Protein-coupled P2Y Receptors Research Articles

Purinergic Signalling in the Cochlea

The mammalian cochlea is the sensory organ of hearing with a delicate, highly organised structure that supports unique operating mechanisms. ATP release from the secretory tissues of the cochlear lateral wall (stria vascularis) triggers numerous physiological responses by activating P2 receptors in sensory, supporting and neural tissues. Two families of P2 receptors, ATP-gated ion channels (P2X receptors) and G protein-coupled P2Y receptors, activate intracellular signalling pathways that regulate cochlear development, homeostasis, sensory transduction, auditory neurotransmission and response to stress. Of particular interest is a purinergic hearing adaptation, which reflects the critical role of the P2X2 receptor in adaptive cochlear response to elevated sound levels. Other P2 receptors are involved in the maturation of neural processes and frequency selectivity refinement in the developing cochlea. Extracellular ATP signalling is regulated by a family of surface-located enzymes collectively known as "ectonucleotidases" that hydrolyse ATP to adenosine. Adenosine is a constitutive cell metabolite with an established role in tissue protection and regeneration. The differential activation of A1 and A2A adenosine receptors defines the cochlear response to injury caused by oxidative stress, inflammation, and activation of apoptotic pathways. A1 receptor agonism, A2A receptor antagonism, and increasing adenosine levels in cochlear fluids all represent promising therapeutic tools for cochlear rescue from injury and prevention of hearing loss.

P2RY14 Is a Potential Biomarker of Tumor Microenvironment Immunomodulation and Favorable Prognosis in Patients With Head and Neck Cancer

The tumor microenvironment (TME) has a crucial role in tumor development, progression, and treatment response. Yet, the exact interaction between cancer biology and the TME is not fully understood. The following study analyzed the correlation between immune/stromal/estimate scores and survival prognosis in head and neck squamous cell carcinoma (HNSC) using a bioinformatic method. As a result, a predictive biomarker, UDP-glucose-specific G(i) protein-coupled P2Y receptor (P2RY14), was discovered. The potential role of P2RY14-driven signaling pathways in the immune-remodeling of TME was then investigated. Briefly, low immune scores were associated with unfavorable prognosis and clinical-stage, larger tumor size, and the down-regulation of P2RY14 in HNSC patients. In addition, the survival analysis showed that HNSC patients with high expression had longer survival than patients with low expression from both TCGA databases and our own patients. We further discovered that P2RY14 is involved in the immune activity in the TME of HNSC; a downregulation of P2RY14 resulted in being an indicator for the conversion of TME status (from immune-dominant to metabolic-dominant status). The intersection analysis of genes co-expressed with P2RY14 indicated that the T-cell receptor signaling pathway and PD-L1 expression and PD-1 checkpoint pathway were candidate signaling pathways driven by the P2RY14 gene in HNSC. Further investigation of immune-associated signaling pathways regulated by P2RY14 may help HNSC patients gain higher immunotherapy benefits.

Control of Macrophage Inflammation by P2Y Purinergic Receptors.

Macrophages comprise a phenotypically and functionally diverse group of hematopoietic cells. Versatile macrophage subsets engage to ensure maintenance of tissue integrity. To perform tissue stress surveillance, macrophages express many different stress-sensing receptors, including purinergic P2X and P2Y receptors that respond to extracellular nucleotides and their sugar derivatives. Activation of G protein-coupled P2Y receptors can be both pro- and anti-inflammatory. Current examples include the observation that P2Y14 receptor promotes STAT1-mediated inflammation in pro-inflammatory M1 macrophages as well as the demonstration that P2Y11 receptor suppresses the secretion of tumor necrosis factor (TNF)-α and concomitantly promotes the release of soluble TNF receptors from anti-inflammatory M2 macrophages. Here, we review macrophage regulation by P2Y purinergic receptors, both in physiological and disease-associated inflammation. Therapeutic targeting of anti-inflammatory P2Y receptor signaling is desirable to attenuate excessive inflammation in infectious diseases such as COVID-19. Conversely, anti-inflammatory P2Y receptor signaling must be suppressed during cancer therapy to preserve its efficacy.

Structural and Functional Features of the P2X4 Receptor: An Immunological Perspective.

Extracellular nucleotides are important mediators of activation, triggering various responses through plasma membrane P2 and P1 receptors. P2 receptors are further subdivided into ionotropic P2X receptors and G protein-coupled P2Y receptors. P2X4 is an ATP-gated cation channel broadly expressed in most tissues of the body. Within the P2X family, P2X4 has a unique subcellular distribution, being preferentially localized in lysosomes. In these organelles, high ATP concentrations do not trigger P2X4 because of the low pH. However, when the pH increases to 7.4, P2X4 can be stimulated by intra-lysosomal ATP, which is in its active, tetra-anionic form. Elucidation of P2X4, P2X3 and P2X7 structures has shed some light on the functional differences between these purinergic receptors. The potential interaction between P2X4 and P2X7 has been extensively studied. Despite intensive effort, it has not been possible yet to determine whether P2X4 and P2X7 interact as heterotrimers or homotrimers at the plasma membrane. However, several publications have shown that functional interactions between P2X4 and P2X7 do occur. Importantly, these studies indicate that P2X4 potentiates P2X7-dependent activation of inflammasomes, leading to increased release of IL-1β and IL-18. The role of P2X4 in various diseases could be beneficial or deleterious even though the pathophysiological mechanisms involved are still poorly defined. However, in diseases whose physiopathology involves activation of the NLRP3 inflammasome, P2X4 was found to exacerbate severity of disease. The recent production of monoclonal antibodies specific for the human and mouse P2X4, some of which are endowed with agonist or antagonist properties, raises the possibility that they could be used therapeutically. Analysis of single nucleotide polymorphisms of the human P2RX4 gene has uncovered the association of P2RX4 gene variants with susceptibility to several human diseases.

Update of P2Y receptor pharmacology: IUPHAR Review 27.

Eight G protein-coupled P2Y receptor subtypes respond to extracellular adenine and uracil mononucleotides and dinucleotides. P2Y receptors belong to the δ group of rhodopsin-like GPCRs and contain two structurally distinct subfamilies: P2Y1 , P2Y2 , P2Y4 , P2Y6 , and P2Y11 (principally Gq protein-coupled P2Y1 -like) and P2Y12-14 (principally Gi protein-coupled P2Y12 -like) receptors. Brain P2Y receptors occur in neurons, glial cells, and vasculature. Endothelial P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptors induce vasodilation, while smooth muscle P2Y2 , P2Y4 , and P2Y6 receptor activation leads to vasoconstriction. Pancreatic P2Y1 and P2Y6 receptors stimulate while P2Y13 receptors inhibits insulin secretion. Antagonists of P2Y12 receptors, and potentially P2Y1 receptors, are anti-thrombotic agents, and a P2Y2 /P2Y4 receptor agonist treats dry eye syndrome in Asia. P2Y receptor agonists are generally pro-inflammatory, and antagonists may eventually treat inflammatory conditions. This article reviews recent developments in P2Y receptor pharmacology (using synthetic agonists and antagonists), structure and biophysical properties (using X-ray crystallography, mutagenesis and modelling), physiological and pathophysiological roles, and present and potentially future therapeutic targeting.

P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction.

Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren’s syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.

P2Y receptors in neuropathic pain

This review summarizes and evaluates the relationship between neuropathic pain and P2Y receptors from inception to 2019. Purinergic receptors have been well studied in recent years using various molecular biological methods. The main research objective of this review is to determine the association of P2Y1, P2Y2, P2Y6, P2Y12 and P2Y13 receptors with neuropathic pain. This review includes the most comprehensive subtypes of P2Y that related to neuropathic pain and the current therapeutic method of neuropathic pain. G protein-coupled P2Y receptors are located on neurons, astrocytes, oligodendrocytes and microglial cells and regulate neurotransmission. Nerve injury is the prime reason for abnormal regulation of P2Y receptor mRNA expression, subsequently, inducing neuropathic pain. Neuropathic pain is a type of chronic pain that is divided into peripheral, central and mixed. Numerous studies demonstrated a positive correlation between the expression level of P2Y receptors and neuropathic pain generation. Also, several reports showed that P2Y short hairpin RNA (shRNA) and P2Y antagonist can be used as an analgesic to relieve neuropathic pain via decreasing P2Y receptor expression level and neural cell activation. However, the transformation process from basic experiments to clinical applications is a long process. Current deficiencies and future research directions are discussed at the end of this review.

Reviewing the role of P2Y receptors in specific gastrointestinal cancers.

Extracellular nucleotides are important intercellular signaling molecules that were found enriched in the tumor microenvironment. In fact, interfering with G protein-coupled P2Y receptor signaling has emerged as a promising therapeutic alternative to treat aggressive and difficult-to-manage cancers such as those affecting the gastrointestinal system. In this review, we will discuss the functions of P2Y receptors in gastrointestinal cancers with an emphasis on colorectal, hepatic, and pancreatic cancers. We will show that P2Y2 receptor up-regulation increases cancer cell proliferation, tumor growth, and metastasis in almost all studied gastrointestinal cancers. In contrast, we will present P2Y6 receptor as having opposing roles in colorectal cancer vs. gastric cancer. In colorectal cancer, the P2Y6 receptor induces carcinogenesis by inhibiting apoptosis, whereas P2Y6 suppresses gastric cancer tumor growth by reducing β-catenin transcriptional activity. The contribution of the P2Y11 receptor in the migration of liver and pancreatic cancer cells will be compared to its normal inhibitory function on this cellular process in ciliated cholangiocytes. Hence, we will demonstrate that the selective inhibition of the P2Y12 receptor activity in platelets was associated to a reduction in the risk of developing colorectal cancer and metastasis formation. We will succinctly review the role of P2Y1, P2Y4, P2Y13, and P2Y14 receptors as the knowledge for these receptors in gastrointestinal cancers is sparse. Finally, redundant ligand selectivity, nucleotide high lability, cell context, and antibody reliability will be presented as the main difficulties in defining P2Y receptor functions in gastrointestinal cancers.

Integrity of Actin-Network Is Involved in Uridine 5′-Triphosphate Evoked Store-Operated CaM2+ Entry in Bovine Adrenocortical Fasciculata Cells

Store-operated Ca2+ entry channels (SOCs) play an important role in the regulation of diverse non-excitable cell functions. However, the precise mechanism of SOCs activation is still controversial. Uridine 5′-triphosphate (UTP) was shown to induce Ca2+ entry in a dihydropyridines-insensitive manner and accelerated steroidogenesis in bovine adrenocortical fasciculata cells (BAFCs) via the Gq/11 protein-coupled P2Y2 receptor. Therefore we investigated whether UTP is involved in SOCs activation and the mechanism of UTP-induced SOCs activation. Fura 2-loaded BAFCs were used for the measurement of intracellular concentration of Ca2+ ([Ca2+]i) mobilization. Extracellular UTP evoked Ca2+ release from intracellular stores followed by an increase in Ca2+ entry. The Ca2+ influx elicited by UTP was inhibited not by nifedipine, but by Zn2+, Cd2+, and Ni2+ (potency order: Zn2+ > Cd2+ >> Ni2+), and the effect of UTP was also attenuated by a phospholipase C inhibitor (U73122). These results indicate that UTP activates SOCs in BAFCs. The increase in [Ca2+]i by UTP was attenuated by ML-9, a myosin-light chain kinase inhibitor, and calmodulin inhibitors, W-7 and E6 berbamine, in a concentration-dependent manner. These reagents depolymerized actin filaments with rhodamine staining in BAFCs. Cytochalasin D also inhibited UTP-activated SOCs and depolymerized actin filaments. From these results, we proposed that calcium/calmodulin dependent myosin-light chain kinase is involved in the mobilization of actin filaments and the integrity of actin-network plays an important role in UTP-induced SOCs activation in BAFCs.

Profiling of a suramin-derived compound library at recombinant human P2Y receptors identifies NF272 as a competitive but non-selective P2Y2 receptor antagonist.

Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y2 receptor antagonist with a Ki of 19μM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 receptors. Up to 250μM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.

Salvianolic acids from antithrombotic Traditional Chinese Medicine Danshen are antagonists of human P2Y1 and P2Y12 receptors

Many hemorheologic Traditional Chinese Medicines (TCMs) that are widely-used clinically lack molecular mechanisms of action. We hypothesized that some of the active components of hemorheologic TCMs may function through targeting prothrombotic P2Y1 and/or P2Y12 receptors. The interactions between 253 antithrombotic compounds from TCM and these two G protein-coupled P2Y receptors were evaluated using virtual screening. Eleven highly ranked hits were further tested in radioligand binding and functional assays. Among these compounds, salvianolic acid A and C antagonized the activity of both P2Y1 and P2Y12 receptors in the low µM range, while salvianolic acid B antagonized the P2Y12 receptor. These three salvianolic acids are the major active components of the broadly-used hemorheologic TCM Danshen (Salvia militorrhiza), the antithrombotic molecular mechanisms of which were largely unknown. Thus, the combination of virtual screening and experimental validation identified potential mechanisms of action of multicomponent drugs that are already employed clinically.

Effect of Low Androgen Status on the Expression of P2Y Receptors in the Corpus Cavernosum of Rats

To determine whether low androgen status impacts erectile function by regulating the expression of the G protein-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, and P2Y6) in the corpus cavernosum penis of rats. A total of 36 healthy, 8-week-old, male Sprague-Dawley rats were randomly allocated into 6 groups: 4 weeks of androgen replacement therapy post castration (group A); 4 weeks post castration (group B); 4 weeks post sham operation (group C); 8 weeks of androgen replacement therapy post castration (group D); 8 weeks post castration (group E); and 8 weeks post sham operation (group F). The ratio of maximum intracavernous pressure/mean arterial pressure was measured for every group and detected the expression of P2Y1, P2Y2, P2Y4, and P2Y6 in the rat corpus cavernosum penis of every group. The ratio of maximum intracavernous pressure/mean arterial pressure significantly declined in the castration group compared with the control group (P <.01). The expression of P2Y1, P2Y2, P2Y4, and P2Y6 and the ratio of the phosphorylated endothelial nitric oxide synthase (eNOS)/eNOS proteins were significantly lower in the castration group vs treatment or control (P <.01) whereas they were significantly lower in the group of 8 weeks vs 4 weeks post castration (P <.05). The rat serum testosterone levels in every group were positively correlated with the protein levels of P2Y1, P2Y2, P2Y4, and P2Y6. Downregulation of the expression of the P2Y1, P2Y2, P2Y4, and P2Y6 receptors that reduces the ratio of phosphorylated eNOS/eNOS and eNOS activity may be one of the important mechanisms of erectile dysfunction caused by low androgen status.

Molecular Recognition of Agonists and Antagonists by the Nucleotide-Activated G Protein-Coupled P2Y2 Receptor.

A homology model of the nucleotide-activated P2Y2R was created based on the X-ray structures of the P2Y1 receptor. Docking studies were performed, and receptor mutants were created to probe the identified binding interactions. Mutation of residues predicted to interact with the ribose (Arg110) and the phosphates of the nucleotide agonists (Arg265, Arg292) or that contribute indirectly to binding (Tyr288) abolished activity. The Y114F, R194A, and F261A mutations led to inactivity of diadenosine tetraphosphate and to a reduced response of UTP. Significant reduction in agonist potency was observed for all other receptor mutants (Phe111, His184, Ser193, Phe261, Tyr268, Tyr269) predicted to be involved in agonist recognition. An ionic lock between Asp185 and Arg292 that is probably involved in receptor activation interacts with the phosphate groups. The antagonist AR-C118925 and anthraquinones likely bind to the orthosteric site. The updated homology models will be useful for virtual screening and drug design.

From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor

The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.

ATP evokes Ca2+ signals in cultured foetal human cortical astrocytes entirely through G protein-coupled P2Y receptors.

Extracellular ATP plays important roles in coordinating the activities of astrocytes and neurons, and aberrant signalling is associated with neurodegenerative diseases. In rodents, ATP stimulates opening of Ca2+‐permeable channels formed by P2X receptor subunits in the plasma membrane. It is widely assumed, but not verified, that P2X receptors also evoke Ca2+ signals in human astrocytes. Here, we directly assess this hypothesis. We showed that cultured foetal cortical human astrocytes express mRNA for several P2X receptor subunits (P2X4, P2X5, P2X6) and G protein‐coupled P2Y receptors (P2Y1, P2Y2, P2Y6, P2Y11). In these astrocytes, ATP stimulated Ca2+ release from intracellular stores through IP 3 receptors and store‐operated Ca2+ entry. These responses were entirely mediated by P2Y1 and P2Y2 receptors. Agonists of P2X receptors did not evoke Ca2+ signals, and nor did ATP when Ca2+ release from intracellular stores and store‐operated Ca2+ entry were inhibited. We conclude that ATP‐evoked Ca2+ signals in cultured human foetal astrocytes are entirely mediated by P2Y1 and P2Y2 receptors, with no contribution from P2X receptors.

Purinergic P2Y receptors: Molecular diversity and implications for treatment of cardiovascular diseases.

Purinergic signaling, mediated mainly by G protein-coupled P2Y receptors (P2YRs), is now attracting attention as a new therapeutic target for preventing or treating cardiovascular diseases. Observations using mice with genetically modified P2YRs and/or treated with a pharmacological P2YR inhibitor have helped us understand the physiological and pathological significance of P2YRs in the cardiovascular system. P2YR-mediated biological functions are predominantly activated by mononucleotides released from non-adrenergic, non-cholinergic nerve endings or non-secretory tissues in response to physical stress or cell injury, though recent studies have suggested the occurrence of ligand-independent P2YR function through receptor-receptor interactions (oligomerization) in several biological processes. In this review, we introduce the functions of P2YRs and possible dimerization with G protein-coupled receptors (GPCRs) in the cardiovascular system. We focus especially on the crosstalk between uridine nucleotide-responsive P2Y6R and angiotensin (Ang) II type1 receptor (AT1R) signaling, and introduce our recent finding that the P2Y6R antagonist MRS2578 interrupts heterodimerization between P2Y6R and AT1R, thereby reducing the risk of AT1R-stimulated hypertension in mice. These results strongly suggest that targeting P2Y6R oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases.

P2Y2 Receptor Functions in Cancer: A Perspective in the Context of Colorectal Cancer.

Purinergic signaling has recently emerged as a network of signaling molecules, enzymes and receptors that coordinates the action and behavior of cancerous cells. Extracellular adenosine 5' triphosphate activates a plethora of P2 nucleotide receptors that can putatively modulate cancer cell proliferation, survival and dissemination. In this context, the G protein-coupled P2Y2 receptor was identified as one of the entities coordinating the cellular and molecular events that characterize cancerous cells. In this chapter, we will look at the contribution of the P2Y2 receptor in cancer outcomes and use this information to demonstrate that the P2Y2 receptor represents a drug target of interest in the setting of colorectal cancer, for which the role and function of this receptor is poorly defined. More particularly, we will review how the P2Y2 receptor modulates cancer cell proliferation and survival, while promoting cell dissemination and formation of metastases. Finally, we will investigate how the P2Y2 receptor can contribute to the detrimental development of drug resistance that is often observed in cancerous cells.

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

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

P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction

It is known that adenosine 5′-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12–13 receptors, 2-methylthioadenosine 5′-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12–13 antagonists AR-C69931MX or 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5′-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.

Role of P2 Receptors as Modulators of Rat Eosinophil Recruitment in Allergic Inflammation.

ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step was to investigate the expression and functionality of the P2X receptors by patch clamping, our results showed a potency ranking order of ATP>ATPγS> 2meSATP> ADP> αβmeATP> βγmeATP>BzATP> UTP> UDP>cAMP. This data suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency (UTP>ATPγS> ATP > UDP> ADP >2meSATP > αβmeATP) suggests the presence of P2Y2, P2Y4, P2Y6 and P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did chemotaxis assays to verify whether nucleotides could induce migration. After 1 or 2 hours of incubation, ATP increased migration of eosinophils, as well as ATPγS, a less hydrolysable analogue of ATP, while suramin a P2 blocker abolished migration. In keeping with this idea, we tested whether these receptors are implicated in the migration of eosinophils to an inflammation site in vivo, using a model of rat allergic pleurisy. In fact, migration of eosinophils has increased when ATP or ATPγS were applied in the pleural cavity, and once more suramin blocked this effect. We have demonstrated that rat eosinophils express P2X and P2Y receptors. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo, an effect blocked by suramin.