Role Of Purinergic Signaling Research Articles

The Complexity of Purinergic Signaling During Toxoplasma Infection.

Toxoplasmosis is a neglected disease caused by infection by the protozoan Toxoplasma gondii. One-third of the global population is expected to be by infected T. gondii. In Europe and North America, most infections do not induce disease, except in the context of immunosuppression. However, in endemic regions such Central and South America, infections induce severe ocular and potentially lethal disease, even in immunocompetent individuals. The immune response against T. gondii infection involves components of innate immunity even in the chronic phase of the disease, including dangerous signal molecules such as extracellular nucleotides. Purinergic signaling pathways include ionotropic and metabotropic receptors activated by extracellular nucleotides that are divided into P2X, P2Y, and A1 receptor families. The activation of purinergic signaling impacts biological systems by modulating immune responses to intracellular pathogens such as T. gondii. Ten years ago, purinergic signaling in the T. gondii infection was reported for the first time. In this review, we update and summarize the main findings regarding the role of purinergic signaling in T. gondii infection; these include in vitro findings: the microbicidal effect of P2Y and P2X7 activation phagocytic cells and parasite control by P2X7 activation in non-phagocytic cells; and in vivo findings: the promotion of early pro-inflammatory events that protect the host in acute and chronic models.

Vesicular ATP release from hepatocytes plays a role in the progression of nonalcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is becoming a growing public health problem along with the increase of metabolic syndrome worldwide. Extracellular nucleotides are known to serve as a danger signal by initiating purinergic signaling in many inflammatory disorders, although the role of purinergic signaling in the progression of NASH remains to be clarified. Vesicular nucleotide transporter (VNUT) is a key molecule responsible for vesicular ATP release to initiate purinergic signaling. Here, we studied the role of VNUT in the progression of nonalcoholic steatohepatitis. VNUT was expressed in mouse hepatocytes and associated, at least in part, with apolipoprotein B (apoB)-containing vesicles. High glucose stimulation evoked release of appreciable amount of ATP from hepatocytes, which disappeared in hepatocytes of Vnut knockout (Vnut−/−) mice. Glucose treatment also stimulated triglyceride secretion from hepatocytes, which was inhibited by PPADS and MRS211, antagonists of P2Y receptors, and clodronate, a VNUT inhibitor, and was significantly reduced in Vnut−/− mice. In vivo, postprandial secretion of triglyceride from hepatocytes was observed, while the serum triglyceride level was significantly reduced in Vnut−/− mice. On a high-fat diet, the liver of wild type mice exhibited severe inflammation, fibrosis, and macrophage infiltration, which is similar to NASH in humans, while this NASH pathology was not observed in Vnut−/− mice. These results suggest that VNUT-mediated vesicular ATP release regulates triglyceride secretion and involves in chronic inflammation in hepatocytes. Since blockade of vesicular ATP release protects against progression of steatohepatitis, VNUT may be a pharmacological target for NASH.

Purinergic signalling in spinal pain processing.

Purinergic signalling plays important roles in somatosensory and nociceptive transmission in the dorsal horn of the spinal cord under physiological and pathophysiological conditions. Physiologically, ATP mediates excitatory postsynaptic responses in nociceptive transmission in the superficial dorsal horn, and in transmission of innocuous primary afferent inputs in the deep dorsal horn. Additionally, extracellular conversion of ATP to adenosine mediates inhibitory postsynaptic responses from Pacinian corpuscle afferents, and is implicated in analgesia caused by transcutaneous electrical nerve stimulation in humans. In terms of pathological pain, P2X4 receptors de novo expressed on dorsal horn microglia are implicated in pain hypersensitivity following peripheral nerve injury. There is evidence that involvement of such P2X4 receptors is sexually dimorphic, occurring in males but not in females. Thus, the roles of purinergic signalling in physiological and pathological pain processing are complex and remain an ever-expanding field of research.

Evidences of G Coupled-Protein Receptor (GPCR) Signaling in the human Malaria Parasite Plasmodium falciparum for Sensing its Microenvironment and the Role of Purinergic Signaling in Malaria Parasites.

The nucleotides were discovered in the early 19th century and a few years later, the role of such molecules in energy metabolism and cell survival was postulated. In 1972, a pioneer work by Burnstock and colleagues suggested that ATP could also work as a neurotransmitter, which was known as the "purinergic hypothesis". The idea of ATP working as a signaling molecule faced initial resistance until the discovery of the receptors for ATP and other nucleotides, called purinergic receptors. Among the purinergic receptors, the P2Y family is of great importance because it comprises of G proteincoupled receptors (GPCRs). GPCRs are widespread among different organisms. These receptors work in the cells' ability to sense the external environment, which involves: to sense a dangerous situation or detect a pheromone through smell; the taste of food that should not be eaten; response to hormones that alter metabolism according to the body's need; or even transform light into an electrical stimulus to generate vision. Advances in understanding the mechanism of action of GPCRs shed light on increasingly promising treatments for diseases that have hitherto remained incurable, or the possibility of abolishing side effects from therapies widely used today.

The anti-inflammatory effect of resistance training in hypertensive women: the role of purinergic signaling

Essential arterial hypertension triggers a chronic inflammatory process that seems to be linked to purinergic signaling. Physical exercise exhibit anti-inflammatory properties and is able to modulates purinergic system. The aim of this study was to evaluate the effect of 6 months of resistance training on inflammatory markers, purinergic system components, hemodynamic and anthropometric parameters in hypertensive woman. A total of 31 hypertensive group and 28 normotensive (control group) middle-aged sedentary women were submitted to 6 months of resistance training. All measurements and blood collection were carried out before (pretest), after 3 months and after 6 months (posttest) of training. Purinergic enzymes [nucleoside triphosphate diphosphohydrolase (NTPDase) and adenosine deaminase] were assessed in lymphocytes; IL-6, IL-10, ATP and C-reactive protein levels were measured in serum. Six months of resistance training was able to significantly reduce blood pressure (BP), IL-6, C-reactive protein, ATP levels as well as NTPDase and adenosine deaminase activities in hypertensive group. Physical training was also able to increase IL-10 levels in hypertensive group. A positive correlation was found between BP, enzyme activities and levels of ATP and IL-6. A negative correlation was found between BP and IL-10. Positive correlation was found between NTPDase and IL-6 levels (P < 0.05) as well as ATP levels and IL-6 levels. Our findings demonstrated the relationship between purinergic signaling and inflammation in hypertension and suggests that resistance training serve as tool to reduce inflammation in hypertensive woman by modulating purinergic system.

Innate immunity orchestrates the mobilization and homing of hematopoietic stem/progenitor cells by engaging purinergic signaling-an update.

Bone marrow (BM) as an active hematopoietic organ is highly sensitive to changes in body microenvironments and responds to external physical stimuli from the surrounding environment. In particular, BM tissue responds to several cues related to infections, strenuous exercise, tissue/organ damage, circadian rhythms, and physical challenges such as irradiation. These multiple stimuli affect BM cells to a large degree through a coordinated response of the innate immunity network as an important guardian for maintaining homeostasis of the body. In this review, we will foc++us on the role of purinergic signaling and innate immunity in the trafficking of hematopoietic stem/progenitor cells (HSPCs) during their egression from the BM into peripheral blood (PB), as seen along pharmacological mobilization, and in the process of homing and subsequent engraftment into BM after hematopoietic transplantation. Innate immunity mediates these processes by engaging, in addition to certain peptide-based factors, other important non-peptide mediators, including bioactive phosphosphingolipids and extracellular nucleotides, as the main topic of this review. Elucidation of these mechanisms will allow development of more efficient stem cell mobilization protocols to harvest the required number of HSPCs for transplantation and to accelerate hematopoietic reconstitution in transplanted patients.

Abstract P3-01-27: Ectonucleotidase ATP hydrolysis facilitates breast cancer bone metastasis

Abstract Bone is the most common site for breast cancer metastasis. Osteocytes are the most abundant bone cell and can signal nearby cells by releasing ATP through connexin 43 hemichannels. We previously found that this signaling pathway hindered breast cancer growth in bone. In this study, we investigated the role of purinergic signaling on breast cancer bone metastasis in patient samples. Surprisingly, no ATP receptors were differentially expressed in bone metastases compared to other sites. However, enzymes that degrade ATP into adenosine (CD39/ENTPD1 and CD73/NT5E) were upregulated, as was the ADORA3 adenosine receptor (Limma, p=0.037, 0.016, 0.0019, respectively). We next investigated whether this expression signature in primary tumors predicts bone metastasis. In primary ER+ breast cancer, which predominately metastasizes to bone, high expression of these genes is correlated with lower distant metastasis-free survival (HR = 1.97, 1.49, 2.03, logrank p=0.0017, 0.0004, 0.0012). Moreover, this expression pattern is correlated with a bone metastasis signature (Spearman R=0.28, p=1.2 × 10−20). In a pharmacologic screen of 750 cancer cell lines, breast cancer lines and especially ER+ ones, are uniquely sensitive to a pan-adenosine receptor inhibitor (Limma, p=2.9 × 10−5) and ADORA3/2B inhibitor (p=5.4 × 10−5). Our findings suggest that an expression program to convert ATP to adenosine facilitates bone metastasis and begins in the primary tumor. ADORA3 inhibition is proposed as a pharmacologic strategy for preventing or treating bone metastasis. Furthermore, bone metastases should be analyzed in anti-CD39 and CD73 clinical trials. Citation Format: Daniel B Shropshire, Jaime Benavides, Jean X Jiang. Ectonucleotidase ATP hydrolysis facilitates breast cancer bone metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-27.

The Purinergic System as a Pharmacological Target for the Treatment of Immune-Mediated Inflammatory Diseases

Immune-mediated inflammatory diseases (IMIDs) encompass a wide range of seemingly unrelated conditions, such as multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases, asthma, chronic obstructive pulmonary disease, and systemic lupus erythematosus. Despite differing etiologies, these diseases share common inflammatory pathways, which lead to damage in primary target organs and frequently to a plethora of systemic effects as well. The purinergic signaling complex comprising extracellular nucleotides and nucleosides and their receptors, the P2 and P1 purinergic receptors, respectively, as well as catabolic enzymes and nucleoside transporters is a major regulatory system in the body. The purinergic signaling complex can regulate the development and course of IMIDs. Here we provide a comprehensive review on the role of purinergic signaling in controlling immunity, inflammation, and organ function in IMIDs. In addition, we discuss the possible therapeutic applications of drugs acting on purinergic pathways, which have been entering clinical development, to manage patients suffering from IMIDs.

Novel Evidence That the Ectonucleotidases CD39 and CD73, Which Are Expressed on Hematopoietic Stem/Progenitor Cells (HSPCs), Regulate Mobilization and Homing - Studies in CD39-/- and CD73-/- Mice and with Small-Molecule CD39 and CD73 Inhibitors

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by extracellular nucleotides (EXNs), including most importantly the purine ATP and its nucleoside metabolite, adenosine. Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. ATP, the most important EXN, is secreted from cells through pannexin and connexin channels and is processed in the extracellular space to ADP, AMP and finally adenosine by the cell-surface-expressed ectonucleotidases CD39 and CD73. We recently reported that the ATP-adenosine axis regulates the mobilization of hematopoietic stem progenitor cells (HSPCs) (Leukemia 2018, in press,doi: 10.1038/s41375-018-0122-0). Hypothesis. We hypothesized that the ectonucleotidases CD39 and CD73 are important modulators of the ATP-adenosine axis and are new and underappreciated modulators, not only of mobilization but also of bone marrow (BM) homing of HSPCs. Materials and Methods. To shed more light on the role of purinergic signaling in the trafficking of HSPCs, we i) phenotyped murine and human HSPCs for expression of CD39 and CD73 ectonucleotidases by FACS, ii) performed mobilization and homing studies in CD39-/- and CD73-/- mice, and iii) blocked CD39 and CD73 activity by employing the small-molecule inhibitors ARL 67156 and AMP-CP, respectively. CD39- and CD73-deficient mice were mobilized with G-CSF or AMD3100, and wild type (WT) animals were exposed to CD39 and CD73 inhibitors in addition to mobilizing agents. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. To address the involvement of CD39 and CD73 expressed on the surface of HSPCs or in the hematopoietic microenvironment, we created irradiation chimeras that were subsequently mobilized with G-CSF or AMD3100. Bone marrow (BM) transplantation studies were performed by transplanting WT mice with WT BM cells or BM cells from ectonucleotidase-deficient mice. Homing efficiency was evaluated by determining i) the number of fluorochrome-labeled cells in BM 24 hours after transplantation ii) the number of CFU-S and CFU-GM progenitors that had engrafted in BM on day 12, and iii) the recovery of peripheral blood counts. Results. We found that CD39 and CD73 are expressed by murine and human HSPCs and process ATP to adenosine, which as we observed, is a novel and potent inhibitor of HSPC trafficking. In support of this finding, CD73-/- and CD39-/- mice, with reduced extracellular adenosine levels, mobilize HSPCs much better than their normal control littermates. A similar effect was observed in WT mice exposed to CD39 and CD73 small-molecule inhibitors during mobilization. Studies in irradiation chimeras revealed that this effect depended on expression of CD39 and CD73 on the surface of HSPCs and not in the BM microenvironment. Moreover, adenosine also inhibited the homing of HSPCs to BM, as cells from CD39-/- or CD73-/- mice engrafted better than WT cells. Furthermore, homing was also inhibited in the presence of exogenously added adenosine. That ectonucleotidase-deficient mice engrafted BM cells from WT littermates as efficiently as did WT recipients indicates the involvement of HSPC-expressed CD39 and CD73 in regulating the homing process. Conclusions. We demonstrate for the first time that HSPC-expressed CD39 and CD39 ectonucleotidases modulate the trafficking of HSPCs. By processing ATP to adenosine, these enzymes negatively affect both the mobilization and the homing processes. Based on this finding, future strategies employing small-molecule inhibitors of both ectonucleotidases could find practical application in improving mobilization in poor mobilizers and accelerating the engraftment of HSPCs after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Pannexin 1 sustains the electrophysiological responsiveness of retinal ganglion cells

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.

Purinergic Signaling in Mast Cell Degranulation and Asthma.

Mast cells are responsible for the majority of allergic conditions. It was originally thought that almost all allergic events were mediated directly only via the high-affinity immunoglobulin E receptors. However, recent evidence showed that many other receptors, such as G protein-coupled receptors and ligand-gated ion channels, are also directly involved in mast cell degranulation, the release of inflammatory mediators such as histamine, serine proteases, leukotrienes, heparin, and serotonin. These mediators are responsible for the symptoms in allergic conditions such as allergic asthma. In recent years, it has been realized that purinergic signaling, induced via the activation of G protein-coupled adenosine receptors and P2Y nucleotide receptors, as well as by ATP-gated P2X receptors, plays a significant role in mast cell degranulation. Both adenosine and ATP can induce degranulation and bronchoconstriction on their own and synergistically with allergens. All three classes of receptors, adenosine, P2X and P2Y are involved in tracheal mucus secretion. This review will summarize the currently available knowledge on the role of purinergic signaling in mast cell degranulation and its most relevant disease, asthma.

4-Aminopyridine based amide derivatives as dual inhibitors of tissue non-specific alkaline phosphatase and ecto-5′-nucleotidase with potential anticancer activity

Ecto-nucleotidase members i.e., ecto-5′-nucleotidase and alkaline phosphatase, hydrolyze extracellular nucleotides and play an important role in purinergic signaling. Their overexpression are implicated in a variety of pathological states, including immunological diseases, bone mineralization, vascular calcification and cancer, and thus they represent an emerging drug targets. In order to design potent and selective inhibitors, new derivatives of 4-aminopyridine have been synthesized (10a-10m) and their structures were established on the basis of spectral data. The effect of nature and position of substituent was interestingly observed and justified on the basis of their detailed structure activity relationships (SARs) against both families of ecto-nucleotidase. Compound 10a displayed significant inhibition (IC50 ± SEM = 0.25 ± 0.05 µM) that was found ≈168 fold more potent as compared to previously reported inhibitor suramin (IC50 ± SEM = 42.1 ± 7.8 µM). This compound exhibited 6 times more selectivity towards h-TNAP over h-e5′NT. The anticancer potential and mechanism were also established using cell viability assay, flow cytometric analysis and nuclear staining. Molecular docking studies were also carried out to gain insight into the binding interaction of potent compounds within the respective enzyme pockets and herring-sperm DNA.

AMP and adenosine are both ligands for adenosine 2B receptor signaling

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A2BR). A2BR is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A2BR signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A2BR.The computational modeling suggested that AMP interacts with more favorable energy to A2BR compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A2BR, indicating preferential signaling via the Gq pathway. Therefore, a putative AMP-A2BR interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential Gq activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI.

Role of purinergic signalling and proinflammatory cytokines in diabetes

Extracellular purines activate P1 adenosine receptors and P2 nucleotide receptors. These receptors are pre­sent on the pancreatic islet cells as well as on hepato­cytes, adipocytes, pancreatic blood vessels and nerves. ATP is released together with insulin from b-cell gran­ules in response to a rapid decrease in blood glucose levels. The ATP-dependent P2X receptor activation on pancreatic b-cells results in a positive autocrine signal and subsequent insulin secretion. Adenosine, through activation of P1 receptors present on adipocytes and pancreatic islet cells, inhibits the release of insulin. Adenosine activates A 2B receptors thereby stimulating production of IL-6 and other cytokines, which increases insulin resistance. Interleukin-6 also plays an important role in diabetes. In type 2 diabetes and obesity, the long-term increase of IL-6 concentration in blood above 5 pg/mL leads to the chronic and permanent increase in expression of SOCS3, contributing to the increase in insulin resistance in cells of the skeletal muscles, liver and adipose tissue. In diabetes there is an increased synthesis and release of pro-inflammatory cytokines, which cause the damage of the pancreatic islet cells, and in type 2 diabetes cause the development of insulin resistance. Ecto-enzymes metabolizing nucleotides are involved in the termination of the nucleotide signalling pathway and play the key role in regulation of extracel­lular ATP concentration. Ecto-NTPDases in cooperation with 5’-nucleotidase may significantly increase ecto-adenosine concentration. NTPDase3 activity has only been demonstrated on Langerhans cells. NTPDase3 may influence the secretion of insulin by hydrolysing adenine nucleotides. In diabetes the pro-inflammatory cytokines such as interleukin 1b (IL-1b), tumour ne­crosis factor-a (TNF-a) and interferon-g (IFN-g), as well as pancreatic derived factor PANDER are involved in the apoptosis of pancreatic b-cells. This causes distur­bance of the balance between pro-inflammatory and protective cytokines. We believe that neutralization of pro-inflammatory cytokines, especially interleukin 1b, with the IL-1 receptor antagonist (IL-1Ra) and/or IL-1b antibodies might cause the reduction of the inflamma­tory process in pancreas islets, normalize concentration of glucose in blood and decrease the insulin resistance. (Clin Diabetol 2017; 6, 3: 90–100)

Purinergic Receptor Expression and Potential Association with Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Development.

Due to recent progress in production of human embryonic stem cell-derived oligodendrocyte progenitor cells (hESC-OPCs) for ameliorating myelin disease such as multiple sclerosis (MS) and the role of purinergic signaling in OPCs development, we avaluated the profile of purinergic receptors expression during development of OPCs from hESC. In this experimental study, we used reverse transcription and quantitative polymerase chain reaction (RT-qPCR) to obtain more information about potential roles of purinergic receptors during in vitro production of hESC-OPCs. We first determined the expression level of different subtypes of purinergic receptors in hESCs, embryoid bodies (EBs), and hESC-OPCs. The effects of A1 adenosine receptor (A1AR) activation on hESC-OPCs development were subsequently examined. hESCs and OPCs had different mRNA expression levels of the AR subtypes. ARs mRNA were expressed in the EB stage, except for A2AAR. We observed expressions of several P2X (P2X1, 2, 3, 4, 5, 7) and P2Y (P2Y1, 2, 4, 6, 11-14) genes in hESCs. hESC-OPCs expressed different subtypes of P2X (P2X1, 2, 3,4,5,7) and P2Y (P2Y1, 2, 4, 6, 11-14). Except for P2X1 and P2X6, all other P2X and P2Y purinergic receptor subtypes expressed in EBs. We also indicate that A1AR might be involved in modulating gene expression levels of cell cycle regulators in an agonist and/or dose-dependent manner. Elucidation of the expression pattern of purinergic receptors and the effects of different subtypes of these receptors in hESC-OPCs may have a promising role in future cell-based therapy or drug design for demyelinating disease.

Purinergic Signalling and Neurological Diseases: An Update.

Purinergic signalling, i.e. ATP as an extracellular signalling molecule and cotransmitter in both peripheral and central neurons, is involved in the physiology of neurotransmission and neuromodulation. Receptors for purines have been cloned and characterised, including 4 subtypes of the P1(adenosine) receptor family, 7 subtypes of the P2X ion channel nucleotide receptor family and 8 subtypes of the P2Y G protein-coupled nucleotide receptor family. The roles of purinergic signalling in diseases of the central nervous system and the potential use of purinergic compounds for their treatment are attracting increasing attention. In this review, the focus is on the findings reported in recent papers and reviews to update knowledge in this field about the involvement of purinergic signalling in Alzheimer's, Parkinson's and Huntington's diseases, multiple sclerosis, amyotrophic lateral sclerosis, degeneration and regeneration after brain injury, stroke, ischaemia, inflammation, migraine, epilepsy, psychiatric disorders, schizophrenia, bipolar disorder, autism, addiction, sleep disorders and brain tumours. The use in particular of P2X7 receptor antagonists for the treatment of neurodegenerative diseases, cancer, depression, stroke and ischaemia, A2A receptor antagonists for Parkinson's disease and agonists for brain injury and depression and P2X3 receptor antagonists for migraine and seizures has been recommended. P2Y receptors have also been claimed to be involved in some central nervous disorders.

Role of purinergic signaling in experimental pneumococcal meningitis

Excessive neutrophilic inflammation contributes to brain pathology and adverse outcome in pneumococcal meningitis (PM). Recently, we identified the NLRP3 inflammasome/interleukin (IL)-1β pathway as a key driver of inflammation in PM. A critical membrane receptor for NLRP3 inflammasome activation is the ATP-activated P2 purinoceptor (P2R) P2X7. Thus, we hypothesized involvement of ATP and P2Rs in PM. The functional role of ATP was investigated in a mouse meningitis model using P2R antagonists. Brain expression of P2Rs was assessed by RT-PCR. ATP levels were determined in murine CSF and cell culture experiments. Treatment with the P2R antagonists suramin or brilliant blue G did not have any impact on disease course. This lack of effect might be attributed to meningitis-associated down-regulation of brain P2R expression and/or a drop of cerebrospinal fluid (CSF) ATP, as demonstrated by RT-PCR and ATP analyses. Supplemental cell culture experiments suggest that the reduction in CSF ATP is, at least partly, due to ATP hydrolysis by ectonucleotidases of neutrophils and macrophages. In conclusion, this study suggests that ATP-P2R signaling is only of minor or even no significance in PM. This may be explained by down-regulation of P2R expression and decreased CSF ATP levels.

P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Urate Crystals.

Human neutrophils (polymorphonuclear leukocytes [PMNs]) generate inflammatory responses within the joints of gout patients upon encountering monosodium urate (MSU) crystals. Neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid of gout patients. The detailed mechanism of MSU crystal-induced NET formation remains unknown. Our goal was to shed light on possible roles of purinergic signaling and neutrophil migration in mediating NET formation induced by MSU crystals. Interaction of human neutrophils with MSU crystals was evaluated by high-throughput live imaging using confocal microscopy. We quantitated NET levels in gout synovial fluid supernatants and detected enzymatically active neutrophil primary granule enzymes, myeloperoxidase, and human neutrophil elastase. Suramin and PPADS, general P2Y receptor blockers, and MRS2578, an inhibitor of the purinergic P2Y6 receptor, blocked NET formation triggered by MSU crystals. AR-C25118925XX (P2Y2 antagonist) did not inhibit MSU crystal-stimulated NET release. Live imaging of PMNs showed that MRS2578 represses neutrophil migration and blocked characteristic formation of MSU crystal-NET aggregates called aggregated NETs. Interestingly, the store-operated calcium entry channel inhibitor (SK&F96365) also reduced MSU crystal-induced NET release. Our results indicate that the P2Y6/store-operated calcium entry/IL-8 axis is involved in MSU crystal-induced aggregated NET formation, but MRS2578 could have additional effects affecting PMN migration. The work presented in the present study could lead to a better understanding of gouty joint inflammation and help improve the treatment and care of gout patients.

The role of purinergic signaling in development of irritant dermatitis of acrodermatitis enteropathica

The role of purinergic signaling in development of irritant dermatitis of acrodermatitis enteropathica

Abstract 353: P2Y 2 Nucleotide Receptor Overexpression: Letting Blind Cardiac Progenitor Cells ‘See’ Again

Heart failure (HF) is a leading cause of death in the US due to limited capability of adult mammalian heart to regenerate after myocardial infarction (MI). Autologous stem cell therapy holds promise for promoting regeneration of injured heart but stem cells derived from diseased organs exhibit poor proliferative, migratory and survival capabilities. Empowering cardiac-derived progenitor cells (CPC) with prosurvival genes has been attempted. However, molecular mechanisms by which stem cells detect stress signals to subsequently initiate appropriate regenerative responses are poorly understood. In this regard, purinergic receptors represent a major detector for extracellular nucleotides released during injury/stress and serve as an intracellular platform harboring numerous signaling pathways that regulate proinflammatory and regenerative responses required for the healing process. Despite the established roles of purinergic signaling in cardiovascular diseases, it has not been well-defined in CPCs. This study shows, for the first time, that the majority of P2 purinergic receptors are expressed and exhibit functional responses to ATP and UTP in human CPCs (hCPC) isolated from HF patients. The G protein-coupled P2Y 2 R is a pivotal stress detector that senses ATP and UTP accumulated in extracellular space after injury and mediates regenerative responses in various injury models, including MI model, and in stem cells from diverse origins. Interestingly, hCPCs with relatively slower growth kinetics and enhanced senescence show dramatic decreases in P2Y 2 receptor (P2Y 2 R) expression compared to fast-growing hCPCs, consistent with our hypothesis that overexpressing P2Y 2 R enables diseased hCPCs to better detect stress stimuli and react with the proper regenerative responses. Along this line, P2Y 2 R stimulation with UTP enhances hCPC proliferation and migration. Interestingly, preliminary results demonstrate that UTP treatment induces YAP activation and nuclear shuttling. Moreover, inhibition of YAP/TEAD interaction impairs UTP-induced proliferation and migration revealing a novel link between extracellular nucleotides released during cardiac ischemia and Hippo signaling that has been recently implicated in cardiac regeneration.