Purinergic signaling: a potential therapeutic target for depression and chronic pain

The comorbid mechanism of depression and chronic pain has been a research hotspot in recent years. Until now, the role of purinergic signals in the comorbid mechanism of depression and chronic pain has not been fully understood. This review mainly summarizes the research results published in PubMed during the past 5 years and concludes that purinergic signaling is a potential therapeutic target for comorbid depression and chronic pain, and the purinergic receptors A1, A2A, P2X3, P2X4, and P2X7and P2Y₆, P2Y₁, and P2Y₁₂ may be important factors. The main potential pathways are as follows: A1 receptor-related G protein-dependent activation of introverted K⁺ channels (GIRKs), A2A receptor-related effects on the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and MAPK/nuclear factor-κB (NF-κB) pathways, P2X3 receptor-related effects on dorsal root ganglia (DRG) excitability, P2X4 receptor-related effects on proinflammatory cytokines and inflammasome activation, P2X7 receptor-related effects on ion channels, the NLRP3 inflammasome and brain-derived neurotrophic factor (BDNF), and P2Y receptor-related effects on the phospholipase C (PLC)/inositol triphosphate (IP3)/Ca²⁺ signaling pathway. We hope that the conclusions of this review will provide key ideas for future research on the role of purinergic signaling in the comorbid mechanism of depression and chronic pain.     

Adenosine A1 receptor: Functional receptor-receptor interactions in the brain

Over the past decade, many lines of investigation have shown that receptor-mediated signaling exhibits greater diversity than previously appreciated. Signal diversity arises from numerous factors, which include the formation of receptor dimers and interplay between different receptors. Using adenosine A₁ receptors as a paradigm of G protein-coupled receptors, this review focuses on how receptor-receptor interactions may contribute to regulation of the synaptic transmission within the central nervous system. The interactions with metabotropic dopamine, adenosine A_2A, A₃, neuropeptide Y, and purinergic P2Y₁ receptors will be described in the first part. The second part deals with interactions between A₁Rs and ionotropic receptors, especially GABA_A, NMDA, and P2X receptors as well as ATP-sensitive K⁺ channels. Finally, the review will discuss new approaches towards treating neurological disorders.     

Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<Subscript>2</Subscript> receptor signaling

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O₂ atmosphere and addition of CoCl₂, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O₂ induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y₂ and adenosine A₁ receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y₂ (but not A₁) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y₂ receptor signaling.     

Differential expression of P2Y receptors in the rat cochlea during development

Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y₂ and P2Y₄ protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca²⁺-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.     

Pharmacochemistry of the platelet purinergic receptors

Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y₁ and P2Y₁₂ receptors, a P2Y₁₄ receptor (GPR105) of unknown function, and anti-aggregatory A_2A and A_2B adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure–activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and non-nucleotide ligands at the P2Y₁, P2Y₁₂, and P2Y₁₄ receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y₁₂ receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y₁₂ receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y₁ receptor or the P2Y₁₂ receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A_2A AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A_2A agonist CGS21680 and the P2Y₁ receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.     

The P2Y<Subscript>1</Subscript> receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y₁ receptor (P2Y₁R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y₁R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y₁R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y₁R protein expression. In addition, assays with the Ca²⁺ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca²⁺ modulation. P2Y₁R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y₁ receptor signaling.     

P2 purinergic receptor modulation of cytokine production

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y₆ receptor mediated impact on interleukin (IL)-8 production, (2) P2Y₁₁ receptor-mediated affects on IL-12/23 output, and (3) P2X₇ receptor mediated IL-1β posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases.     

P2 purinergic receptor modulation of cytokine production

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y₆ receptor mediated impact on interleukin (IL)-8 production, (2) P2Y₁₁ receptor-mediated affects on IL-12/23 output, and (3) P2X₇ receptor mediated IL-1β posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases.     

P2Y2 Nucleotide Receptor-Mediated Responses in Brain Cells

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G_q protein-coupled P2Y₂ nucleotide receptor subtype (P2Y₂R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y₂R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y₂R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y₂R activation increases astrocyte migration. P2Y₂R activation by UTP increases the expression in astrocytes of α_Vβ_3/5 integrins that bind directly to the P2Y₂R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y₂R, an interaction required for G_o and G₁₂ protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y₂R expression is increased by stimulation with interleukin-1β (IL-1β), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric β-amyloid peptide (Aβ_1-42), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y₂Rs in neurons. Data with rPCNs suggest that P2Y₂R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y₂Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y₂Rs in glial cells can promote neuroprotective responses, suggesting that P2Y₂Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.     

Pro- and anti-inflammatory macrophages express a sub-type specific purinergic receptor profile

Extracellular nucleotides act as danger signals that orchestrate inflammation by purinergic receptor activation. The expression pattern of different purinergic receptors may correlate with a pro- or anti-inflammatory phenotype. Macrophages function as pro-inflammatory M1 macrophages (M1) or anti-inflammatory M2 macrophages (M2). The present study found that murine bone marrow-derived macrophages express a unique purinergic receptor profile during in vitro polarization. As assessed by real-time polymerase chain reaction (PCR), Gαs-coupled P1 receptors A2A and A2B are upregulated in M1 and M2 compared to M0, but A2A 15 times higher in M1. The ionotropic P2 receptor P2X₅ is selectively upregulated in M1- and M2-polarized macrophages. P2X₇ is temporarily expressed in M1 macrophages. Metabotropic P2Y receptors showed a distinct expression profile in M1 and M2-polarized macrophages: Gαq coupled P2Y₁ and P2Y₆ are exclusively upregulated in M2, whereas Gαi P2Y₁₃ and P2Y₁₄ are overexpressed in M1. This consequently leads to functional differences between M1 and M2 in response to adenosine di-phosphate stimulation (ADP): In contrast to M1, M2 showed increased cytoplasmatic calcium after ADP stimulation. In the present study we show that bone marrow-derived macrophages express a unique repertoire of purinergic receptors. We show for the first time that the repertoire of purinergic receptors is highly flexible and quickly adapts upon pro- and anti-inflammatory macrophage differentiation with functional consequences to nucleotide stimulation.     

P1 receptors and cytokine secretion

Evidence has accumulated in the last three decades to suggest tissue protection and regeneration by adenosine in multiple different cell types. Adenosine produced in hypoxic or inflamed environments reduces tissue injury and promotes repair by receptor-mediated mechanisms. Among other actions, regulation of cytokine production and secretion by immune cells, astrocytes and microglia (the brain immunocytes) has emerged as a main mechanism at the basis of adenosine effects in diseases characterized by a marked inflammatory component. Many recent studies have highlighted that signalling through A₁ and A_2A adenosine receptors can powerfully prevent the release of pro-inflammatory cytokines, thus inhibiting inflammation and reperfusion injury. However, the activation of adenosine receptors is not invariably protective of tissues, as signalling through the A_2B adenosine receptor has been linked to pro-inflammatory actions which are, at least in part, mediated by increased release of pro-inflammatory cytokines from epithelial cells, astrocytes and fibroblasts. Here, we discuss the multiple actions of P1 receptors on cytokine secretion, by analyzing, in particular, the role of the various adenosine receptor subtypes, the complex reciprocal interplay between the adenosine and the cytokine systems, their pathophysiological significance and the potential of adenosine receptor ligands as new anti-inflammatory agents.     

MicroRNA: Crucial modulator in purinergic signalling involved diseases

Both microRNAs (miRNAs) and purinergic signalling are widely and respectively expressed in various tissues of different organisms and play vital roles in a variety of physiological and pathological processes. Here, we reviewed the current publications contributed to the relationship of miRNAs and purinergic signalling in cardiovascular diseases, gastrointestinal diseases, neurological diseases, and ophthalmic diseases. We tried to decode the miRNAs-purinergic signalling network of purinergic signalling involved diseases. The evidence indicated that more than 30 miRNAs (miR-22, miR-30, miR-146, miR-150, miR-155, miR-187, etc.) directly or indirectly modulate P1 receptors (A₁, A_2A, A_2B, A₃), P2 receptors (P2X1, P2X3, P2X4, P2X7, P2Y2, P2Y6, P2Y12), and ecto-enzymes (CD39, CD73, ADA2); P2X7 and CD73 could be modulated by multiple miRNAs (P2X7: miR-21, miR-22, miR-30, miR-135a, miR-150, miR-186, miR-187, miR-216b; CD73: miR-141, miR-101, miR-193b, miR-340, miR-187, miR-30, miR-422a); miR-187 would be the common miRNA to modulate P2X7 and CD73.     

P2Y6 Receptor-Mediated Proinflammatory Signaling in Human Bronchial Epithelia

P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y₆ receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl⁻ secretion via both Ca²⁺- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca²⁺ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y₆ receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca²⁺, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y₆ receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such “cytokine networks” play an important role in sustaining the airway inflammatory disease.     

Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth,Neovascularization, Angiogenesis and Macrophage Infiltration

CD73 (ecto-5''-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A₁, A_2A, and A₃ in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A₁AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A₃AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy.     

P2Y6 Receptor Potentiates Pro-Inflammatory Responses in Macrophages and Exhibits Differential Roles in Atherosclerotic Lesion Development

Background

P2Y₆, a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y₆ deficiency on atherosclerosis.

Methodology/Principal Findings

Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y₆ receptors, showed that exogenous expression of P2Y₆ induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y₆-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y₆ and in acute peritonitis models of inflammation. To evaluate the role of P2Y₆ in atherosclerotic lesion development, we used P2Y₆-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y₆ receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y₆xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y₆ deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice.

Conclusions

P2Y₆ receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y₆ deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y₆ in vascular disease pathophysiologies, such as aneurysm formation.     

NCI-H295R,a Human Adrenal Cortex-Derived Cell Line,Expresses Purinergic Receptors Linked to Ca2+-Mobilization/Influx and Cortisol Secretion

Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A_2A and A_2B), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10–1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1–1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10–100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.     

Purinergic interplay between erythrocytes and platelets in diabetes-associated vascular dysfunction

Cardiovascular complications in diabetes are the leading causes for high morbidity and mortality. It has been shown that alteration of purinergic signaling contributes to diabetes-associated cardiovascular complications. Red blood cells (RBCs) and platelets play a fundamental role in regulation of oxygen transport and hemostasis, respectively. Of note, these cells undergo purinergic dysfunction in diabetes. Recent studies have established a novel function of RBCs as disease mediators for the development of endothelial dysfunction in type 2 diabetes (T2D). RBC-released ATP is defective in T2D, which has implication for induction of vascular dysfunction by dysregulating purinergic signaling. Platelets are hyperactive in diabetes. ADP-mediated P2Y₁ and P2Y₁₂ receptor activation contributes to platelet aggregation and targeting P2Y receptors particularly P2Y₁₂ receptor in platelets is effective for the treatment of cardiovascular events. In contrast to other P2Y₁₂ receptor antagonists, platelet-targeting drug ticagrelor has potential to initiate purinergic signaling in RBCs for the beneficial cardiovascular outcomes. It is increasingly clear that altered vascular purinergic signaling mediated by various nucleotides and nucleoside contributes to diabetes-associated vascular dysfunction. However, the contribution of complex purinergic networks between RBCs and platelets to the vascular dysfunction in diabetes remains unclear. This study discusses the possible interplay of RBCs and platelets via the purinergic network for diabetes-associated vascular dysfunction.     

Blockade of murine T cell activation by antagonists of P2Y6 and P2X7 receptors

Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y₆ antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X₇ antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y₆ and P2X₇ receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca²⁺ in T cells. The P2X₇ and P2Y₆ receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y₆ and P2X₇ receptors contributes to T cell activation via TCR.