Adenosine Triphosphate Receptor Research Articles

Increased Purinergic Signaling in Human Dental Pulps With Inflammatory Pain is Sex-Dependent

An enhanced understanding of neurotransmitter systems contributing to pain transmission aids in drug development, while the identification of biological variables like age and sex helps in the development of personalized pain management and effective clinical trial design. This study identified enhanced expression of purinergic signaling components specifically in painful inflammation, with levels increased more in women as compared to men. Inflammatory dental pain is common and potentially debilitating; as inflammation of the dental pulp can occur with or without pain, it provides a powerful model to examine distinct pain pathways in humans. In control tissues, P2X3 and P2X2 receptors colocalized with PGP9.5-positive nerves. Expression of the ecto-nucleotidase NTPDase1 (CD39) increased with exposure to extracellular adenosine triphosphate (ATP), implying CD39 acted as a marker for sustained elevation of extracellular ATP. Both immunohistochemistry and immunoblots showed P2X2, P2X3, and CD39 increased in symptomatic pulpitis, suggesting receptors and the ATP agonist were elevated in patients with increased pain. The increased expression of P2X3 and CD39 was more frequently observed in women than men. In summary, this study identifies CD39 as a marker for chronic elevation of extracellular ATP in fixed human tissue. It supports a role for increased purinergic signaling in humans with inflammatory dental pain and suggests the contribution of purines shows sexual dimorphism. This highlights the potential for P2X antagonists to treat pain in humans and stresses the need to consider sex in clinical trials that target pain and purinergic pathways. PerspectiveThis article demonstrates an elevation of ATP-marker CD39 and of ATP receptors P2X2 and P2X3 with inflammatory pain and suggests the rise is greater in women. This highlights the potential for P2X antagonists to treat pain and stresses the consideration of sexual dimorphism in studies of purines and pain.

Effect of Yiyuan moxibustion on urodynamics and TRPV4/ATP/P2X5 signal pathway of bladder tissue in rats with neurogenic bladder after sacral cord injury.

To observe the effect of Yiyuan moxibustion on urodynamics and the expressions of transient receptor potential vanilloid 4 (TRPV4), adenosine triphosphate (ATP), tyrosine protein kinase KIT (C-Kit) and adenosine triphosphate receptor P2X5 in bladder tissue of rats with detrusor reflex-free neurogenic bladder (NB) after sacral cord injury (SCI), so as to explore its mechanism in promoting the recovery of urination function of NB rats. Female SD rats were randomly divided into sham operation, model, Yiyuan moxibustion, Yiyuan moxibustion+inhibitor (combination) and inhibitor groups, with 12 rats in each group. The model of detruser reflex-free NB after sacral SCI was established by modified Hassan Shaker spinal cord transection method. The behavioral score of Basso Beasttie Bresnahan (BBB) and urodynamic indexes were used to evaluate the model of rats after operation. Fifteen days after modeling, Yiyuan moxibustion was applied to "Shenque" (CV8) and "Guanyuan" (CV4) for 20 min, once daily for 14 days. Rats of the inhibitor and combination groups were given intravesical instillation of HC067047 (1 mL, 1 μmol/L, 30 min). After the interventions, urodynamics was used to evaluate the bladder function of rats. HE staining was used to observe the morphology of bladder tissue. ATP content in bladder tissue was detected by colorimetric method. The positive expression rates of C-Kit and their receptor P2X5 in bladder tissue were observed by immunofluorescence double labeling method, and TRPV4, C-Kit, and P2X5 protein expression levels in bladder tissue were detected by Western blot. Compared with the sham operation group, the maximum bladder capacity and bladder compliance of rats in the model group were increased (P<0.01), the leak point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and the protein expression levels of TRPV4, C-Kit, P2X5 in bladder tissue were decreased (P<0.01). In comparison with the model and combination groups, the Yiyuan moxibustion group showed a decrease in maximum bladder capacity and bladder compliance (P<0.01), an increase in leakage point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and TRPV4, C-Kit, and P2X5 protein expression levels (P<0.01, P<0.05)；However, these indicators showed opposite trends in the inhibitor group (P<0.01, P<0.05). Yiyuan moxibustion can improve the urodynamics and bladder function in rats with bladder detrusor nonreflective after SCI, which may be related to its effect in activating the TRPV4 channel in bladder tissue, promoting the release of ATP from bladder epithelium, thus increasing the expression of bladder Cajal interstitial cells and their purinergic P2X5 receptors.

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

Inhibition of P2X4 and P2X7 receptors improves histological and behavioral outcomes after experimental traumatic brain injury in rats.

Release of large amounts of adenosine triphosphate (ATP), a gliotransmitter, into the extracellular space by traumatic brain injury (TBI) is considered to activate the microglia followed by release of inflammatory cytokines resulting in excessive inflammatory response that induces secondary brain injury. The present study investigated whether antagonists of ATP receptors (P2X4 and/or P2X7) on microglia are beneficial for reducing the post-injury inflammatory response that leads to secondary injury, a prognostic aggravation factor of TBI. Adult male Sprague-Dawley rats were subjected to cortical contusion injury (CCI) and randomly assigned to injury and drug treatment conditions, as follows: i) No surgical intervention (naïve group); ii) dimethyl sulfoxide treatment after CCI (CCI-control group); iii) 5-BDBD (antagonist of P2X4 receptor) treatment after CCI (CCI-5-BDBD group); iv) CCI-AZ11645373 (antagonist of P2X7 receptor) treatment after CCI (CCI-AZ11645373 group); v) or 5-BDBD and AZ11645373 treatment after CCI (CCI-5-BDBD + AZ11645373 group). In the CCI-5-BDBD, CCI-AZ11645373, and CCI-5-BDBD + AZ11645373 groups, expression of activated microglia was suppressed in the ipsilateral cortex and hippocampus 3 days after the CCI. Western blotting with ionized calcium-binding adaptor molecule 1 antibody revealed that administration of CCI-5-BDBD and/or CCI-AZ11645373 suppressed expression of microglia and reduced expression of inflammatory cytokine mRNA 3 days after the CCI. Furthermore, the plus maze test, which reflects the spatial memory function and involves the hippocampal function, showed improvement 28 days after secondary injury to the hippocampus. These findings confirmed that blocking the P2X4 and P2X7 receptors, which are ATP receptors central in gliotransmission, suppresses microglial activation and subsequent cytokine expression after brain injury, and demonstrates the potential as an effective treatment for reducing secondary brain injury.

Hindbrain Adenosine 5-Triphosphate (ATP)-Purinergic Signaling Triggers LH Surge and Ovulation via Activation of AVPV Kisspeptin Neurons in Rats.

Ovulation disorders are a serious problem for humans and livestock. In female rodents, kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are responsible for generating a luteinizing hormone (LH) surge and consequent ovulation. Here, we report that adenosine 5-triphosphate (ATP), a purinergic receptor ligand, is a possible neurotransmitter that stimulates AVPV kisspeptin neurons to induce an LH surge and consequent ovulation in rodents. Administration of an ATP receptor antagonist (PPADS) into the AVPV blocked the LH surge in ovariectomized (OVX) rats treated with a proestrous level of estrogen (OVX + high E2) and significantly reduced the ovulation rate in proestrous ovary-intact rats. AVPV ATP administration induced a surge-like LH increase in OVX + high E2 rats in the morning. Importantly, AVPV ATP administration could not induce the LH increase in Kiss1 KO rats. Furthermore, ATP significantly increased intracellular Ca2+ levels in immortalized kisspeptin neuronal cell line, and coadministration of PPADS blocked the ATP-induced Ca2+ increase. Histologic analysis revealed that the proestrous level of estrogen significantly increased the number of P2X2 receptor (an ATP receptor)-immunopositive AVPV kisspeptin neurons visualized by tdTomato in Kiss1-tdTomato rats. The proestrous level of estrogen significantly increased varicosity-like vesicular nucleotide transporter (a purinergic marker)-immunopositive fibers projecting to the vicinity of AVPV kisspeptin neurons. Furthermore, we found that some hindbrain vesicular nucleotide transporter-positive neurons projected to the AVPV and expressed estrogen receptor α, and the neurons were activated by the high E2 treatment. These results suggest that hindbrain ATP-purinergic signaling triggers ovulation via activation of AVPV kisspeptin neurons.SIGNIFICANCE STATEMENT Ovulation disorders, which cause infertility and low pregnancy rates, are a serious problem for humans and livestock. The present study provides evidence that adenosine 5-triphosphate, acting as a neurotransmitter in the brain, stimulates kisspeptin neurons in the anteroventral periventricular nucleus, known as the gonadotropin-releasing hormone surge generator, via purinergic receptors to induce the gonadotropin-releasing hormone/luteinizing hormone surge and ovulation in rats. In addition, histologic analyses indicate that adenosine 5-triphosphate is likely to be originated from the purinergic neurons in the A1 and A2 of the hindbrain. These findings may contribute to new therapeutic controls for hypothalamic ovulation disorders in humans and livestock.

Effect of extracellular adenosine triphosphate hydrolysis by apyrase on bleomycin-induced circulating and alveolar mononuclear phagocyte activation and lung inflammation.

Effect of extracellular adenosine triphosphate hydrolysis by apyrase on bleomycin-induced circulating and alveolar mononuclear phagocyte activation and lung inflammation.

P2 Receptor Signaling in Motor Units in Muscular Dystrophy.

The purine signaling system is represented by purine and pyrimidine nucleotides and nucleosides that exert their effects through the adenosine, P2X and P2Y receptor families. It is known that, under physiological conditions, P2 receptors play only a minor role in modulating the functions of cells and systems; however, their role significantly increases under some pathophysiological conditions, such as stress, ischemia or hypothermia, when they can play a dominant role as a signaling molecule. The diversity of P2 receptors and their wide distribution in the body make them very attractive as a target for the pharmacological action of drugs with a new mechanism of action. The review is devoted to the involvement of P2 signaling in the development of pathologies associated with a loss of muscle mass. The contribution of adenosine triphosphate (ATP) as a signal molecule in the pathogenesis of a number of muscular dystrophies (Duchenne, Becker and limb girdle muscular dystrophy 2B) is considered. To understand the processes involving the purinergic system, the role of the ATP and P2 receptors in several models associated with skeletal muscle degradation is also discussed.

Mast Cell-Associated Serotonin in Acupoint Contributes to Acupuncture Analgesia in Arthritis Rats by Mediating ATP Release.

The activation of subcutaneous mast cells (MCs) helps to trigger the analgesic effect induced by acupuncture (AP), a traditional oriental therapy, that has been gradually accepted worldwide. This work aimed to reveal whether the serotonin (5-hydroxytryptamine, 5-HT) released from MCs plays an important role in this process, which has a controversial effect in the mechanism of pain. In vivo tests, a 20-min session of AP was applied at Zusanli acupuncture point (acupoint) of acute ankle arthritis rats. Pain thresholds of the injured hindpaw were assessed to reflect the pain state, and the targeting substances in the interstitial space of the treated acupoint were sampled by microdialysis. In vitro experiments, exogenous 5-HT (exo-5-HT) was introduced to mediate adenosine triphosphate (ATP) release from cultured MCs. Needling promoted 5-HT accumulation at the Zusanli acupoint, which was prevented by sodium cromolyn. AP's analgesic effect was suppressed by the inhibition of 5-HT receptors at the acupoint, especially 5-HT1A subtype. In vitro tests, mechanical perturbation mimicking needling stimulation induced MCs to release 5-HT. 1 μM and 10 μM of exo-5-HT facilitated ATP release, which was restrained by blocking of 5-HT1 receptors rather than 5-HT3 receptors. As 5-HT, ATP and adenosine were also transiently accumulated in the treated acupoint during needling. Promoting ATP hydrolysis or activation adenosine A1 receptors duplicated AP analgesic effect. Finally, the inhibition of ATP receptors by suramin or pyridoxal phosphate-6-azo tetrasodium salt hydrate (PPADS) prevented AP analgesic effect. Our results suggest that MC-associated 5-HT release at acupoints contributes to AP analgesia, and the mediation of ATP secretion through 5-HT1A receptors might be the underlying mechanism at play. ATP could facilitate adenosine production or the propagation of needling signals.

Blocking P2X7 by intracerebroventricular injection of P2X7-specific nanobodies reduces stroke lesions

BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1β release after incubation with the P2X7-specific nbs.ResultsTransgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1β release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood–brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size.ConclusionMechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.

2-hydroxyethyl methacrylate-derived reactive oxygen species stimulate ATP release via TRPA1 in human dental pulp cells

Extracellular ATP (adenosine triphosphate) and transient receptor potential ankyrin 1 (TRPA1) channels are involved in calcium signaling in odontoblasts and dental pain. The resin monomer 2-hydroxyethyl methacrylate (HEMA), used in dental restorative procedures, is related to apoptotic cell death via oxidative stress. Although the TRPA1 channel is highly sensitive to reactive oxygen species (ROS), the effect of HEMA-induced ROS on ATP release to the extracellular space and the TRPA1 channel has not been clarified in human dental pulp. In this study, we investigated the extracellular ATP signaling and TRPA1 activation by HEMA-derived ROS in immortalized human dental pulp cells (hDPSC-K4DT). Among the ROS-sensitive TRP channels, TRPA1 expression was highest in undifferentiated hDPSC-K4DT cells, and its expression levels were further enhanced by osteogenic differentiation. In differentiated hDPSC-K4DT cells, 30 mM HEMA increased intracellular ROS production and ATP release, although 3 mM HEMA had no effect. Pretreatment with the free radical scavenger PBN (N-tert-butyl-α-phenylnitrone) or TRPA1 antagonist HC-030031 suppressed HEMA-induced responses. These results suggest that ROS production induced by a higher dose of HEMA activates the TRPA1 channel in human dental pulp cells, leading to ATP release. These findings may contribute to the understanding of the molecular and cellular pathogenesis of tertiary dentin formation and pain in response to dental biomaterials.

ATP Secretion and Metabolism in Regulating Pancreatic Beta Cell Functions and Hepatic Glycolipid Metabolism.

Diabetes (DM), especially type 2 diabetes (T2DM) has become one of the major diseases severely threatening public health worldwide. Islet beta cell dysfunctions and peripheral insulin resistance including liver and muscle metabolic disorder play decisive roles in the pathogenesis of T2DM. Particularly, increased hepatic gluconeogenesis due to insulin deficiency or resistance is the central event in the development of fasting hyperglycemia. To maintain or restore the functions of islet beta cells and suppress hepatic gluconeogenesis is crucial for delaying or even stopping the progression of T2DM and diabetic complications. As the key energy outcome of mitochondrial oxidative phosphorylation, adenosine triphosphate (ATP) plays vital roles in the process of almost all the biological activities including metabolic regulation. Cellular adenosine triphosphate participates intracellular energy transfer in all forms of life. Recently, it had also been revealed that ATP can be released by islet beta cells and hepatocytes, and the released ATP and its degraded products including ADP, AMP and adenosine act as important signaling molecules to regulate islet beta cell functions and hepatic glycolipid metabolism via the activation of P2 receptors (ATP receptors). In this review, the latest findings regarding the roles and mechanisms of intracellular and extracellular ATP in regulating islet functions and hepatic glycolipid metabolism would be briefly summarized and discussed.

Extracellular ATP Limits Homeostatic T Cell Migration Within Lymph Nodes.

Whereas adenosine 5’-triphosphate (ATP) is the major energy source in cells, extracellular ATP (eATP) released from activated/damaged cells is widely thought to represent a potent damage-associated molecular pattern that promotes inflammatory responses. Here, we provide suggestive evidence that eATP is constitutively produced in the uninflamed lymph node (LN) paracortex by naïve T cells responding to C-C chemokine receptor type 7 (CCR7) ligand chemokines. Consistently, eATP was markedly reduced in naïve T cell-depleted LNs, including those of nude mice, CCR7-deficient mice, and mice subjected to the interruption of the afferent lymphatics in local LNs. Stimulation with a CCR7 ligand chemokine, CCL19, induced ATP release from LN cells, which inhibited CCR7-dependent lymphocyte migration in vitro by a mechanism dependent on the purinoreceptor P2X7 (P2X7R), and P2X7R inhibition enhanced T cell retention in LNs in vivo. These results collectively indicate that paracortical eATP is produced by naïve T cells in response to constitutively expressed chemokines, and that eATP negatively regulates CCR7-mediated lymphocyte migration within LNs via a specific subtype of ATP receptor, demonstrating its fine-tuning role in homeostatic cell migration within LNs.

Clonal osteoblastic cell lines with CRISPR/Cas9-mediated ablation of Pit1 or Pit2 show enhanced mineralization despite reduced osteogenic gene expression.

Multiple actions of extracellular Pi on the skeletal cells are likely to be partly mediated by type III sodium/phosphate (Na+/Pi) cotransporters Pit1 and Pit2, although the details are not fully understood. In the current study, to determine the roles of Pit1 and Pit2 in osteoblasts, we generated Pit1-knockout (KO) and Pit2-KO osteoblastic cells by applying CRISPR/Cas9 genome editing to an osteoblastic cell line MC3T3-E1 subclone 4. The extracellular Pi level was increased in the Pit1-KO and Pit2-KO clones due to the reduced Pi uptake. Interestingly, in vitro mineralization was accelerated in the Pit1-KO and Pit2-KO clones, although the induction of the expression of osteogenic marker genes was suppressed. In the cells before mineralization, extracellular levels of pyrophosphate (PPi) and adenosine triphosphate (ATP) were increased in the Pit1-KO and Pit2-KO clones, which might be attributable to the reduced expression and activity of tissue-nonspecific alkaline phosphatase (TNSALP). A 24-h treatment with high Pi reduced the expression and activity of TNSALP, suggesting that the suppression of TNSALP in the Pit1-KO and Pit2-KO clones was caused by the increased availability of extracellular Pi. Lentiviral gene transfer of Pit1 and Pit2 restored the changes observed in Pit1-KO and Pit2-KO clones, respectively. The expressions of P2Y2 and P2X7 which encode receptors for extracellular ATP were altered in the Pit1-KO and Pit2-KO clones, suggesting an influence on purinergic signaling. In mineralized cells after long-term culture, intracellular levels of PPi and ATP were higher in the Pit1-KO and Pit2-KO clones. Taken together, ablation of Pit1 or Pit2 in this osteoblastic cell model led to accelerated mineralization, suppressed TNSALP and altered the levels of extracellular and intracellular PPi and ATP, which might be partly mediated by changes in the availability of extracellular Pi.

Adenosine A2B receptor down-regulates metabotropic glutamate receptor 5 in astrocytes during postnatal development.

Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca2+ imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca2+ responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca2+ responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A2B receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca2+ imaging from Adora2b knockout mice indicated that the A2B receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A2B receptor increased the number of excitatory synapse in developmental stage. Taken together, the A2B receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.

Inflammatory effect of Bothropstoxin-I from Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor.

Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from Bothrops jararacussu venom, is a K49-phospholipase A2 (PLA2) that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing interleukin (IL)-1β and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1β production induced by BthTX-I in peritoneal macrophages (PMs) requires caspase 1/11, ASC and NLRP3 and is dependent on adenosine 5'-triphosphate (ATP)-induced K+ efflux and P2X7 receptor (P2X7R). BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages (HMDMs) were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

Neuroprotective and neuro-rehabilitative effects of acute purinergic receptor P2X4 (P2X4R) blockade after ischemic stroke

Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke are lacking. P2X4R, a receptor for adenosine triphosphate (ATP), regulates activation of myeloid immune cells (infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. However, over-stimulation of P2X4Rs due to excessive ATP release from dying or damaged neuronal cells can contribute to ischemic injury. Therefore, we pharmacologically inhibited P2X4R to limit the over-stimulated myeloid cell immune response and improve both acute and chronic stroke recovery. We subjected 8–12-week-old male and female wild type mice to a 60 min right middle cerebral artery occlusion (MCAo) followed by 3 or 30 days of reperfusion. We performed histological, RNA sequencing, behavioral (sensorimotor, anxiety, and depressive), and biochemical (Evans blue dye extravasation, western blot, quantitative PCR, and flow cytometry) analyses to determine the acute (3 days after MCAo) and chronic (30 days after MCAo) effects of P2X4R antagonist 5-BDBD (1 mg/kg P.O. daily x 3 days post 4 h of MCAo) treatment. 5-BDBD treatment significantly (p < .05) reduced infarct volume, neurological deficit (ND) score, levels of cytokine interleukin-1 beta (IL-1β) and blood brain barrier (BBB) permeability in the 3-day group. Chronically, 5-BDBD treatment also conferred progressive recovery (p < .05) of motor balance and coordination using a rotarod test, as well as reduced anxiety-like behavior over 30 days. Interestingly, depressive-type behavior was not observed in mice treated with 5-BDBD for 3 days. In addition, flow cytometric analysis revealed that 5-BDBD treatment decreased the total number of infiltrated leukocytes, and among those infiltrated leukocytes, pro-inflammatory cells of myeloid origin were specifically reduced. 5-BDBD treatment reduced the cell surface expression of P2X4R in flow cytometry-sorted monocytes and microglia without reducing the total P2X4R level in brain tissue. In summary, acute P2X4R inhibition protects against ischemic injury at both acute and chronic time-points after stroke. Reduced numbers of infiltrating pro-inflammatory myeloid cells, decreased surface P2X4R expression, and reduced BBB disruption are likely its mechanism of neuroprotection and neuro-rehabilitation.

ATP mediates the interaction between human blastocyst and endometrium.

ObjectiveEmbryo implantation needs a reciprocal interaction between competent embryo and receptive endometrium. Adenosine triphosphate (ATP) produced by stressed or injured cells acts as an important signalling molecule. This study aims to investigate whether adenosine triphosphate (ATP) plays an important role in the dialogue of human blastocyst‐endometrium.Materials and methodsThe concentration of lactate was analysed in culture medium from human embryos collected from in vitro fertilization patients. Extracellular ATP was measured by ATP Bioluminescent Assay Kit. Ishikawa cells and T‐HESCs were treated with ATP, ATP receptor antagonist, ATP hydrolysis enzyme or inhibitors of ATP metabolic enzymes. The levels of gene expression were evaluated by real‐time PCR and immunoassay.ResultsWe showed that injured human endometrial epithelial cells could rapidly release ATP into the extracellular environment as an important signalling molecule. In addition, blastocyst‐derived lactate induces the release of non‐lytic ATP from human endometrial receptive epithelial cells via connexins. Extracellular ATP stimulates the secretion of IL8 from epithelial cells to promote the process of in vitro decidualization. Extracellular ATP could also directly promote the decidualization of human endometrial stromal cells via P2Y‐purinoceptors. More importantly, the supernatants of injured epithelial cells clearly induce the decidualization of stromal cells in time‐dependent manner.ConclusionOur results suggest that ATP should play an important role in human blastocyst‐endometrium dialogue for the initiation of decidualization.

Danger-associated extracellular ATP counters MDSC therapeutic efficacy in acute GVHD

AbstractMyeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD), donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer, resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen–induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival, indicating in vivo biologic effects, no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1β (IL-1β), we expected that IL-1β KO donor MDSCs would be superior in subverting GVHD, but such MDSCs proved inferior relative to WT. IL-1β release and IL-1 receptor expression was required for optimal MDSC function, and exogenous IL-1β added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1β could diminish survival in GVHD. However, loss of inflammasome activation and IL-1β release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1β signaling, improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum, the ATP release site, synergized with WT MDSCs, as did regulatory T-cell infusion, which we showed reduced but did not eliminate MDSC inflammasome activation, as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments.

P2X4R silence suppresses glioma cell growth through BDNF/TrkB/ATF4 signaling pathway.

Purinergic receptor P2X 4 (P2X4R), a member of purinergic channels family and a subtype of ionotropic adenosine triphosphate receptors, plays a critical role in tumorigenesis. Evidence suggested that P2X4R is expressed in rat C6 glioma model, however, its role and the underlying mechanism of action are still unclear in human glioblastoma multiforme (GBM). In the current study, our aim is to examine the function and the molecular basis of P2X4R in GBM. We first observed that GBM cells, U251, T98, U87, U373, and A172 were all high expressed P2X4R, when compared with the normal human astrocytes (NHA) cells. To gain the function of P2X4R, P2X4R silence cells were constructed by transfection with P2X4R small interfering RNA (siRNA). We found that P2X4R deletion impeded T98 and U87 cell viability and proliferation, and further studies indicated that cell apoptosis and caspase-3 activity was increased in T98 and U87 cell transfected with P2X4R siRNA. Subsequently, we confirmed that P2X4R silence suppressed brain-derived neurotrophic factor (BDNF), Trk receptor tyrosine kinases (TrkB), and activating transcription factor 4 (ATF4) expression in T98 and U87 cells. And P2X4R siRNA-induced ATF4-expression inhibition dependent on BDNF/TrkB signaling pathway. The impact of P2X4R silence on T98 and U87 cell growth and apoptosis was reversed by ATF4 overexpression. In summary, this study provides the first evidence that P2X4R plays important roles in GBM cell growth and apoptosis.

Purinergic receptor Y2 (P2Y2)- dependent VCAM-1 expression promotes immune cell infiltration in metabolic syndrome.

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y2. The gene expression of ATP receptor P2Y2 did not change in several tissues in the course of obesity, but was increased within epididymal fat. Adipose tissue from P2Y 2-/- mice consuming high-fat diet (HFD) contained less crown-like structures with a reduced frequency of adipose tissue macrophages (ATMs). This was likely due to decreased leukocyte migration because of missing VCAM-1 exposition on P2Y2 deficient hypertrophic adipose tissue endothelial cells. Accordingly, P2Y 2-/- mice showed blunted traits of the metabolic syndrome: they gained less weight compared to P2Y 2+/+ controls, while intake of food and movement behaviour remained unchanged. Liver and adipose tissue were smaller in P2Y 2-/- animals. Insulin tolerance testing (ITT) performed in obese P2Y 2-/- mice revealed a better insulin sensitivity as well as lower plasma C-peptide and cholesterol levels. We demonstrate that interfering with somatic P2Y2 signalling prevents excessive immune cell deposition in diet-induced obesity (DIO), both attenuating adipose tissue inflammation and ameliorating the metabolic phenotype. Thus, blocking the P2Y2 cascade may be a promising strategy to limit metabolic disease and its sequelae.