Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^?/? mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^?/? mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^−/− mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^−/− mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Synthesis and SAR development of novel P2X7 receptor antagonists for the treatment of pain: part 1

Structure-activity relationship (SAR) efforts around our initial lead compound 1 led to the identification of potent P2X₇ receptor antagonists with improved pharmacokinetic profiles. These compounds were potent and selective at the P2X₇ receptor in both human and rodent. Compound (entry 31) exhibited oral efficacy in the rat MIA and CCI pain models.     

P2X(7) receptor activation causes phosphatidylserine exposure in human erythrocytes

Activation of cation channels causes erythrocyte phosphatidylserine (PS) exposure and cell shrinkage. Human erythrocytes express the P2X₇ receptor, an ATP-gated cation channel. The two most potent P2X₇ agonists, BzATP and ATP, stimulated PS exposure in human erythrocytes. Other nucleotides also induced erythrocyte PS exposure with an order of agonist potency of BzATP > ATP > 2MeSATP > ATPγS; however neither ADP nor UTP had an effect. ATP induced PS exposure in erythrocytes in a dose-dependent fashion with an EC₅₀ of ∼75 μM. BzATP- and ATP-induced erythrocyte PS exposure was impaired by oxidised ATP, as well as in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X₇ receptor. ATP-induced PS exposure in erythrocytes was not significantly altered in the presence of EGTA excluding a role for extracellular Ca²⁺. These results show that P2X₇ activation by extracellular ATP can induce PS exposure in erythrocytes.     

Synthesis and SAR development of novel P2X7 receptor antagonists for the treatment of pain: part 2

Novel P2X₇ antagonists were developed using a purine scaffold. These compounds were potent and selective at the P2X₇ receptor in human and rodent as well as efficacious in rodent pain models. Compound 15a was identified to have oral potency in several pain models in rodent similar to naproxen, gabapentin and pregabalin. Structure–activity relationship (SAR) development and results of pain models are presented.     

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X₇ antagonists. These compounds were assayed for activity at both the human and rat P2X₇ receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X₇ receptors. Compounds 12 and 38 displayed hP2X₇pIC₅₀s >7.8 with less than 2-fold difference in potency at the rP2X₇.     

Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter

A region 2 kb upstream of exon 1 of the P2X7 gene was sequenced using DNA from nine healthy individuals who exhibited three different ATP response phenotypes (i.e. high, low and interferon gamma-inducible). Five single nucleotide polymorphisms were identified within the nine donor promoter sequences but none were associated with a specific ATP response phenotype. A P2X7 loss of function polymorphism (1513 in exon 13) was also screened for within donor DNA but no response associations were identified. ATP response phenotype was positively associated with P2X(7) receptor expression, as assessed by flow cytometry, but not with any identified receptor or promoter gene polymorphisms.     

Identification of an intracellular microdomain of the P2X7 receptor that is crucial in basolateral membrane targeting in epithelial cells

We investigated membrane targeting of the P2X₇ receptor (P2X₇R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein μ1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X₇R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process.

Structured summary

MINT-8055849:Beta-catenin (uniprotkb:B6V8E6) and P2X7R (uniprotkb:Q64663) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Identification and characterization of splice variants of the human P2X7 ATP channel

The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing. Two of these variants (one lacking the first transmembrane domain, the second lacking the entire cytoplasmic tail) were compared to the full-length channel. Real-time PCR analysis demonstrated that both variants were expressed in various tissues and that the cytoplasmic tail deleted variant is highly expressed. Deletion of the first transmembrane domain resulted in a non-functional channel. Deletion of the cytoplasmic tail did not affect ion movement but severely affected the ability to form a large pore and to induce activation of caspases.     

Different properties of P2X7 receptor in hippocampal and cortical astrocytes

P2X₇ receptor is a ligand-gated ion channel, which can induce the opening of large membrane pores. Here, we provide evidence that the receptor induces pore formation in astrocytes cultured from cortex, but not from the hippocampus. Furthermore, P2X₇ receptor activation promptly induces p38 mitogen-activated protein kinase (MAPK) phosphorylation in cortical but not in hippocampal astrocytes. Given the role of p38 MAPK activation in pore opening, these data suggest that defective coupling of the receptor to the enzyme could occur in hippocampal cultures. The different capabilities of the receptor to open membrane pores cause relevant functional consequences. Upon pore formation, caspase-1 is activated and pro-IL1-β is cleaved and released extracellularly. The receptor stimulation does not result in interleukin-1beta secretion from hippocampal astrocytes, although the pro-cytokine is present in the cytosol of lipopolysaccharide-primed cultures. These results open the possibility that activation of P2X₇ receptors differently influences the neuroinflammatory processes in distinct brain regions.     

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.     

P2X7 receptor expression is decreased in epithelial cancer cells of ectodermal, uro-genital sinus, and distal paramesonephric duct origin

The P2X₇ receptor is an important regulator of epithelial cell growth. The aim of the present study was to better understand the biological significance of P2X₇ receptor expression in normal and cancer human epithelial tissues. P2X₇ receptor and messenger RNA (mRNA) levels were determined in human tissues containing epithelial dysplastic, pre- or early cancerous, and cancer cells, and the levels were compared to those in the corresponding normal epithelial cells within the same tissue of the same case. P2X₇ receptor levels were determined by quantification of immunoreactivity specific to the functional (full-length) P2X₇ receptor, and P2X₇ mRNA levels were determined by real-time polymerase chain reaction. P2X₇ receptor levels in cancer cells were similar (colon adenocarcinoma) or greater (thyroid papillary carcinoma) than those in the corresponding normal cells. In contrast, in cancer cells of the ectocervix (squamous), endocervix and endometrium (adenocarcinoma), urinary bladder (transitional cell carcinoma), and breast (ductal and lobular adenocarcinomas), P2X₇ receptor levels were lower by about twofold than those in the corresponding normal epithelial cells. Similarly, P2X₇ mRNA levels were lower in uterine, bladder, and breast cancer epithelial tissues by about fourfold than those in the corresponding normal tissues. In addition, P2X₇ receptor levels were decreased already in dysplastic ectocervical cells and pre- or early cancerous endometrial and bladder cells. The data suggest that in epithelia originating from the ectoderm, the uro-genital sinus, and the distal paramesonephric duct, decreased expression of the P2X₇ receptor precedes or coincides with neoplastic changes in those tissues.     

The role of P2X7 in pain and inflammation

The P2X₇ purinoceptor is unique amongst the P2X receptor family in that its activation is able to stimulate the release of mature, biologically active interleukin-1β (IL-1β), as well as a variety of other proinflammatory cytokines. Coupled with the predominate localisation of this receptor to immunocytes of haemopoetic origin, this receptor is an obvious candidate to play a major and pivotal role in processes of pain and inflammation. Using genetically modified animals that lack the P2X₇ receptor, several investigators have shown that these mice do indeed demonstrate a blunted inflammatory response, and fail to develop pain following both inflammatory and neuropathic insult. These animals also show altered cytokine production in response to inflammatory stimulus, which is far broader than merely modulation of IL-1β release. In this short article, we review the role of the P2X₇ receptor in modulating the release of cytokines and other mediators, and discuss the findings made from P2X₇ receptor-deficient animals. As well as highlighting outstanding questions regarding this intriguing receptor, we also speculate as to the potential therapeutic benefit of P2X₇ receptor modulation.     

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X₁–P2X₇) have been identified. Most of the P2X₁ receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X₁ receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X₁ receptors. In the present study, we examined the effect of gintonin on P2X₁ receptor channel activity. P2X₁ receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X₁ receptors induced large inward currents (I _ATP ), but repetitive ATP treatments induced a rapid desensitization of I _ATP . Gintonin treatment after P2X₁ receptor desensitization potentiated I _ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I _ATP . Gintoninmediated I _ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X₁ receptor greatly attenuated the gintonin-mediated I _ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X₁ receptor through a phosphoinositide-dependent pathway.     

Trehalose uptake through P2X7 purinergic channels provides dehydration protection

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.     

P2X7 receptors mediate NADH transport across the plasma membranes of astrocytes

NADH plays critical roles in mitochondrial functions and energy metabolism. There has been no study demonstrating that NADH can be transported across the plasma membranes of cells. In this study we tested our hypothesis that NADH can be transported across the plasma membranes of astrocytes by a P2X7 receptor (P2X7R)-mediated mechanism. We found that treatment of astrocytes with NADH led to increases in both intracellular NADH and NAD+. Three lines of studies suggest that P2X7R mediates the NADH transport into astrocytes: the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) blocked the NADH transport; RNAi knockdown of P2X7R led to decreased NADH transport; and transfection of HEK293 cells with mouse P2X7R cDNA led to increased NADH transport. Collectively, our study provides the first direct evidence demonstrating that NADH can be transported across the plasma membranes of astrocytes by a P2X7R-mediated mechanism. Our study also suggests a novel approach for manipulating intracellular NADH and NAD+ levels.     

Inhibition of the human P2X7 receptor by a novel protein tyrosine kinase antagonist

A panel of 18 protein tyrosine kinase antagonists were tested for their inhibitory effect on human P2X₇ receptor-mediated ⁸⁶Rb⁺ (K⁺) efflux. The most potent compound (compound P), a phthalazinamine derivative and an inhibitor of vascular endothelial growth factor receptor kinase, blocked ATP-induced ⁸⁶Rb⁺-efflux in human B-lymphocytes and erythrocytes by 76% and 66%, respectively. This inhibition was dose-dependent in both cell types with an IC₅₀ of ∼5 μM. Kinetic analysis showed compound P was a non-competitive inhibitor of P2X₇. This compound also inhibited ATP-induced ethidium⁺ influx into B-lymphocytes and P2X₇-transfected-HEK-293 cells, as well as ATP-induced ⁸⁶Rb⁺-efflux from canine erythrocytes. Externally, but not internally, applied compound P impaired ATP-induced inward currents in P2X₇-transfected-HEK-293 cells. This study demonstrates that a novel protein tyrosine kinase antagonist directly impairs native and recombinant human P2X₇ receptors. The data suggests that antagonists which target ATP-binding sites of kinases may potentially block the P2X₇ receptor.     

The role of P2X7 receptors in tissue fibrosis: a brief review

Many previous studies have demonstrated that P2X₇ receptors (P2X₇Rs) have a pleiotropic function in different pathological conditions and could represent a novel target for the treatment of a range of diseases. In particular, recent studies have explored the role of P2X₇R in fibrosis, the pathological outcome of most chronic inflammatory diseases. The aim of this review is to discuss the biological features of P2X₇R and summarize the current knowledge about the putative role of the P2X₇R in triggering fibrosis in a wide spectrum of organs such as the lung, kidney, liver, pancreas, and heart.     

Lack of evidence for direct phosphorylation of recombinantly expressed P2X2 and P2X3 receptors by protein kinase C

P2X₃ and P2X₂₊₃ receptors are present on sensory neurons, where they contribute not only to transient nociceptive responses, but also to hypersensitivity underlying pathological pain states elicited by nerve injuries. Increased signalling through P2X₃ and P2X₂₊₃ receptors may arise from an increased routing to the plasma membrane and/or gain of function of pre-existing receptors. An obvious effector mechanism for functional modulation is protein kinase C (PKC)-mediated phosphorylation, since all P2X family members share a conserved consensus sequence for PKC, TXR/K, within the intracellularly located N-terminal domain. Contradictory reports have been published regarding the exact role of this motif. In the present study, we confirm that site-directed elimination of the potential phosphor-acceptor threonine or the basic residue in the P+2 position of the TXR/K sequence accelerates desensitization of P2X₂ receptors and abolishes P2X₃ receptor function. Moreover, the PKC activator phorbol 12-myristate 13-acetate increased P2X₃ (but not P2X₂) receptor-mediated currents. Biochemically, however, we were unable to demonstrate by various experimental approaches a direct phosphorylation of wild-type P2X₂ and P2X₃ receptors expressed in both Xenopus laevis oocytes and HEK293 cells. In conclusion, our data support the view that the TXR/K motif plays an important role in P2X function and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes. The underlying mechanism, however, is unlikely to involve direct PKC-mediated P2X receptor phosphorylation.     

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca²⁺]_i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X₃ subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg₂₊-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X₇ receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca²⁺]_i measurements in single synaptic terminals. Additionally, the P2X₇ receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.