Role Of Adenosine A2A Receptors Research Articles

Impaired autophagic flux is associated with the severity of trauma and the role of A2AR in brain cells after traumatic brain injury

Recent studies have shown that after traumatic brain injury (TBI), the number of autophagosomes is markedly increased in brain cells surrounding the wound; however, whether autophagy is enhanced or suppressed by TBI remains controversial. In our study, we used a controlled cortical impact system to establish models of mild, moderate and severe TBI. In the mild TBI model, the levels of autophagy-related protein 6 (Beclin1) and autophagy-related protein 12 (ATG12)-autophagy-related protein 5 (ATG5) conjugates were increased, indicating the enhanced initiation of autophagy. Furthermore, the level of the autophagic substrate sequestosome 1 (SQSTM1) was decreased in the ipsilateral cortex. This result, together with the results observed in tandem mRFP-GFP-LC3 adeno-associated virus (AAV)-infected mice, indicates that autophagosome clearance was also increased after mild TBI. Conversely, following moderate and severe TBI, there was no change in the initiation of autophagy, and autophagosome accumulation was observed. Next, we used chloroquine (CQ) to artificially impair autophagic flux in the injured cortex of the mild TBI model and found that the severity of trauma was obviously exacerbated. In addition, autophagic flux and trauma severity were significantly improved in adenosine A2A receptor (A2AR) knockout (KO) mice subjected to moderate TBI. Thus, A2AR may be involved in regulating the impairment of autophagic flux in response to brain injury. Our findings suggest that whether autophagy is increased after TBI is associated with whether autophagic flux is impaired, and the impairment of autophagic flux exacerbates the severity of trauma. Furthermore, A2AR may be a target for alleviating the impairment in autophagic flux after TBI.

Antipsychotic-Like Efficacy of Dopamine D2 Receptor-Biased Ligands is Dependent on Adenosine A2A Receptor Expression.

Dopamine D2 receptor (D2R) activation triggers both G protein- and β-arrestin-dependent signaling. Biased D2R ligands activating β-arrestin pathway have been proposed as potential antipsychotics. The ability of D2R to heteromerize with adenosine A2A receptor (A2AR) has been associated to D2R agonist-induced β-arrestin recruitment. Accordingly, here we aimed to demonstrate the A2AR dependence of D2R/β-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between β-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of D2R/A2AR homomers and heteromers, we demonstrated that the D2R agonists quinpirole and UNC9994 could promote β-arrestin-2 recruitment only when A2AR/D2R heteromers were expressed. Subsequently, the role of A2AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and A2AR-/- mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in A2AR-/- mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that D2R/A2AR heteromerization facilitates D2R β-arrestin recruitment, and furthermore, reveal a pivotal role for A2AR in the antipsychotic-like activity of the β-arrestin-biased D2R ligand, UNC9994.

Reduced coronary reactive hyperemia in mice was reversed by the soluble epoxide hydrolase inhibitor (t-AUCB): Role of adenosine A2A receptor and plasma oxylipins

Coronary reactive hyperemia (CRH) protects the heart against ischemia. Adenosine A2AAR–deficient (A2AAR−/−) mice have increased expression of soluble epoxide hydrolase (sEH); the enzyme responsible for breaking down the cardioprotective epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). sEH–inhibition enhances CRH, increases EETs, and modulates oxylipin profiles. We investigated the changes of oxylipins and their impact on CRH in A2AAR−/− and wild type (WT) mice. We hypothesized that the attenuated CRH in A2AAR−/− mice is mediated by changes in oxylipin profiles, and that it can be reversed by either sEH- or ω-hydroxylases–inhibition. Compared to WT mice, A2AAR−/− mice had attenuated CRH and changed oxylipin profiles, which were consistent between plasma and heart perfusate samples, including decreased EET/DHET ratios, and increased hydroxyeicosatetraenoic acids (HETEs). Plasma oxylipns in A2AAR−/− mice indicated an increased proinflammatory state including increased ω-terminal HETEs, decreased epoxyoctadecaenoic/dihydroxyoctadecaenoic acids (EpOMEs/DiHOMEs) ratios, increased 9-hydroxyoctadecadienoic acid, and increased prostanoids. Inhibition of either sEH or ω-hydroxylases reversed the reduced CRH in A2AAR−/− mice. In WT and sEH−/− mice, blocking A2AAR decreased CRH. These data demonstrate that A2AAR–deletion was associated with changes in oxylipin profiles, which may contribute to the attenuated CRH. Also, inhibition of sEH and ω-hydroxylases reversed the reduction in CRH.

Toll-like receptors, NF-κB, and IL-27 mediate adenosine A2A receptor signaling in BTBR T+ Itpr3tf/J mice

Autism is a predominant neurodevelopmental disorder characterized by impaired communication, social deficits, and repetitive behaviors. Recent research has proposed that the impairment of innate immunity may play an important role in autism. Toll-like receptors (TLRs) are potential therapeutic targets against neuroinflammation. The BTBR T+ Itpr3tf/J (BTBR) mouse is a well-known model of autism, showing repetitive behaviors such as cognitive inflexibility and increased grooming as compared to C57BL/6 (B6) mice. Adenosine A2A receptor (A2AR) signaling is involved in inflammation, brain injury, and lymphocyte infiltration into the CNS, but the role of A2AR in autism remains unknown. We investigated the effect of A2AR antagonist SCH 5826 (SCH) and agonist CGS 21680 (CGS) on the expression levels of TLRs, IL-27, NF-κB p65, and IκBα in BTBR mice. Treatment of BTBR mice with SCH increased the percentage of splenic CD14+TLR2+ cells, CD14+TLR3+ cells, CD14+TLR4+ cells, and decreased the percentage of CD14+IL-27+ cells, as compared to the untreated BTBR mice. Our results reveal that BTBR mice treated with CGS had reversal of SCH-induced immunological responses. Moreover, mRNA and protein expression analyses confirmed increased expression of TLR2, TLR3, TLR4, and NF-κB p65 in brain tissue, and decreased IL-27 and IκBα expression following SCH treatment, as compared to the untreated-BTBR and CGS-treated BTBR mice. Together, these results suggest that the A2AR agonist corrects neuroimmune dysfunction observed in BTBR mice, and thus has the potential as a therapeutic approach in autism.

Blockade of adenosine A2A receptor enhances CD8+ T cells response and decreases regulatory T cells in head and neck squamous cell carcinoma

BackgroundCancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC.MethodsThe expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model.ResultsImmunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4+Foxp3+ regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4+ Foxp3+ Tregs and enhanced the anti-tumor response of CD8+ T cells.ConclusionsThese results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.

Hypoxic postconditioning attenuates apoptosis via activation of adenosine A2a receptors on dermal microvascular endothelial cells of human flaps

BackgroundIn recent years, many studies have demonstrated that endogenous adenosine induced by ischemia postconditioning reduces apoptosis in various models, but no study has clearly elucidated the effects of hypoxic postconditioning (HPC) in human dermal microvascular endothelial cells (HDMECs) of flaps, and the subtype of adenosine receptors involved remains unknown. In our study, we sought to identify the role of adenosine A2a receptor in the antiapoptotic effects of HPC. Materials and methodsThe HDMECs were isolated from human upper eyelid tissue. After hypoxia for 6 h, the HDMECs were either abruptly reperfused with medium for 12 h (reoxygenation) or postconditioned by three cycles of 5 min of transitory reoxygenation and 5 min of rehypoxia followed by 11.5 h of reoxygenation. CGS-21680 was used as an adenosine A2a agonist. ResultsAdenosine A2a receptors were found in the cytoplasm of HDMECs. Hypoxia/reoxygenation (H/R) resulted in severe injury in HDMECs as evidenced by an increase in apoptosis percentage and an increase in expressions of apoptosis-related proteins (the ratio of Bax/Bcl-2 and caspase-3), which can be significantly attenuated by HPC treatment or exposure to CGS-21680, a selective adenosine A2a receptor agonist (all P values < 0.05). Meanwhile, HPC treatment or CGS-21680 significantly activated A2a receptors compared with the hypoxia/reoxygenation group (P < 0.05 versus P < 0.05, respectively). Statistical analysis showed that the increase of cell apoptosis closely correlated inversely with the increase of relative protein level of adenosine A2a receptors (r2 = 0.8177, P < 0.0001). ConclusionsHPC protects against apoptosis induced by reoxygenation via activation of adenosine A2a receptors on HDMECs.

The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center

T follicular helper (TFH) cells have been shown to be critically required for the germinal center (GC) reaction where B cells undergo class switch recombination and clonal selection to generate high affinity neutralizing antibodies. However, detailed knowledge of the physiological cues within the GC microenvironment that regulate T cell help is limited. The cAMP-elevating, Gs protein-coupled A2a adenosine receptor (A2aR) is an evolutionarily conserved receptor that limits and redirects cellular immunity. However, the role of A2aR in humoral immunity and B cell differentiation is unknown. We hypothesized that the hypoxic microenvironment within the GC facilitates an extracellular adenosine-rich milieu, which serves to limit TFH frequency and function, and also promotes immunosuppressive T follicular regulatory cells (TFR). In support of this hypothesis, we found that following immunization, mice lacking A2aR (A2aRKO) exhibited a significant expansion of T follicular cells, as well as increases in TFH to TFR ratio, GC T cell frequency, GC B cell frequency, and class switching of GC B cells to IgG1. Transfer of CD4 T cells from A2aRKO or wild type donors into T cell-deficient hosts revealed that these increases were largely T cell-intrinsic. Finally, injection of A2aR agonist, CGS21680, following immunization suppressed T follicular differentiation, GC B cell frequency, and class switching of GC B cells to IgG1. Taken together, these observations point to a previously unappreciated role of GS protein-coupled A2aR in regulating humoral immunity, which may be pharmacologically targeted during vaccination or pathological states in which GC-derived autoantibodies contribute to the pathology.

The Mediating Role of A2A Adenosine Receptors in the Mitochondrial Pathway of Apoptotic Hippocampal Cell Death, Following the Administration of MDMA in Rat.

Introduction:The 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a popular recreational drug and a major source of substance abuse, which ultimately leads to sensations of well-being, elation and euphoria, moderate derealization/depersonalization, and cognitive disruptions, as well as intense sensory awareness. The mechanisms involved in memory impairment induced by MDMA are not completely understood.Methods:The current study used 40 Sprague-Dawley rats, weighted 200 to 250 g. Experiments were performed in four groups, each containing 10 rats. The first group of rats was used as the control, treated with dimethyl sulfoxide (DMSO). The second group was treated with MDMA. The third group was treated with MDMA and CGS (the adenosine A2A receptor agonist, 2-[p-(2-carboxyethyl) phenethylamino]-5′-N-ethylcarboxamidoadenosine) (CGS 21680) and the fourth group was treated with MDMA and SCH (the A2A receptor antagonist [7-(2-phenylethyl)-5-amino-2-(2-furyl-) pyrazolo-[4, 3-e]-1, 2, 4 triazolo [1,5-] pyrimidine]) (SCH 58261). The drugs in all groups were administrated intraperitoneally (i.p.) once a day for 7 days. In 5 rats of each group, following perfusion, samples were taken from hippocampi to investigate apoptosis. Accordingly, the samples were stained using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit, and studied by light microscopy. In other rats, fresh tissue was also removed to study the expression of bax and bcl-2 by Western blotting technique.Results:It was observed that the coadministration of MDMA with CGS reduced bax expression and prevented apoptosis of hippocampal cells. The coadministration of MDMA and SCH increased bax expression, and also increased the frequency of hippocampal cell apoptosis.Conclusion:The results of the current study showed that administration of CGS with MDMA decreased the common side effects associated with MDMA.

The Role of Adenosine A2A Receptor, CYP450s, and PPARs in the Regulation of Vascular Tone.

Adenosine is an endogenous mediator involved in a myriad of physiologic functions, including vascular tone regulation. It is also implicated in some pathologic conditions. Four distinct receptor subtypes mediate the effects of adenosine, such as its role in the regulation of the vascular tone. Vascular tone regulation is a complex and continuous process which involves many mechanisms and mediators that are not fully disclosed. The vascular endothelium plays a pivotal role in regulating blood flow to and from all body organs. Also, the vascular endothelium is not merely a physical barrier; it is a complex tissue with numerous functions. Among adenosine receptors, A2A receptor subtype (A2AAR) stands out as the primary receptor responsible for the vasodilatory effects of adenosine. This review focuses on important effectors of the vascular endothelium, including adenosine, adenosine receptors, EETs (epoxyeicosatrienoic acids), HETEs (hydroxyeicosatetraenoic acids), PPARs (peroxisome proliferator-activated receptors), and KATP channels. Given the impact of vascular tone regulation in cardiovascular physiology and pathophysiology, better understanding of the mechanisms affecting it could have a significant potential for developing therapeutic agents for cardiovascular diseases.

Blunted dynamics of adenosine A2A receptors is associated with increased susceptibility to Candida albicans infection in the elderly.

Opportunistic gut infections and chronic inflammation, in particular due to overgrowth of Candida albicans present in the gut microbiota, are increasingly reported in the elder population. In aged, adult and young mice, we now compared the relative intestinal over-colonization by ingested C. albicans and their translocation to other organs, focusing on the role of adenosine A2A receptors that are a main stop signal of inflammation. We report that elderly mice are more prone to over-colonization by C. albicans than adult and young mice. This fungal over-growth seems to be related with higher growth rate in intestinal lumen, independent of gut tissues invasion, but resulting in higher GI tract inflammation. We observed a particularly high colonization of the stomach, with increased rate of yeast-to-hypha transition in aged mice. We found a correlation between A2A receptor density and tissue damage due to yeast infection: comparing with young and adults, aged mice have a lower gut A2A receptor density and C. albicans infection failed to increase it. In conclusion, this study shows that aged mice have a lower ability to cope with inflammation due to C. albicans over-colonization, associated with an inability to adaptively adjust adenosine A2A receptors density.

Effect of adenosine A2A receptor antagonists on motor disorders induced by 6-hydroxydopamine in rat.

To investigate the role of adenosine A2A receptors on 6-OHDA-induced motor disorder in rat. In order to induce experimental model of Parkinson's disease, 6-hydoxydopamine (8 μg/rat) was injected unilaterally into the SNc. After three weeks as a recovery period, 6-OHDA-induced bradykinesia and balance disturbances were assessed by using beam traversal test 10, 30 and 60 minutes after intraperitoneal injections of the drugs (caffeine, SCH58261). The results showed that 6-OHDA (8 μg/rat, Intra-SNc) induced motor disorders of Parkinson's disease and increased elapsed time in the beam test (p<0.001). Injection of caffeine (30 mg/kg, i.p.) and SCH58261 (2 mg/kg, i.p.) attenuated elapsed time on beam (p<0.01 and p<0.001). We showed that acute administration of caffeine and SCH 58261 can improve the 6-OHDA-induced bradykinesia and motor disturbance. Adenosine A2AR antagonists improve 6-OHDA-motor deficit and this effect seems to be mediated by the inhibition of A2A presynaptic receptors in substantia nigra pars compacta.

Role of adenosine A2A receptor in cerebral ischemia reperfusion injury: Signaling to phosphorylated extracellular signal-regulated protein kinase (pERK1/2)

Following brain ischemia reperfusion (IR), the dramatic increase in adenosine activates A2AR to induce further neuronal damage. Noteworthy, A2A antagonists have proven efficacious in halting IR injury, however, the detailed downstream signaling remains elusive. To this end, the present study aimed to investigate the possible involvement of phospho-extracellular signal-regulated kinase (pERK1/2) pathway in mediating protection afforded by the central A2A blockade. Male Wistar rats (250–270g) subjected to bilateral carotid occlusion for 45min followed by a 24-h reperfusion period showed increased infarct size corroborating histopathological damage, memory impairment and motor incoordination as well as increased locomotor activity. Those events were mitigated by the unilateral intrahippocampal administration of the selective A2A antagonist SCH58261 via a decrease in pERK1/2 downstream from diacyl glycerol (DAG) signaling. Consequent to pERK1/2 inhibition, reduced hippocampal microglial activation, glial tumor necrosis factor-alpha (TNF-α) and brain-derived neurotropic factor (BDNF) expression, glutamate (Glu), inducible nitric oxide synthase (iNOS) and thiobarbituric acid reactive substances (TBARS) were evident in animals receiving SCH58261. Additionally, the anti-inflammatory cytokine interleukin-10 (IL-10) increased following nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). Taken all together, these events suppressed apoptotic pathways via a reduction in cytochrome c (Cyt. c) as well as caspase-3 supporting a crucial role for pERK1/2 inhibition in consequent reduction of inflammatory and excitotoxic cascades as well as correction of the redox imbalance.

Adenosine A2A Receptors Modulate α-Synuclein Aggregation and Toxicity.

Abnormal accumulation of aggregated α-synuclein (aSyn) is a hallmark of sporadic and familial Parkinson's disease (PD) and related synucleinopathies. Recent studies suggest a neuroprotective role of adenosine A2A receptor (A2AR) antagonists in PD. Nevertheless, the precise molecular mechanisms underlying this neuroprotection remain unclear. We assessed the impact of A2AR blockade or genetic deletion (A2AR KO) on synaptic plasticity and neuronal cell death induced by aSyn oligomers. We found that impairment of LTP associated with aSyn exposure was rescued in A2AR KO mice or upon A2AR blockade, through an NMDA receptor-dependent mechanism. The mechanisms underlying these effects were evaluated in SH-SY5Y cells overexpressing aSyn and rat primary neuronal cultures exposed to aSyn. Cell death in both conditions was prevented by selective A2AR antagonists. Interestingly, blockade of these receptors did not interfere with aSyn oligomerization but, instead, reduced the percentage of cells displaying aSyn inclusions. Altogether, our data raise the possibility that the well-documented effects of A2AR antagonists involve the control of the latter stages of aSyn aggregation, thereby preventing the associated neurotoxicity. These findings suggest that A2AR represent an important target for the development of effective drugs for the treatment of PD and related synucleinopathies.

Adenosine A2A Receptors Mediate Anti-Inflammatory Effects of Electroacupuncture on Synovitis in Mice with Collagen-Induced Arthritis

To study the role of adenosine A2A receptor (A2AR) in mediating the anti-inflammatory effect of electroacupuncture (EA) on synovitis in collagen-induced arthritis (CIA), C57BL/6 mice were divided into five treatment groups: Sham-control, CIA-control, CIA-EA, CIA-SCH58261 (A2AR antagonist), and CIA-EA-SCH58261. All mice except those in the Sham-control group were immunized with collagen II for arthritis induction. EA treatment was administered using the stomach 36 and spleen 6 points, and stimulated with a continuous rectangular wave for 30 min daily. EA treatment and SCH58261 were administered daily from days 35 to 49 (n = 10). After treatment, X-ray radiography of joint bone morphology was established at day 60 and mouse blood was collected for ELISA determination of tumor necrosis factor alpha (TNF-α) levels. Mice were sacrificed and processed for histological examination of pathological changes of joint tissue, including hematoxylin-eosin staining and immunohistochemistry of A2AR expression. EA treatment resulted in significantly reduced pathological scores, TNF-α concentrations, and bone damage X-ray scores. Importantly, the anti-inflammatory and tissue-protective effect of EA treatment was reversed by coadministration of SCH58261. Thus, EA treatment exerts an anti-inflammatory effect resulting in significant protection of cartilage by activation of A2AR in the synovial tissue of CIA.

The role of adenosine A2A receptors of the preoptic area in somnogenic activity of 70 kDa protein in pigeons

The effects of adenosine A2A receptor antagonist microinjections into the hypothalamic ventrolateral preoptic area (VLPA) on the natural sleep-wake cycle and somnogenic activity of a 70 kDa heat shock protein (Hsp70) are analyzed electrophysiologically in birds, specifically, pigeons (Columba livia) for the first time. We report that: (1) at the beginning of the rest period, microinjections of 8-(3-Chlorostyryl)caffeine (ChC), an adenosine A2A receptor antagonist, into VLPA increase wake duration and inhibited sleep in a dose-dependent manner; (2) Hsp70 injections into VLPA evoke the somnogenic effect manifested as an increase in total slow-wave sleep (SWS) duration and enhancement of the mechanisms responsible for SWS triggering and maintenance; (3) ChC blockade of adenosine A2A receptors inhibits Hsp70-induced SWS. These data suggest that adenosine A2A receptors located in VLPA are involved in the modulation of the natural sleep-wake cycle and promotion of the Hsp70 somnogenic effect in pigeons. It is hypothesized that the somnogenic effect of Hsp70 is mediated by the modulating influence of this chaperone on the functioning of adenosine receptor proteins.

Role of adenosine A2A receptor in ischemia reperfusion injury: signaling to extracellular signal‐regulated protein kinase (663.6)

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischemia. Out of the four known adenosine receptor subtypes (A1, A2A, A2B and A3), the A2A is of critical importance in stroke. Although, both A2A agonists and antagonists are proven protective during ischemia reperfusion (IR) injury, however, ,the mechanisms by which A2A receptors are noxious during IR remain elusive. The objective of the present study was to investigate the role of central A2A receptor in modulating cellular as well as behavioral responses to IR injury and the possible involvement of ERK signalling pathway. Hence, we hereby report that bilateral carotid occlusion for 45 min followed by 24h reperfusion period resulted in a significant memory impairment, motor incoordination as well as increased locomotor activity that were not affected by unilateral intrahippocampal injection of a selective A2A agonist CGS21680, but conversely mitigated by the selective A2A antagonist SCH58261. On the cellular level, IR increased the pERK, NFkB, cytochrome c, BDNF, Nrf2 and TNF‐α content in the hippocampus. With the exception of Nrf2 level, treatment with SCH58261 significantly reversed these effects, however, CGS21680 treatment resulted in a non‐significant change from IR insult. Accordingly these results suggest a possible protective role of the central A2A receptor blockade in IR injury with a prospective involvement of p42/44 pathway.

Adenosine A2A receptor and retinal pathological neovascularization in mice

Objective To investigate the role of adenosine A2A receptor plays in retinal pathological neovascularization in mice.Methods A total of 202 mice were divided into room-air group (n =66) and oxygen induced retinopathy (OIR) group (n=136).Among room-air group,there were 18 A2A knock-out (KO) mice (KO subgroup) and 24 C57BL/6 mice as wide type (wide type subgroup).OIR group were divided into OIR control subgroup (n =48),A2A-OIR subgroup (n =24) and Caffeine-OIR subgroup (n =64).The retinal neovascularization of OIR group was induced by oxygen.The pathological neovascularization was determined by retinal sections.Fluorescent quantitative polymerase chain reaction (PCR) was used to measure the mRNA expression of A2A and vascular endothelial growth factor (VEGF).0.1,0.3,1.0 g/L Caffeine was dissolve in drinking water of lactating females in Caffeine-OIR subgroup,non-perfusion areas of retina in mice at the age of 0-17,0-7,7-17,7-12,and 12-17 days were analyzed in different dosage and when the dosage as 1.0 g/L.Results Compared with OIR control subgroup,the retinal non-perfusion areas and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in A2A-OIR subgroup were reduced significantly (t =7.694,7.747; P ＜0.001).Compared with wide type subgroup,the level of A2A and VEGF mRNA in OIR control subgroup increased significantly (t=4.036,2.230; P＜0.05).Compared with OIR control subgroup,the level of VEGF mRNA in A2A-OIR subgroup decreased significantly (t=3.122,P＜0.01).Compared with OIR control subgroup,the retinal non perfusion areas in mice at the dosage of 0.1 and 1.0 g/L (t=2.397,4.533) and at the age of 0-17,0-7 days when the dosage as 1.0 g/L (t=4.070,2.399) were reduced significantly (P＜0.05).Conclusions The expression of adenosine A2A receptor increases in oxygeninduced retinal pathological neovascularization..Adenosine A2A receptor may regulate the expression of VEGF.A2A receptor inactivation can inhibit oxygen-induced retinal pathological neovascularization. Key words: Retinal neovascularization/physiopathology; Receptor, adenosine A2A; Caffeine

The Presence of Adenosine A2a Receptor in Thyrocytes and Its Involvement in Graves' IgG-Induced VEGF Expression

Goitrogenesis in Graves' disease (GD) has been attributed to anti-TSH receptor antibody stimulation. Recently, a role for adenosine A2a receptor (A2aR) in goiter formation was reported in the thyroglobulin-A2aR transgenic mice. However, it is unclear whether A2aR is expressed in the thyroid and whether it is associated with the pathogenesis of goiter in GD. Here, we confirmed the expression of A2aR in FRTL-5 cells, primary normal human thyrocytes (both sexes were used without regard to sex), and thyroid tissue (both sexes were used without regard to sex) by PCR, Western blotting, immunohistochemistry, and immunofluorescence. After treatments with A2aR-specific agonist 2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine or GD IgG, the mRNA and protein levels of vascular endothelial growth factor (VEGF), a growth factor related to goitrogenesis, were evaluated along with upstream signaling pathways. A2aR activation and GD IgG promoted the expression of VEGF in thyrocytes, which was accompanied by the activation of cAMP/protein kinase A/phosphorylated-cAMP-response element-binding protein, peroxisome proliferator-activated receptor γ coactivator-1α, and hypoxia-inducible factor-1α. The changes induced by GD IgG were partially abrogated by A2aR small interfering RNA and an A2aR antagonist. These results were supported by data on the goiter samples from the thyrotropin receptor adenovirus-induced GD mouse model (female). These data demonstrate that GD IgG could up-regulate the VEGF expression through A2aR, indicating a potential mechanism for goitrogenesis in GD.

Potential role of A2A adenosine receptor in traumatic optic neuropathy

In traumatic optic neuropathy (TON), apoptosis of retinal ganglion cells is closely related to the local production of reactive oxygen species and inflammatory mediators from activated microglial cells. Adenosine receptor A2A (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in TON. In the present study, we examined the role of A2AAR in retinal complications associated with TON. Initial studies in wild-type mice revealed that treatment with the A2AAR agonist resulted in marked decreases in the TON-induced microglial activation, retinal cell death and releases of reactive oxygen species and pro-inflammatory cytokines TNF-α and IL-6. To further assess the role of A2AAR in TON, we studied the effects of A2AAR ablation on the TON-induced retinal abnormalities. A2AAR−/− mice with TON showed a significantly higher mRNA level of TNF-α, Iba1-1 in retinal tissue, and ICAM-1 expression in retinal sections compared with wild-type mice with TON. To explore a potential mechanism by which A2AAR-signaling regulates inflammation in TON, we performed additional studies using hypoxia- or LPS-treated microglial cells as an in vitro model for TON. Activation of A2AAR attenuates hypoxia or LPS-induced TNF-α release and significantly repressed the inflammatory signaling, ERK in the activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in TON and suggests that the retinal protective effect of A2AAR is mediated by attenuating the inflammatory response that occurs in microglia via interaction with MAPKinase pathway.

Role of adenosine A2A receptor in organ-specific vascular reactivity following hemorrhagic shock in rats

BackgroundPrevious studies have demonstrated differences among organs in terms of shock-induced vascular reactivity and a role for adenosine A2A receptors (A2ARs) in protection against ischemia/reperfusion injury. However, the contributions of A2ARs to organ-specific vascular reactivity and the protection of vascular responsiveness following shock are currently unknown. MethodsWe investigated the role of A2ARs in different arteries, including the left femoral artery (LFA), thoracic aorta (TA), superior mesenteric artery (SMA), right renal artery (RRA), pulmonary artery (PA), and middle cerebral artery (MCA), in hemorrhagic-shock rats. ResultsThe vascular reactivities of the LFA, SMA, RRA, and MCA increased slightly during early shock and then gradually decreased, whereas those of the PA and TA decreased from the start of shock. Different blood vessels lost vascular reactivity at different rates compared with controls; the LFA had the highest rate of loss (64.51%), followed by the SMA (44.69%), TA (36.06%), PA (37.83%), and RRA (32.33%), whereas the MCA had the lowest rate (18.45%). The rate of loss of vascular reactivity in the different vessels was negatively correlated with A2AR expression levels in normal and shock conditions. The highly selective A2AR agonist CGS 21680 significantly improved vascular reactivity, hemodynamic parameters, and animal survival, whereas the specific antagonist SCH58261 further decreased the shock-induced reduction in vascular reactivity and hemodynamic parameters. ConclusionsA2ARs are involved in the regulation and protection of vascular reactivity following shock. A2AR activation may have a beneficial effect on hemorrhagic shock by improving vascular reactivity and hemodynamic parameters.