P2X Receptor Family Research Articles

Antagonism of the ATP-gated P2X7 receptor inhibits the proliferation of hepatocellular carcinoma cells.

The P2X7 receptor, an ATP-gated ion channel which belongs to the P2X receptor family, plays critical roles in recognizing extracellular adenosine 5'-triphosphate (ATP) and is widely expressed in most tumor cells as well as inflammatory cells. Previously, the P2X7 receptor has been demonstrated to modulate the progression of various malignancies, including glioblastoma, pancreatic cancer, lung cancer, leukemia, and lymphoma. However, the biological function and prognostic values of P2X7 receptor in hepatocellular carcinoma remain to be determined. Here, we investigated the expression level of P2X7 receptor in patients with hepatocellular carcinoma. Then MTS and EdU assays were carried out to study the role of P2X7 receptor blockade in the proliferation of hepatocellular carcinoma cells. In addition, the underlying mechanism was further elucidated by bulk RNAseq. Compared to the control group, the P2X7 receptor was significantly up-regulated in the hepatocellular carcinoma group. Interestingly, A740003 and A438079, two selective antagonists at P2X7 receptor, significantly blocked Ca2+ influx and decreased the proliferative rate of hepatocellular carcinoma cells. Furthermore, the expression level of chondroitin sulfate synthase 1 (CHSY1), an enzyme that mediates the polymerization step of chondroitin sulfate, was reduced by both A740003 and A438079. In conclusion, inhibition of the P2X7 receptor attenuated the proliferation of hepatocellular carcinoma cells, and this process was largely modulated by CHSY1. Thus, our findings reveal a previously unknown role for P2X7 receptor in the proliferation of hepatocellular carcinoma cells and imply that the P2X7 receptor may represent a new target for the treatment of hepatocellular carcinoma.

A Second Drug Binding Site in P2X3.

Purinergic P2X3 receptors form trimeric cation-gated channels, which are activated by extracellular ATP. P2X3 plays a crucial role in chronic cough and affects over 10% of the population. Despite considerable efforts to develop drugs targeting P2X3, the highly conserved structure within the P2X receptor family presents obstacles for achieving selectivity. Camlipixant, a potent and selective P2X3 antagonist, is currently in phase III clinical trials. However, the mechanisms underlying receptor desensitization, ion permeation, principles governing antagonism, and the structure of P2X3 when bound to camlipixant remain elusive. In this study, we established a stable cell line expressing homotrimeric P2X3 and utilized a peptide scaffold to purify the complex and determine its structure using cryo-electron microscopy (cryo-EM). P2X3 binds to camlipixant at a previously unidentified drug-binding site and functions as an allosteric inhibitor. Structure-activity studies combined with modeling and simulations have shed light on the mechanisms underlying the selective targeting and inhibition of P2X3 by camlipixant, distinguishing it from other members of the P2X receptor family.

Graph neural networks-based prediction of drug gene association of P2X receptors in periodontal pain

The P2X7 receptor, a member of the P2X receptor family, plays a crucial role in various physiological processes, particularly pain perception. Its expression across immune, neuronal, and glial cells facilitates the release of pro-inflammatory molecules, thereby influencing pain development and maintenance, as evidenced by its association with pulpitis in rats. Notably, P2X receptors such as P2X3 and P2X7 are pivotal in dental pain pathways, making them promising targets for novel analgesic interventions. Leveraging graph neural networks (GNNs) presents an innovative approach to model graph data, aiding in the identification of drug targets and prediction of their efficacy, complementing advancements in genomics and proteomics for therapeutic development. In this study, 921 drug-gene interactions involving P2X receptors were accessed through https://www.probes-drugs.org/. These interactions underwent meticulous annotation, preprocessing, and subsequent utilization to train and assess GNNs. Furthermore, leveraging Cytoscape, the CytoHubba plugin, and other bioinformatics tools, gene expression networks were constructed to pinpoint hub genes within these interactions. Through analysis, SLC6A3, SLC6A2, FGF1, GRK2, and PLA2G2A were identified as central hub genes within the context of P2X receptor-mediated drug-gene interactions. Despite achieving a 65 percent accuracy rate, the GNN model demonstrated suboptimal predictive power for gene-drug interactions associated with oral pain. Hence, further refinements and enhancements are imperative to unlock its full potential in elucidating and targeting pathways underlying oral pain mechanisms.

Transplantation of olfactory ensheathing cells can alleviate neuroinflammatory responses in rats with trigeminal neuralgia

Trigeminal neuralgia (TN) is a common form of facial pain, which primarily manifests as severe pain similar to facial acupuncture and electric shock. Olfactory ensheathing cells (OECs) are glial cells with high bioactivity; these cells are essential for the periodic regeneration of the olfactory nerve and have been utilized for the repair of nerve injuries. A member of the P2X receptor family, P2X7R, is an ion channel type receptor that has been confirmed to participate in various pain response processes. In this study, we transplanted OECs into trigeminal nerve–model rats with distal infraorbital nerve ligation to observe the therapeutic effect of transplanted OECs in rats. Additionally, we utilized the P2X7R-specific inhibitor brilliant blue G (BBG) to study the therapeutic mechanisms of cell transplantation. The facial mechanical pain threshold of these rats significantly increased following cell transplantation. The immunohistochemistry, immunoblotting, and RT-qPCR results demonstrated that the levels of P2X7R, (NOD)-like receptor protein-3 (NLRP3), nuclear factor-κB (NF-κB), interleukin (IL)-1β, and IL-18 in the trigeminal ganglion of rats treated with OEC transplantation or BBG treatment were significantly lower than those in the injured group without treatment. Overall, our results demonstrate that OEC transplantation can alleviate TN in rats, and it can reduce the expression of P2X7R related inflammatory factors in TN rats, reducing neuroinflammatory response in TG.

A shared mechanism for TNP-ATP recognition by members of the P2X receptor family

P2X receptors (P2X1–7) are non-selective cation channels involved in many physiological activities such as synaptic transmission, immunological modulation, and cardiovascular function. These receptors share a conserved mechanism to sense extracellular ATP. TNP-ATP is an ATP derivative acting as a nonselective competitive P2X antagonist. Understanding how it occupies the orthosteric site in the absence of agonism may help reveal the key allostery during P2X gating. However, TNP-ATP/P2X complexes (TNP-ATP/human P2X3 (hP2X3) and TNP-ATP/chicken P2X7 (ckP2X7)) with distinct conformations and different mechanisms of action have been proposed. Whether these represent species and subtype variations or experimental differences remains unclear. Here, we show that a common mechanism of TNP-ATP recognition exists for the P2X family members by combining enhanced conformation sampling, engineered disulfide bond analysis, and covalent occupancy. In this model, the polar triphosphate moiety of TNP-ATP interacts with the orthosteric site, while its TNP-moiety is deeply embedded in the head and dorsal fin (DF) interface, creating a restrictive allostery in these two domains that results in a partly enlarged yet ion-impermeable pore. Similar results were obtained from multiple P2X subtypes of different species, including ckP2X7, hP2X3, rat P2X2 (rP2X2), and human P2X1 (hP2X1). Thus, TNP-ATP uses a common mechanism for P2X recognition and modulation by restricting the movements of the head and DF domains which are essential for P2X activation. This knowledge is applicable to the development of new P2X inhibitors.

Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) are competitive antagonists of the human P2X3 receptor.

Introduction: The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products, and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents. Methods: To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology. Results: We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 and 76.7µM, respectively). A weaker inhibition of hP2X1R, hP2X4R, and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) inhibited hP2X3R, rP2X3R, and hP2X7R (IC50 values of 221µM, 264.1µM, and ∼900µM, respectively), calling into question its use as a non-selective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP application or increasing concentrations of the agonist α,β-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results suggest a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R, and hP2X7R. Discussion: Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac, which are rarely reached in the therapeutic range, may play a minor role in analgesia compared to the high-potency cyclooxygenase inhibition but may explain the known side effect of taste disturbances caused by diclofenac.

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.

Novel evidence that the P2X1 purinergic receptor-Nlrp3 inflammasome axis orchestrates optimal trafficking of hematopoietic stem progenitors cells.

Our previous research demonstrated P2X purinergic receptors as important extracellular adenosine triphosphate (eATP) sensing receptors promoting the trafficking of hematopoietic stem progenitor cells (HSPCs). Accordingly, mice deficient in expression of P2X4 and P2X7 receptors turned out to mobilize poorly HSPCs. Similarly, defective expression of these receptors on transplanted HSPCs or in the bone marrow (BM) microenvironment of graft recipient mice led to defective homing, engraftment, and delayed hematopoietic reconstitution. This correlated with decreased activation of intracellular pattern recognition receptor Nlrp3 inflammasome. The P2X receptor family consists of seven purinergic receptors (P2X1-7) and we noticed that in addition to P2X4 and P2X7, HSPCs also highly express rapidly signaling the P2X1 receptor. Therefore, we asked if P2X1 receptor is also involved in HSPCs trafficking. We employed in vitro and in vivo murine models to study the role of P2X1 receptor blocked on HSPCs or bone marrow microenvironment cells by specific small molecular inhibitor NF499. First, we performed in vitro cell migration assays of bone marrow mononuclear cells (BMMNCs) isolated from normal mice that were exposed to NF499 and compared them to unexposed control cells. Next, in experiments in vivo we mobilized mice exposed to NF499 with G-CSF or AMD3100 and compared mobilization to control unexposed animals. Flow cytometry was employed to identify cell populations and clonogenic assays to enumerate the number of mobilized clonogenic progenitors. Similarly, in homing and engraftment experiments BMMNCs or recipient mice were exposed to NF499 and we evaluated homing and engraftment of transplanted cells by enumerating the number of cells labeled with fluorochromes in recipient mice BM and by evaluating the number of clonogenic progenitors in BM and spleen 24 hours and 12 days after transplantation. We also evaluated the potential involvement of Nlrp3 inflammasome in P2X1 receptor-mediated HSPCs trafficking. We report that the functional P2X1 receptor is highly expressed on murine and human HSPCs. We could demonstrate that the P2X1 receptor promotes the trafficking of murine cells in Nlrp3 inflammasome-dependent manner. Mice after exposure to P2X1 receptor inhibitor poorly mobilized HSPCs from the bone marrow into the peripheral blood. Mice transplanted with BMNNCs exposed to NF499 or recipient mice pretreated with this inhibitor demonstrated defective homing and engraftment as compared to control animals transplanted with cells not exposed to P2X1 inhibitor. Similar effects were noticed for control recipient mice that were not exposed to NF499. This study demonstrates for the first time the novel role of the P2X1 receptor in HSPCs trafficking in the mouse. Furthermore, it supports an important role of purinergic signaling engaging its downstream target Nlrp3 inflammasome in the mobilization, homing and engraftment of HSPCs.

The long β2,3-sheets encoded by redundant sequences play an integral role in the channel function of P2X7 receptors

P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened β2- and β3-sheets and their linker (loop β2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer β2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer β2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype.

ATP as a cotransmitter in sympathetic and parasympathetic nerves - another Burnstock legacy

Geoff Burnstock created an outstanding scientific legacy that includes identification of adenosine 5′-triphosphate (ATP) as an inhibitory neurotransmitter in the gut, the discovery and characterisation of a large family of purine and uridine nucleotide-sensitive ionotropic P2X and metabotropic P2Y receptors and the demonstration that ATP is as an excitatory cotransmitter in autonomic nerves. The evidence for cotransmission includes that: 1) ATP is costored with noradrenaline in synaptic vesicles in postganglionic sympathetic nerves innervating smooth muscle tissues, including the vas deferens and most arteries. 2) When coreleased with noradrenaline, ATP acts at postjunctional P2X1 receptors to elicit depolarisation, Ca2+ influx, Ca2+ sensitisation and contraction. 3) ATP is also coreleased with acetylcholine from postganglionic parasympathetic nerves innervating the urinary bladder, where it stimulates postjunctional P2X1 receptors, and a second, as yet unidentified site to evoke contraction of detrusor smooth muscle. In both systems membrane-bound ecto-enzymes and soluble nucleotidases released from postganglionic nerves dephosphorylate ATP and so terminate its neurotransmitter actions. Currently, the most promising potential area of therapeutic application relating to cotransmission is treatment of dysfunctional urinary bladder. This family of disorders is associated with the appearance of a purinergic component of neurogenic contractions. This component is an attractive target for drug development and targeting it may be a rewarding area of research.

P2X7 Receptors in Neurodegeneration: Potential Therapeutic Applications From Basic to Clinical Approaches.

Purinergic receptors play important roles in central nervous system (CNS), where the bulk of these receptors are implicated in neuroinflammatory responses and regulation of cellular function of neurons, microglial and astrocytes. Within the P2X receptor family, P2X7 receptor is generally known for its inactivity in normal conditions and activation by moderately high concentrations (>100 μM) of extracellular adenosine 5′-triphosphate (ATP) released from injured cells as a result of brain injury or pathological conditions. Activation of P2X7R contributes to the activation and proliferation of microglia and directly contribute to neurodegeneration by provoking microglia-mediated neuronal death, glutamate-mediated excitotoxicity, and NLRP3 inflammasome activation that results in initiation, maturity and release of the pro-inflammatory cytokines and generation of reactive oxygen and nitrogen species. These components of the inflammatory response play important roles in many neural pathologies and neurodegeneration disorders. In CNS, expression of P2X7R on microglia, astrocytes, and oligodendrocytes are upregulated under neuroinflammatory conditions. Several in vivo studies have demonstrated beneficial effects of the P2X7 receptor antagonists in animal model systems of neurodegenerative diseases. A number of specific and selective P2X7 receptor antagonists have been developed, but only few of them have shown efficient brain permeability. Finding potent and selective P2X7 receptor inhibitors which are also CNS penetrable and display acceptable pharmacokinetics (PK) has presented challenges for both academic researchers and pharmaceutical companies. In this review, we discuss the role of P2X7 receptor function in neurodegenerative diseases, the pharmacological inhibition of the receptor, and PET radiopharmaceuticals which permit non-invasive monitoring of the P2X7 receptor contribution to neuroinflammation associated with neurodegeneration.

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.

A Novel Underappreciated Role for the Extracellular Adenosine Triphosphate (ATP)-P2X4 Purinergic Receptor Axis in the Homing and Engraftment of HSPCs

Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but when released from activated cells into the extracellular space as extracellular ATP (eATP) it becomes a crucial mediator of the purinergic signaling network. Purinergic receptors for extracellular nucleotides (EXNs), expressed on the surface of all cells in the body, are represented by the P1, P2X, and P2Y receptor families, which are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for eATP signaling and consists of seven members (P2X1-7). We found that human and murine hematopoietic stem progenitor cells (HSPCs) highly express two members of this family, the P2X4 and P2X7 receptors. We recently reported that both are involved in optimal mobilization of HSPCs by activating Nlrp3 inflammasome (Leukemia 2020 Jun;34(6):1512-1523 and Stem Cell Rev Rep. 2019 Jun;15(3):391-403). We also reported that the P2X7 receptor expressed on the surface of HSPCs facilitates the homing and engraftment of HSPCs by increasing their responsiveness to SDF-1 gradients. Interestingly, it has been proposed that both receptors heterodimerize to exert optimal activity. Hypothesis. Since, the P2X4 and P2X7 receptors show several similar biological effects in non-hematopoietic cells, we became interested in the role of the P2X4 receptor in homing and engraftment of HSPCs.Materials and Methods. To test this hypothesis, we isolated SKL cells from P2X4-KO mice and tested them for migration in response to BM chemoattractants, including the major homing factor SDF-1. Next, we tested the short- and long-term homing of mouse BM cells after exposure to the P2X4-specific inhibitor PBS12054 in normal mice by evaluating the number of donor-derived PKH67-labeled BMMNCs and CFU-GM clonogenic progenitors isolated from recipient mouse BM 24 hours after transplantation as well as the number of day-12 colony-forming units in spleen (CFU-S) and day-12 CFU-GM clonogenic progenitors. These data were confirmed in transplant studies employing P2X4-KO bone marrow cells. In parallel, we also evaluated the recovery kinetics of leukocytes and blood platelets in the PB of transplanted animals. Finally, we also perturbed P2X4 expression in transplanted mice with PBS12054 and studied the effect on homing and engraftment of normal BM cells, as described above. Results. We found that P2X4-KO mouse HSPCs have a defect in migration in response to BM chemoattractants involved in BM homing, including the major homing factor SDF-1 as well as the supportive factors S1P and eATP. Perturbation of P2X4 expression on the surface of HSPCs led to significant defective homing and engraftment of HSPCs. Moreover, inhibition of P2X4 in the recipient mouse BM microenvironment had a similar effect. Conclusions. We identified for the first time the role of eATP-P2X4 signaling in the homing and engraftment of HSPCs. To explain this result, we conclude that the eATP-P2X4 axis is, like the eATP-P2X7 axis, a potent activator of Nlrp3 inflammasomes and that defective eATP-P2X4 signaling impairs the role of purinergic signaling and the Nlrp3 inflammasome in homing and engraftment. Moreover, our results show a similar homing and engraftment phenotype for P2X4-KO mice as that seen in P2X7-KO animals, which provides functional support for the proposed dimerization of P2X7 with P2X4 receptors and the necessary presence of both receptors for optimal function. This question is currently being addressed in our laboratory by employing the fluorescence resonance energy transfer (FRET) technique. Finally, we provide additional evidence that, in addition to SDF-1 and S1P, eATP and purinergic signaling involving P2X4 and P2X7 receptors is an important and underappreciated regulator of HSPC trafficking and a potential target for molecular optimization of both processes. Disclosures No relevant conflicts of interest to declare.

Geoffery Burnstock's influence on the evolution of P2X3 receptor pharmacology.

I first met Geoffery Burnstock in 1985 while working as a young post-doctoral fellow for Michael Williams at CIBA-Geigy Corporation. During that time, we were researching the role of adenosine in stroke and cardiovascular disease. These research efforts crystallized into a drug discovery program aimed at developing selective adenosine receptor agonists as cardioprotective agents. The first potent and selective A2A receptor agonist, CGS-21680, was discovered as part of this effort [1]. This high-affinity agonist was successfully radiolabeled [2] and quantitative receptor autoradiography studies demonstrated that A2A receptors were discretely localized in the rat basal ganglia [3]. These data were considered controversial at the time since other radioligand binding studies with less selective ligands had indicated a much broader expression of the A2A receptor in the central nervous system [4]. During my first discussions with Geoff, I had the opportunity to share some of these early data. Needless to say, that as a newcomer to the field, I was a bit nervous about discussing our work with such a pioneering scientist. I remember Geoff’s supportive enthusiasm for our new data and his encouragement not to let existing dogma hinder our exploration into this area of purine pharmacology. His open-minded approach and unfettered excitement for new ideas has served as an inspirational guidepost throughout my research career. Shortly after I joined the Neuroscience Discovery team at Abbott Laboratories in 1995, a new drug discovery effort targeting novel analgesic mechanisms as potential non-opioid interventions for chronic neuropathic and inflammatory pain was initiated. During an internal scientific research governance meeting, a photomicrograph from Geoff’s UCL team showing the discrete expression of P2X3 mRNA selectively localized on peripherin-positive small diameter sensory nerves was shared ahead its publication [5]. Geoff’s willingness to share these exciting data indicating a potential specific role for a single member of the emerging P2X receptor family in nociceptive neurotransmission provided sufficient evidence to initiate a specific drug discovery research effort on the nociceptive role of P2X3 receptors. In retrospect, the notion that highly selective expression of individual members of the emerging purine receptor superfamilies (e.g., A2A receptors in brain and P2X3 receptors on peripheral nerves) could drive physiologically relevant neuromodulation is striking. Furthermore, it served as the scientific foundation of much of my drug discovery research activities for the better part of two decades. Below is a brief overview of the historical development P2X3 receptor pharmacology, much of which was directly driven by Geoff’s interactions with a wide variety of academic and industrial purinergic investigators over the years. Specific questions and insights provided by Geoff on our work are highlighted to illustrate the significant impact Geoff had on our work.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

Drosophila taste neurons as an agonist-screening platform for P2X receptors

The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, here we demonstrate the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. We demonstrate that wild-type rat P2X2 expressed in Drosophila is fully functional (ATP EC50 8.7 µM), and that screening of small (2 µl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. We have determined agonist potency and specificity profiles for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists ATPγS and ATPαS, we were also able to identify a novel agonist, the synthetic analogue 2-fluoro-ATP, and to confirm its agonist activity on rat P2X2 receptors expressed in human cells. These data validate our Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists.

Differential Expression of the Metabotropic P2Y Receptor Family in the Cortex Following Status Epilepticus and Neuroprotection via P2Y1 Antagonism in Mice.

Purinergic signaling via P2 receptors is now widely accepted to play a critical role during increased states of hyperexcitability and seizure-induced pathology. In the setting of seizures and epilepsy, most attention has been paid to investigating the fast-acting ATP-gated P2X receptor family. More recent evidence has now also provided compelling evidence of an involvement of the slower-acting P2Y receptor family during seizures. This includes data demonstrating expression changes of P2Y receptors in the hippocampus following acute seizures and during epilepsy and anticonvulsive properties of P2Y-targeting drugs; in particular drugs targeting the P2Y1 subtype. Seizures, however, also involve damage to extra-hippocampal brain regions such as the cortex, which is thought to contribute to the epileptic phenotype. To analyze expressional changes of the P2Y receptor family in the cortex following status epilepticus and to determine the impact of drugs interfering with P2Y1 signaling on cortical damage, we used a unilateral mouse model of intraamygdala kainic acid-induced status epilepticus. Analysis of cortical tissue showed that status epilepticus leads to a global up-regulation of the P2Y receptor family in the cortex including P2Y1, P2Y2, P2Y4, and P2Y6, with the P2Y1 and P2Y4 receptor subtypes showing the strongest increase. Supporting a detrimental role of P2Y1 activation during status epilepticus, treatment with the P2Y1 agonist MRS2365 exacerbated high frequency high amplitude spiking, synonymous with injury-causing electrographic activity, and treatment with the P2Y1 antagonists MRS2500 protected against seizure-induced cortical damage. Suggesting P2Y1-mediated effects are predominantly due to increased microglia activation, treatment with the broad-spectrum anti-inflammatory drug minocycline abolished the observed neuroprotective effects of P2Y1 antagonism. In conclusion, our results further support a role for P2Y1-mediated signaling during seizure generation and seizure-induced neurodegeneration, suggesting P2Y1-targeting therapies as novel treatment for drug-refractory status epilepticus.

Molecular Mechanisms of Calcium Signaling During Phagocytosis.

Calcium (Ca2+) is a ubiquitous second messenger involved in the regulation of numerous cellular functions including vesicular trafficking, cytoskeletal rearrangements and gene transcription. Both global as well as localized Ca2+ signals occur during phagocytosis, although their functional impact on the phagocytic process has been debated. After nearly 40years of research, a consensus may now be reached that although not strictly required, Ca2+ signals render phagocytic ingestion and phagosome maturation more efficient, and their manipulation make an attractive avenue for therapeutic interventions. In the last decade many efforts have been made to identify the channels and regulators involved in generating and shaping phagocytic Ca2+ signals. While molecules involved in store-operated calcium entry (SOCE) of the STIM and ORAI family have taken center stage, members of the canonical, melastatin, mucolipin and vanilloid transient receptor potential (TRP), as well as purinergic P2X receptor families are now recognized to play significant roles. In this chapter, we review the recent literature on research that has linked specific Ca2+-permeable channels and regulators to phagocytic function. We highlight the fact that lipid mediators are emerging as important regulators of channel gating and that phagosomal ionic homeostasis and Ca2+ release also play essential parts. We predict that improved methodologies for measuring these factors will be critical for future advances in dissecting the intricate biology of this fascinating immune process.

Novel Evidence That Extracellular Adenosine Triphosphate (ATP), As a Purinergic Signaling Mediator, Activates Mobilization By Engaging a P2X4 Ligand-Gated Cation Channel Receptor Expressed on the Surface of Hematopoietic and Innate Immunity Cells

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 ATP and other extracellular nucleotides (EXNs). 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. Of all these receptors, the P2X receptor family is most highly specific for ATP signaling and consists of seven members (P2X1-7). We recently reported that by activating the P2X7 receptor ATP activates the Nlrp3 inflammasome in hematopoietic cells and significantly enhances G-CSF-induced mobilization of hematopoietic stem progenitor cells (HSPCs, Leukemia 2018, 32:1920-1931). Hypothesis. Since P2X7-KO mice still mobilize some HSPCs, we hypothesized that, in addition to the P2X7 receptor, other receptors from this family also play a role in the mobilization process.Materials and Methods. RT-PCR and FACS were employed to phenotype human and murine bone marrow (BM) mononuclear cells (MNCs) as well as sorted HSCs for expression of P2X receptors. Mobilization studies were performed in wild type mice in which the P2X4 receptor was blocked by the specific small-molecule inhibitor PSB12054 and in P2X7-KO animals. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. We also evaluated activation of the Nlrp3 inflammasome as well as other inflammasomes expressed in hematopoietic cells, including Aim2, Nlrp1a, Nlrp1b, and Nlrc4. Results. We found that, of all the receptors of the P2X family, two members, P2X7 and P2X4, are highly expressed on the surface of murine and human MNCs and HSPCs (Figure 1a). Activation of both receptors on the surface of innate immunity CD11b+Gr-1+ cells resulted in activation of the Nlrp3, Nlrp1a, Nlrp1b, and Aim2 inflammasomes. Inhibition of the P2X4 receptor by a specific small-molecule inhibitor decreased G-CSF- and AMD3100-induced mobilization in wild type mice (Figure 1b). Interestingly, we observed differences during AMD3100 mobilization, namely that while P2X7-KO deficiency resulted in decreased G-CSF-induced mobilization and did not affect AMD3100 mobilization efficiency, P2X4 inhibition, by contrast, led to a profound decrease in both G-CSF- and AMD3100-induced mobilization efficiency. Conclusions. Our results further support an important role for purinergic signaling in the mobilization of HSPCs. We also demonstrated that P2X7 and P2X4 receptors, which are highly expressed on human and murine hematopoietic cells, are involved in G-CSF-induced mobilization by activating the Nlrp3, Nlrp1a, Nlrp1b and Aim2 inflammasomes. By contrast, AMD3100-induced mobilization requires expression of P2X4 but not P2X7 receptors, which implies the involvement of different mechanisms and indicates that P2X7 deficiency can be compensated by the P2X4 receptor. Currently, we are performing RNASeq studies to identify differences in the G-CSF- and AMD3100-induced pathways in the mobilization process. These investigations will shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in cases of poor mobilizers. Figure 1 Disclosures No relevant conflicts of interest to declare.

Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors

The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration–response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC50 values of 0.76 μM, 22 μM, 763 μM or 118 μM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 μM ATA did not compete with [3H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration–response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αβ-methylene-ATP-sensitive P2X1 and P2X3 receptors.