Adenosine A2A Receptor Agonist CGS21680 Research Articles

An agonist of the adenosine A2A receptor, CGS21680, promotes corneal epithelial wound healing via the YAP signalling pathway.

The adenosine A2A receptor (A2AR) is involved in various physiological and pathological processes in the eye; however, the role of the A2AR signalling in corneal epithelial wound healing is not known. Here, the expression, therapeutic effects and signalling mechanism of A2AR in corneal epithelial wound healing were investigated using the A2AR agonist CGS21680. A2AR localization and expression during wound healing in the murine cornea were determined by immunofluorescence staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The effect of CGS21680 on corneal epithelial wound healing in the lesioned corneal and cultured human corneal epithelial cells (hCECs) by modulating cellular proliferation and migration was critically evaluated. The role of Hippo-YAP signalling in mediating the CGS21680 effect on wound healing by pharmacological inhibition of YAP signalling was explored. A2AR expression was up-regulated after corneal epithelial injury. Topical administration of CGS21680 dose-dependently promoted corneal epithelial wound healing in the injured corneal epithelium by promoting cellular proliferation. Furthermore, CGS21680 accelerated the cellular proliferation and migration of hCECs in vitro. A2AR activation promoted early up-regulation and later down-regulation of YAP signalling molecules, and pharmacological inhibition of YAP signalling reverted CGS21680-mediated wound healing effect in vivo and in vitro. A2AR activation promotes wound healing by enhancing cellular proliferation and migration through the YAP signalling pathway. A2ARs play an important role in the maintenance of corneal epithelium integrity and may represent a novel therapeutic target for facilitating corneal epithelial wound healing.

Functional Interaction between Adenosine A2A and mGlu5 Receptors Mediates STEP Phosphatase Activation and Promotes STEP/mGlu5R Binding in Mouse Hippocampus and Neuroblastoma Cell Line.

(1) Background: Recently, we found that adenosine A2A receptor (A2AR) stimulation results in an increase in STEP phosphatase activity. In order to delve into the mechanism through which A2AR stimulation induced STEP activation, we investigated the involvement of mGlu5R since it is well documented that A2AR and mGlu5R physically and functionally interact in several brain areas. (2) Methods: In a neuroblastoma cell line (SH-SY5Y) and in mouse hippocampal slices, we evaluated the enzymatic activity of STEP by using a para-nitrophenyl phosphate colorimetric assay. A co-immunoprecipitation assay and a Western blot analysis were used to evaluate STEP/mGlu5R binding. (3) Results: We found that the A2AR-dependent activation of STEP was mediated by the mGlu5R. Indeed, the A2AR agonist CGS 21680 significantly increased STEP activity, and this effect was prevented not only by the A2AR antagonist ZM 241385, as expected, but also by the mGlu5R antagonist MPEP. In addition, we found that mGlu5R agonist DHPG-induced STEP activation was reversed not only by the mGlu5R antagonist MPEP but also by ZM 241385. Finally, via co-immunoprecipitation experiments, we found that mGlu5R and STEP physically interact when both receptors are activated (4) Conclusions: These results demonstrated a close functional interaction between mGlu5 and A2A receptors in the modulation of STEP activity.

Adenosine A2A Receptor Regulates microRNA-181b Expression in Aorta: Therapeutic Implications for Large-Artery Stiffness.

Background The identification of large-artery stiffness as a major, independent risk factor for cardiovascular disease-associated morbidity and death has focused attention on identifying therapeutic strategies to combat this disorder. Genetic manipulations that delete or inactivate the translin/trax microRNA-degrading enzyme confer protection against aortic stiffness induced by chronic ingestion of high-salt water (4%NaCl in drinking water for 3 weeks) or associated with aging. Therefore, there is heightened interest in identifying interventions capable of inhibiting translin/trax RNase activity, as these may have therapeutic efficacy in large-artery stiffness. Methods and Results Activation of neuronal adenosine A2A receptors (A2ARs) triggers dissociation of trax from its C-terminus. As A2ARs are expressed by vascular smooth muscle cells (VSMCs), we investigated whether stimulation of A2AR on vascular smooth muscle cells promotes the association of translin with trax and, thereby increases translin/trax complex activity. We found that treatment of A7r5 cells with the A2AR agonist CGS21680 leads to increased association of trax with translin. Furthermore, this treatment decreases levels of pre-microRNA-181b, a target of translin/trax, and those of its downstream product, mature microRNA-181b. To check whether A2AR activation might contribute to high-salt water-induced aortic stiffening, we assessed the impact of daily treatment with the selective A2AR antagonist SCH58261 in this paradigm. We found that this treatment blocked aortic stiffening induced by high-salt water. Further, we confirmed that the age-associated decline in aortic pre-microRNA-181b/microRNA-181b levels observed in mice also occurs in humans. Conclusions These findings suggest that further studies are warranted to evaluate whether blockade of A2ARs may have therapeutic potential in treating large-artery stiffness.

Knockdown of adenosine A2A receptors in hippocampal neurons prevents post-TBI fear memory retrieval

The formation of fear memory is crucial in emotional disorders such as PTSD and anxiety. Traumatic brain injury (TBI) can cause emotional disorders with dysregulated fear memory formation; however, their cross-interaction remains unclear and hurdled the treatment against TBI-related emotional disorders. While adenosine A2A receptor(A2AR) contributes to the physiological regulation of fear memory, this study aimed to evaluate the A2AR role and possible mechanisms in post-TBI fear memory formation using a craniocerebral trauma model, genetically modified A2AR mutant mice, and pharmacological A2AR agonist CGS21680 and antagonist ZM241385. Our finding showed (i) TBI enhanced mice freezing levels (fear memory) at seven days post-TBI; (ii) The A2AR agonist CGS21680 enhanced the post-TBI freezing levels; conversely, the A2AR antagonist ZM241385 reduced mice freezing level; further (iii) Genetic knockdown of neuronal A2AR in the hippocampal CA1, CA3, and DG regions reduced post-TBI freezing levels, while A2AR knockout in DG region yielded the most reduction in fear memory; finally, (iv) AAV-CaMKII-Cre virus-mediated DG deletion of A2AR on excitatory neurons led to a significant decreased freezing levels post-TBI. These findings indicate that brain trauma increases fear memory retrieval post-TBI, and A2AR on DG excitatory neurons plays a crucial role in this process. Importantly, inhibition of A2AR attenuates fear memory enhancement, which provides a new strategy to prevent fear memory formation/enhancement after TBI.

PDE1 inhibition and adenosine A2A receptor activation synergistically potentiates spontaneous calcium waves and transients in mouse ventricular myocytes

Abstract Background The phosphodiesterase-1C (PDE1C) and the adenosine A2A receptor (A2AR) are located in a macromolecular cluster and expected to modulate cAMP-dependent regulation of the intracellular calcium homeostasis in cardiac myocytes. In this contect, atrial fibrillation has been associated with a high incidence of spontanoeus arrhythmogenic calcium waves caused by excessive A2AR activation. On the other hand, inhibition of PDE1C and A2AR activation has also been reported to produce a synergistic and protective effect on cardiomyocyte survival, but little is known about the corresponding effects on the intracellular calcium homeostasis. Purpose This study, therfore, aimed to investigate how PDE1 inhibition and/or A2AR activation modulate sarcoplasmic reticulum (SR) calcium homeostasis in mouse ventricular myocytes. Methods Mouse ventricular myocytes (47 cells from 20 mice) were loaded with the calcium indicator Rhod-2 and spontaneous calcium release events at rest or caffeine induced calcium transients were visualized using resonance-scanning confocal microscopy at a frame rate of 150 Hz. The SR calcium content was estimated as the ratio of the peak fluorescence and the fluoresence the end of a 10 mM caffeine pulse (20s). Calcium leak was estimated as ratio of the flourescence emmision at baseline before and at the end of the caffeine pulse. Results The incidence of spontaneous calcium waves or transients increased from 0.7±0.3 events/min in control to 1.1±0.5 events/min and 3.4±1.0 events/min (p<0.01) after inhibition of PDE1 with the selective antagonist ITI-214 and the A2AR agonist CGS-21680 respectively. Concomitant PDE1 inhibition and A2AR activation synergistically increased the event frequency to 8.4±2.3 events/min (p<0.05). There were no significant differences in the corresponding SR calcium contents (4.4±0.3, 4.5±0.4, 4.7±0.4 and 4.1±0.2 for control, ITI-214, CGS-21680 and ITI-214+CGS-21680 respectively). However, there was a parallel increase in calcium leak from 8.7±4.2% in control to 21.8±9.8% with ITI-214, 17.6±7.2% with CGS-21680 and 37.5±11.1% when myocytes were exposed to both compounds (p<0.05), suggesting that the higher incidence of spontaneous calcium waves and transients observed upon PDE1 inhibition and/or A2AR activation is likely caused by a lowering of the threshold for spontaneous SR calcium release rather than by SR calcium overloading. Conclusion PDE1 inhibition exacerbates the incidence of spontaneous arrhythmogenic SR calcium release events in myocytes with high levels of A2AR activation, suggesting that PDE1 inhibition under these conditions increases the risk of arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation

The role of dorsomedial striatum adenosine 2A receptors in the loss of goal-directed behaviour.

Adenosine A2A receptors (A2AR) in the dorsal striatum have been implicated in goal-directed behaviour. While activation of these receptors with several methods has resulted in an insensitivity to outcome devaluation, particular explanations for how they disrupt behaviour have not been explored. We both confirm a role for A2A receptors in goal-directed responding and evaluate additional behavioural aspects of goal-directed control to more fully understand the role of A2A receptors in instrumental behaviour. To examine the effects of the adenosine A2A agonist CGS-21680 in the DMS on response-outcome encoding, updating representations of outcome value and on the ability to inhibit behaviour when reward is not available. Male rats were trained to lever press for food reward. The A2AR agonist CGS-21680 was infused into the dorsomedial striatum either before an outcome devaluation test, prior to training with two distinct response-outcome associations or prior to a test of discriminative stimulus control over instrumental performance. Intra-DMS administration of CGS-21680 impaired sensitivity to outcome devaluation. CGS-21680 treatment did not impair acquisition of specific response-outcome associations, selective control of responding based on the presence of stimuli that signaled when reward was or was not available, discrimination between stimuli or lever choices nor did it influence the effect of devaluation on the amounts of food eaten in a consumption test. CGS-21680 impairs the ability to modulate responding based on recent changes to outcome value, an effect that is not accounted for by impairments in behavioural inhibition, discrimination, encoding the specific outcome of a response or the effectiveness of specific satiety.

Choroid plexus-selective inactivation of adenosine A2A receptors protects against T cell infiltration and experimental autoimmune encephalomyelitis

BackgroundMultiple sclerosis (MS) is one of the most common autoimmune disorders characterized by the infiltration of immune cells into the brain and demyelination. The unwanted immunosuppressive side effect of therapeutically successful natalizumab led us to focus on the choroid plexus (CP), a key site for the first wave of immune cell infiltration in experimental autoimmune encephalomyelitis (EAE), for the control of immune cells trafficking. Adenosine A2A receptor (A2AR) is emerging as a potential pharmacological target to control EAE pathogenesis. However, the cellular basis for the A2AR-mediated protection remains undetermined.MethodsIn the EAE model, we assessed A2AR expression and leukocyte trafficking determinants in the CP by immunohistochemistry and qPCR analyses. We determined the effect of the A2AR antagonist KW6002 treatment at days 8–12 or 8–14 post-immunization on T cell infiltration across the CP and EAE pathology. We determined the critical role of the CP-A2AR on T cell infiltration and EAE pathology by focal knock-down of CP-A2AR via intracerebroventricular injection of CRE-TAT recombinase into the A2ARflox/flox mice. In the cultured CP epithelium, we also evaluated the effect of overexpression of A2ARs or the A2AR agonist CGS21680 treatment on the CP permeability and lymphocytes migration.ResultsWe found the specific upregulation of A2AR in the CP associated with enhanced CP gateway activity peaked at day 12 post-immunization in EAE mice. Furthermore, the KW6002 treatment at days 8–12 or 8–14 post-immunization reduced T cell trafficking across the CP and attenuated EAE pathology. Importantly, focal CP-A2AR knock-down attenuated the pathogenic infiltration of Th17+ cells across the CP via inhibiting the CCR6–CCL20 axis through NFκB/STAT3 pathway and protected against EAE pathology. Lastly, activation of A2AR in the cultured epithelium by A2AR overexpression or CGS21680 treatment increased the permeability of the CP epithelium and facilitated lymphocytes migration.ConclusionThese findings define the CP niche as one of the primary sites of A2AR action, whereby A2AR antagonists confer protection against EAE pathology. Thus, pharmacological targeting of the CP-A2AR represents a novel therapeutic strategy for MS by controlling immune cell trafficking across CP.

Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is widely known for its multiple systems damage, especially neurocognitive deficits in children. Since their discovery, adenosine A2A receptors (A2ARs) have been considered as key elements in signaling pathways mediating neurodegenerative diseases such as Huntington's and Alzheimer's, as well as cognitive function regulation. Herein, we investigated A2AR role in cognitive impairment induced by chronic intermittent hypoxia (CIH). Mice were exposed to CIH 7 h every day for 4 weeks, and intraperitoneally injected with A2AR agonist CGS21680 or A2AR antagonist SCH58261 half an hour before IH exposure daily. The 8-arm radial arm maze was utilized to assess spatial memory after CIH exposures.To validate findings using pharmacology, the impact of intermittent hypoxia was investigated in A2AR knockout mice. CIH-induced memory dysfunction was manifested by increased error rates in the radial arm maze test. The behavioral changes were associated with hippocampal pathology, neuronal apoptosis, and synaptic plasticity impairment. The stimulation of adenosine A2AR exacerbated memory impairment with more serious neuropathological damage, attenuated long-term potentiation (LTP), syntaxin down-regulation, and increased BDNF protein. Moreover, apoptosis-promoting protein cleaved caspase-3 was upregulated while anti-apoptotic protein Bcl-2 was downregulated. Consistent with these findings, A2AR inhibition with SCH58261 and A2AR deletion exhibited the opposite result. Overall, these findings suggest that A2AR plays a critical role in CIH-induced impairment of learning and memory by accelerating hippocampal neuronal apoptosis and reducing synaptic plasticity. Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice.

Adenosine A2a Receptors Improve Hypoxic Pulmonary Arterial Hypertension Via Mitochondrial ATP-sensitive Potassium Channels

IntroductionThis study is aimed to explore the effects of Adenosine A2a receptors (A2aR) on hypoxia-induced pulmonary hypertension (HPH) via mitochondrial ATP-sensitive potassium channels (MitoKATP) in vivo and in vitro.Material and methodsUsing wild-type (WT) and A2aR-deficient (A2aR-/-) mice; hypoxic pulmonary artery smooth muscle cells (PASMCs) were induced by a 24-hours hypoxia exposure. Mice and PASMCs were treated with the A2aR agonist CGS21680, MitoKATP blocker 5-hydroxydecanoic acid sodium salt (5HD), or MitoKATP agonist diazoxide. Mitochondrial morphology was observed by electron microscopy. The mitochondrial membrane potential (Δψm); invasive hemodynamic parameters; right ventricular (RV) hypertrophy index; pulmonary arterial remodeling index; proliferative and apoptotic indexes; protein expression levels of A2aR, Bax, Bcl-2, and Caspase-9; and release of cytochrome C from the mitochondria to the cytoplasm were measured.ResultsIn vitro, hypoxia induced the opening of MitoKATP. The up-regulation of A2aR reduced the opening of MitoKATP, and the blocking of MitoKATP or activating A2aR promoted mitochondria-dependent apoptosis of PASMCs. In vivo, compared with WT mice, A2aR-/- mice displayed increased RV systolic pressure, RV hypertrophy index, and pulmonary arterial remodeling index. The expression levels of Bax, cytochrome C, and Caspase-9 were higher and Bcl-2 expression was lower in A2aR-/- mice than in WT mice. CGS21680 could reverse hypoxia-induced hemodynamic changes, RV hypertrophy, and pulmonary arterial remodeling as well as abnormal proliferation and apoptosis resistance in WT mice with pulmonary hypertension (PH).ConclusionsA2aR induced the mitochondrial-dependent apoptosis pathway and inhibited PASMC proliferation by blocking MitoKATP, thereby inhibiting pulmonary vascular structural remodeling and reducing PH.

Adenosine A2A receptor inhibition reduces synaptic and cognitive hippocampal alterations in Fmr1 KO mice

In fragile X syndrome (FXS) the lack of the fragile X mental retardation protein (FMRP) leads to exacerbated signaling through the metabotropic glutamate receptors 5 (mGlu5Rs). The adenosine A2A receptors (A2ARs), modulators of neuronal damage, could play a role in FXS. A synaptic colocalization and a strong permissive interaction between A2A and mGlu5 receptors in the hippocampus have been previously reported, suggesting that blocking A2ARs might normalize the mGlu5R-mediated effects of FXS. To study the cross-talk between A2A and mGlu5 receptors in the absence of FMRP, we performed extracellular electrophysiology experiments in hippocampal slices of Fmr1 KO mouse. The depression of field excitatory postsynaptic potential (fEPSPs) slope induced by the mGlu5R agonist CHPG was completely blocked by the A2AR antagonist ZM241385 and strongly potentiated by the A2AR agonist CGS21680, suggesting that the functional synergistic coupling between the two receptors could be increased in FXS. To verify if chronic A2AR blockade could reverse the FXS phenotypes, we treated the Fmr1 KO mice with istradefylline, an A2AR antagonist. We found that hippocampal DHPG-induced long-term depression (LTD), which is abnormally increased in FXS mice, was restored to the WT level. Furthermore, istradefylline corrected aberrant dendritic spine density, specific behavioral alterations, and overactive mTOR, TrkB, and STEP signaling in Fmr1 KO mice. Finally, we identified A2AR mRNA as a target of FMRP. Our results show that the pharmacological blockade of A2ARs partially restores some of the phenotypes of Fmr1 KO mice, both by reducing mGlu5R functioning and by acting on other A2AR-related downstream targets.

The Adenosine A2A Receptor Agonist Accelerates Bone Healing and Adjusts Treg/Th17 Cell Balance through Interleukin 6.

The aim of this study was to explore the effect of adenosine A2A receptor agonists on fracture healing and the regulation of the immunity system after bone fracture. We implanted fibrin gel containing adenosine A2A receptor agonist CGS 21680/inhibitor ZM 241385/saline locally in rat tibial fracture models, finding that the adenosine A2A receptor agonist could promote fracture healing. At the same time, the adenosine A2A receptor agonist decreased the level of IL-6 in blood and the fracture area, increased Treg cells, and decreased Th17 cells in blood of bone fracture rats. Further, tibial fracture rats implanted with the adenosine A2A receptor agonist gel were injected with IL-6. We found that IL-6 could reverse the effect of adenosine A2A receptor agonists on fracture healing and Treg/Th17 cells in blood. Through the above results, we believe that the adenosine A2A receptor agonist can promote fracture healing and regulate Treg/Th17 cells in blood of rats with fractures. These effects are related to IL-6.

Acute cocaine treatment enhances the antagonistic allosteric adenosine A2A-dopamine D2 receptor\u2013receptor interactions in rat dorsal striatum without increasing significantly extracellular dopamine levels

BackgroundAntagonistic adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) receptor–receptor interactions have previously been demonstrated in A2AR–D2R heteroreceptor complexes in the rat dorsal striatum. They mainly involve a reduction of affinity in the high-affinity component of the D2R agonist binding site upon activation in vivo of the A2AR by an A2AR agonist. Upon cocaine self-administration, this antagonistic A2AR–D2R interaction disappeared in the dorsal striatum.MethodsIn the current experiments, it was tested whether such modifications in the antagonistic A2AR–D2R receptor–receptor interactions can develop also after an acute systemic injection of a low cocaine dose (1 mg/kg; sc).ResultsMicrodialysis experiments indicated that acute cocaine did not significantly alter the extracellular dopamine levels in the dorsal striatum of the awake Wistar rats. Competition dopamine receptor binding experiments demonstrated that in the acute cocaine group, the A2AR agonist CGS-21680 produced significantly larger increases in the D2R Ki, High values (reduction of high-affinity) versus the saline-injected (i.e. control) group. Furthermore, in the dorsal striatum membrane preparation from acute cocaine-injected rats, CGS-21680 also produced significant increases in the D2R Ki, Low values (reduction of low-affinity) and in the proportion of D2Rs in the high-affinity state (RH). Such significant effects were not observed with CGS-21680 in the control group.ConclusionsThe molecular mechanism involved in the acute cocaine-induced increase in the antagonistic allosteric A2AR–D2R receptor–receptor interactions may be an increased formation of higher-order complexes A2AR–D2R-sigma1R in which cocaine by binding to the sigma1R protomer also allosterically enhances the inhibitory A2AR–D2R interaction in this receptor complex.

Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT

Hypertensive cardiac remodeling is a constellation of abnormalities that includes cardiomyocyte hypertrophy and death and tissue fibrosis. Adenosine is a long-known vasodilator, through interacting with its four cell surface receptor subtypes in cardiovascular system. However, it is unclear that whether adenosine A2A receptor (A2AR) activation is involved in the cardiac remodeling in hypertension. WT mice were utilized to induce DOCA-salt sensitive hypertension and received A2AR agonist CGS21680 or antagonist KW6002 treatment. Cardiac functional phenotyping measurement by echocardiography showed that CGS21680 improved cardiac dysfunction in DOCA-salt mice. Moreover, CGS21680 reduced cardiomyocyte hypertrophy, cardiac inflammation and fibrosis. However, iBAT depletion surgery induces dramatic cardiac remodeling in DOCA-salt mice, and the protective function of CGS21680 was blocked without intact iBAT. Mechanistically, A2AR agonist CGS21680 increased iBAT-derived fibroblast growth factor 21 (FGF21). Our data suggest that activation of A2AR could be a potential therapeutic strategy in preventing heart damage in hypertension.

Adenosine A2A receptor stimulation restores cell functions and differentiation in Niemann-Pick type C-like oligodendrocytes

Niemann Pick type C (NPC) disease is a rare neurovisceral disorder. Mutations in npc1 gene induce an intracellular accumulation of unesterified cholesterol in the endosomal/lysosomal system causing cell death. We recently showed that stimulation of adenosine A2A receptors (A2AR) restores cholesterol accumulation in late endosomes/lysosomes in human NPC fibroblasts and neural cell lines transiently transfected with NPC1 siRNA, suggesting that these receptors might be targeted to contrast the disease. Since NPC1 disease is characterized by dysmyelination and maturational arrest of oligodendrocyte progenitors (OPs), in this study, we investigated whether A2AR stimulation could promote oligodendrocyte differentiation and myelin formation, thus overcoming these important neurological abnormalities. We developed a NPC1 pharmacological model, in which primary cultures of OPs are exposed to a cholesterol transport inhibitor to induce a NPC1-like phenotype characterized by several typical features such as (i) cholesterol accumulation, (ii) altered mitochondrial morphology and membrane potential, (iii) defect of autophagy and (iv) maturation arrest. The A2AR agonist CGS21680 normalized all NPC1-like features. The ability of CGS21680 of rescuing OP from maturational arrest and promoting their differentiation to mature OL, suggests that A2AR stimulation might be exploited to correct dysmyelination in NPC1, further supporting their therapeutic potential in the disease.

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.

Effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion injury in diabetic rats: the relationship with autophagy

Objective To evaluate the effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion (I/R) injury in diabetic rats and the relationship with autophagy. Methods Clean-grade healthy male Sprague-Dawley rats, aged 6 weeks, weighing 200-250 g, were studied.The diabetic rat model was established by intraperitoneal injection of 1% streptozotocin 60 mg/kg.Forty diabetic rats were divided into 4 groups (n=10 each) using a random number table method: sham operation group (group Sham), I/R group (group I/R), I/R plus adenosine A2A receptor agonist CGS21680 group (group CGS), and I/R plus CGS21680 plus adenosine A2A receptor antagonist ZM241385 group (group CGS+ ZM). Myocardial I/R was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120-min reperfusion.Adenosine A2A receptor agonist CGS21680 10 μg/100g was intravenously injected at 10 min before reperfusion in group CGS.CGS21680 10 ug/100g and ZM241385 0.2 mg/kg were intravenously injected sequentially at 10 min before reperfusion in group CGS+ ZM.Blood samples were obtained at the end of reperfusion for determination of concentrations of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct volume (by TTC staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3 Ⅱ, p62 and Beclin-1 (by Western blot). LC3 Ⅱ/LC3 Ⅰ ratio was calculated. Results Compared with group Sham, the serum CK-MB, LDH and cTnI concentrations and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was up-regulated, and the LC3 Ⅱ/LC3 Ⅰ ratio was increased in group I/R (P 0.05). Compared with group CGS, the concentrations of serum CK-MB, LDH and cTnI and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in group CGS+ ZM (P<0.05). Conclusion Activating adenosine A2A receptors can mitigate myocardial I/R injury, and the mechanism may be related to enhancing autophagy in diabetic rats. Key words: Receptor, adenosine A2A; Autophagy; Diabetes mellitus; Myocardial reperfusion injury

Balance between dopamine and adenosine signals regulates the PKA/Rap1 pathway in striatal medium spiny neurons

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration.

Enhancing endogenous adenosine A2A receptor signaling induces slow-wave sleep without affecting body temperature and cardiovascular function

Insomnia is one of the most common sleep problems with an estimated prevalence of 10%–15% in the general population. Although adenosine A2A receptor (A2AR) agonists strongly induce sleep, their cardiovascular effects preclude their use in treating sleep disorders. Enhancing endogenous A2AR signaling, however, may be an alternative strategy for treating insomnia, because adenosine levels in the brain accumulate during wakefulness. In the present study, we found that 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, denoted A2AR positive allosteric modulator (PAM)-1, enhanced adenosine signaling at the A2AR and induced slow wave sleep (SWS) without affecting body temperature in wild-type male mice after intraperitoneal administration, whereas the SWS-inducing effect of this benzoic acid derivative was abolished in A2AR KO mice. In contrast to the A2AR agonist CGS 21680, the A2AR PAM-1 did not affect blood pressure or heart rate. These findings indicate that enhancing A2AR signaling promotes SWS without cardiovascular effects. Therefore, small molecules that allosterically modulate A2ARs could help people with insomnia to fall asleep.

The promotion of tissue engineering blood vessel patency by CGS21680 through regulating pro-inflammatory activities of endothelial progenitor cell.

The mobilization and homing of endothelial progenitor cells (EPCs) contribute to the rapid endothelialization of tissue engineering blood vessel (TEBV). Inflammation can affect TEBV patency, and monocytes/macrophages (MM) are the main effector cells. But it is not clear how EPCs interact with MM after TEBV transplantation. Our results showed acellular materials would not directly cause acute and severe inflammatory responses but activate E-selectin expression in homing EPCs, gradually promoting the polarization of MM to the M1. Adenosine A2a receptor agonist CGS21680 promoted the secretion of more proangiogenic factors from MM, inducing EPC migration and mobilization. CGS21680 could inhibit MM polarization to the M1 type through the down-regulation of EPC proinflammatory molecules, such as E-selectin. Chitosan/(2-hydroxypropyl)-β-cyclodextrin nanoparticles were prepared to control the release of CGS-21680 and then modified to TEBVs through layer-by-layer assembly. Animal experiments showed that this TEBV can maintain patency for 6 months and good endothelialization was observed. In summary, our results showed the regulation of EPC pro-inflammatory activities is a new approach to enhance TEBV patency. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2634-2642, 2018.

An adenosine�A1R-A2aR imbalance regulates low glucose/hypoxia-induced microglial activation, thereby contributing to oligodendrocyte damage through NF-κB and CREB phosphorylation

Microglial activation-mediated inflammatory damage to oligodendrocytes is a key step in the etiology of ischemic white matter lesions. The adenosineA1receptor (A1R) and adenosineA2areceptor (A2aR) have been reported to regulate the activation of microglia, however, the underlying mechanisms remain elusive. Thus, the present study used a microglia/oligodendrocyte co‑culture model exposed to low glucose/hypoxia, and treated with agonists/antagonists of A1R and A2aR to investigate the role of A1R and A2aR. Changes in A1R and A2aR expression and inflammatory cytokine secretion by the microglia, and oligodendrocyte damage, after exposure were examined. Low glucose/hypoxia induced a higher elevation of A1R than A2aR. In addition, activation of A1R inhibited A2aR protein expression and vice versa. The A1R antagonist DPCPX (100nM) and A2aR agonist CGS 21680 (100nM) inhibited microglial activation, reduced the production of inflammatory cytokines and attenuated oligodendrocyte damage, along with elevating the levels of phosphorylated nuclear factor (NF)‑κB and cyclic adenosine monophosphate response element binding protein (CREB). These data indicate that an A1R‑A2aR imbalance is able to modulate low glucose‑induced microglial activation and the cellular immune response through altering NF‑κB and CREB phosphorylation. This suggests that rebalancing A1R‑A2aR is a promising approach for treating white matter injury.