P2X7 receptors regulate engulfing activity of non-stimulated resting astrocytes

We previously demonstrated that P2X7 receptors (P2X7Rs) expressed by cultured mouse astrocytes were activated without any exogenous stimuli, but its roles in non-stimulated resting astrocytes remained unknown. It has been reported that astrocytes exhibit engulfing activity, and that the basal activity of P2X7Rs regulates the phagocytic activity of macrophages. In this study, therefore, we investigated whether P2X7Rs regulate the engulfing activity of mouse astrocytes. Uptake of non-opsonized beads by resting astrocytes derived from ddY-mouse cortex time-dependently increased, and the uptaken beads were detected in the intracellular space. The bead uptake was inhibited by cytochalasin D (CytD), an F-actin polymerization inhibitor, and agonists and antagonists of P2X7Rs apparently decreased the uptake. Spontaneous YO-PRO-1 uptake by ddY-mouse astrocytes was reduced by the agonists and antagonists of P2X7Rs, but not by CytD. Down-regulation of P2X7Rs using siRNA decreased the bead uptake by ddY-mouse astrocytes. In addition, compared to in the case of ddY-mouse astrocytes, SJL-mouse astrocytes exhibited higher YO-PRO-1 uptake activity, and their bead uptake was significantly greater. These findings suggest that resting astrocytes exhibit engulfing activity and that the activity is regulated, at least in part, by their P2X7Rs.     

The human cathelicidin LL-37 modulates the activities of the P2X7 receptor in a structure-dependent manner

Extracellular ATP, released at sites of inflammation or tissue damage, activates the P2X(7) receptor, which in turn triggers a range of responses also including cell proliferation. In this study the ability of the human cathelicidin LL-37 to stimulate fibroblast growth was inhibited by commonly used P2X(7) blockers. We investigated the structural requirements of the growth-promoting activity of LL-37 and found that it did not depend on helix sense (the all-d analog was active) but did require a strong helix-forming propensity in aqueous solution (a scrambled analog and primate LL-37 orthologs devoid of this property were inactive). The involvement of P2X(7) was analyzed using P2X(7)-expressing HEK293 cells. LL-37 induced proliferation of these cells, triggered Ca(2+) influx, promoted ethidium bromide uptake, and synergized with benzoyl ATP to enhance the pore and channel functions of P2X(7). The activity of LL-37 had an absolute requirement for P2X(7) expression as it was blocked by the P2X(7) inhibitor KN-62, was absent in cells lacking P2X(7), and was restored by P2X(7) transfection. Of particular interest, LL-37 led to pore-forming activity in cells expressing a truncated P2X(7) receptor unable to generate the non-selective pore typical of the full-length receptor. Our results indicate that P2X(7) is involved in the proliferative cell response to LL-37 and that the structural/aggregational properties of LL-37 determine its capacity to modulate the activation state of P2X(7).     

The P2X(7) receptor-mediated phospholipase D activation is regulated by both PKC-dependent and PKC-independent pathways in a rat brain-derived Type-2 astrocyte cell line, RBA-2.

The aim of this study was to characterize the regulatory mechanisms of the P2X(7) receptor (P2X(7)R)-mediated phospholipase D (PLD) activation in a rat brain-derived Type-2 astrocyte cell line, RBA-2. A time course study revealed that activation of P2X(7)R resulted in a choline and not phosphorylcholine formation, suggesting that activation of P2X(7)R is associated with the phosphatidylcholine-PLD (PC-PLD) in these cells. GF 109203X, a selective protein kinase C (PKC) inhibitor, partially inhibited the P2X(7)R-mediated PLD activation, while blocking the phorbol 12-myristate 13-acetate (PMA)-stimulated PLD activity. In addition, PMA synergistically activated the P2X(7)R-mediated PLD activity. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the P2X(7)R-activated PLD, while KN62, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, was less effective, whereas the mitogen-activated protein kinase (MAPK) inhibitor PD98059 was ineffective. No additive inhibitory effects were found by simultaneous treatment of GF 109203X and KN62 on P2X(7)R-activated PLD. Taken together, these results demonstrate that both PKC-dependent and PKC-independent signaling pathways are involved in the regulation of P2X(7)R-mediated PLD activation. Additionally, CaMKII may participate in the PKC-dependent pathway, and tyrosine kinase may play a pivotal role on both PKC-dependent and PKC-independent pathways in the P2X(7)R-mediated PLD activation in RBA-2 cells.     

Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca²⁺ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.     

Paroxetine suppresses recombinant human P2X7 responses

P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC₅₀ of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC₅₀ of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14⁺ monocytes was suppressed with trifluoperazine and paroxetine.     

Synthesis and in vitro activity of 1-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-1H-1,2,4-triazol-5-amine and 4-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-4H-1,2,4-triazol-3-amine P2X7 antagonists

A novel series of aminotriazole-based P2X(7) antagonists was synthesized, and their structure-activity relationships (SAR) were investigated for activity at both human and rat P2X(7) receptors. Most compounds showed greater potency at the human receptor although several analogs were discovered with potent activity (pIC(50) > or = 7.5) at both human and rat P2X(7).     

P2X purinergic receptor channel expression and function in bovine aortic endothelium

We examined bovine aortic endothelial cells (BAECs) for the functional expression of P2X receptors, the ATP-gated cation channels. We identified the P2X subtypes present in BAECs using RT-PCR. mRNA was present for only three of seven family members: P2X4, P2X5, and P2X7. We then characterized agonist-activated currents in whole cell and outside-out patch recordings using 2-methyl-thio-ATP (MeSATP) as a P2X4 and P2X5 receptor agonist and 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP) as a P2X7 receptor agonist. MeSATP (10-20 microM) produced current with characteristics of P2X4 receptors. The current was an inwardly rectifying current, reversed near 0 mV, slowly desensitized, was not blocked by suramin (300 microM) or reactive blue (60 microM), and had a single channel conductance of 36 pS. BzATP (10-100 microM), on the other hand, activated a 9-pS channel with sustained activity in the continued presence of the agonist. BzATP-activated current was blocked by reactive blue (60 microM) and by suramin (approximately 50% block at 300 microM). We confirmed, by immunocytochemistry, the presence of P2X4 and P2X7 protein. The agonists failed, however, to induce significant uptake of the large molecule YO-PRO, indicating the lack of pore development that has been demonstrated for P2X7 and P2X4 in response to agonist in some cell types.     

The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity

P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.     

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.     

Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

Background

Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.

Principal Findings

P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.

Conclusions

In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.     

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.     

Rottlerin inhibits P2X(7) receptor-stimulated phospholipase D activity in chronic lymphocytic leukaemia B-lymphocytes

Phospholipase D (PLD) is a ubiquitous enzyme that can be activated by extracellular adenosine 5'-triphosphate (ATP) or phorbol 12-myristate 13-acetate (PMA) in B-lymphocytes from subjects with chronic lymphocytic leukaemia (CLL). In this study, ATP- but not PMA-induced PLD stimulation in CLL B-lymphocytes was abolished in the presence of an anti-P2X(7) receptor monoclonal antibody, as well as in B-lymphocytes from CLL subjects homozygous for the Glu(496) to Ala loss-of-function P2X(7) polymorphism. Rottlerin, an inhibitor of novel protein kinase C (PKC) isoforms, but not GF 109203X, an inhibitor of conventional PKC isoforms, impaired the ATP-stimulated PLD activity in CLL B-lymphocytes. In contrast, both inhibitors impaired PLD activity stimulated by PMA, a known mediator of PKC activation. The inhibition of P2X(7)-stimulated PLD activity by rottlerin was attributed to a target downstream of P2X(7) activation, as the ATP-mediated (86)Rb(+) efflux from CLL B-lymphocytes was not altered in the presence of rottlerin. Our results indicate a possible role for novel PKC isoforms in the regulation of P2X(7)-mediated PLD activity.     

P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases,caspases, and IL-1 beta release

In response to ATP binding, the P2X7R facilitates cation channel activation, nonspecific pore formation, rapid changes in plasma membrane morphology, and secretion of IL-1 beta from LPS-primed macrophages. To investigate the relationship between the P2X7R-dependent changes in plasma membrane organization and the release of IL-1 beta, we generated time-lapse movies of ATP-stimulated BAC1 murine macrophages in conjunction with biochemical analyses of IL-1 beta release. Similar image analyses in human embryonic kidney 293 cells expressing recombinant P2X7R (HEK-P2X7) permitted comparison of P2X7R-dependent effects in macrophage vs nonmacrophage backgrounds. Whereas HEK-P2X7 cells exhibit zeiotic blebbing within 5 min of ATP treatment, BAC1 macrophages initiated a distinct "tethered" blebbing 10 min after ATP addition. This blebbing was comparably induced by the P2X7R-selective agonist BzATP and was blocked by P2X7R inhibitors KN-62 and oxidized ATP. Blebbing was initiated at ATP concentrations > or = 3 mM, but optimal IL-1 beta release occurred at 1 mM ATP. P2X7R-dependent blebbing was abrogated in the presence of Rho-effector kinase inhibitors Fasudil and Y-27632, but ATP-induced IL-1 beta release was unaffected. ATP-induced activation of RhoA could be detected in both HEK-P2X7 cells and BAC1 murine macrophages. Thus, P2X7R activation signals distinct, novel membrane blebbing events (dependent on RhoA activation and Rho-effector kinase activity) and simultaneously initiates release of IL-1 beta. Our observations that blebbing and IL-1 beta release are dissociable suggest these events occur via parallel rather than convergent signaling pathways.     

P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E₂ (PGE₂) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE₂ release and was attenuated by inhibition of either phospholipase A₂ or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.     

Somatic and axonal effects of ATP via P2X2 but not P2X7 receptors in rat thoracolumbar sympathetic neurones

Excitatory ATP responses in rat cultured thoracolumbar sympathetic neurones are mediated by somatic P2X(2) receptors. The present study investigated a possible role of axonal P2X(2) as well as P2X(7) receptors on the same preparation. Confocal laser scanning microscopy demonstrated P2X(2) and P2X(7) immunoreactivity along the axons as well as P2X(7) immunoreactivity surrounding the cell nuclei. P2X(7) mRNA expression was detected in individual neurones using a single-cell RT-PCR approach. Adenosine triphosphate (ATP) caused a significant increase in axonal Ca(2+) concentration which was dependent on external Ca(2+) but insensitive to depletion of the cellular Ca(2+) pools by cyclopiazonic acid. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 micro m) virtually abolished the ATP response, whereas brilliant blue G (0.1 micro m), a selective P2X(7) receptor antagonist, had no effect. Dibenzoyl-ATP (BzATP; 100 micro m) induced a much smaller increase in axonal [Ca(2+)] concentration than ATP at equimolar concentrations. The response to BzATP was distinctly reduced by PPADS but not by brilliant blue G. The overall pharmacological profile of the axonal P2X receptors resembled closely that of the somatic P2X(2) receptors. In conclusion, the present data suggest the occurrence of axonal excitatory P2X(2) receptors in thoracolumbar sympathetic neurones. However, the functional significance of axonal and (peri)-nuclear P2X(7) receptors has still to be proven.     

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.     

8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages

Extracellular adenosine 5′-triphosphate (ATP) triggers the P2X7 receptor (P2X7R) ionic channel to stimulate the release of the interleukin-IL-1β cytokine into macrophages. The current study explored the reaction of six structurally diverse triazole derivatives on P2X7-mediated dye uptake into murine peritoneal macrophages. P2X7R activity determined by ATP-evoked fluorescent dye uptake. Triazole derivatives toxicity measured using dextran rhodamine exclusion based colorimetric assay. A740004 and BBG, both P2X7R antagonist, inhibited ATP-induced dye uptake. In contrast, the derivatives 5a, 5b, 5e, and 5f did not diminish P2X7R activity in concentrations until 100?µM. 5c and 5d analogs caused a potent inhibitory activity on P2X7-induced dye uptake. Dextran Rhodamine exclusion measurements after 24?h of continuous treatment with triazole derivatives indicated a moderated toxicity for all molecules. In conclusion, this study showed that a series of new hybrid 1,2,3-triazolic naphthoquinones reduces P2X7R-induced dye uptake into murine macrophages. In silico analysis indicates a good pharmacokinetic profile and molecular docking results of these analogs indicate the potential to bind into an allosteric site located into the P2X7R pore and juxtaposed with the ATP binding pocket. In this manner, the compounds 5c and 5d may be used as a scaffold for new P2X7R inhibitors with reduced toxicity, and good anti-inflammatory activity.     

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.     

Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.