Purinergic P2X7 Receptor Research Articles

MEDIATORS OF THE SYMPATHETIC NERVOUS SYSTEM ATP AND NORADRENALINE IN THE MODULATION OF ANTIBODY FORMATION DURING DEEP COOLING OF THE ORGANISM

In experiments on rats, the participation of co-mediators of the sympathetic nervous system in the suppressive effect of deep cooling on the antibody-forming function of the spleen was studied. Studied: 1) the effect of deep cooling (decrease in deep temperature by 3–4°С), 2) the introduction of the mediator of the sympathetic nervous system norepinephrine (NE, 1 mg/ml), 3) its co-transmitter ATP (0.01 mg/ml and 10 mg/ml) and 4) P2X-purinergic receptor blocker PPADS on the number of antibody-forming cells of the spleen in response to immunization with sheep erythrocytes. Deep cooling as well as ATP inhibited, while NE stimulated antibody formation in the spleen. Blockade of P2X-purinergic receptors by PPADS stimulated antibody formation in the norm. Against the background of blockade of P2X-purinergic receptors, the inhibitory effect of ATP and deep cooling on antibody formation was not manifested. The results obtained indicate the opposite effects of co-mediators of the sympathetic nervous system on antibody formation in the spleen and suggest that the inhibitory effect of cold on antibody formation in the spleen occurs with the participation of ATP through P2X-purinergic receptors.

The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection.

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.

Dynamical changes in the expression of GABAergic and purinergic components occur during the polarization of THP-1 monocytes to proinflammatory macrophages

The monocytes are key components of innate immunity, as they can differentiate into phagocytic cells or macrophages with proinflammatory or anti-inflammatory phenotypes. The gamma-aminobutyric acid (GABA) and adenosine triphosphate (ATP), two known neurotransmitters, are two environmental signals that contribute to the differentiation of monocytes into macrophages and their subsequent polarization into proinflammatory M1 and anti-inflammatory M2 macrophages. Although monocytes and macrophages express proteins related to GABA and ATP-mediated response (GABAergic and purinergic systems, respectively), it is unknown whether changes in their expression occur during monocyte activation or their differentiation and polarization into macrophages. Therefore, we evaluated the expression levels of GABAergic and purinergic signaling components in the THP-1 monocyte cell line and their changes during monocyte activation, differentiation, and polarization to M1 proinflammatory macrophages. Our results showed that activated monocytes are characterized by increased expression of two GABAergic components, the GABA transporter 2 (GAT-2) and the glutamic acid decarboxylase (GAD)-67, an enzyme involved in GABA synthesis. Also, monocytes showed a pronounced expression of the purinergic receptors P2X4 and P2X7. Interestingly, during differentiation, monocytes increased the expression of the β2 subunit of GABA A-type receptor (GABA-AR), while the purinergic receptors P2X1 and P2X1del were reduced. In contrast, proinflammatory M1 macrophages showed a reduced expression in the α4 subunit of GABA-AR and GAD67, while P2X4 and P2X7 were overexpressed. These results indicate that dynamical changes in the GABAergic and purinergic components occur during the transition from monocytes to macrophages. Since GABA and ATP are two neurotransmitters, our results suggest that monocytes and macrophages respond to neurotransmitter-induced stimulation and may represent a path of interaction between the nervous and immune systems during peripheral inflammation and neuroinflammation development.

Genetic Variations in the Purinergic P2X7 Receptor Are Associated with the Immune Response to Ocular Toxoplasmosis in Colombia.

Ocular toxoplasmosis (OT) is characterized by inflammation within the eye and is the most recognized clinical manifestation of toxoplasmosis. The objective of this study was to identify new single-nucleotide polymorphisms (SNPs) in the P2RX7 gene that may have significance in the immune response to OT in Colombian patients. A case-control study was conducted to investigate the associations between SNPs (rs1718119 and rs2230912) in the P2RX7 gene and OT in 64 Colombian patients with OT and 64 controls. Capillary electrophoresis was used to analyze the amplification products, and in silico algorithms were employed to predict deleterious SNPs. Stability analysis of amino acid changes indicated that both mutations could lead to decreased protein structure stability. A nonsynonymous SNP, Gln460Arg, located in the long cytoplasmic tail of the receptor, showed a significant association with OT (Bonferroni correction (BONF) = 0.029; odds ratio OR = 3.46; confidence interval CI: 1.05 to 11.39), while no significant association between rs1718119 and OT risk was observed. Based on the 3D structure analysis of the P2RX7 protein trimer, it is hypothesized that an increase in the flexibility of the cytoplasmic domain of this receptor could alter its function. This SNP could potentially serve as a biomarker for identifying Colombian patients at risk of OT.

Challenges with retention and recovery of impurities containing acidic moieties during analytical UHPLC method development and validation for gefapixant freebase

Gefapixant citrate is a P2X3 purinergic receptor antagonist developed for the treatment of chronic cough. Gefapixant freebase is the penultimate intermediate in the commercial manufacturing route for gefapixant citrate and contains a complex impurity profile consisting of acidic and basic analytes. A UHPLC method was developed for assay and purity determination of gefapixant freebase utilizing a Waters Acquity Charged Surface Hybrid (CSH) C18 column (2.1 mm I.D. x 10 cm length, 1.7 µm particle size) with 0.1 % phosphoric acid and acetonitrile as the mobile phases. Method optimization was performed using ACD Labs LC Simulator to achieve baseline separation of all impurities and the method was successfully validated. During routine use and method transfer for gefapixant freebase, an increase in retention time for impurities containing strongly acidic functional groups, and poor recovery of a sulfinic acid impurity were observed. Subsequent investigation determined that the CSH column aging resulting from exposure of the column packing to the acidic mobile phase was the root cause for these behaviors. The mechanism of peak-shifting was further investigated using model compounds and determined to be due to an increase in ionic interactions with the CSH stationary phase with routine column use. The increase in ionic interactions was demonstrated to correlate with the charge state of the analyte. Poor recovery for the sulfinic acid impurity was attributed to increased peak tailing for this single impurity on older columns. This knowledge was leveraged to establish additional system suitability requirements to monitor column performance for the lifecycle of the analytical procedure.

Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling.

Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC50 of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

A review of the pathophysiology and the role of ion channels on bronchial asthma.

Asthma is one of the main non-communicable chronic diseases and affects a huge portion of the population. It is a multifactorial disease, classified into several phenotypes, being the allergic the most frequent. The pathophysiological mechanism of asthma involves a Th2-type immune response, with high concentrations of allergen-specific immunoglobulin E, eosinophilia, hyperreactivity and airway remodeling. These mechanisms are orchestrated by intracellular signaling from effector cells, such as lymphocytes and eosinophils. Ion channels play a fundamental role in maintaining the inflammatory response on asthma. In particular, transient receptor potential (TRP), stock-operated Ca2+ channels (SOCs), Ca2+-activated K+ channels (IKCa and BKCa), calcium-activated chloride channel (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR), piezo-type mechanosensitive ion channel component 1 (PIEZO1) and purinergic P2X receptor (P2X). The recognition of the participation of these channels in the pathological process of asthma is important, as they become pharmacological targets for the discovery of new drugs and/or pharmacological tools that effectively help the pharmacotherapeutic follow-up of this disease, as well as the more specific mechanisms involved in worsening asthma.

P2X7 receptor blockade reduces pyroptotic inflammation and promotes phagocytosis in Vibrio vulnificus infection.

Vibrio vulnificus, a gram-negative bacterium, causes serious wound infections and septicemia. Once it develops into early phase sepsis, hyperinflammatory immune responses result in poor prognosis in patients. The present study aimed to examine the possible underlying pathogenic mechanism and explore potential agents that could protect against V. vulnificus cytotoxicity. Here, we report that infection of mouse macrophages with V. vulnificus triggers antiphagocytic effects and pyroptotic inflammation via ATP-mediated purinergic P2X7 receptor (P2X7R) signaling. V. vulnificus promoted P2X7-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 translocation, modulating the expression of the inflammasome sensor NLR family pyrin domain containing 3 (NLRP3), adaptor apoptosis-associated speck-like protein containing a card (ASC), and pyroptotic protein gasdermin D (GSDMD) in mouse macrophages. V. vulnificus induced the NLRP3/caspase-1 inflammasome signaling complex expression that drives GSDMD transmembrane pore formation and secretion of interleukin (IL)-1β, IL-18, and macrophage inflammatory protein-2 (MIP-2). This effect was blocked by P2X7R antagonists, indicating that the P2X7R mediates GSDMD-related pyroptotic inflammation in macrophages through the NF-κB/NLRP3/caspase-1 signaling pathway. Furthermore, blockade of P2X7R reduced V. vulnificus-colony-forming units in the spleen, immune cell infiltration into the skin and lung tissues, and serum concentrations of IL-1β, IL-18, and MIP-2 in mice. These results indicate that P2X7R plays a vital role in mediating phagocytosis by macrophages and pyroptotic inflammation during V. vulnificus infection and provides new opportunities for therapeutic intervention in bacterial infections.

Cross Talk on P2X4 Purinergic Receptors and Neuropathic Pain

Cross Talk on P2X4 Purinergic Receptors and Neuropathic Pain

Expression of the Purinergic P2X7 Receptor in Murine MOPC315.BM Myeloma Cells

The adenosine-5’ triphosphate (ATP)-gated, ion channel, P2X receptor superfamily has seven members expressed by many cancer types. Subtype 7 (P2X7 receptor) is expressed consistently at levels higher than in comparatively healthy tissues. Moreover, transcript variant heterogeneity is associated with drug resistance. We have previously described the role of the P2X7 receptor in myeloma, a rare blood disease that uniquely presents with aggressive bone destruction. In this study, we used known agonists of the P2X7 receptor to induce calcium influx and YO-PRO-1 uptake in murine MOPC315.BM myeloma cells as readouts of P2X7 receptor-mediated channel activation and pore formation, respectively. Neither ATP- nor BzATP-induced calcium influx and YO-PRO-1 indicated an absence of the P2X7 receptor function on MOPC315.BM cells. TaqMan revealed low (Ct > 35) P2rx7 but high P2rx4 gene expression in MOPC315.BM; the latter was downregulated with BzATP treatment. The concomitant downregulation of CD39/Entpd1, Icam-1, and Nf-kb1 and the upregulation of Casp-1 genes regulated during purinergic signaling and with established roles in myeloma progression suggest P2RX4-mediated survival adaptation by cancer cells. Further studies are needed to characterize the P2RX4 pharmacology on MOPC315.BM since transcriptional regulation may be utilized by cancer cells to overcome the otherwise toxic effects of high extracellular ATP.

Purinergic neurotransmission receptor P2X4 silencing alleviates intracerebral hemorrhage-induced neuroinflammation by blocking the NLRP1/Caspase-1 pathway

This study is performed to explore the role of P2X4 in intracerebral hemorrhage (ICH) and the association between P2X4 and the NLRP1/Caspase-1 pathway. The mouse ICH model was established via collagenase injection into the right basal ganglia. P2X4 expression in brain tissues was knocked down via intracerebroventricular injection with adeno-associated virus (AAV) harboring shRNA against shP2X4. The gene expression of P2X4 and protein levels related to NLRP1 inflammasome were detected using qRT-PCR and Western blot analysis, respectively. Muramyl dipeptide (an activator of NLRP1) was used to activate NLRP1 in brain tissues. ICH induced high expression of P2X4 in mouse brain tissues. The knockdown of P2X4 alleviated short- and long-term neurological deficits of ICH mice, as well as inhibited the tissue expression and serum levels of pro-inflammatory cytokines, including TNF-α, interleukin (IL)-6, and IL-1β. Additionally, the expressions of NLRP1, ASC, and pro-Caspase-1 were down-regulated upon P2X4 silencing. Moreover, neurological impairment and the expression and secretion of cytokines after P2X4 silencing were aggravated by the additional administration of MDP. P2X4 knockdown represses neuroinflammation in brain tissues after ICH. Mechanistically, P2X4 inhibition exerts a neuroprotective effect in ICH by blocking the NLRP1/Caspase-1 pathway.

Autophagy may protect the brain against prolonged consequences of headache attacks: A narrative/hypothesis review.

To assess the potential of autophagy in migraine pathogenesis. The interplay between neurons and microglial cells is important in migraine pathogenesis. Migraine-related effects, such as cortical spreading depolarization and release of calcitonin gene-related peptide, may initiate adenosine triphosphate (ATP)-mediating pro-nociceptive signaling in the meninges causing headaches. Such signaling may be induced by the interaction of ATP with purinergic receptor P2X 7 (P2X7R) on microglial cells leading to a Ca2+ -mediated pH increase in lysosomes and release of autolysosome-like vehicles from microglial cells indicating autophagy impairment. A search in PubMed was conducted with the use of the terms "migraine," "autophagy," "microglia," and "degradation" in different combinations. Impaired autophagy in microglia may activate secretory autophagy and release of specific proteins, including brain-derived neurotrophic factor (BDNF), which can be also released through the pores induced by P2X7R activation in microglial cells. BDNF may be likewise released from microglial cells upon ATP- and Ca2+ -mediated activation of another purinergic receptor, P2X4R. BDNF released from microglia might induce autophagy in neurons to clear cellular debris produced by oxidative stress, which is induced in the brain as the response to migraine-related energy deficit. Therefore, migraine-related signaling may impair degradative autophagy, stimulate secretory autophagy in microglia, and degradative autophagy in neurons. These effects are mediated by purinergic receptors P2X4R and P2X7R, BDNF, ATP, and Ca2+ . Different effects of migraine-related events on degradative autophagy in microglia and neurons may prevent prolonged changes in the brain related to headache attacks.

Design, Synthesis, and Biological Evaluation of Novel P2X7 Receptor Antagonists for the Treatment of Septic Acute Kidney Injury.

Sepsis-associated acute kidney injury (AKI) is a serious clinical problem, without effective drugs. Abnormal activation of the purinergic P2X7 receptor (P2X7R) in septic kidneys makes its antagonist a promising therapeutic approach. Herein, a series of novel P2X7R antagonists were designed, synthesized, and structurally optimized. Based on in vitro potency in human/mouse P2X7R using HEK293 cells, hepatic microsomal stability, and pharmacokinetic and preliminary in vivo assessments, compound 14a was identified by respective human and mouse P2X7R IC50 values of 64.7 and 10.1 nM, together with favorable pharmacokinetic properties. Importantly, 14a dose-dependently alleviated kidney dysfunction and pathological injury in both lipopolysaccharide (LPS)- and cecal ligation/perforation (CLP)-induced septic AKI mice with a good safety profile. Mechanistically, 14a could suppress NLRP3 inflammasome activation to inhibit the expression of cleaved caspase-1, gasdermin D, IL-1β, and IL-18 in the injured kidneys of septic mice. Collectively, these results highlighted that P2X7R antagonist 14a exerted a therapeutic potential against septic AKI.

P2X7 signaling influences the production of pro-resolving and pro-inflammatory lipid mediators in alveolar macrophages derived from individuals with asthma.

Few new therapeutics exist to target airway inflammation in mild-to-moderate asthma. Alveolar macrophages regulate airway inflammation by producing proresolving eicosanoids. We hypothesized that stimulation of the purinergic receptor P2X7 in macrophages from individuals with asthma produces eicosanoids associated with airway inflammation and resolution, and that these responses are predicted, in part, by P2X7 pore function. Study subjects were recruited in an Institutional Review Board (IRB)-approved study. Alveolar macrophages were recovered from bronchoalveolar lavage fluid following bronchoscopy. Purinergic receptor classification was performed using flow cytometry and fluorescent cell assay. Macrophages were stimulated in vitro and eicosanoids were measured via ELISA or enzyme immunoassay (EIA) in the presence and absence of P2X7-specific agonist [2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate tri(triethylammonium) salt (Bz-ATP)] and antagonist (AZD9056). Functional P2X7 pore status was confirmed in a live cell assay using P2X7-specific agonists and antagonists. Alveolar macrophages produced increased quantities of the oxylipins lipoxin A4 (LXA4), resolvin D1 (RvD1), and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) following stimulation with Bz-ATP compared with vehicle controls, responses that were attenuated in the presence of the P2X7-selective antagonist, AZD9056. LXA4 and RvD1 production was greatest at 1 h, whereas 15(S)-HETE was maximally produced 24 h. Prostaglandin E-2 and resolvin E1 were minimally produced by P2X7 activation, indicating differential signaling pathways involved in eicosanoid production in alveolar macrophages derived from individuals with asthma. The early production of the proresolving eicosanoids, LXA4 and resolvin D1, is regulated by P2X7, whereas generation of the proinflammatory eicosanoid, 15(S)-HETE, is only partially regulated through P2X7 signaling and reaches maximal production after the peak in proresolving eicosanoids.NEW & NOTEWORTHY Alveolar macrophages obtained from individuals with asthma produce soluble lipid mediators in response to P2X7 purinergic receptor signaling. Proinflammatory mediators may contribute to asthma exacerbations but proresolving mediators may help with resolution of asthma loss of control. These specialized proresolving lipid mediators may serve as future potential therapeutics for asthma exacerbation resolution and recovery.

P2X7 purinergic receptor modulates dentate gyrus excitatory neurotransmission and alleviates schizophrenia-like symptoms in mouse

ATP-gated P2X7 receptors (P2X7Rs) play a crucial role in brain disorders. However, how they affect normal and pathological synaptic transmission is still largely unclear. Here, by using whole-cell patch-clamp technique to record AMPA- and NMDA receptor-mediated excitatory postsynaptic currents (s/mEPSCs) in dentate gyrus granule cells (DG GCs), we revealed a modulation by P2X7Rs of presynaptic sites, especially originated from entorhinal cortex (EC)-GC path but not the mossy cell (MC)-GC path. The involvement of P2X7Rs was confirmed using a pharmacological approach. Additionally, the acute activation of P2X7Rs directly elevated calcium influx from EC-GC terminals. In postnatal phencyclidine (PCP)-induced mouse model of schizophrenia, we observed that P2X7R deficiency restored the EC-GC synapse alteration and alleviated PCP-induced symptoms. To summarize, P2X7Rs participate in the modulation of GC excitatory neurotransmission in the DG via EC-GC pathway, contributing to pathological alterations of neuronal functions leading to neurodevelopmental disorders.

Machine learning discriminates P2X7-mediated intracellular calcium sparks in human-induced pluripotent stem cell-derived neural stem cells

Adenosine triphosphate (ATP) is an extracellular signaling molecule that mainly affects the pathophysiological situation in the body and can be sensed by purinergic receptors, including ionotropic P2X7. Neuronal stem cells (NSCs) remain in adult neuronal tissues and can contribute to physiological processes via activation by evoked pathophysiological situations. In this study, we revealed that human-induced pluripotent stem cell-derived NSCs (iNSCs) have ATP-sensing ability primarily via the purinergic and ionotropic receptor P2X7. Next, to develop a machine learning (ML)-based screening system for food-derived neuronal effective substances and their effective doses, we collected ATP-triggered calcium responses of iNSCs pretreated with several substances and doses. Finally, we discovered that ML was performed using composite images, each containing nine waveform images, to achieve a better ML model (MLM) with higher precision. Our MLM can correctly sort subtle unidentified changes in waveforms produced by pretreated iNSCs with each substance and/or dose into the positive group, with common mRNA expression changes belonging to the gene ontology signatures.

Tetracyclic 1,4-Naphthoquinone Thioglucoside Conjugate U-556 Blocks the Purinergic P2X7 Receptor in Macrophages and Exhibits Anti-Inflammatory Activity In Vivo.

P2X7 receptors (P2X7Rs) are ligand-gated ion channels that play a significant role in inflammation and are considered a potential therapeutic target for some inflammatory diseases. We have previously shown that a number of synthetic 1,4-naphthoquinones are capable of blocking P2X7Rs in neuronal and macrophage cells. In the present investigation, we have demonstrated the ability of the tetracyclic quinone-thioglucoside conjugate U-556, derived from 1,4-naphthoquinone thioglucoside, to inhibit ATP-induced Ca2+ influx and YO-PRO-1 dye uptake, which indicates blocking P2X7R in RAW 264.7 macrophages. This process was accompanied by the inhibition of ATP-induced reactive oxygen species production in macrophages, as well as the macrophage survival strengthening under ATP toxic effects. Nevertheless, U-556 had no noticeable antioxidant capacity. Naphthoquinone-thioglucoside conjugate U-556 binding to the extracellular part of the P2X7R was confirmed by SPR analysis, and the kinetic characteristics of this complex formation were established. Computer modeling predicted that U-556 binds the P2X7R allosteric binding site, topographically similar to that of the specific A438079 blocker. The study of biological activity in in vivo experiments shows that tetracylic conjugate significantly reduces inflammation provoked by carrageenan. The data obtained points out that the observed physiological effects of U-556 may be due to its ability to block the functioning of the P2X7R.

P2X7R/AKT/mTOR signaling mediates high glucose-induced decrease in podocyte autophagy.

Diabetic nephropathy is one of the leading causes of end-stage renal disease worldwide. In our study we found that Adenosine triphosphate (ATP) content was significantly increased in the urine of diabetic mice. We examined the expression of all purinergic receptors in the renal cortex and found that only purinergic P2X7 receptor (P2X7R) expression was significantly increased in the renal cortex of wild-type diabetic mice and that the P2X7R protein partially co-localized with podocytes. Compared with P2X7R(-/-) non-diabetic mice, P2X7R(-/-) diabetic mice showed stable expression of the podocyte marker protein podocin in the renal cortex. The renal expression of microtubule associated protein light chain 3 (LC-3II) in wild-type diabetic mice was significantly lower than in wild-type controls, whereas the expression of LC-3II in the kidneys of P2X7R(-/-) diabetic mice was not significantly different from that of P2X7R(-/-) non-diabetic mice. In vitro, high glucose induced an increase in p-Akt/Akt, p-mTOR/mTOR and p62 protein expression along with a decrease in LC-3II levels in podocytes, whereas after transfection with P2X7R siRNA, Phosphorylated protein kinase B (p-Akt)/Akt, Phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR, and p62 expression were restored and LC-3II expression was increased. In addition, LC-3II expression was also restored after inhibition of Akt and mTOR signaling with MK2206 and rapamycin, respectively. Our results suggest that P2X7R expression is increased in podocytes in diabetes, and that P2X7R is involved in the inhibition of podocyte autophagy by high glucose, at least in part through the Akt-mTOR pathway, thereby exacerbating podocyte damage and promoting the onset of diabetic nephropathy. Targeting P2X7R may be a potential treatment for diabetic nephropathy.

P2RX7 gene knockout or antagonism reduces angiotensin II-induced hypertension, vascular injury and immune cell activation.

Extracellular ATP is elevated in hypertensive mice and humans and may trigger immune activation through the purinergic receptor P2X7 (P2RX7) causing interleukin-1β production and T-cell activation and memory T-cell development. Furthermore, P2RX7 single nucleotide polymorphisms (SNP) are associated with hypertension. We hypothesized that P2RX7 activation contributes to hypertension and cardiovascular injury by promoting immune activation. Male wild-type and P2rx7-/- mice were infused or not with angiotensin II (AngII) for 14 days. A second group of AngII-infused wild-type mice were co-infused with the P2RX7 antagonist AZ10606120 or vehicle. BP was monitored by telemetry. Cardiac and mesenteric artery function and remodeling were assessed using ultrasound and pressure myography, respectively. T cells were profiled in thoracic aorta/perivascular adipose tissue by flow cytometry. Associations between SNPs within 50 kb of P2RX7 transcription, and BP or hypertension were modeled in 384 653 UK Biobank participants. P2rx7 inactivation attenuated AngII-induced SBP elevation, and mesenteric artery dysfunction and remodeling. This was associated with decreased perivascular infiltration of activated and effector memory T-cell subsets. Surprisingly, P2rx7 knockout exaggerated AngII-induced cardiac dysfunction and remodeling. Treatment with a P2RX7 antagonist reduced BP elevation, preserved mesenteric artery function and reduced activated and effector memory T cell perivascular infiltration without adversely affecting cardiac function and remodeling in AngII-infused mice. Three P2RX7 SNPs were associated with increased odds of DBP elevation. P2RX7 may represent a target for attenuating BP elevation and associated vascular damage by decreasing immune activation.

P2X7-Receptor Pathway Involvement in the Anti-Inflammatory Activity of Medicinal Plants.

Medicinal plants used in European folk medicine attached to Lamiales, Gentianales or Asterales orders are used to treat inflammatory disorders. Many targets have been identified but to date, implication of purinergic receptor P2X7 activation has not yet been investigated. We managed to evaluate the protective effect on P2X7 activation by plant extracts used as anti-inflammatory in European folk medicine by the YO-PRO-1 uptake dye in vitro bioassay. Results revealed that among our selected plants, species from Scrophularia and Plantago genus were able to decrease significantly P2X7 activation (>50 % at 0.1 and 1 μg/mL). UPLC/MS, dereplication and metabolomic analysis of Scrophularia extracts, allowed us to identify the cinnamoyl-iridoid harpagoside as putative inhibitor of P2X7 activation. These results open a new research field regarding the anti-inflammatory mechanism of cinnamoyl-iridoids bearing plants, which may involve the P2X7 receptor.