Mouse P2X7 Receptor Research Articles

Synthetic 1,4-Naphthoquinones inhibit P2X7 receptors in murine neuroblastoma cells

The P2X7 receptor (P2X7R) is an ATP-gated ion channel and potential therapeutic target for new drug development. In this study, we synthesized a series of new 1,4-naphthoquinone (1,4-NQ) derivatives and investigated their antagonistic effects against mouse P2X7R. We explored the ability of the tested substances to block ATP-induced Ca2+ influx into mouse Neuro-2a cells and selected the four most effective substances: the 1,4-naphthoquinone thioglucosides U-548 and U-557 and their tetracyclic conjugates U-286 and U-556. Biological analysis of these compounds revealed significant in vitro inhibition of murine P2X7R. This inhibition resulted in marked blockade of ethidium bromide (EtBr) and YO-PRO-1 fluorescent dye uptake, pronounced decreases in ROS and NO production and protection of neuronal cell viability against the toxic action of high ATP concentrations. In silico analysis indicated favorable molecular docking results of these 1,4-NQs, pointing to their potential to bind in an allosteric site located in the extracellular region of P2X7R. These findings suggest compounds U-286, U-548, U-556 and U-557 as potential scaffolds for the design of new P2X7R blockers and drugs effective against neuropathic pain and neurodegenerative diseases.

Design, synthesis and evaluation in an LPS rodent model of neuroinflammation of a novel 18F-labelled PET tracer targeting P2X7

BackgroundThe P2X7 receptor has been shown to play a fundamental role in the initiation and sustenance of the inflammatory cascade. The development of a novel fluorine-18 PET tracer superior and with a longer half-life to those currently available is a promising step towards harnessing the therapeutic and diagnostic potential offered by this target. Inspired by the known antagonist A-804598, the present study outlines the design via molecular docking, synthesis and biological evaluation of the novel P2X7 tracer [18F]EFB. The tracer was radiolabelled via a three-step procedure, in vitro binding assessed in P2X7-transfected HEK293 and in B16 cells by calcium influx assays and an initial preclinical evaluation was performed in a lipopolysaccharide (LPS)-injected rat model of neuroinflammation.ResultsThe novel tracer [18F]EFB was synthesised in 210 min in 3–5% decay-corrected radiochemical yield (DC RCY), >99% radiochemical purity (RCP) and >300 GBq/μmol and fully characterised. Functional assays showed that the compound binds with nM Ki to human, rat and mouse P2X7 receptors. In vivo, [18F]EFB displayed a desirable distribution profile, and while it showed low blood–brain barrier penetration, brain uptake was quantifiable and displayed significantly higher mean longitudinal uptake in inflamed versus control rat CNS regions.Conclusions[18F]EFB demonstrates strong in vitro affinity to human and rodent P2X7 and limited yet quantifiable BBB penetration. Considering the initial promising in vivo data in an LPS rat model with elevated P2X7 expression, this work constitutes an important step in the development of a radiotracer useful for the diagnosis and monitoring of clinical disorders with associated neuroinflammatory processes.

Species Difference in Sensitivity of Human and Mouse P2X7 Receptors to Inhibitory Effects of Divalent Metal Cations.

P2X7 receptor (P2X7R), a purinergic receptor, is involved in pathophysiological events such as inflammation and cell death, and thus is an attractive target for therapeutic approaches. It is reported that divalent metal cations (DMCs) inhibit P2X7R activation and that there are species differences in their inhibitory effects. To extrapolate the findings in experimental animals to humans, these species differences have to be clarified, but species differences in the sensitivity of P2X7R to DMCs between man and mouse have not been demonstrated. Here we performed direct comparison of the inhibitory effects of DMCs on human and mouse P2X7R activation. Cell lines constitutively expressing human and mouse P2X7R were used, and their P2X7R activation was evaluated as means of YO-PRO-1 dye uptake. MgCl2, NiCl2, ZnCl2, CuCl2 and CaCl2 dose-dependently decreased agonist-induced YO-PRO-1 uptake via both human and mouse P2X7Rs. Apparent differences in the inhibitory profiles for NiCl2 and CaCl2 between them were found, and the IC50 values of DMCs were in the order of CaCl2>MgCl2>NiCl2≈ZnCl2>CuCl2 for both human and mouse P2X7Rs. In this study, we demonstrate that human P2X7R exhibits different sensitivity to nickel and calcium compared with the case of the mouse one, while there is no species difference in the sensitivity of their P2X7Rs to magnesium, zinc and copper, suggesting that the effects of magnesium, zinc and copper on P2X7R-associated pathophysiological events in humans might be predicted from those in mice.

Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors

In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.

Expression, assembly and function of novel C‐terminal truncated variants of the mouse P2X7 receptor: re‐evaluation of P2X7 knockouts

Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7(-/-) mice (Pfizer KO). Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function.

Splice-variants of the P2X7 receptor reveal differential agonist-dependence and functional coupling with pannexin-1

P2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca(2+) and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner.

Mammalian P2X7 receptor pharmacology: comparison of recombinant mouse, rat and human P2X7 receptors

Acute activation of P2X7 receptors rapidly opens a non-selective cation channel. Sustained P2X7 receptor activation leads to the formation of cytolytic pores, mediated by downstream recruitment of hemichannels to the cell surface. Species- and single-nucleotide polymorphism-mediated differences in P2X7 receptor activation have been reported that complicate understanding of the physiological role of P2X7 receptors. Studies were conducted to determine pharmacological differences between human, rat and mouse P2X7 receptors. Receptor-mediated changes in calcium influx and Yo-Pro uptake were compared between recombinant mouse, rat and human P2X7 receptors. For mouse P2X7 receptors, wild-type (BALB/c) and a reported loss of function (C57BL/6) P2X7 receptor were also compared. BzATP [2,3-O-(4-benzoylbenzoyl)-ATP] was more potent than ATP in stimulating calcium influx and Yo-Pro uptake at rat, human, BALB/c and C57BL/6 mouse P2X7 receptors. Two selective P2X7 receptor antagonists, A-740003 and A-438079, potently blocked P2X7 receptor activation across mammalian species. Several reported P2X1 receptor antagonists [e.g. MRS 2159 (4-[(4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl}-2-pyridinyl)azo]-benzoic acid), PPNDS and NF279] blocked P2X7 receptors. NF279 fully blocked human P2X7 receptors, but only partially blocked BALB/c P2X7 receptors and was inactive at C57BL/6 P2X7 receptors. These data provide new insights into P2X7 receptor antagonist pharmacology across mammalian species. P2X7 receptor pharmacology in a widely used knockout background mouse strain (C57BL/6) was similar to wild-type mouse P2X7 receptors. Several structurally novel, selective and competitive P2X7 receptor antagonists show less species differences compared with earlier non-selective antagonists.

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC 50 = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED 50 = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED 50 = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.

Amino Acid Residues in the P2X7 Receptor that Mediate Differential Sensitivity to ATP and BzATP

Agonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 microM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.

A Truncated P2X7 Receptor Variant (P2X7-j) Endogenously Expressed in Cervical Cancer Cells Antagonizes the Full-length P2X7 Receptor through Hetero-oligomerization

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.

A Role for Mitogen-activated Protein KinaseErk1/2 Activation and Non-selective Pore Formation in P2X7 Receptor-mediated Thymocyte Death

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. Thus, extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation was found to be required for cell lysis, and both events were independent of ATP-induced calcium influx. P2X7R-dependent thymocyte death involved the chronological activation of Src family tyrosine kinase(s), phosphatidylinositol 3-kinase, the mitogen-activated protein (MAP) kinase(Erk1/2) module, and the proteasome. Although independent of this signaling cascade, non-selective pore formation may modulate ATP-mediated thymocyte death. These results therefore suggest a role for both activation of MAP kinase(Erk1/2) and non-selective pore opening in P2X7R-induced thymocyte death.

Regulation of the P2X7 Receptor Permeability to Large Molecules by Extracellular Cl– and Na+

Upon continuous stimulation, the pore of the monovalent cation-selective P2X7 receptor (P2X7R) expands to accommodate large molecules such as N-methyl-D-glucamine (NMDG+). How the change in P2X7R permeability is regulated is not known. Here we report that extracellular Cl- (Cl-(o)) regulates the outward current, whereas extracellular Na+ (Na+(o)) regulates the inward current of large molecules by P2X7Rs. The P2X7R-mediated current was measured in parotid acinar and duct cells of wild type and P2X7R-/- mice and in HEK293 cells expressing the human or mouse P2X7R isoforms. In symmetrical NaCl, triethylammonium chloride, and NMDG+ chloride solutions, the P2X7R current followed a linear current/voltage relationship. In symmetrical NaCl, removal of Cl-(o) reduced the inward Na+ current by approximately 35% and the outward Na+ current by only 10%. By contrast, in the absence of Na+(i) and the presence of Na+(o) or NMDG+(o), the removal of Cl-(o) reduced the inward Na+ or NMDG+ currents by 35% but the outward NMDG+ current by >95%. The effect of Cl-(o) was half-maximal at approximately 60 mm. Reducing Cl-(i) from 150 to 10 mm did not reproduce the effects of Cl-(o). All currents were eliminated in P2X7R-/- cells and reproduced by expressing the P2X7Rs in HEK cells. These findings suggest that Cl-(o) primarily regulates the outward P2X7R current of large molecules. When cells dialyzed with NMDG+ were stimulated in the presence of Na+(o), subsequent removal of Na+(o) resulted in a strongly outward rectifying NMDG+ current, indicating maintained high selectivity for Na+ over NMDG+. During continuous incubation in Na+-free medium, the permeability of the P2X7Rs to NMDG+ gradually increased. On the other hand, when the cells were incubated in symmetrical NMDG+ and only then stimulated with ATP, the NMDG+ current by P2X7Rs followed a linear current/voltage relationship and did not change with time. These findings suggest that the P2X7R has a "Na+(o) memory" and that Na+(o) regulates the inward permeability of P2X7Rs to large molecules. The novel regulation of P2X7R outward and inward permeability to large molecules by Cl-(o) and Na+(o), respectively, may have an important protective function, particularly in secretory epithelial cells.

Extracellular ATP Induces Cytokine Expression and Apoptosis through P2X7 Receptor in Murine Mast Cells

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor.

The P2X7 receptor (P2X(7)R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X(7)R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X(7)R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X(7)R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X(7)R. These P2X(7)R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation.

Characteristics of ATP-Induced Current Through P2X7 Receptor in NG108-15 Cells: Unique Antagonist Sensitivity and Lack of Pore Formation

ATP activates the mouse P2X7 receptor and induces a nonselective-cation current in NG108-15 cells. We investigated the effects of five receptor antagonists on the ATP-induced nonselective-cation current through P2X7 receptor (I(NS.P2X7)) in NG108-15 cells. Nonselective P2 receptor antagonists, RB-2, PPADS and suramin inhibited the I(NS.P2X7) with IC50 values of 4.3, 53 and 40 microM, respectively. However, KN-04, which is a potent antagonist of human P2X7 receptors but is not that of rat P2X7 receptors, had only a weak blocking effect. Furthermore, oxidized-ATP (300 microM), an antagonist of the P2X7 receptor-mediated pore-formation, did not affect the I(NS.P2X7). Prolonged ATP application did not increase the membrane permeability to large molecules, N-methyl-D-glucamine or Yo-Pro-1, indicating that pore-formation was not promoted by the P2X7 receptor activation in NG108-15 cells. These results suggest that antagonist sensitivities and pore-forming properties of the P2X7 receptors in NG108-15 cells are different from those of other cells types.