Enzyme Apyrase Research Articles

Protection from environmental enteric dysfunction and growth improvement in malnourished newborns by amplification of secretory IgA

Environmental enteric dysfunction (EED) is a condition associated with malnutrition that can progress to malabsorption and villous atrophy. Severe EED results in linear growth stunting, slowed neurocognitive development, and unresponsiveness to oral vaccines. Prenatal exposure to malnutrition and breast feeding by malnourished mothers replicates EED. Pups are characterized by deprivation of secretory IgA (SIgA) and altered development of the gut immune system and microbiota. Extracellular ATP (eATP) released by microbiota limits T follicular helper (Tfh) cell activity and SIgA generation in Peyer's patches (PPs). Administration of a live biotherapeutic releasing the ATP-degrading enzyme apyrase to malnourished pups restores SIgA levels and ameliorates stunted growth. SIgA is instrumental in improving the growth and intestinal immune competence of mice while they are continuously fed a malnourished diet. The analysis of microbiota composition suggests that amplification of endogenous SIgA may exert a dominant function in correcting malnourishment dysbiosis and its consequences on host organisms, irrespective of the actual microbial ecology.

Mechanosensitive ion channel Piezo1 mediates mechanical ventilation-exacerbated ARDS-associated pulmonary fibrosis

IntroductionPulmonary fibrosis is a major cause of the poor prognosis of acute respiratory distress syndrome (ARDS). While mechanical ventilation (MV) is an indispensable life-saving intervention for ARDS, it may cause the remodeling process in lung epithelial cells to become disorganized and exacerbate ARDS-associated pulmonary fibrosis. Piezo1 is a mechanosensitive ion channel that is known to play a role in regulating diverse physiological processes, but whether Piezo1 is necessary for MV-exacerbated ARDS-associated pulmonary fibrosis remains unknown. ObjectivesThis study aimed to explore the role of Piezo1 in MV-exacerbated ARDS-associated pulmonary fibrosis. MethodsHuman lung epithelial cells were stimulated with hydrochloric acid (HCl) followed by mechanical stretch for 48 h. A two-hitmodel of MV afteracidaspiration-inducedlunginjuryin mice was used. Mice were sacrificed after 14 days of MV. Pharmacological inhibition and knockout of Piezo1 were used to delineate the role of Piezo1 in MV-exacerbated ARDS-associated pulmonary fibrosis. In some experiments, ATP or the ATP-hydrolyzing enzyme apyrase was administered. ResultsThe stimulation of human lung epithelial cells to HCl resulted in phenotypes of epithelial-mesenchymal transition (EMT), which were enhanced by mechanical stretching. MV exacerbated pulmonary fibrosis in mice exposed to HCl. Pharmacologicalinhibitionorknockout of Piezo1 attenuated the MV-exacerbated EMT process and lung fibrosis in vivo and in vitro. Mechanistically, the observed effects were mediated by Piezo1-dependent Ca2+ influx and ATP release in lung epithelial cells. ConclusionsOur findings identify a key role for Piezo1 in MV-exacerbated ARDS-associated pulmonary fibrosis that is mediated by increased ATP release in lung epithelial cells. Inhibiting Piezo1 may constitute a novelstrategyfor the treatment of MV-exacerbated ARDS-associated pulmonary fibrosis.

Spontaneous calcium responses of SF2 rat dental epithelial cells stably expressing the calcium sensor G-GECO.

Genetically-encoded calcium indicators such as G-GECO are useful for studying Ca2+ responses during long-term processes. In this study, we employed a lentiviral vector and established a rat dental epithelial cell line that stably expressed G-GECO (SF2-G-GECO). Ca2+ imaging analysis under cell culture conditions revealed that SF2-G-GECO cells exhibited spontaneous Ca2+ responses, which could be classified into the following three major patterns depending on the cell density: localized Ca2+ responses at cell protrusions at a low density, a cell-wide spread of Ca2+ responses at a medium density, and Ca2+ responses in clusters of 3-20 cells at a high density. The P2Y receptor inhibitor suramin (10 μM), the ATP-degrading enzyme apyrase (5 units/mL), and the fibroblast growth factor (FGF) receptor inhibitor FIIN-2 (1 μM) decreased the frequency of spontaneous Ca2+ responses. These results indicate that ATP and FGF are involved in the spontaneous Ca2+ responses. SF2 cells differentiate into ameloblasts via interactions with mesenchymal cells. Therefore, SF2-G-GECO cells are expected to be a useful tool for studying the functions of Ca2+ responses in regulating gene expression during tooth development.

Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

Development of a label-free ATP image sensor for analyzing spatiotemporal patterns of ATP release from biological tissues

Abstract We investigated the output characteristics of a label-free, 37.3 μm pitch, 128 × 128 pixel adenosine-5'-triphosphate (ATP) image sensor, in which different thicknesses of ATP-degrading enzyme apyrase were deposited using the poly-ion complex (PIC) method. ATP was detected based on the changes in the concentration of hydrogen ions (H+) generated by enzymatic reactions. Enzyme films of different thicknesses were formed on the surface of the sensor by controlling the volumes of the PIC solution. A reduction in the enzyme film thickness (0.2 μm thickness), improved the sensitivity of the ATP sensor, and a clear image than that obtained with a sensor with a thicker film (2.3 μm thickness) was obtained. Additionally, the detection limit for the 0.2-μm-thick sensor was as low as 10 μM, whereas that of the 2.3-μm thick sensor was 100 μM. To examine ATP release from biological tissues, we placed a mouse hippocampal slice on the sensor with the 0.2-μm-thick film and electrically stimulated the nerve fiber (i.e., the Schaffer collateral). The neuronal stimulation evoked significant changes in the output signals of the hippocampal slice but not in the sensor area which was not in contact with tissue. An elevation in the output signal was initiated at the stimulated site with a maximum increment of 5 mV, and the signal traveled along the neuronal fibers. By reducing the film thickness, we successfully developed a high-sensitivity label-free ATPo image sensor with a high spatiotemporal resolution, which allows imaging of endogenous ATPo released from the hippocampus.

Changes in Gene Expression of the MCU Complex Are Induced by Electrical Stimulation in Adult Skeletal Muscle

The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 μM exogenous ATP produces the same effect as 20-Hz ES. Moreover, the use of apyrase in resting conditions (without ES) increased mRNA levels of MCU, pointing out the importance of extracellular ATP concentration over MCU mRNA levels. The use of xestospongin B (inhibitor of IP3 receptors) also prevented the decrease of mRNA levels of MCU, MICU1, MICU2, and EMRE mediated by a low-frequency ES. Our results show that the MCU complex can be regulated by electrical stimuli in a frequency-dependent manner. The changes observed in mRNA levels may be related to changes in the mitochondria, associated with the phenotypic transition from a fast- to a slow-type muscle, according to the described effect of this stimulation frequency on muscle phenotype. The decrease in mRNA levels of the MCU complex by exogenous ATP and the increase in MCU levels when basal ATP is reduced with the enzyme apyrase indicate that extracellular ATP may be a regulator of the MCU complex. Moreover, our results suggest that this regulation is part of the axes linking low-frequency stimulation with ATP/IP3/IP3R.

Mechanism of Human Platelet Activation by Endotoxic Glycolipid-Bearing Mutant Re595 of Salmonella minnesota

The mechanism through which human blood platelets interact with gram-negative bacteria with well-defined structural variations in endotoxic lipopolysaccharide was studied. Secretion of 14C-serotonin and aggregation of platelets separated from plasma proteins were observed on challenge with rough mutant Re595 of Salmonella minnesota possessing a glycolipid outer layer composed of Lipid A and 2-keto-3-deoxyoctonate (KDO) but lacking heptose phosphate in the core and O-polysaccharide in its outer portion. Both 14C-serotonin secretion and platelet aggregation were concentration-dependent, with a half-maximum response at the ratio of one bacterial colony-forming unit (CFU) to two platelets. The aggregation of human platelets induced by mutant Re595 was divalent cation-dependent and required secretion of ADP and fibrinogen from platelet storage granules because it was inhibited by chelators, by the ADP-splitting enzyme apyrase, and by monospecific antifibrinogen Fab fragments. The synthetic peptide analog of the platelet receptor recognition site on the gamma chain of fibrinogen, gamma 400-411, inhibited platelet aggregation induced by mutant Re595 (IC50 160 mumol/L), whereas serotonin secretion was unaffected. Tetrapeptide, RGDS, analogous to human fibrinogen alpha chain (alpha 572-575) and to the cell adhesion site of fibronectin, also inhibited aggregation induced by mutant Re595 (IC50 60 mumol/L). Secretion of 14C-serotonin was preceded by a very rapid phosphorylation of a platelet protein of mol wt 47,000, which is associated with protein kinase C activation. Myosin light chain (mol wt 20,000) was also phosphorylated. Both phosphoproteins were dephosphorylated while secretion was reaching maximum. Furthermore, release of 3H-arachidonic acid from platelet phospholipids and generation of thromboxane B2 via the cyclooxygenase pathway were observed. Inhibition of this pathway with acetylsalicylic acid (10(-4) mol/L) or indomethacin (5 X 10(-4) mol/L) reduced 14C-serotonin secretion and platelet aggregation. The role of Lipid A in the interaction of mutant Re595 with human platelets was deduced from the inhibitory effect of the Lipid A-binding protein present in Limulus amebocyte lysate. Likewise, polymyxin B, known to complex with Lipid A, was inhibitory. The reactivity of mutant Re595 toward platelets was attenuated by mild acid hydrolysis, during which KDO was dissociated from the glycolipid, and by alkaline hydrolysis, which breaks ester-linked fatty acids in Lipid A. In contrast to mutant Re595, strain S218 of S minnesota bearing "complete" endotoxic lipopolysaccharide did not induce secretion and aggregation of human platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Five‐, Four‐ and Three‐Dentate Europium Chelates for Anion Sensing and Their Applicability to Enzymatic Dephosphorylation Reactions

AbstractThe design of anion‐sensitive probes with sufficient sensitivity and selectivity is a demanding task in analytical sciences and chemical sensor technology. The reversible binding of anions to lanthanide centers is a possible approach for the development of molecular anion sensors, as reversibility is a prerequisite for continuous sensing and monitoring of enzymatic reactions. Some anion species lead to a strong increase of luminescence intensities and lifetimes by the replacement of luminescence quenching water molecules, though the selectivity of the luminescence response is still a major problem. We synthesized a series of positively charged pyridyl‐based multidentate europium complexes (five‐, four‐ and three dentate) including sensitizing chromophores and studied their luminescence intensity and lifetime responses to different polyphosphates, pyrophosphate, phosphate anions, and carboxyanions. The results revealed that the number and symmetry of the binding sites have a significant impact on the response. The five‐dentate complex was used for the real‐time monitoring of the activity of the ATP hydrolyzing enzyme apyrase.

ATP promotes immunosuppressive capacities of mesenchymal stromal cells by enhancing the expression of indoleamine dioxygenase.

IntroductionMSCs are often found within tumors, promote cancer progression and enhance metastasis. MSCs can act as immuosuppressive cells, partially due to the expression of the enzyme indoleamine dioxygenase (IDO) which converts tryptophan to kynurenine. Decreased concentration of tryptophan and increased kynurenine, both interfere with effective immune response. Damage associated molecular patterns (DAMPs) including ATP are found within the tumor microenvironment, attract MSCs, and influence their biology.MethodsBone marrow derived MSCs were exposed to ATP for 4 days, in the presence of 100 ng IFNγ/mL. Intracellular expression of IDO in MSCs was assessed by FACS. Conditioned media from thus stimulated MSCs was analyzed for kynurenine content and its suppressive effect on lymphocyte proliferation. Apyrase or P2 × 7‐receptor antagonist (AZ 11645373) were applied in order to inhibit ATP induced effect on MSCs.ResultsWe demonstrate, that ATP at concentrations between 0.062 and 0.5 mM increases dose dependently the expression of IDO in MSCs with subsequent increased kynurenine concentrations within the supernatant at about 60%. This effect could be abolished completely in the presence of ATP degrading enzyme (apyrase) or when MSCs were pretreated with a P2 × 7‐receptor antagonist (AZ 11645373). Consistently, supernatants from MSCs stimulated with ATP, inhibited lymphocyte proliferation from 65% to 16%.ConclusionsWe characterized ATP as a DAMP family member responsible for necrosis‐induced immunomodulation. Given the increased concentration of DAMPs within tumor tissue and the fact that DAMPs can act as chemotattractants to MSCs, our results have implications for therapeutic strategies targeting the tumor microenvironment.

Shear Stress Induces Transverse Ca2+ Waves via Autocrine Activation of P2X Purinoceptors in Rat Atrial Myocytes

Atrial myocytes are exposed to high shear stress during blood regurgitation and high intra-atrial pressure due to valve diseases and heart failure, since such disturbances disrupt endocardium. We have previously reported that shear stress induces two types of global Ca2+ waves in atrial myocytes, longitudinal and transverse Ca2+ waves (T-waves) (Biophys J 2012;102(3, Suppl 1);227a), and that the longitudinal wave is triggered by Ca2+ release via P2Y1 purinoceptor-inositol 1,4,5-trisphosphate receptor signaling (J Physiol 2015;593:5091-5109). Here, we investigated cellular mechanism for the generation of T-wave in atrial cells under shear stress. Shear stress of ∼16 dyn/cm2 was applied onto single myocytes using micro fluid-jet, and two-dimensional confocal Ca2+ imaging was performed. Shear stress-induced T-waves were observed repetitively under 3-4 min intervals between the stimuli, and occurred at ∼1 event per 10 s. They were eliminated by inhibition of the voltage-gated Na+ or Ca2+ channels, or ryanodine receptors, suggesting that the T-wave is mediated by action potential-triggered Ca2+ release. Blockades of key stretch signaling molecules, stretch-activated channel, Na+-Ca2+ exchange, and NADPH oxidase, did not suppress T-wave generation by shear. However, shear-induced T-wave generation was abolished by pre-incubation of cells with external ATP-metabolizing enzyme apyrase, the gap junction blocker carbenoxolone, or with P2X purinoceptor antagonist iso-PPADS. Inhibition of P2Y1 purinergic signaling that mediates the longitudinal Ca2+ wave under shear did not attenuate the occurrence of T-waves. Our data suggest that shear stress induces activation of P2X purinoceptors via gap junction-mediated ATP release, thereby triggering action potential with subsequent T-wave in atrial myocytes.

Reduction of the foreign body response and neuroprotection by apyrase and minocycline in chronic cannula implantation in the rat brain.

Implantation of electrodes or cannulae into the brain is accompanied by a tissue response referred to as foreign body response. Adenosine triphosphate (ATP) is one of the signalling molecules released by injured cells which mediate the chemoattraction of microglial cells. The constitutive release of pro-inflammatory and cytotoxic substances by microglial cells in chronic implants exacerbates neuronal cell death and the immune response. This study aimed to interfere with the initial events of the foreign body response in order to mitigate neurotoxicity and inflammation. For this purpose, the ATP-hydrolysing enzyme apyrase and the antibiotic minocycline with a broad range of anti-inflammatory, anti-apoptotic and glutamate-antagonist properties were locally infused during cannula implantation in the caudal forelimb area of the motor cortex in Lister Hooded rats. The rats' motor performance was assessed in a skilled reaching task and the distribution of neurons and glial cells in the vicinity of the implant was examined 2 and 6weeks post-implantation. Apyrase as well as minocycline increased the number of surviving neurons and reduced microglial activation. Moreover, minocycline improved the motor performance and, additionally, caused a temporary reduction in astrogliosis, suggesting it as a possible therapeutic candidate to improve the biocompatibility of chronic brain implants.

Better blood thinner, without bleeding

Cardiovascular Disease Blood thinners prevent heart attacks and strokes by making it harder for blood to clot, but these drugs can put patients at risk of dangerous bleeding. Now Moeckle et al. describe an enzyme that can prevent clots without this perilous side effect. They engineered the enzyme apyrase to remove the pro-clotting molecule ADP from the blood quickly. In dogs and mice with heart attacks, apyrase stopped blood cells from aggregating, the first step in forming a clot. At the highest dose, the animals suffered less heart damage and did not bleed excessively. In comparison, clopidogrel, a blood thinner used currently in patients, protected the heart less well and did cause excessive bleeding. Sci. Transl. Med. , [ 6 , 248ra105 (2014)][1] [1]: http://stm.sciencemag.org/content/6/248/248ra105.full

Cytosolic K+ and extracellular ATP as regulators of NLRP3 inflammasome activation and the IL-1β secretion response of macrophages to crystalline stimuli. (114.9)

Abstract The crystalline particulate Alum has widely been used as an adjuvant in vaccines. Alum and other crystalline particulates also stimulate NLRP3/caspase-1 inflammasome assembly and secretion of mature IL-1β via poorly understood mechanisms that involve decreases in cytosolic [K+]. This study characterized mechanisms for K+ efflux in LPS-primed murine bone marrow-derived macrophages (BMDM) stimulated with either Imject Alum or the Na+, K+-ATPase inhibitor ouabain. Treatment with Alum for 3 h induced robust secretion of IL-1β that was similar in the absence or presence of antagonists of the P2X7 receptor, an ATP-gated K+ efflux channel known to regulate NLRP3 inflammasome assembly. However, the ATP-scavenging enzyme apyrase markedly reduced this IL-1β secretion, suggesting that NLRP3 activation and IL-1β release induced by Alum crystals is at least partially dependent on a P2X receptor subtype(s) other than P2X7. Current experiments are testing possible involvement of P2X4 receptor channels. Treatment of LPS-primed BMDMs with ouabain for 6 h also induced significant release of IL-1β. We hypothesize that ouabain causes NLRP3 activation and IL-1β secretion by inhibiting the steady-state Na+, K+ATPase activity which counteracts the slow efflux of cytosolic K+ via leak/rectifier-type K+ channels.

Microfluidic bead-based enzymatic primer extension for single-nucleotide discrimination using quantum dots as labels

This study reports the development of an on-chip enzyme-mediated primer extension process based on a microfluidic device with microbeads array for single-nucleotide discrimination using quantum dots as labels. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. The applied allele-specific primer extension method employed a nucleotide-degrading enzyme (apyrase) to achieve specific single-nucleotide detection. Based on the apyrase-mediated allele-specific primer extension with quantum dots as labels, on-chip single-nucleotide discrimination was demonstrated with high discrimination specificity and sensitivity (0.5pM, signal/noise>3) using synthesized target DNA. The chip-based signal enhancement for single-nucleotide discrimination resulted in 200 times higher sensitivity than that of an off-chip test. This microfluidic device successfully achieved simultaneous detection of two disease-associated single-nucleotide polymorphism sites using polymerase chain reaction products as target. This apyrase-mediated microfluidic primer extension approach combines the rapid binding kinetics of homogeneous assays of suspended microbeads array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for single-base analysis with small reagent consumption, short assay time, and parallel detection.

Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells

Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell-cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from -60 to +100 mV; threshold was correlated with extracellular K+, so that at 10 mM K+, channels began to open at -30 mV. Evaluation of cell-cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10-20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 μM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 μM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K+ provides a mechanism for enhancement of ATP release under pathological conditions.

ATP secreted by endothelial cells blocks CX3CL1-elicited natural killer cell chemotaxis and cytotoxicity via P2Y11 receptor activation

AbstractEndothelial cells (ECs) represent a major source of actively secreted adenosine triphosphate (ATP). Natural killer (NK) cells can mediate vascular injury in several pathologic conditions, including cytomegalovirus infection and vascular leak syndrome. We studied NK-cell expression of P2 receptors and the role of these nucleotide receptors in the regulation of endothelial-NK cell cross-talk. NK cells from healthy subjects expressed P2Y1,2,4,6,11,12,13,14 and P2X1,4,5,6,7 receptors. NK cells stimulated with ATP, but not uridine triphosphate, increased intracellular Ca2+ and chemokinesis. Moreover, ATP, but not uridine triphosphate, inhibited NK chemotaxis in response to CX3CL1, whereas chemotaxis to CXCL12 was increased. CX3CL1 elicited killing of human umbilical vein ECs and human coronary artery ECs by NK cells. However, in the presence of ATP, CX3CL1 failed to stimulate killing of ECs. Such inhibitory effect was lost on exogenous addition of the ATP-hydrolyzing enzyme apyrase or by pharmacologic inhibition of the P2Y11R, and correlated with increased intracellular cyclic adenosine monophosphate concentrations induced by ATP or other P2Y11R agonists, including NAD+. Extracellular ATP regulates NK-cell cytotoxicity via P2Y11R activation, protecting ECs from CX3CL1-elicited NK cell–mediated killing. These findings point out the P2Y11R as a potential target for pharmacologic intervention aimed at reducing NK-mediated vascular injury.

Lack of the purinergic receptor P2X7 results in resistance to contact hypersensitivity

Sensitization to contact allergens requires activation of the innate immune system by endogenous danger signals. However, the mechanisms through which contact allergens activate innate signaling pathways are incompletely understood. In this study, we demonstrate that mice lacking the adenosine triphosphate (ATP) receptor P2X(7) are resistant to contact hypersensitivity (CHS). P2X(7)-deficient dendritic cells fail to induce sensitization to contact allergens and do not release IL-1β in response to lipopolysaccharide (LPS) and ATP. These defects are restored by pretreatment with LPS and alum in an NLRP3- and ASC-dependent manner. Whereas pretreatment of wild-type mice with P2X(7) antagonists, the ATP-degrading enzyme apyrase or IL-1 receptor antagonist, prevents CHS, IL-1β injection restores CHS in P2X(7)-deficient mice. Thus, P2X(7) is a crucial receptor for extracellular ATP released in skin in response to contact allergens. The lack of P2X(7) triggering prevents IL-1β release, which is an essential step in the sensitization process. Interference with P2X(7) signaling may be a promising strategy for the prevention of allergic contact dermatitis.

Extracellular ATP and the P2X7receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

BackgroundDuring pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.MethodsWe evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.ResultsRepeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.ConclusionsHere we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.

Extracellular ATP Is a Danger Signal Activating P2X7 Receptor in Lung Inflammation and Fibrosis

Pulmonary fibrosis is a devastating as yet untreatable disease. We previously investigated the endogenous mediators released on lung injury and showed that uric acid is a danger signal activating Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in lung inflammation and fibrosis (Gasse et al., Am J Respir Crit Care Med 2009;179:903-913). Here we address the role of extracellular adenosine triphosphate (eATP) in pulmonary inflammation and fibrosis. ATP was quantified in bronchoalveolar lavage fluid (BALF) of control subjects and patients with idiopathic pulmonary fibrosis. The contribution of eATP as a danger signal was assessed in a murine model of lung fibrosis induced by airway-administered bleomycin (BLM), an intercalating agent that causes DNA strand breaks. Fibrotic patients have elevated ATP content in BALF in comparison with control individuals. In mice, we report an early increase in eATP levels in BALF on BLM administration. Modulation of eATP levels with the ATP-degrading enzyme apyrase greatly reduced BLM-induced inflammatory cell recruitment, lung IL-1β, and tissue inhibitor of metalloproteinase (TIMP)-1 production, while administration of ATP-γS, a stable ATP derivative, enhanced inflammation. P2X(7) receptor-deficient mice presented dramatically reduced lung inflammation, with reduced fibrosis markers such as lung collagen content and matrix-remodeling proteins TIMP-1 and matrix metalloproteinase-9. The acute inflammation depends on a functional pannexin-1 hemichannel protein. In vitro, ATP is released by pulmonary epithelial cells on BLM-induced stress and this is partly dependent on the presence of functional P2X(7) receptor and pannexin-1 hemichannel. ATP released from BLM-injured lung cells constitutes a major endogenous danger signal that engages the P2X(7) receptor/pannexin-1 axis, leading to IL-1β maturation and lung fibrosis.

Astrocytes Control Breathing Through pH-Dependent Vesicular Release of Atp

Extracellular signalling mediated by ATP has been associated with central chemosensory function. It has been shown that increase in pCO2 in the arterial blood triggers an immediate release of ATP from the chemosensitive regions located on the ventral surface of the medulla oblongata (VMS).Using in vitro and in vivo preparations and novel genetically encoded Ca2+ indicator based on cyclically permutated GFP - Case 12, we show that astrocytes in the ventral regions of the medulla oblongata are highly sensitive to changes in pH. Decrease in extracellular pH from 7.4 to 7.2 induced transient increases in [Ca2+]i in astrocytes from dissociated neuro-glial cultures prepared from the VMS, in ventral astrocytes of organotypic brainstem slice cultures and in acute horizontal brainstem slices of adult rats, as well as on the VMS in anaesthetized and artificially ventilated rats. ATP receptor antagonists such as MRS2179, PPADS and TNP-ATP effectively blocked [Ca2+]i responses evoked by lowering pH in VMS astrocytes. ATP hydrolyzing enzyme apyrase completely prevented propagation of [Ca2+]i excitation in VMS astrocytes evoked by lowering external pH. These data suggest that Ca2+ responses induced by lowering external pH are mediated by ATP release and subsequent activation of P2 receptors. Inhibitors of vesicular transport brefeldine A and bafilomycin A both effectively abolished [Ca2+]i excitation of VMS astrocytes evoked by lowering external pH. Incubation of astrocytes with FM 1-46 dye (vesicular marker) leads to selective labelling of intracellular vesicles. Acidification of external medium induced decrease in the fluorescence intensity of vesicles labelled with FM 1-43. These data suggest that VMS astrocytes respond to a decrease in pH by releasing ATP via vesicular exocytosis.