrin receptor DCC plays critical roles in many cellular processes, including axonal outgrowth and migration, angiogenesis, and apoptosis, but the molecular basis of DCC-mediated signaling is largely unclear. ERK2, a member of the MAPK family, is one of the few proteins known to be involved in DCC-mediated signaling. Here, we report that ERK2 directly interacts with DCC, and the ERK2-binding region was mapped to the conserved intracellular P1 domain of the receptor. The structure of ERK2 in complex with the P1 domain of DCC reveals that DCC contains a MAPK docking motif. The docking of the P1 domain onto ERK2 physically positions several phosphorylation sites of DCC in the vicinity of the kinase active site. We further show that the docking interaction between the P1 domain and ERK2 is essential for the ERK2-mediated phosphorylation of DCC. We conclude that DCC signaling is directly coupled with MAPK signaling cascades.     

Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell death via a caspase-1-dependent mechanism, independently of cytokine production.

ATP stimulation of cell surface P2X7 receptors results in cytolysis and cell death of macrophages. Activation of this receptor in bacterial lipopolysaccharide (LPS)-activated macrophages or monocytes also stimulates processing and release of the cytokine interleukin-1beta(IL-1beta) through activation of caspase-1. The cytokine interleukin 18 (IL-18) is also cleaved by caspase-1 and shares pro-inflammatory characteristics with IL-1beta. The objective of the present study was to test the hypothesis that IL-1beta, IL-18, and/or caspase-1 activation contribute directly to macrophage cell death induced by LPS and ATP. Macrophages were cultured from normal mice or those in which genes for the P2X7 receptor, IL-1beta, IL-1alpha, IL-18, or caspase-1 had been deleted. Our data confirm the importance of the P2X7 receptor in ATP-stimulated cell death and IL-1beta release from LPS-primed macrophages. We demonstrate that prolonged stimulation with ATP leads to cell death, which is partly dependent on LPS priming and caspase-1, but independent of cytokine processing and release. We also provide evidence that LPS priming of macrophages makes them highly susceptible to the toxic effects of brief exposure to ATP, which leads to rapid cell death by a mechanism that is dependent on caspase-1 but, again, independent of cytokine processing and release.     

The use of phage display technique for the isolation of androgen receptor interacting peptides with (F/W)XXL(F/W) and FXXLY new signature motifs

Early studies suggested that the signature motif, LXXLL, within steroid hormone receptor p160 coregulators may play important roles for the mediation of receptor-coregulator interaction. Interestingly, several androgen receptor (AR) coregulators, such as ARA70 and ARA55, may not use such a unique motif to mediate their coregulator activity. Here we apply the phage display technique to identify some new signature motifs, (F/W)XXL(F/W) and FXXLY (where F is phenylalanine, W is tryptophan, L is leucine, Y is tyrosine, and X is any amino acid) that can influence the interaction between AR and AR coregulators. Sequence analyses found that several AR coregulators, such as ARA70, ARA55, ARA54, and FHL2, contain FXXL(F/Y) motifs. Both glutathione S-transferase pull-down assays and transient transfection reporter assays demonstrate that these AR coregulators may use the FXXL(F/Y) motif to interact with AR and exert their AR coregulator activity. Exchanging the amino acid of Phe, Trp, or Tyr in this newly identified signature motif cluster may influence these peptides to interact with AR. The motif-containing peptides, as well as ARA70 or ARA54, may require selective flanking sequences for the better interaction with AR. In addition to influencing the AR transactivation, these motifs in AR-interacting peptides/proteins were also able to influence the AR N-/C-terminal interaction. Together, our data suggest that AR interacting peptides and/or AR coregulators may utilize the (F/W)XXL(F/W) and FXXLY motifs to mediate their interaction with AR and exert their influences on the AR transactivation.     

Involvement of the purinergic P2X7 receptor in the formation of multinucleated giant cells

Multinucleated giant cells (MGC), a hallmark of chronic inflammatory reactions, remain an enigma of cell biology. There is evidence implicating the purinergic P2X7 receptor in the fusion process leading to MGC. To investigate this, we used HEK 293 cells stably transfected with either 1) the full-length rat P2X7 receptor (P2X7 cells), 2) a rat P2X7 receptor lacking the C-terminal domain (P2X7TC), or 3) a mock vector, and rat alveolar macrophages (MA) expressing the native receptor. P2X7 cells cultured in serum-free medium formed increased numbers of MGC and displayed a higher fusion index compared with mock transfectants. Stimulation of P2X7 pore-forming activity in P2X7 cells by polymyxin B (PMB) further increased significantly the formation of MGC. Conversely, blockers of P2X-receptors including oxidized ATP, brilliant blue G, and pyridoxal phosphate-6-azophenyl-2'-4'-disulfonic acid inhibited significantly MGC formation in both unstimulated and PMB-stimulated P2X7-transfected cells. In contrast, cells transfected with the truncated P2X7TC were devoid of pore-forming activity, did not respond to PMB stimulation, and failed to form enhanced numbers of MGC, thus behaving as mock transfectants. As found for P2X7-transfected cells, PMB also potentiated dose-dependently the formation of multinucleated MA by rat alveolar MA. Pretreatment with oxidized ATP abrogated the PMB stimulatory effects. Together, these data demonstrate unequivocally the participation of P2X7 receptor in the process of MGC formation. Our study also provides evidence suggesting that stimulation of the P2X7 receptor pathway in MA may mediate increased formation of MGC during chronic inflammatory reactions.