Activation Of A2A Receptors Research Articles

Carbamazepine Attenuates Astroglial L-Glutamate Release Induced by Pro-Inflammatory Cytokines via Chronically Activation of Adenosine A2A Receptor

Carbamazepine (CBZ) binds adenosine receptors, but detailed effects of CBZ on astroglial transmission associated with adenosine receptor still need to be clarified. To clarify adenosinergic action of CBZ on astroglial transmission, primary cultured astrocytes were acutely or chronically treated with CBZ, proinflammatory cytokines (interferon γ (IFNγ) and tumor necrosis factor α (TNFα)), and adenosine A2A receptor (A2AR) agonist (CGS21680). IFNγ and TNFα increased basal, adenophostin-A (AdA)-evoked, and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)-evoked astroglial L-glutamate releases. In physiological condition, CGS21680 increased basal astroglial L-glutamate release but glutamate transporter inhibition prevented this CGS21680 action. CBZ did not affect basal release, whereas glutamate transporter inhibition generated CBZ-induced glutamate release. Furthermore, AdA-evoked and AMPA-evoked releases were inhibited by CBZ but were unaffected by CGS21680. Contrary to physiological condition, chronic administrations of IFNγ and TNFα enhanced basal, AdA-, and AMPA-evoked releases, whereas IFNγ and TNFα decreased and increased CGS21680-evoked releases via modulation A2AR expression. Both chronic administration of CGS21680 and CBZ suppressed astroglial L-glutamate release responses induced by chronic cytokine exposer. Especifically, chronic administration of CBZ and CGS21680 prevented the reduction and elevation of A2AR expression by respective IFNγ and TNFα. These findings suggest that A2AR agonistic effects of CBZ contribute to chronic prevention of pathomechanisms developments of several neuropsychiatric disorders associated with proinflammatory cytokines.

Indirect Medium Spiny Neurons in the Dorsomedial Striatum Regulate Ethanol-Containing Conditioned Reward Seeking.

Adenosine 2A receptor (A2AR)-containing indirect medium spiny neurons (iMSNs) in the dorsomedial striatum (DMS) contribute to reward-seeking behaviors. However, those roles for ethanol-seeking behaviors remain unknown. To investigate ethanol-seeking behaviors, we used an ethanol-containing reward (10% ethanol and 10% sucrose solution; 10E10S). Upon conditioning with 10E10S, mice that initially only preferred 10% sucrose, not 10E10S, showed a stronger preference for 10E10S. Then, we investigated whether the manipulation of the DMS-external globus pallidus (GPe) iMSNs circuit alters the ethanol-containing reward (10E10S) seeking behaviors using the combination of pharmacologic and optogenetic approaches. DMS A2AR activation dampened operant conditioning-induced ethanol-containing reward, whereas A2AR antagonist abolished the effects of the A2AR agonist and restored ethanol-containing reward-seeking. Moreover, pre-ethanol exposure potentiated the A2AR-dependent reward-seeking. Interestingly, mice exhibiting ethanol-containing reward-seeking showed the reduction of the DMS iMSNs activity, suggesting that disinhibiting iMSNs decreases reward-seeking behaviors. In addition, we found that A2AR activation reversed iMSNs neural activity in the DMS. Similarly, optogenetic stimulation of the DMS-GPe iMSNs reduced ethanol-containing reward-seeking, whereas optogenetic inhibition of the DMS-GPe iMSNs reversed this change. Together, our study demonstrates that DMS A2AR and iMSNs regulate ethanol-containing reward-seeking behaviors.SIGNIFICANCE STATEMENT Our findings highlight the mechanisms of how operant conditioning develops the preference of ethanol-containing conditioned reward. Mice exhibiting ethanol-containing reward-seeking showed a reduction of the indirect medium spiny neuronal activity in the dorsomedial striatum. Pharmacological activation of adenosine A2A receptor (A2AR) or optogenetic activation of indirect medium spiny neurons dampened operant conditioned ethanol-containing reward-seeking, whereas inhibiting this neuronal activity restored ethanol-containing reward-seeking. Furthermore, repeated intermittent ethanol exposure potentiated A2AR-dependent reward-seeking. Therefore, our finding suggests that A2AR-containing indirect medium spiny neuronal activation reduces ethanol-containing reward-seeking, which may provide a potential therapeutic target for alcohol use disorder.

Abstract 3278: EOS100850 potently restores adenosine A2Areceptor-dependent suppression of T cell function in the adenosine rich tumor microenvironment

Abstract Adenosine is a potent immunosuppressive metabolite that is often found elevated in the extracellular tumor microenvironment (TME). We determined concentrations of extracellular adenosine in different patient-derived xenografts (PDX) from 7 different histological types, in which extracellular median adenosine concentrations were shown to range from 0.5 to 45 µM, with the overall median adenosine concentration being about 4.5 µM. Adenosine concentration in the non-tumorous subcutaneous space in mice was measured at about 0.5 µM. Adenosine in the TME is generated mainly by the concerted action of the ectonucleotidases CD39 and CD73. The expression of these enzymes across various cancer types was evaluated by flow cytometry in dissociated human tumor biopsies. CD73 and CD39 were strongly expressed by multiple tumor-infiltrating T cell types. Tumor-associated myeloid cells mostly expressed CD39 and high frequencies of EpCAM+ tumor cells strongly expressed CD73. These data strongly suggest that adenosine levels could be further increased compared to non-immune-infiltrated PDX.Adenosine activates 4 G protein-coupled receptor subtypes, of which the adenosine A2A receptor in particular suppresses innate and adaptive immune cell responses leading to suppression of anti-tumor immunity. Among the 4 adenosine receptors, we confirmed the A2A receptor as the main adenosine receptor expressed in CD4+ and CD8+ T cells, natural killer cells, monocytes, and dendritic cells. Stimulation of these immune cell subsets further increased A2A receptor expression. A2B receptor was expressed at very low levels in stimulated CD4+ and CD8+ T cells and in monocytes and immature DCs. A1 and A3 receptors were hardly detected in these subsets of immune cells. EOS100850, a highly potent and selective A2A receptor antagonist, was characterized in various in vitro functional assays. A2A receptor activation by a selective agonist suppressed priming of mouse OVA-specific OT1 T cells and subsequent antigen-specific CD8+ T cell cytotoxicity in a co-culture assay of effector CD8+ T cells and target cancer cells. This A2A receptor-mediated immune suppression was potently and dose-dependently reversed by EOS100850. In a mixed lymphocyte reaction between human dendritic cells and T cells, EOS100850 blocked the adenosine-dependent inhibition of T cell proliferation and secretion of IFNg, TNFa and IL-2 in a dose-dependent manner. In conclusion, extracellular adenosine as well as adenosine pathway components were strongly present across multiple tumor types, and across multiple tumor-associated cell types. In addition, EOS100850, a highly potent A2A receptor antagonist, reversed A2A receptor-mediated suppression of T cell priming, cytotoxicity, cytokine production and proliferation. Citation Format: Erica Houthuys, Paola Basilico, Veronique Bodo, Margreet Brouwer, Michel Detheux, Gregory Driessens, Bruno Gomes, Annelise Hermant, Catherine Hoofd, Florence Lambolez, Xavier Leroy, Reece Marillier, Chiara Martinoli, Marjorie Mercier, Florence Nyawouame, Shruthi Prasad, Ariane Scoumanne, Stefano Crosignani. EOS100850 potently restores adenosine A2Areceptor-dependent suppression of T cell function in the adenosine rich tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3278.

Abstract 4147: EOS100850, an A2Areceptor antagonist with prolonged pharmacodynamic activity, mediates the generation ofspecific durable immune responses in a murine breast cancer model

Abstract We and others have shown that extracellular adenosine (Ado), acting predominantly through the A2A receptor (A2AR), mediates immunosuppression through different modes of action. These include suppression of Th1 responses and cytotoxicity as well as increasing the activity of Tregs and MDSC. We therefore developed EOS100850, a novel, non-brain penetrant and highly selective inhibitor of A2AR with sub-nanomolar Ki. As compared to other A2AR antagonist in development, EOS100850 maintain its potency in high Ado environment (>10uM). To better characterize the Ado levels found in the tumor microenvironment, we first assessed extra-cellular Ado concentrations by microdialysis. Concentrations measured in mouse and human tumors were elevated compared to normal skin and confirmed that immune cells infiltrating the tumor microenviroment are confronted with elevated suppressive Ado levels. To demonstrate and compare the ability of EOS100850 to inhibit A2AR signaling in vivo with other molecules in development, we measured the in vivo pharmacodynamic (PD) modulation of A2AR activity by investigating its ability to inhibit CREB phosphorylation (pCREB) following activation of the Ado receptor by NECA, an A2AR agonist. While EOS100850 demonstrated complete inhibition of pCREB 30 minutes after oral gavage at a dose as low as 0.1 mg/kg, other A2AR inhibitors required much higher concentrations to achieve a similar effect. Remarkably, 24 hours after dosing, more than 60% of inhibition of pCREB was still observed, at a dose of 10 mg/kg when the EOS100850 antagonist was no longer detectable in the plasma. These results demonstrate that EOS100850 has a PD activity that extends well beyond its PK, which can be explained by a long residence time. To demonstrate the in vivo antitumor efficacy of EOS100850, we used the murine syngeneic breast cancer model, EMT-6. EOS100850 administered in combination with anti-CTLA-4 mAb increased the number of complete responders (CR) and significantly reduced tumor growth compared with anti-CTLA-4 single agent treatment. Furthermore, the CR were protected against re-inoculation with EMT6 but not irrelevant CT26 tumor. Lastly, flow cytometry and RNAseq analysis, performed on tumors taken during the course of treatment, confirmed the potential of EOS100850 as single agent or in combination with anti-CTLA4 to induce a pro-inflammatory response that correlate with tumor regression. This best-in-class profile of EOS100850 A2A receptor antagonist supports its progression into clinical development. Citation Format: Romain Pirson, Anne-Catherine Michaux, Diane Jamart, Julie Preillon, Kim Frederix, Paola Basilico, Chiara Martinoli, Xavier Leroy, Gregory Driessens, Erica Houthuys, Scott Chappel, Stefano Crosignani, Reece Marillier. EOS100850, an A2Areceptor antagonist with prolonged pharmacodynamic activity, mediates the generation ofspecific durable immune responses in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4147.

Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events.

The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.

Activation of adenosine A2a receptor accelerates and A2a receptor antagonist reduces intermittent hypoxia induced PC12 cell injury via PKC-KATP pathway

Obstructive sleep apnea hypopnea syndrome (OSAHS) is associated with multiple system diseases. Neurocognitive dysfunction resulting from central nervous system complications has been reported, especially in children with OSAHS. Chronic intermittent hypoxia is accepted to be the major pathophysiological mechanism of OSAHS. Adenosine plays an important role in cellular function via interactions with its receptors. A2a receptor has been recognized as a factor involved in neuroprotection. However, the role of adenosine A2a receptor in intermittent hypoxia induced cellular injury is not completely understood. In this study, we aim to investigate the underlying mechanisms of A2a receptor mediated cellular damage caused by intermittent hypoxia in PC12 cells. We found that activated A2a receptor by CGS21680 decreased cellular viability, increased PKC as well as ATP-sensitive potassium channel (KATP) subunits expression Kir6.2 and SUR1. Inhibition of A2a receptor by SCH58261 increased cellular viability, suppressed PKC and SUR1 expression level, ultimately showing a protective role in PC12 cells. Moreover, we observed that CHE, which is an antagonist of PKC, downregulated Kir6.2 and SUR1 expression and increased cellular viability. Additionally, we found that A2a receptor activation induced cell injury was associated with increased Cleaved-Caspase 3 expression, which can be decreased by inhibition of A2a receptor or PKC. In conclusion, our findings indicate that A2a receptor induced KATP expression by PKC activation and plays a role in accelerating PC12 cells injury induced by intermittent hypoxia exposure via A2a-PKC-KATP signal pathway mediated apoptosis.

Adenosine A2areceptor agonist CGS21680 treatment attenuates cardiopulmonary bypass‑associated inflammatory lung injury in juvenile rats.

The adenosineA2areceptor agonist CGS21680 has been suggested to act as an anti‑inflammatory agent that protects against cardiopulmonary bypass (CPB)‑induced organ injury. However, the therapeutic effects of CGS21680 for CPB‑induced lung injury have not been comprehensively evaluated. Using a juvenile rat model, the present study was designed to evaluated whether CGS21680 attenuates CPB‑induced lung injury. Our juvenile rat CPB model was established by 60min CPB with or without CGS21680 pretreatment (100µg/kg, in the CPB priming solution). Rats in the Sham group only underwent cannulation and heparinization. Serum and pulmonary levels of inflammatory markers and histological features of pulmonary tissues were analyzed. All juvenile rats survived following CPB. Significantly elevated serum levels of tumor necrosis factor‑α (TNF‑α), myeloperoxidase(MPO) and interleukin‑1β(IL‑1β), and decreased glutathione peroxidase (GSH‑PX) levels were observed in the CPB group compared to the Sham group (all P<0.05). TNF‑α, MPO and IL‑1β were significantly decreased, while GSH‑PX was markedly increased in the CGSgroup when compared to the CPBgroup. Consistently, pulmonary tissues from rats in the CPBgroup showed considerable amounts of damaged pneumocytes, severe edema, and increased alveolar macrophages, and significantly higher lung injury scores compared to the controls. Collectively, these changes were all further attenuated by CGS21680. Pretreatment with CGS21680 before CPB attenuated pulmonary injury, which may be related to the anti‑inflammatory effects of CGS21680 downstream of A2areceptor activation.

Adenosine A2a receptor promotes lymphangiogenesis and lymph node metastasis

ABSTRACT The formation of new lymphatic vessels, or lymphangiogenesis, is a critical step of the tissue repair program. In pathological conditions involving chronic inflammation or tumorigenesis, this process is often dysregulated and can contribute to disease progression. Yet, lymphangiogenesis is still incompletely understood. In this study, we identified A2a adenosinergic signaling as an important regulator of inflammatory and tumor-associated lymphangiogenesis. Using Adora2a (A2a)-deficient mice, we demonstrated that A2a signaling was involved in the formation of new lymphatic vessels in the context of peritoneal inflammation. We also demonstrated that tumor-associated and sentinel lymph node lymphangiogenesis were impaired in A2a-deficient mice, protecting them from lymph node metastasis. Notably, A2a signaling in both hematopoietic and non-hematopoietic cells contributed to sentinel lymph node metastasis. In A2a-deficient tumor-draining lymph nodes, impaired lymphangiogenesis was associated with a reduced accumulation of B cells and decreased VEGF-C levels. Supporting a role for non-hematopoietic A2a signaling, we observed that primary murine lymphatic endothelial cells (LEC) predominantly expressed A2a receptor and that A2a signaling blockade altered LEC capillary tube formation in vitro. Finally, we observed that Adora2a, Nt5e and Entpd1 gene expression positively correlated with Lyve1, Pdpn and Vegfc in several human cancers, thereby supporting the notion that adenosine production and A2a receptor activation might promote lymphangiogenesis in human tumors. In conclusion, our study highlights a novel pathway regulating lymphangiogenesis and further supports the use of A2a or adenosine blocking agents to inhibit pathological lymphangiogenesis in cancers and block the dissemination of tumor cells through the lymphatic system.

Differential Effects of Limonene on Inflammation via Activation of A2A and A2B Adenosine Receptors in Asthma

Limonene a mono-terpene flavonoid that is anti-inflammatory in asthma. We have shown that limonene induces bronchodilation and lowers inflammation via A2A receptor activation (FASEB J April 2017 31:820.1) in a mouse model of asthma. However, our data showed that limonene's effects on lowering neutrophils was occurring through a different pathway, as limonene reduced neutrophils in the absence of the A2A receptor. We hypothesized that limonene may also activate A2B receptors based on its similar binding interactions with both A2AAR and A2BAR using a structural homology model. We investigated the effects of limonene on lung inflammation and airway reactivity in A2AAR knock-out (A2AKO) and wild-type (WT) mice. Mice were divided into control (CON) and allergen sensitized-challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13 (n=6 mice per group). Limonene was administered as an inhalation prior to allergen challenges in one group of allergic mice for both WT and KO (SEN+LIM). The role of A2B receptors was studied by treating SEN A2AKO mice with BAY 606583 (selective A2B agonist; 150mg/kg i.p.). Differential BAL cell analysis showed that limonene reduced levels of eosinophils in WT SEN (70.66±2.6% in SEN vs. 28.66±1.45% in SEN+LIM, p<0.05) while no difference was observed in A2AKO (76.33±1.35% in SEN vs 79±0.69% in SEN+LIM). However, neutrophils were significantly lowered in A2AKO (from 19±1.02 in SEN A2AKO to 10±0.4 in SEN+LIM) in BAL. SEN A2AKO treated with BAY 606583 showed similar reduction in the number of neutrophils (10±2.1%) compared to A2AKO SEN treated with limonene (10±0.4). No significant difference was seen in other inflammatory cells sensitized A2AKO mice treated with BAY606583. Tracheal ring reactivity was also studied using isolated organ bath system. Contraction to methacholine (MCh; 0.1μM) in isolated trachea was significantly higher in SEN tissues (225.99±67.34% vs 5.57±19.74% in CON). Treatment with limonene significantly reduced the tracheal reactivity (80.68±20% in SEN+LIM vs SEN; p<0.05). In A2AKO tissues, limonene did not have any effect to reduce MCh reactivity. SCH58651(A2A receptor antagonist) blunted the effects of limonene on tracheal reactivity. CGS-21680 (A2A receptor agonist) had very little effect on tracheal relaxation in SEN WT tissue while SEN+LIM trachea had relaxation to CGS 21680. These data suggest that limonene reduces tracheal reactivity to MCh and eosinophilia via an A2AAR-dependent pathway, while reduction in airway neutrophilia occurs vai an A2BAR-dependent pathway. Support or Funding Information Institutional Grant, Long Island University D-limonene binds to A2A adenosine receptor (A2AAR) and decreases the eosinophilia and airway hyper reactivity induced by methacholine. In the absence of A2AAR (A2AKO), d-limonene can bind to A2BAR and decreases airway neutrophil count in bronchoalveolar lavage This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurochemical Plasticity of Phrenic Motor Neuron Adenosine 2A Receptors: Effects of Cervical Spinal Injury and Intermittent Hypoxia

Cervical spinal adenosine 2A receptor activation is necessary for certain forms of spinal respiratory motor plasticity, such as phrenic long‐term facilitation elicited by severe acute intermittent hypoxia (AIH; Nichols et al., 2012) or moderate AIH with acute cervical spinal injury (cSCI; Navarette‐Opazo et al., 2016). Whereas cervical spinal A2A receptor activation is sufficient to elicit phrenic motor facilitation (pMF; Golder et al., 2008), it undermines other, serotonin‐dependent forms of pMF via cross‐talk inhibition (Hoffman et al., 2010; Navarette‐Opazo et al., 2017). Thus, A2A receptors exert complex effects on phrenic motor plasticity. However, it is not known if conditions such as cSCI and/or repetitive exposure to intermittent hypoxia elicit neurochemical plasticity in A2A receptor expression (and associated phrenic motor plasticity). Thus, we characterized A2A receptor expression in phrenic motor neurons after chronic cSCI and prolonged exposure to different protocols of intermittent hypoxia. A2A receptor expression was determined in male Sprague Dawley rats with and without C2 spinal hemisection (C2Hx; 12 wks post‐injury) that were exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min 10.5% O2 episodes with 5‐min intervals per day; 1.5 hrs/day); 3) moderate CIH (CIH 5/5: 5‐min 10.5% O2 episodes, 5‐min intervals; 8 hrs/day); and 4) high dose CIH (CIH 2/2: 2‐min 10.5% O2 episodes, 2 min intervals; 8 hrs/day). Rats were then perfused, and cervical spinal cords sectioned (40μm). C3 to C5 sections were processed for A2A receptor and Cholera toxin B subunit (CtB, injected 14 days before C2Hx). Using a custom MATLAB algorithm, optical density in CtB‐positive phrenic motor neurons was quantified. Statistical analysis was performed using MANOVA (SAS JMP). C2Hx decreased A2A receptor expression in phrenic motor neurons (p&lt;0.05). Intermittent hypoxia exerted protocol‐specific effects on A2A receptor expression (CIH 2/2 &gt; CIH 5/5 &gt; daily AIH; p=0.014). Although intermittent hypoxia appeared to increase A2A receptor expression more after C2Hx, this effect was only marginally significant (injury/treatment interaction; p=0.061). The dual effects of injury and intermittent hypoxia on A2A receptor expression suggest that adenosine receptor plasticity may be important in the development of new therapeutic strategies to improve breathing after spinal cord injury.Support or Funding InformationSupported by: NIH T32 HD043730 (LLA) OT2OD023854 (SPARC), NIH K12 HD055929 (EGR), McKnight Brain Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts.

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A2A receptors (A2ARs) and Wingless/Integrated-β-catenin pathways. Cytosolic β-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular β-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) β-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular β-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 μM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 β-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular β-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, β-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts.

Ischemia postconditioning protects dermal microvascular endothelial cells of rabbit epigastric skin flaps against apoptosis via adenosine A2a receptors

Background: It has been shown that endogenous adenosine-induced by ischemia postconditioning attenuates apoptosis in recent studies; however, they focus only on parenchymal cells. The detailed mechanism has not been clearly clarified in any research and the subtype of adenosine receptors involved remains unknown. In our study, dermal microvascular endothelial cells (DMECs) are used to explore the role of adenosine A2a receptor in the anti-apoptotic effects of ischemic postconditioning.Material and methods: The epigastric skin flaps of rabbits were elevated. After 4 h of ischemia, the flaps were either abruptly reperfused or postconditioned by six cycles of brief reperfusion (15s) and re-ischemia (15s). Adenosine A2a receptor agonist (CGS-21680) and antagonist (ZM-241385) were used separately in other groups. The apoptosis-related proteins and adenosine A2a receptors were determined by immunohistochemical staining. Then apoptosis index was calculated by TUNEL.Results: Ischemia/reperfusion caused severe damages in DMECs of flaps as demonstrated by an increase in apoptosis index and an increase in expressions of apoptosis-related proteins, which can be significantly attenuated by IPC treatment or exposure to a selective adenosine A2a receptor agonist (all p values <.05). Meanwhile, the anti-apoptosis effects of IPC can be blocked by a selective adenosine A2a receptor antagonist. Statistical analysis revealed that the increase of apoptosis index closely correlated inversely with the relative increase of adenosine A2a receptors (p < .0001).Conclusions: Ischemia postconditioning protects DMECs of rabbit skin flap against apoptosis via activation of adenosine A2a receptors.

Extracellular adenosine and slow-wave sleep are increased after ablation of nucleus accumbens core astrocytes and neurons in mice

Sleep and wakefulness are controlled by a wide range of neuronal populations in the mammalian brain. Activation of adenosine A2A receptor (A2AR)-expressing neurons in the nucleus accumbens (NAc) core promotes slow-wave sleep (SWS). The neuronal mechanism by which activation of NAc A2AR neurons induces SWS, however, is unknown. We hypothesized that the ability of NAc activation to induce sleep is mediated by the classic somnogen adenosine, which can be formed by various processes in all types of cells. Here, to investigate whether astrocytes are involved in the ability of the NAc to regulate SWS, we ablated glial fibrillary acidic protein (GFAP)-positive cells in the NAc core of mice by virus-mediated expression of diphtheria toxin (DT) receptors and intraperitoneal administration of DT. Analysis of electroencephalogram and electromyogram recordings of DT-treated wild-type mice revealed that SWS was remarkably increased at 1 week after DT treatment, whereas sleep-wake behavior was unchanged in DT-treated A2AR knockout mice. Cell ablation was associated with an increased number of GFAP-positive cells and activation of microglia in the NAc. In-vivo microdialysis revealed significantly increased levels of extracellular adenosine in the NAc at 1 week after DT treatment. Our findings suggest that elevated adenosine levels in the NAc core promote SWS by acting on A2ARs and provide the first evidence that adenosine is an endogenous candidate for activating NAc A2AR neurons that have the ability to induce SWS.

CAMP-producing chemogenetic and adenosine A2a receptor activation inhibits the inwardly rectifying potassium current in striatal projection neurons

Adenosine A2a receptors (A2aRs) are highly and selectively expressed in D2-medium spiny neurons (D2-MSNs) that also express a high level of dopamine D2 receptors (D2Rs). However, it was not established how A2aR activity affects D2-MSN excitability, let alone the ion channels involved. We have performed two sets of experiments to determine the potential A2aR agonistic effects on D2-MSN intrinsic excitability and the underlying ion channel mechanism. First, we have used the cAMP-producing, Gαs/olf coupled designer receptors exclusively activated by designer drug (Gs-DREADDs) to phenocopy cAMP-stimulating A2aR activation. We found that activation of Gs-DREADD inhibited the inwardly rectifying potassium current (Kir)–a key regulator of MSN excitability, caused a depolarization, increased input resistance, and substantially increased the intrinsic excitability of MSNs such that depolarizing inputs evoked many more action potentials. Second, we have determined that A2aR agonism produced these same excitatory effects on D2-MSN intrinsic excitability and spike firing, although at lower magnitudes than those induced by Gs-DREADD activation; furthermore, these A2aR-triggered excitatory effects were intact in the presence of a D2R antagonist. Taken together, these results clearly establish that in striatal D2-MSNs, A2aR activation can independently inhibit Kir and increase intrinsic excitability and spike and neurotransmitter output; our results also indicate that Gs-DREADD can serve as a broadly useful positive control for neurotransmitter receptors that increase intracellular cAMP levels and hence facilitate the determination of the cellular effects of these neurotransmitter receptors.

PDRN, a Biologic Agonist of the Adenosine A &lt;sub&gt;2A&lt;/sub&gt; Receptor, Ameliorates Imiquimod-Induced Psoriasis Through NF-κB Pathway Inhibition and Wnt/β-Catenin Signalling Modulation

Background: Inflammation is a key hallmark of psoriasis and pro-inflammatory cytokines are produced by the transcription factor Nuclear factor-κB (NF-κB). Canonical Wnt/β-catenin pathway blunts the immune-mediated inflammatory cascade in psoriasis. Adenosine A2A receptor activation either blocks NF-κB and boosts the Wnt/β-catenin signalling. PDRN (Polydeoxyribonucleotide) is a biologic agonist of the A2A receptor and its effects were studied in an experimental model of psoriasis. Methods: Psoriasis-like lesions were induced by a topical daily application of imiquimod cream (IMQ; 62.5 mg/day for 7 days) on the shaved back skin of mice. Animals were randomly assigned to the following groups: Sham psoriasis challenged with vaseline cream; IMQ animals challenged with imiquimod and IMQ animals challenged with IMQ and treated with PDRN (8 mg/kg/ip). In histological experiments an additional arm of IMQ animals was treated with PDRN plus istradefylline (KW6002; 25 mg/kg/ip) as an A2A antagonist. Findings: PDRN administration reduced squamous lesions, erythema, epidermal thickness and acanthosis, trying to restore a normal skin architecture. Istradefylline administration abrogated PDRN positive effects, thus pointing out the mechanistic role of the A2A receptor. PDRN decreased pNF-κB and TNF-α IL-6 and IL-12 expression compared to untreated psoriatic animals. Furthermore, A2Areceptor activation prompted Wnt signalling, reduced IL-2 expression and increased IL-10 mRNA expression in psoriatic skin. Finally, PDRN reduced CD3+ cells in superficial psoriatic dermis. Interpretations: PDRN anti-psoriasis potential may be linked to a dual mode of action: NF-κB pathway inhibition and Wnt/β-catenin signalling stimulation. Funding: The work has been performed with Departmental funding assigned to FS. Competing Interest Declaration: The authors declare that the research was conducted in the absence of any commercial or financial conflict of interest. Ethics: The procedures were evaluated and approved by the Ethics Committee of the University of Messina (OPBA, #820/2016).

Anti-aggregation effect on platelets of Indiplon a hypnotic sedative non-benzodiazepine drug

Cardiovascular diseases are one of the main public health problems, and many of them, their pathophysiology involves alterations in platelet activity. Platelet activation is an essential event that is regulated by the intracellular concentrations of Ca2+ and cAMP. Interestingly, it has been shown that the activation of adenosine A2A receptors increases cAMP levels and produces the inhibition of platelet aggregation, which appears as a potential target for regulation of platelet activity. Therefore, we tried to activate A2A receptors using Indiplon, a drug developed for the treatment of insomnia, and analyze its effect on platelet activity in vitro. Our results indicate that Indiplon is able to interact in silico with the adenosine A2A receptor (ΔGbind of -73.321 kcal/mol, similar to that obtained with adenosine), which is involved in the regulation of platelet cAMP levels. In functional studies using PRP, a reduction in platelet aggregation induced by ADP was observed in the presence of Indiplon at 500 μM with a percentage of inhibition 70%, where the use of specific inhibitors (ZM241385 and MSX-2) of the A2A receptor also blocked these effects reducing the percentage of inhibition to 41% and 34.1%, respectively. Also, the use of Indiplon produced a decrease in the expression in the membrane of P-selectin. Thus, Indiplon acts as an A2A receptor agonist and whose activation results in inhibition of platelet aggregation and activation, showing a possible cardiovascular protective role.

Resveratrol-mediated reversal of changes in purinergic signaling and immune response induced by Toxoplasma gondii infection of neural progenitor cells.

The effects of Toxoplasma gondii during embryonic development have not been explored despite the predilection of this parasite for neurons and glial cells. Here, we investigated the activation of the purinergic system and proinflammatory responses during congenital infection by T. gondii. Moreover, neuroprotective and neuromodulatory properties of resveratrol (RSV), a polyphenolic natural compound, were studied in infected neuronal progenitor cells (NPCs). For this study, NPCs were isolated from the telencephalon of infected mouse embryos and subjected to neurosphere culture in the presence of EGF and FGF2. ATP hydrolysis and adenosine deamination by adenosine deaminase activity were altered in conditions of T. gondii infection. P2X7 and adenosine A2A receptor expression rates were augmented in infected NPCs together with an increase of proinflammatory (INF-γ and TNF-α) and anti-inflammatory (IL-10) cytokine gene expression. Our results confirm that RSV counteracted T. gondii-promoted effects on enzymes hydrolyzing extracellular nucleotides and nucleosides and also upregulated P2X7 and A2A receptor expression and activity, modulating INF-γ, TNF-α, and IL-10 cytokine production, which plays an integral role in the immune response against T. gondii.

Effects of intra-accumbal or intra-prefrontal cortex microinjections of adenosine 2A receptor ligands on responses to cocaine reward and seeking in rats

Rationale and objectivesMany studies indicated that adenosine via its A2A receptors influences the behavioral effects of cocaine by modulating dopamine neurotransmission. The hypothesis was tested that A2A receptors in the nucleus accumbens (NAc) or the prefrontral cortex (PFc) may modulate cocaine reward and/or cocaine seeking behavior in rats.MethodsThe effects of local bilateral microinjections of the selective A2A receptor agonist CGS 21680 or the A2A receptor antagonists KW 6002 and SCH 58261 were investigated on cocaine self-administration on reinstatement of cocaine seeking.ResultsThe intra-NAc shell, but not intra-infralimbic PFc, administration of CGS 21680 significantly reduced the number of active lever presses and the number of cocaine (0.25 mg/kg) infusions. However, tonic activation of A2A receptors located in the NAc or PFc did not play a role in modulating the rewarding actions of cocaine since neither KW 6002 nor SCH 58261 microinjections altered the cocaine (0.5 mg/kg) infusions. The intra-NAc but not intra-PFc microinjections of CGS 21680 dose- dependently attenuated the reinstatement of active lever presses induced by cocaine (10 mg/kg, i.p.) and the drug-associated combined conditioned stimuli using the subthreshold dose of cocaine (2.5 mg/kg, i.p.). On the other hand, the intra-NAc pretreatment with SCH 58261, but not with KW 6002, given alone evoked reinstatement of cocaine seeking behavior.ConclusionThe results strongly support the involvement of accumbal shell A2A receptors as a target, the activation of which exerts an inhibitory control over cocaine reward and cocaine seeking.

Adenosine deaminase inhibition suppresses progression of 4T1 murine breast cancer by adenosine receptor-dependent mechanisms.

The activity of a cell‐surface ecto‐adenosine deaminase (eADA) is markedly increased in the endothelial activation and vascular inflammation leading to decreased adenosine concentration and alterations in adenosine signalling. Depending on the specific pathway activated, extracellular purines mediate host cell response or regulate growth and cytotoxicity on tumour cells. The aim of this study was to test the effects of adenosine deaminase inhibition by 2′deoxycoformycin (dCF) on the breast cancer development. dCF treatment decreased a tumour growth and a final tumour mass in female BALB/c mice injected orthotopically with 4T1 cancer cells. dCF also counteracted cancer‐induced endothelial dysfunction in orthotopic and intravenous 4T1 mouse breast cancer models. In turn, this low dCF dose had a minor effect on immune stimulation exerted by 4T1 cell implantation. In vitro studies revealed that dCF suppressed migration and invasion of 4T1 cells via A2a and A3 adenosine receptor activation as well as 4T1 cell adhesion and transmigration through the endothelial cell layer via A2a receptor stimulation. Similar effects of dCF were observed in human breast cancer cells. Moreover, dCF improved a barrier function of endothelial cells decreasing its permeability. This study highlights beneficial effects of adenosine deaminase inhibition on breast cancer development. The inhibition of adenosine deaminase activity by dCF reduced tumour size that was closely related to the decreased aggressiveness of tumour cells by adenosine receptor‐dependent mechanisms and endothelial protection.

Abstract 251: The Role of Adenosine Derived From Mesenchymal Stromal Cell on Neutrophil Extracellular Trap Formation in Myocardial Ischemia Reperfusion

Introduction: In response to tissue damage or infection, neutrophils expel their chromatin into the extracellular space in a thrombotic and inflammatory process called neutrophil extracellular traps(NETs). There is increasing evidence that NETs play a role in myocardial ischemia/reperfusion (MI/R) injury. NETs are found in thrombi aspirated from coronary arteries, atherosclerotic plaques, and deep vein thrombosis. However, nothing is known about the regulation of NETs in vivo. We hypothesized that adenosine, well know for anti-inflammatory effects, reduces NET formation. After MI/R, adenosine is made from conversion of ATP released from damaged cells. Methods: Neutrophils were isolated from human venous blood and stimulated to produce NETs by PMA, a PKC agonist and is well known to induce robust NET formation and serves as a positive control. NETs were quantified by Sytox green fluorescent to quantify extracellular DNA and immunohistochemical staining for citrullinated histone H3, a specific marker of NETs. Results: Neutrophils treated with PMA increased NET production (1.81fold vs unstimulate groups, p&lt;0.001, n=27-28). Neutrophils treated with adenosine (10uM) had a 30.2% decrease NET formation (p&lt;0.001, n=15). There are four adenosine receptors called A1, A2a, A2b, and A3. We used agonists and antagonists’ strategy to check probable receptors involved in suppression NETs signal. We find both A2a receptor agonist-CGS 21680 and A2b agonist-BAY 60-6583 can suppress NETs formation (29.6% and 29.3% respectively p&lt;0.001, n=5-6). But only A2a antagonist-ZM241385 can reverse adenosine’s inhibition (13.8%v.s.24.7%, p&lt;0.05, n=6). Conclusion: Adenosine can downregulate NET formation in vitro likely through A2a receptor activation. Future studies will investigate A2a receptor using genetic knock out, as well as in vivo significance of NET inhibition in MI/R.