P2X7 receptor antagonists display agonist-like effects on cell signaling proteins

Background

The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca²⁺]_i. We report that some agents that can block P2X₇R receptors also promote diverse P2X₇R-independent effects on cell signaling.

Methods

We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X₇R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.

Results

With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated ⁴⁵Ca²⁺ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X₇R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.

Conclusions

Several agents used as P2X₇R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

General significance

Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Extracellular adenosine activates P1 receptors (A₁, A_2A, A_2B, A₃) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4⁺CD62L⁺ naïve murine T cells under Treg-skewing conditions. Antagonists of A₁ and A_2B receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A₁ and A_2B receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A₁ antagonist, but not A_2B antagonist, suppressed T cell activation. We conclude that A₁ and A_2B receptors are both involved in differentiation to Treg, but through different mechanisms. Since A_2B antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A_2B receptor would facilitate tumor immunity.     

Investigation of the functional expression of purine and pyrimidine receptors in porcine isolated pancreatic arteries

Receptors for purines and pyrimidines are expressed throughout the cardiovascular system. This study investigated their functional expression in porcine isolated pancreatic arteries. Pancreatic arteries (endothelium intact or denuded) were prepared for isometric tension recording and preconstricted with U46619, a thromboxane A₂ mimetic; adenosine-5′-diphosphate (ADP), uridine-5′-triphosphate (UTP) and MRS2768, a selective P2Y₂ agonist, were applied cumulatively, while adenosine-5′-triphosphate (ATP) and αβ-methylene-ATP (αβ-meATP) response curves were generated from single concentrations per tissue segment. Antagonists/enzyme inhibitors were applied prior to U46619 addition. ATP, αβ-meATP, UTP and MRS2768 induced vasoconstriction, with a potency order of αβ-meATP > MRS2768 > ATP ≥ UTP. Contractions to ATP and αβ-meATP were blocked by NF449, a selective P2X1 receptor antagonist. The contraction induced by ATP, but not UTP, was followed by vasorelaxation. Endothelium removal and DUP 697, a cyclooxygenase-2 inhibitor, had no significant effect on contraction to ATP but attenuated that to UTP, indicating actions at distinct receptors. MRS2578, a selective P2Y₆ receptor antagonist, had no effect on contractions to UTP. ADP induced endothelium-dependent vasorelaxation which was inhibited by MRS2179, a selective P2Y₁ receptor antagonist, or SCH58261, a selective adenosine A_2A receptor antagonist. The contractions to ATP and αβ-meATP were attributed to actions at P2X1 receptors on the vascular smooth muscle, whereas it was shown for the first time that UTP induced an endothelium-dependent vasoconstriction which may involve P2Y₂ and/or P2Y₄ receptors. The relaxation induced by ADP is mediated by P2Y₁ and A_2A adenosine receptors. Porcine pancreatic arteries appear to lack vasorelaxant P2Y₂ and P2Y₄ receptors.     

PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X₇ receptors (P2X₇Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X₇R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X₇R might be expressed in neural progenitor cells (NPCs). P2X₇R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X₇R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X₇Rs induced Ca²⁺ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X₇R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca²⁺ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X₇R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca²⁺ and treatment with blockers for P2X₇R and PKC activity. Stimulation of P2X₇R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X₇R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.     

UTP is not a biased agonist at human P2Y11 receptors

Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y₁₁ receptor, as it increased cytosolic [Ca²⁺], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y₁₁ receptor in greater detail. Intracellular Ca²⁺ was monitored in 1321N1 cells stably expressing human P2Y₁₁ receptors using the Ca²⁺-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca²⁺, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca²⁺ in 1321N1 cells stably expressing the human P2Y₂ receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y₁₁ receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y₁₁ receptor, nor does UTP act as an antagonist.     

P2 receptors and platelet function

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA₂ and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y₁ and P2Y₁₂ receptor subtypes, while the P2X₁ receptor ligand-gated cation channel is activated by ATP. The P2Y₁ receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y₁₂ receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X₁ receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.     

Off-target effect of the Epac agonist 8-pCPT-2′-O-Me-cAMP on P2Y12 receptors in blood platelets

The primary target of the cAMP analogue 8-pCPT-2′-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2′-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2′-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2′O-Me-cAMP binding into the purinergic platelet receptor P2Y₁₂ revealed that the analogue docks similar to the P2Y₁₂ antagonist 2MeSAMP. The 8-pCPT-2′-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2′-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2′-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y₁₂ receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2′-O-Me-cAMP did not affect platelet activation at similar concentrations.     

Modeling P2Y receptor-Ca response coupling in taste cells

Here we elaborated an analytical approach for the simulation of dose-response curves mediated by cellular receptors coupled to PLC and Ca²⁺ mobilization. Based on a mathematical model of purinergic Ca²⁺ signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y₂ and P2Y₄ receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca²⁺. Cellular responses versus concentration of BzATP, a P2Y₂ agonist and a P2Y₄ antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose-response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y₂ and P2Y₁₁ are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose-response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y₂/P2Y₄ homo- and heterodimers. Our computations and analytical analysis of experimental dose-response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y₂ and P2Y₄ receptors operative mostly in the dimeric form.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X₁–P2X₇) have been identified. Most of the P2X₁ receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X₁ receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X₁ receptors. In the present study, we examined the effect of gintonin on P2X₁ receptor channel activity. P2X₁ receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X₁ receptors induced large inward currents (I _ATP ), but repetitive ATP treatments induced a rapid desensitization of I _ATP . Gintonin treatment after P2X₁ receptor desensitization potentiated I _ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I _ATP . Gintoninmediated I _ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X₁ receptor greatly attenuated the gintonin-mediated I _ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X₁ receptor through a phosphoinositide-dependent pathway.     

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

Expression of P2 receptors at sites of chronic inflammation

Extracellular nucleotides have been identified as important signaling molecules. These nucleotides act on the P2 family of receptors that respond by either forming an ion-channel or by activation of a signal transduction cascade, both of which enable a cellular response. Although a role for P2 receptors in inflammation has been implied, the local expression pattern and kinetics of these receptors at sites of inflammation are not known. Therefore, we have studied the expression of the P2 receptors expressed by inflammatory cells or by cells in the vasculature, with special attention to P2X₁R, P2X₇R, P2Y₁R, and P2Y₂R. As a suitable model for studying inflammatory reactions, we have employed the foreign body reaction (FBR), a sterile inflammatory reaction induced by implanting degradable cross-linked dermal sheep collagen disks subcutaneously in the rat. We show that, in the vasculature, the expression of P2X₇R, P2Y₁R, and P2Y₂R increase until day 2. The expression of P2X₇R and P2Y₁R on macrophages and giant cells increased during the course of the inflammatory reaction which was studied for 21 days. The expression of the P2Y₂R on macrophages and giant cells inside the foreign body increases with time, whereas the expression on macrophages in the surrounding tissue is maximal at day 5. The expression of P2X₁R remains at a constant low level. The upregulation of P2X₇R, P2Y₁R, and P2Y₂R over time suggests a regulatory function for these receptors in inflammation.     

Copper inhibits P2Y2-dependent Ca signaling through the effects on thapsigargin-sensitive Ca stores in HTC hepatoma cells

Purinergic P2Y₂ G-protein coupled receptors play a key role in the regulation of hepatic Ca²⁺ signaling by extracellular ATP. The concentration of copper in serum is about 20 μM. Since copper accumulates in the liver in certain disease states, the purpose of these studies was to assess the effects of copper on P2Y₂ receptors in a model liver cell line. Exposure to a P2Y₂ agonist UTP increased [Ca²⁺]_i by stimulating Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores. Pretreatment of HTC cells for several minutes with copper did not affect cell viability, but potently inhibited increases in [Ca²⁺]_i evoked by UTP and thapsigargin. During this pretreatment, copper was not transported into the cytosol, and inhibited P2Y₂ receptors in a concentration-dependent manner with the IC₅₀ of about 15 μM. These results suggest that copper inhibits P2Y₂ receptors through the effects on thapsigargin-sensitive Ca²⁺ stores by acting from an extracellular side. Further experiments indicated that these effect of copper may lead to inhibition of regulatory volume decrease (RVD) evoked by hypotonic solution. Thus, copper may contribute to defective regulation of purinergic signaling and liver cell volume in diseases associated with the increased serum copper concentration.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^−/− mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^−/− mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^?/? mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^?/? mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Expression and function of P2X(7) receptor and CD39/Entpd1 in patients with type 2 diabetes and their association with biochemical parameters

Chronic inflammation is an important contributor to the insulin resistance observed in type 2 diabetes (T2D). We evaluated the expression and function of the P2X₇ receptor and CD39/Entpd1, molecules involved in the cellular regulation of inflammation, in peripheral blood mononuclear cells from T2D patients, and their correlation with the concentration of HbA1c in blood. T2D patients with deficient metabolic control (DC) showed increased proportion of P2X₇⁺ cells compared with healthy individuals; T2D-DC subjects also displayed higher proportion of CD14⁺, CD4⁺ and CD19⁺ subpopulations of P2X₇⁺ cells when compared with T2D patients with acceptable metabolic control. A significant association was observed between the proportion of P2X₇⁺CD14⁺ cells and blood concentration of LDL-c. In addition, the percentages of CD39⁺ cells and CD39⁺CD19⁺ cells were significantly associated with HbA1c and fasting plasma glucose levels. No changes were observed in the function of P2X₇⁺ cells from T2D patients; however, enhanced CD39/Entpd1 enzyme activity and low serum levels of IL-17 were detected. Therefore, CD39⁺ cells could have a balancing regulatory role in the inflammatory process observed in patients with T2D.     

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na+, K+, 2Cl− cotransport-dependent signaling

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog α,β-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X₁ receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y₆ receptors. Inhibition of Na⁺, K⁺, 2Cl⁻ cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y₆-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y₆-mediated signaling should be examined further.     

Enhancement of Glucose Uptake in Mouse Skeletal Muscle Cells and Adipocytes by P2Y6 Receptor Agonists

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y₆ receptor (P2Y₆R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y₆R activation on glucose uptake in insulin target tissues. An agonist of the P2Y₆R, P¹-(5′-uridine)-P³-(5′-N⁴-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [³H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y₆R antagonist N,N″-1,4-butanediyl-bis[N′-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5′-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y₆R activation by either MRS2957 or native agonist UDP, and the P2Y₆R effect was antagonized by MRS2578. However, in adipocytes from P2Y₆R-knockout mice P2Y₆R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y₆R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.     

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X₇ receptor (P2X₇R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X₇R activation induced Ca²⁺-dependent vesicular glutamate release and significant Ca²⁺-independent glutamate efflux through the P2X₇R itself. In the present study, we investigated the effect of the new selective P2X₇R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca²⁺]_i signals and glutamate release, and evaluated whether P2X₇R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X₇R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X₇R-mediated [Ca²⁺]_i signals. These findings provide compelling evidence of functional presynaptic P2X₇R in cortical nerve terminals.