P2X7 Antagonist Research Articles

Emerging drugs in the treatment of chronic cough

Introduction Chronic cough is a debilitating condition that is among the most common reasons for seeking medical attention yet remains challenging to manage. Identifying an underlying respiratory, nasal or upper gastrointestinal disease triggering cough is the first step in assessment, but once this has been ruled out or adequately treated, many patients remain troubled with chronic cough. Areas covered This narrative review discusses the role of existing treatments and describes the current research landscape for the development of new therapies for chronic cough greater than 8 weeks that is refractory (RCC) or unexplained (UCC). The literature search includes published studies found on pubmed and conference abstracts until 2023. Expert opinion RCC/UCC can occur due to neuronal dysregulation of the vagus nerve or central nervous system. Hence, novel anti-tussives have targeted ion channels involved in the neuronal signaling which triggers cough. Although some therapies targeting receptors such as TRPV1 have failed to show efficacy, P2X3 antagonists have emerged as the most promising therapy for patients impacted by chronic cough. Disease specific therapies such as for idiopathic pulmonary fibrosis are in early development.

Bone marrow-derived mesenchymal stem cells attenuate complete Freund's adjuvant-induced inflammatory pain by inhibiting the expression of P2X3.

Bone marrow-derived mesenchymal stem cells (BMSCs) show a good property for pain treatment by modulating inflammatory response. However, the underlying therapeutic effect and related mechanism of BMSCs on inflammatory pain remain unclear. Therefore, we explored the function and potential mechanism of BMSCs performing in a complete Freund's adjuvant (CFA)-induced inflammatory pain model in this study. Here, BMSCs were injected into the CFA-treated rats, and we used behavioural tests to evaluate the changes in hypersensitivity. High-throughput sequencing was used to screen out the hub genes. Molecular biology experiments were performed to detect the level of P2X3 or inflammatory mediators in rats and observed the distribution of P2X3 in neural cells. Furthermore, the function of the P2X3 was explored via inhibitor and activator experiments. Finally, we found that BMSCs alleviated hyperalgesia and spinal levels of pro-inflammatory factors in CFA-treated rats. High-throughput sequencing showed that P2X3 and P2X7 were identified as hub genes, and only the expression level of P2X3 was significantly down-regulated after BMSCs treatment. Immunohistochemistry showed that P2X3 mainly colocalized with microglia and astrocytes. The levels of P2X3 and pro-inflammatory factors were all significantly reduced after BMSC injection. Moreover, similar attenuation was found in the CFA-treated rats after injecting the P2X3 inhibitor, and a P2X3 antagonist reversed the attenuation induced by the BMSCs. These findings suggest that BMSCs exerted a therapeutic effect on inflammatory pain by inhibiting the expression of P2X3 and the excessive production of inflammatory mediators was associated with an increased P2X3 level and BMSC therapy reverse these effects.

Identification of the molecular determinants of antagonist potency in the allosteric binding pocket of human P2X4.

P2X receptors are a family of ATP-gated cation channels comprising seven subtypes in mammals, which play key roles in nerve transmission, pain sensation and inflammation. The P2X4 receptor in particular has attracted significant interest from pharmaceutical companies due to its physiological roles in neuropathic pain and modulation of vascular tone. A number of potent small-molecule P2X4 receptor antagonists have been developed, including the allosteric P2X4 receptor antagonist BX430, which is approximately 30-fold more potent at human P2X4 compared with the rat isoform. A single amino-acid difference between human and rat P2X4 (I312T), located in an allosteric pocket, has previously been identified as critical for BX430 sensitivity, implying that BX430 binds in this pocket. Using a combination of mutagenesis, functional assay in mammalian cells and in silico docking we confirmed these findings. Induced-fit docking, permitting the sidechains of the amino-acids of P2X4 to move, showed that BX430 could access a deeper portion of the allosteric pocket, and that the sidechain of Lys-298 was important for shaping the cavity. We then performed blind docking of 12 additional P2X4 antagonists into the receptor extracellular domain, finding that many of these compounds favored the same pocket as BX430 from their calculated binding energies. Induced-fit docking of these compounds in the allosteric pocket enabled us to show that antagonists with high potency (IC50 ≤ 100nM) bind deep in the allosteric pocket, disrupting a network of interacting amino acids including Asp-85, Ala-87, Asp-88, and Ala-297, which are vital for transmitting the conformational change following ATP binding to channel gating. Our work confirms the importance of Ile-312 for BX430 sensitivity, demonstrates that the allosteric pocket where BX430 binds is a plausible binding pocket for a series of P2X4 antagonists, and suggests a mode of action for these allosteric antagonists involving disruption of a key structural motif required for the conformational change induced in P2X4 when ATP binds.

Chronic Cough Medical Journey of Participants in a Phase 2b Study for Refractory Chronic Cough

Refractory chronic cough (RCC) is diagnosed through a process of exclusion, based on clinical investigations and trials of therapy. We report the relevant medical history of RCC patients enrolled in a dose-finding study of the P2X3 antagonist BLU-5937.

Burden of Cough in Refractory Chronic Cough Patients Enrolled in A Phase 2b Study of the P2X3 Antagonist BLU-5937

Refractory Chronic Cough (RCC) imposes a significant burden on patients’ quality of life (QoL). Tools such as the Leicester Cough Questionnaire (LCQ) are used to assess this impact of cough. We report the burden at baseline in RCC populations enrolled in a study of the P2X3 antagonist BLU-5937.

Abstract 41: Synthesis And Pharmacological Validation Of Novel Neuroprotective Purinergic Receptor P2X4 Antagonists For The Treatment Of Ischemic Stroke

Background and Purpose: Identification of a novel treatment for ischemic stroke is urgently needed. We previously showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute neuroprotection and thus can be potential drug targets to treat ischemic stroke. Here we synthesized and pharmacologically validated those experimental drugs for the treatment of ischemic stroke. Methods: We first performed the P2X4 receptor structure activity (SAR) relationship based on known heterocyclic antagonist P2X4 antagonists including NP-1815-PX Following extensive modification of the reported synthetic route, we synthesized several analogues that maintained or enhanced antagonist affinity. We used [Ca 2+ ] i influx assay in P2X4R overexpressing human HEK cells to identify potent and specific P2X4R antagonist. Two most potent leads (MRS4719 and 4596 given subcutaneously for 3 days post stroke) were then tested for their neuroprotective activity using middle cerebral artery occlusion (MCAo) model of ischemic stroke (60 min MCAo followed by 3 days of reperfusion) using both sexes of young and aged mice. MRS4719 was also tested in human primary monocyte derived macrophage obtained from the blood of de-identified aged healthy subjects (55±10 years). Results: Analogues MRS 4719 and MRS4596 were most potent at human (h) P2X4R (IC 50 0.503 and 1.38 μM, respectively), and were highly selective versus hP2X1R, hP2X2/3R, hP2X3R). Both MRS 4719 and MRS 4596 showed neuroprotective effects after 3 days of ischemic stroke in a dose dependent manner in young mice (8-12 weeks old). Optimal dose of MRS4719 (1.5 mg/kg/day) also significantly reduced infarct volume (59.6±5.3 vs. 27.47±10.23 mm 3 ; p<0.05) in middle-aged mice (11-12 months). MRS 4719 showed learning and memory enhancing activity when measured after 4 weeks of stroke. MRS 4719 inhibited ATP-induced [Ca 2+ ] i influx in primary human monocyte-derived macrophages, consistent with its blocking effect at endogenously expressed P2X4R. Conclusions: This study indicates MRS4719 is a potent P2X4R antagonist and has translational potential for the treating of ischemic stroke across the age.

Purinergic Receptor Antagonist A438079 Disrupts Benzo[a] pyrene-Mediated IL-1β, CYP1A and CYP1B Transcript Induction Pathway in Zebrafish: Insights into Possible Mechanisms

The deleterious effects of benzo[a]pyrene are mainly due to its metabolites generated by CYP1 metabolism. P2X7 is an important member of purinergic receptors involved in diverse cell signaling cascades, as the inflammasome pathway. Receptor blocking has beneficial effects in several models of inflammatory and neurological diseases. In our study, we show for the first time that the A438079, a “selective” P2X7 antagonist, promotes a downregulation in IL-1β, CYP1A1 and CYP1B1 gene transcription in Danio rerio gills exposed to benzo[a]pyrene. Our results show a modulation of IL- 1β and CYP1 mRNAs, suggesting a possible novel mechanism involving the P2X7 receptor in benzo[a]pyrene-mediated CYP1 induction. Nevertheless, as A438079 was also proven to block the membrane ATP channel pannexin-1, the effects of this compound on downregulating CYPs transcription would also be due to a disruption of Ca2+ influx necessary to activate CYPs transcription by the AhR-Arnt pathway.

Identification of a novel P2X7 antagonist using structure-based virtual screening.

P2X4 and P2X7 receptors are ATP-gated ion channels, which play important roles in neuropathic and inflammatory pain, and as such they are important drug targets in diseases of inflammatory origin. While several compounds targeting P2X4 and P2X7 receptors have been developed using traditional high-throughput screening approaches, relatively few compounds have been developed using structure-based design. We initially set out to develop compounds targeting human P2X4, by performing virtual screening on the orthosteric (ATP-binding) pocket of a molecular model of human P2X4 based on the crystal structure of the Danio rerio receptor. The screening of a library of approximately 300,000 commercially available drug-like compounds led to the initial selection of 17 compounds; however, none of these compounds displayed a significant antagonist effect at P2X4 in a Fluo-4 ATP-induced calcium influx assay. When the same set of compounds was tested against human P2X7 in an ATP-stimulated Yo-Pro1 dye uptake assay, one compound (an indeno(1,2-b)pyridine derivative; GP-25) reduced the response by greater than 50% when applied at a concentration of 30µM. GP-25 displayed an IC50 value of 8.7μM at human P2X7 and 24.4μM at rat P2X7, and was confirmed to be active using whole-cell patch clamp electrophysiology and not cytotoxic. Schild analysis suggested that mode of action of GP-25 was orthosteric. Screening of a further 16 commercially available analogues of GP-25 led to the discovery of five additional compounds with antagonist activity at human P2X7, enabling us to investigate the structure-activity relationship. Finally, docking of the R- and S-enantiomers of GP-25 into the orthosteric pocket of molecular models of human P2X4 and human P2X7 revealed that, while both enantiomers were able to make multiple interactions between their carboxyl moieties and conserved positively charged amino-acids in human P2X7, only the S-enantiomer of GP-25 was able to do this in human P2X4, potentially explaining the lack of activity of GP-25 at this receptor.

P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types.

Adenosine triphosphate (ATP) released from dying cells with high concentrations is sensed as a danger signal by the P2X7 receptor. Sodium iodate (NaIO3) is an oxidative toxic agent, and its retinal toxicity has been used as the model of dry age-related macular degeneration (AMD). In this study, we used NaIO3-treated mice and cultured retinal cells, including BV-2 microglia, 661W photoreceptors, rMC1 Müller cells and ARPE-19 retinal epithelial cells, to understand the pathological action of P2X7 in retinal degeneration. We found that NaIO3 can significantly decrease the photoreceptor function by reducing a-wave and b-wave amplitudes in electroretinogram (ERG) analysis. Optical coherence tomography (OCT) analysis revealed the degeneration of retinal epithelium and ganglion cell layers. Interestingly, P2X7-/- mice were protected from the NaIO3-induced retinopathy and inflammatory NLRP3, IL-1β and IL-6 gene expression in the retina. Hematoxylin and eosin staining indicated that the retinal epithelium was less deteriorated in P2X7-/- mice compared to the WT group. Although P2X7 was barely detected in 661W, rMC1 and ARPE-19 cells, its gene and protein levels can be increased after NaIO3 treatment, leading to a synergistic cytotoxicity of BzATP [2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate tri(triethyleneammonium)salt] and NaIO3 administration in ARPE-19 cells. In conclusion, the paracrine action of the ATP/P2X7 axis via cell-cell communication is involved in NaIO3-induced retinal injury. Our results show that P2X7 antagonist might be a potential therapy in inflammation-related retinal degeneration.

P2X7 receptor antagonist improves gastrointestinal disorders in spontaneously hypertensive rats.

The purinergic system participates in the control of blood pressure. Hypertension promotes the occurrence of gastrointestinal disorders such as intestinal inflammation and gastric emptying delay. This study aimed i) to investigate the participation of the P2X7 receptor blocker Brilliant Blue G (BBG) on gastric emptying of solids and changes in oxidative stress in the gastric fundus, duodenum, and colon of spontaneously hypertensive rats (SHR) and ii) to study the putative relationship of this effect with the renin-angiotensin system. Rats were divided into five groups: Control, SHR, SHR+BBG, SHR+BBG+ATP, and SHR+BBG+ANG II. In the gastrointestinal tract, we assessed gastric emptying (GE) and oxidative stress markers (NOx, MPO, GSH, SOD). We observed a decrease in the GE rate (P<0.05) in SHR vs control rats (21.8±2.0% vs 42.8±3.5%). The decrease in GE was returned (P<0.05) to control levels by BBG in SHR rats (21.8±2.0% vs 41.6±3.2%). Co-administration of ATP or ANG II together with BBG bypassed the effect of the P2X7 antagonist on GE in SHR (P<0.05) (21.9±5.0% vs 25.6±3.0% vs 41.6±3.2%). The MPO activity increased (P<0.05) in the gastric fundus of SHR compared to control rats (6.12±2.26 vs 0.077±0.02 UMPO/mg tissue); this effect was prevented (P<0.05) by BBG (0.55±0.15 vs 6.12±2.26 UMPO/mg tissue). Data demonstrated that blockage of P2X7 receptors with BBG can improve the GE delay and oxidative stress biomarkers in SHR animals. This preventive effect of BBG on GE delay was abrogated by ANG II and ATP, thus prompting crosstalk between renin-angiotensin and the purinergic signaling systems underlying this phenomenon.

Upregulation of CD86 and IL-12 by rhododendrol in THP-1 cells cocultured with melanocytes through ROS and ATP

BackgroundThe tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma. ObjectiveAlthough an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood. MethodsWe hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined. ResultsWhen RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid. ConclusionRD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma.

P2X1 enhances leukemogenesis through PBX3-BCAT1 pathways

How bone marrow niches regulate leukemogenic activities of leukemia-initiating cells (LICs) is unclear. The present study revealed that the metabolic niche component, ATP, efficiently induced ion influx in LICs through its ligand-gated ion channel, P2X1. P2X1 deletion impaired LIC self-renewal capacities and resulted in an approximately 8-fold decrease in functional LIC numbers in a murine acute myeloid leukemia (AML) model without affecting normal hematopoiesis. P2X1 phosphorylation at specific sites of S387 and T389 was essential for sustaining its promoting effects on leukemia development. ATP-P2X1-mediated signaling upregulated the PBX3 level to transactivate BCAT1 to maintain LIC fates. P2X1 knockdown inhibited the proliferation of both human AML cell lines and primary cells. The P2X1 antagonist sufficiently suppressed AML cell proliferation. These results provided a unique perspective on how metabolic niche factor ATP fine-tunes LIC activities, which may benefit the development of strategies for targeting LICs or other cancer stem cells.

Ligand-gated ion channel P2X7 regulates hypoxia-induced factor-1α mediated pain induced by dental pulpitis in the medullary dorsal horn.

Dental pulpitis often induces severe pain, and the molecular immune response is remarkable in both peripheral and central nervous system. Accumulating evidence indicates that activated microglia in the medullary dorsal horn (MDH) contribute to dental pulpitis induced pain. The P2X7 receptor plays an important role in driving pain and inflammatory processes, and its downstream target hypoxia-induced factor-1α (HIF-1α) has a crucial role in maintaining inflammation. However, the relationship between P2X7 and HIF-1α in dental inflammatory pain remains unclear. This study demonstrated that the degree of inflammation in the dental pulp tissue became more severe in a time-dependent manner by establishing a rat dental pulpitis model via pulp exposure. Meanwhile, the expression of P2X7, HIF-1α, IL-1β, and IL-18 in the MDH increased most on the seventh day when the pain threshold was the lowest in the dental pulpitis model. Furthermore, lipopolysaccharides (LPS) increased P2X7-mediated HIF-1α expression in microglia. Notably, the suppression of P2X7 caused less IL-1β and IL-18 release and lower HIF-1α expression, and P2X7 antagonist Brilliant Blue G (BBG) could alleviate pain behaviors of the dental pulpitis rats. In conclusion, our results provide further evidence that P2X7 is a key molecule, which regulates HIF-1α expression and inflammation in dental pulpitis-induced pain.

P2X7 receptor activation impairs antitumour activity of natural killer cells.

A high number of intratumoural infiltrating natural killer (NK) cells is associated with better survival in several types of cancer, constituting an important first line of defence against tumours. Hypoxia in the core of solid tumours induces cellular stress and ATP release into the extracellular space where it triggers purinergic receptor activation on tumour-associated immune cells. The aim of this study was to assess whether activation of the purinergic receptor P2X7 by extracellular ATP plays a role in the NK cells antitumour activity. We carried out in vitro experiments using purified human NK cells triggered through P2X7 by extracellular ATP. NK cell killing activity against the tumour target cells K562 was studied by means of NK cytotoxicity assays. Likewise, we designed a subcutaneous solid tumour in vivo mouse model. In this study we found that human NK cells, expressing a functional plasma membrane P2X7, acquired an anergic state after ATP treatment, which impaired their antitumour activity and decreased IFN-γ secretion. This effect was reversed by specific P2X7 antagonists and pretreatment with either IL-2 or IL-15. Furthermore, genetic P2rx7 knockdown resulted in improved control of tumour size by NK cells. In addition, IL-2 therapy restored the ability of NK cells to diminish the size of tumours. Our results show that P2X7 activation represents a new mechanism whereby NK cells may lose antitumour effectiveness, opening the possibility of generating modified NK cells lacking P2X7 but with improved antitumour capacity.

PRIOR SPECIALIST CONSULTATIONS AND TREATMENTS IN A REFRACTORY CHRONIC COUGH POPULATION ENROLLED IN A PHASE 2B STUDY OF THE P2X3 ANTAGONIST BLU-5937

PRIOR SPECIALIST CONSULTATIONS AND TREATMENTS IN A REFRACTORY CHRONIC COUGH POPULATION ENROLLED IN A PHASE 2B STUDY OF THE P2X3 ANTAGONIST BLU-5937

The inhibitory effect of Yam polysaccharides on acrylamide-induced programmed cell death in RAW 264.7 cells.

Acrylamide has been well known for its neurotoxicity, genotoxicity, carcinogenicity, etc. Recently, the immunotoxicity of acrylamide has been reported by different research groups, although the underlying mechanisms of acrylamide endangering immune systems have not been fully elucidated. In this study, mouse monocyte-macrophage cells model was used to clarify the toxic mechanism of acrylamide and the inhibitory effect of Yam polysaccharides (YPS) on acrylamide-induced damage. We found that acrylamide induced RAW 264.7 cell death in a time- and concentration-dependent manner. After acrylamide (2.0, 3.0, 4.0 mmol/L) treatment for 24 h, cell apoptosis, autophagy, and pyroptosis were observed. However, the levels of autophagy and pyroptosis decreased at a high concentration of acrylamide (4.0 mmol/L). Acrylamide upregulated P2X7 expression, but the P2X7 level was not showing a monotone increasing trend. When the P2X7 antagonist was applied, the effect of acrylamide on autophagy and pyroptosis was weakened. Additionally, acrylamide triggered the occurrence of oxidative stress and a decreased nitric oxide (NO) level. However, reactive oxygen species (ROS) generation, the decrease of heme oxygenase-1 (HO-1) expression, and the increase of inducible nitric oxide synthase (iNOS) expression were reversed by the inhibition of P2X7. Yam polysaccharides (50.0μg/ml) significantly inhibited acrylamide-induced oxidative stress and cell death (including apoptosis, autophagy, and pyroptosis). Yam polysaccharides also effectively reversed the increase of iNOS expression induced by acrylamide. However, Yam polysaccharides promoted the expression of P2X7 rather than prohibit it. These results indicated that acrylamide caused RAW 264.7 cell death due to pro-apoptosis as well as excessive autophagy and pyroptosis. Apoptosis might be more predominant than autophagy and pyroptosis under a higher concentration of acrylamide (4.0 mmol/L). P2X7-stimulated oxidative stress was responsible for acrylamide-induced programmed cell death (PCD), but P2X7 showed limited regulatory effect on apoptosis. Yam polysaccharides with antioxidant activity inhibited acrylamide-induced cell death (apoptosis, autophagy, and pyroptosis), but exerted limited effect on the acrylamide-induced P2X7 expression. These findings would offer an insight into elucidating the immunotoxic mechanism of acrylamide and the potential approaches to control its toxicity.

Purinergic P2X7 receptor antagonist inhibits methamphetamine-induced reward, hyperlocomotion, and cortical IL-7A levels in mice: A role for P2X7/IL-17A crosstalk in methamphetamine behaviors?

P2X7 receptors are dysregulated during psychostimulant exposure. Furthermore, P2X7 receptors enhance endogenous systems (e.g., cytokines, dopamine, and glutamate) that facilitate psychostimulant addiction. Therefore, using mouse locomotor, conditioned place preference (CPP), and intracranial self-stimulation (ICSS) assays, we tested the hypothesis that methamphetamine (METH) reward and acute locomotor activation requires P2X7 receptor activity. We also investigated effects of P2X7 blockade on METH-induced changes in cytokine levels in brain reward regions. A438079 (5, 10, 50 mg/kg), a P2X7 antagonist, did not affect spontaneous locomotor activity but reduced hyperlocomotion caused by acute METH (1 mg/kg) exposure. A438079 (10 mg/kg) also prevented expression of METH CPP without causing aversive or rewarding effects. For ICSS experiments, METH (1 mg/kg) facilitated brain reward function as interpreted from reductions in baseline threshold. In the presence of A438079 (50 mg/kg), METH-induced facilitation of ICSS was reduced. Repeated METH exposure (1 mg/kg × 7 d) caused enhancement of IL-17A levels in the prefrontal cortex (PFC) that was normalized by A438070 (10 mg/kg × 7 d). The present data suggest that P2X7 receptor activity contributes to rewarding and locomotor-stimulant effects of METH through a potential mechanism involving IL-17A, which has recently been implicated in anxiety.

6-Furopyridine Hexamethylene Amiloride Is a Non-Selective P2X7 Receptor Antagonist.

P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.

P2X7-mediated alteration of membrane fluidity is associated with the late stages of age-related macular degeneration.

We have shown deficits in monocyte phagocytosis from patients with age-related macular degeneration (AMD). Cell membrane fluidity is known to affect phagocytic capacity and leucocyte functionality more generally. Therefore, we examined membrane fluidity of peripheral blood leucocytes in human patients with AMD and in the P2X7 null mouse model of AMD using flow cytometry with a fluorescent probe for fluidity, TMA-DPH. The results showed that membrane fluidity was decreased in all leucocyte types of late AMD relative to healthy controls (HC) including monocytes, neutrophils and lymphocytes but this was not apparent in earlier stages of AMD. Further analysis of factors contributing to membrane fluidity indicated that pre-treatment of monocytes and lymphocytes with ATP greatly increased membrane fluidity in humans and mice. Evidence from P2X7 null mice and P2X7 antagonists confirmed that these ATP-driven increases in membrane fluidity were mediated by P2X7 but were not associated with the classic P2X7 functions of pore formation or phagocytosis. Analysis of P2X7 expression indicated that receptor levels were elevated in classic monocytes of late AMD patients, further suggesting the P2X7 may contribute to altered plasma membrane properties. Our findings identified a novel biological function of P2X7 in modulating membrane fluidity of leucocytes and demonstrated reduced membrane fluidity in cellular changes associated with the late stage of AMD.

P2X7 Purinoceptor Affects Ectopic Calcification of Dystrophic Muscles.

Ectopic calcification (EC) of myofibers is a pathological feature of muscle damage in Duchenne muscular dystrophy (DMD). Mineralisation of muscle tissue occurs concomitantly with macrophage infiltration, suggesting a link between ectopic mineral deposition and inflammation. One potential link is the P2X7 purinoceptor, a key trigger of inflammation, which is expressed on macrophages but also up-regulated in dystrophic muscle cells. To investigate the role of P2X7 in dystrophic calcification, we utilised the Dmd mdx-βgeo dystrophin-null mouse model of DMD crossed with a global P2X7 knockout (P2rx7 −/− ) or with our novel P2X7 knockin-knockout mouse (P2x7 KiKo ), which expresses P2X7 in macrophages but not muscle cells. Total loss of P2X7 increased EC, indicating that P2X7 overexpression is a protective mechanism against dystrophic mineralisation. Given that muscle-specific P2X7 ablation did not affect dystrophic EC, this underlined the role of P2X7 receptor expression on the inflammatory cells. Serum phosphate reflected dystrophic calcification, with the highest serum phosphate levels found in genotypes with the most ectopic mineral. To further investigate the underlying mechanisms, we measured phosphate release from cells in vitro, and found that dystrophic myoblasts released less phosphate than non-dystrophic cells. Treatment with P2X7 antagonists increased phosphate release from both dystrophic and control myoblasts indicating that muscle cells are a potential source of secreted phosphate while macrophages protect against ectopic mineralisation. Treatment of cells with high phosphate media engendered mineral deposition, which was decreased in the presence of the P2X7 agonist BzATP, particularly in cultures of dystrophic cells, further supporting a protective role for P2X7 against ectopic mineralisation in dystrophic muscle.