Agonist CGS Research Articles

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

A2A adenosine receptor stimulation regenerates cartilage in osteoarthritis animal model

Purpose: Many studies have been shown that obesity along with joint injury is one of the most common risk factor in the development of osteoarthritis (OA). In a previous study we described that intra-articular injections of liposomal preparations of adenosine completely prevent progression and reverse cartilage loss in post-traumatic OA. TGF-β signaling plays dual and opposing roles in cartilage health and chondrocyte life depending on the signals activated downstream. Activation of downstream signaling pathways for TGF-β leading to localization of phospho-SMAD2/3 associated with maintenance of cartilage. In contrast nuclear localization of phospho-SMAD1/5/8 results in chondrocyte hypertrophy. Here we report that intraarticular injections of liposomal adenosine and A2AR agonist reverses OA in a post-traumatic OA model in rat and in an obesity related mice model. We moreover explore the role of TGF-β signaling in this phenomenon. Methods: Obesity-induced OA model: C57Bl6 mice (5-6 for each group, 12 weeks old) were fed a 60% fat diet (HFF mice) for 3months, after which received intraarticular injections (10 μl) of empty liposomes (Lipo) or liposomes containing the A2AR agonist CGS21680 (Lipo-CGS) or Adenosine (Lipo-Ado) into the knee every 10 days for 4 injections. Post-traumatic OA (PTOA) was induced in Sprague Dawley rats following non-surgical rupture of anterior cruciate ligament (ACL). Four weeks later rats were injected in the knee with 100ul of saline, Lipo or Lipo-CGS every 10 days (6 injections). RNA was isolated from chondrocytes in knee cartilage of rats treated as described above (3 from each group X 3 replicates) and subjected to RNAseq analysis. TC28a2 human chondrocyte cell line was used for in vitro experiments. Results: Lipo-CGS and Lipo-Ado reversed OA in the obesity and post traumatic OA model. Mouse knees had an OARSI score of 5.17±1.84 before treatment. Treatment with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33±0.81 and 1.83±0.98, respectively, p<0.001 vs pre-treatment; figure 1). In the PTOA model the OARSI score significantly decreases after Lipo-CGS and Lipo-Ado treatment compare to the saline group (OARSI: 1.28±0.39; 1.42±0.69; 2.97±0.0.75 respectively; p<0.05). Analysis of the transcriptome suggests that the treatment of Lipo-CGS promotes the up-regulation of genes involved in proliferation process and downregulations of genes responsible of apoptosis, cartilage catabolism and chondrocyte hypertrophy (Figure 2). TGF-β expression was increased in deep layers of cartilage in the Lipo-CGS-treated rats and there was notable nuclear localization of phospho-SMAD2/3 in these chondrocytes. In contrast, phospho-SMAD1/5/8 was expressed in the nuclei of chondrocytes in the saline and LIPO-treated rats but not in the LIPO-CGS treated rats. Identical changes were observed in the knees of obese mice. To determine whether the effect of A2AR stimulation on TGF-β signaling was direct or indirect we studied the effect of CGS21680 on nuclear phospho-SMAD expression in TC28a2 cells and found that CGS21680 increased nuclear phospho-SMAD2/3 and reduced nuclear phospho-SMAD1/5/8, as detected by immunofluorescence. Conclusions: Administration of an A2AR agonist to established OA knees reverses OA in rats and mice and shifts TGF-β signaling from ALK1/SMAD1/5/8 to ALK5/SMAD2/3 in OA chondrocytes after activation of A2AR in 2 OA animal models.

Adenosine 2a Receptor Signal Blockade of Murine Autoimmune Arthritis via Inhibition of Pathogenic Germinal Center-Follicular Helper T Cells.

CD4 germinal center (GC)-follicular helper T (Tfh) cells are important in the pathogenesis of autoimmune arthritis. Previous studies have shown that adenosine 2a receptor (A2aR; Adora2a) signaling can divert CD4 T cells away from the GC-Tfh cell lineage during the primary response to foreign antigens. This study was undertaken to examine the effects of A2aR signaling on CD4 T cells during the recognition of self antigen in a murine model of autoimmune arthritis. Wild-type and Adora2a-deficient mouse KRN T cell receptor-transgenic CD4 T cells specific for glucose-6-phosphate isomerase (GPI)/I-Ag7 were transferred into immunodeficient Tcra-/- I-Ag7 -expressing mice to induce arthritis. Recipients were then treated with either the selective A2aR agonist CGS-21680 (CGS) or phosphate buffered saline alone. Severity of disease, autoantibody titers, KRN T cell numbers and phenotype, and GPI-specific isotype class-switched plasmablasts were tracked. CGS treatment inhibited the development of arthritis and differentiation of KRN GC-Tfh cells, blocked the appearance of high-affinity GPI-specific and IgG1 isotype class-switched polyclonal plasmablasts, and led to a reduction in serum titers of anti-GPI IgG1. In addition, therapeutic administration of CGS after the onset of arthritis blocked further disease progression in association with reductions in the number of KRN GC-Tfh cells and anti-GPI IgG1 serum titers. Strong A2aR signaling diverts autoreactive CD4 T cell differentiation away from the GC-Tfh cell lineage, thus reducing help for the differentiation of dangerous autoreactive B cells that promote arthritis. These data in a mouse model of autoimmune arthritis suggest that A2aR and its downstream signaling pathways in CD4 T cells may be promising therapeutic targets for interfering with potentially dangerous autoreactive GC-Tfh cell differentiation.

Abstract P4-06-19: The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines

Abstract Introduction: Trastuzumab (T) is a monoclonal antibody therapy used in the treatment of HER2+ breast cancer. T inhibits HER2 intracellular signalling and is capable of engaging the immune system through ADCC. Adenosine is an important negative regulator of the immune response through its interaction with the A2A receptor (A2AR, ADORA2A). Relieving adenosine-mediated immunosuppression by inhibiting A2AR may improve NK cell-mediated T-ADCC against HER2+ breast cancer cells. In addition, we have previously shown that SKBR3 cells resistant to the EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib are less sensitive to T-ADCC and showed increased A2AR protein levels. This study examines the effects of inhibiting A2AR signalling on NK cell-mediated T-ADCC against treatment naïve HER2+ breast cancer cell lines HCC1954 and SKBR3 and lapatinib and afatinib (irreversible pan-HER-family TKI)-resistant sublines of HCC1954 and SKBR3. Methods: HER2+ breast cancer cell lines SKBR3 and HCC1954 were exposed to afatinib (150nM) or lapatinib (1μM) for 6 months to generate TKI-resistant SKBR3-A and HCC1954-L cell lines. Acid-phosphatase-based proliferation assays were used to confirm resistance to TKI treatment. Western blotting was used to examine A2AR and HER2 protein levels in cell lines. NK cells were isolated from healthy volunteer whole blood by MACSxpress isolation kits. Immune cell-mediated cytotoxicity was determined at a 1:1 (NK cell: TC) ratio over 12 hours using a flow cytometry-based method. Direct cytotoxicity and T-ADCC were determined +/- A2AR agonist CGS21680 (1 μM) and/or A2AR antagonist preladenant (100 nM) for all cell lines. Experiments were carried out three times with three separate volunteer samples with representative results presented. Results: HCC1954-L cells were 5.3-fold resistance to lapatinib (IC50 1.65 μM +/- 0.22 μM) vs. HCC1954 (IC50 0.31 μM +/- 0.15 μM). SKBR3-A cells were 33-fold resistant to afatinib (IC50 0.28 μM +/- 0.006 nM) vs. the parental SKBR3 cell line (IC50 0.009 μM +/- 0.006 μM). SKBR3 and HCC1954 expressed detectable protein levels of A2AR. A2AR and HER2 levels were not significantly changed between parental and resistant cell lines. Levels of direct cytotoxicity and T-ADCC elicited by NK cells were higher against SKBR3-A (p=0.002) and HCC1954-L cells (p=0.0004) than parental cell lines. The A2AR agonist CGS21680 alone had inconsistent effects on direct cytotoxicity and T-ADCC in all cell lines tested. The addition of A2AR antagonist preladenant to CGS21680, but not preladenant alone, increased T-ADCC against the parental HCC1954 cells by 12.7 +/- 3.4% and parental SKBR3 cells by 9.5 +/- 3.6%. T-ADCC levels in the targeted therapy-resistant HCC1954-L and SKBR3-A cell lines were not impacted by the CGS21680/preladenant combination. Conclusions: A HER2-targeted therapy resistance phenotype is associated with increased T-ADCC in the models tested. Inhibition of activated A2AR can increase T-ADCC elicited by NK cells against treatment naïve HER2+ breast cancer cell lines but not TKI-resistant sublines. Further work is warranted to examine the impact of targeting A2AR in HER2+ breast cancer. Citation Format: Gaynor N, Noone J, Monedero J, Murphy EE, O'Gorman DJ, Crown J, Collins DM. The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-19.

Attenuation of adverse effects of noise induced hearing loss on adult neurogenesis and memory in rats by intervention with Adenosine A2A receptor agonist

Hearing loss and cognitive decline are commonly associated with aging and morbidity. Present clinical interest lies in whether peripheral hearing loss promotes cognitive decline and if prophylaxis with selective adenosine receptor agonist CGS21680 effectively mitigates the adverse effects. In the current study, male Sprague Dawley rats weighing 200–250 g m were randomly allocated into three groups: Group 1) rats exposed to 100 dB SPL white noise, 2 h a day for 15 consecutive days, 2) rats supplemented with an adenosine receptor agonist, CGS21680 at 100 μg/kg/day prior to noise exposure and 3) unexposed control rats. Baseline hearing and cognition assessed by auditory brainstem response (ABR) and water maze respectively was undertaken for all the groups. Phalloidin stain and synaptic ribbons count in cochlea, and, Ki67, DCX and NeuN in hippocampus were observed by immunohistochemistry. It was inferred that noise exposed rats showed elevated thresholds of ABR and poorer performances in spatial working memory when compared with controls. On the contrary, CGS21680 administered group exhibited improved ABR and cognitive functions with shorter mean latency and path-length to reach the platform, significant reduction in the noise induced loss of synaptic ribbons count and increased number of Ki67 and doublecortin (DCX) positive cells compared to their noise exposed counterparts. Pharmacologic intervention with selective A2A receptor agonist CGS21680 provided adequate protection from noise by effectively maintaining hearing threshold levels, cell viability in cochlea and hippocampus & functional/intact reference memory.

The Role of CD39/CD73/Ado/A2AR Axis and HIF-1α in Chronic Lymphocytic Leukemia

Introduction: CD39/CD73/ADO/A2A system plays important roles in tumor growth and therapy. Extracellular adenosine (ADO) receptor A2A (A2AR) is the dominant receptor expressed on chronic lymphocytic leukemia (CLL) cells. ADO has been shown to protect CLL cells from spontaneous and drug-induced apoptosis through A2AR activation. Overexpression of hypoxia inducible factor-1α (HIF-1α), an important mediator controlling the expression of a wide variety of apoptotic genes, has been observed in bone marrow leukemic cells from CLL patients. However, the reciprocal action of A2AR and HIF-1α in CLL remains elusive. This study was aimed to explore the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α in CLL.Materials and Methods: Peripheral blood (PB) and bone marrow (BM) samples were collected from 30 healthy volunteers (control) and 20 patients who were diagnosed with CLL for the first time at the Hematology Department in our hospital. CD39/CD73/ADO/A2AR axis-related protein and HIF-1α protein expressions in CLL PB and BM tissues were examined by Western-blot. CD39, CD73, A2AR and HIF-1α expression on CLL cells were also determined by FACS. CLL cell line (Lym-2) was purchased from ATCC and incubated in DMEM medium supplemented with 10% FBS. The protein expressions of CD39, CD73 and A2AR on Lym-2 cells were confirmed by Western-blot. To study the impacts of A2AR activation and inactivation on CLL cells, CLL cells were treated with A2AR agonist CGS21680 or antagonist SCH58261, followed by determination of cell proliferation and HIF-1α protein expression.Results: The protein expressions of CD39, CD73, A2AR and HIF-1α were higher in CLL group than those in control group (BM: CD39 1.471 vs 0.926, CD73 1.097 vs 0.489, A2AR 1.139 vs 0.342 and HIF-1α 0.940 vs 0.362, P<0.05 Figure 1A; PB : CD39 1.809 vs 1.331, CD73 1.039 vs 0.653, A2AR 1.738 vs 1.119 and HIF-1α 1.336 vs 1.010, P<0.05 Figure 1B). Moreover, CD73 and HIF-1α protein expression was found to have relationship with disease stage. Patients who belonged to stage IV (Rai) and stage B/C (Binet) exhibited higher levels of CD73 and HIF-1α than patients who belonged to stage I to III (Rai) and stage A (Binet) . Data from FACS analysis showed that the proportion of CD39+CLL cells was higher than CD73+CLL cells and HIF-1α+CLL cells (P<0.05, Figure 2A). When compared to control group, the proportions of CD39+cells and CD73+ cells were significant higher in CLL group (Figure 2B). Western-blot results showed that Lym-2 cells expressed CD39, CD73 and A2AR simultaneously (Figure 3A). The results from CLL cells and A2AR agonist/antagonist incubation showed that when CGS21680 concentration was of ≥ 10uM, cell proliferation was promoted obviously (Figure 3B). On the contrary, SCH58261 could inhibit CLL cells proliferation when its concentration was of ≥5uM at a concentration dependent manner (Figure 3C). Moreover, HIF-1α protein expression was also affected by A2AR agonist at a concentration dependent manner(0uM 0.851, 10uM 1.116, 20uM 1.420, 50uM 1.41, Figure 3D).Conclusion: Our study showed that ADO produced by CD39 and CD73 could affect CLL cells proliferation and HIF-1α expression through A2AR-mediated mechanisms. The exploration of the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α could help us to find a novel approach to CLL therapy.Disclosures: No relevant conflicts of interest to declare. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

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.

Modulation of Microglial Activation by Adenosine A2a Receptor in Animal Models of Perinatal Brain Injury.

Neuroinflammation has a key role in the pathogenesis of perinatal brain injury. Caffeine, a nonspecific antagonist of adenosine receptors (ARs), is widely used to treat apnea of prematurity and has been linked to a decrease in the incidence of cerebral palsy in premature infants. The mechanisms explaining its neuroprotective effect have not yet been elucidated. The objective of this study was to characterize the expression of adenosine and ARs in two neonatal rat models of neuroinflammation and to determine the effect of A2aR blockade on microglial activation assessed through inflammatory cytokine gene expression. We have used two rat models of microglial activation: the gestational low protein diet (LPD) model, associated with chronic brain injury, and postnatal ibotenate intracerebral injections, responsible for acute excitotoxicity injury. Adenosine blood levels have been measured by Tandem Mass Spectrometry. The expression of ARs in vivo was assessed using qPCR and immunohistochemistry. In vivo models have been replicated in vitro on primary microglial cell cultures exposed to A2aR agonist CGS-21680 or antagonist SCH-58261. The effects of these treatments have been assessed on the M1/M2 cytokine expressions measured by RT-qPCR. LPD during pregnancy was associated with higher adenosine levels in pups at postnatal day 1 and 4. A2aR mRNA expression was significantly increased in both cortex and magnetically sorted microglial cells from LPD animals compared to controls. CD73 expression, responsible for extracellular production of brain adenosine, was significantly increased in LPD cortex and sorted microglia cells. Moreover, CD73 protein level was increased in ibotenate treated animals. In vitro experiments confirmed that LPD or control microglial cells exposed to ibotenate display an increased expression, at both protein and molecular levels, of A2aR and M1 markers (IL-1β, IL-6, iNOS, TNFα). This pro-inflammatory profile was significantly reduced by SCH-58261, which reduces M1 markers in both LPD and ibotenate-exposed cells, with no effect on control cells. In the same experimental conditions, a partial increased of M1 cytokines was observed in response to A2aR agonist CGS-21680. These results support the involvement of adenosine and particularly of its receptor A2aR in the regulation of microglia in two different animal models of neuroinflammation.

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.

Adenosine activates Gαs proteins and inhibits C3a-induced activation of human mast cells

Anaphylatoxin C3a and adenosine receptors (AR) are implicated in the inflammatory process associated with allergic rhinitis and asthma by modifying mast cell (MC) responses. Possible interactions between these G-protein coupled receptor (GPCR) pathways in MCs have not yet been demonstrated. LAD2 human MC were stimulated with C3a in the presence or absence of AR agonists and antagonists and their adhesion, chemotaxis and mediator release were measured. The pan-specific AR agonist, 5′-N-Ethylcarboxamidoadenosine (NECA) inhibited C3a-induced LAD2 cell migration, adhesion, degranulation, production of CCL2, and ERK1/2 phosphorylation. The selective A2A receptor agonist CGS 21680 inhibited C3a-mediated degranulation, while the A2B and A3 receptor agonists BAY 60-6583 and IB-MECA, respectively, had no effect. Moreover, an A2A receptor antagonist SCH 58261 blocked the inhibitory effect of NECA on C3a-induced degranulation, suggesting that inhibition of degranulation was mediated through the A2A receptor. NECA increased intracellular cAMP in C3a-activated mast cells, suggesting that Gαs protein signals are required for adenosine-induced inhibition of C3a-mediated human mast cell activation. The adenylyl cyclase inhibitor SQ 22536 attenuated the inhibitory effect of NECA on C3a-activated degranulation, and the A2A agonist CSG 21680 potentiated the inhibition of mast cell activation mediated by the A2A receptor. Our results suggest that adenosine inhibits C3a-mediated activation of human mast cells, possibly through a Gαs protein-dependent pathway.

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Adenosine A(2A) receptor activation reverses hypoxia‑induced rat pulmonary artery smooth muscle cell proliferation via cyclic AMP‑mediated inhibition of the SDF1‑CXC4 signaling pathway.

The occurrence and the subsequent development of pulmonary arterial hypertension (PAH) involve complicated mechanisms. Of these, the proliferation of pulmonary artery smooth muscle cells (PASMCs) has been indicated to be closely associated with its progression. Therefore, therapeutic methods targeting PASMCs to inhibit proliferation is an effective method for alleviating PAH. The present study was designed to determine the role of the adenosine A(2A) receptor (A2A receptor) in hypoxia‑induced rat PASMC (RPASMC) proliferation. Primary RPASMCs were isolated from the pulmonary artery of adult male SD rats, cultured and used for the following experiments. The mRNA level and protein expression of CXCR4 were measured by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. The cell proliferation of RPASMCs was measured using a cell proliferation assay kit. In the present study, it was demonstrated that the proliferation of RPASMCs was partially mediated by activation of the stromal cell‑derived factor 1 (SDF1)‑CXC chemokine receptor 4 (CXCR4) axis under hypoxic conditions. In addition, SDF1‑α alone upregulated the mRNA and protein expression levels of CXCR4, and stimulated the proliferation of RPASMCs. The protein expression of CXCR4 and the cell proliferation were markedly inhibited by application of A2A receptor agonist CGS21680 or cyclic adenosine monophosphate(cAMP) under hypoxic conditions or treatment with SDF1‑α and was reversed by the A2A receptor antagonist SCH58261 or 8‑bromoadenosine‑3',5'‑cyclic monophosphorothioate. These results demonstrated that the inhibition of SDF1‑CXC4 signaling by the activation of A2A receptor and subsequent increase in the level of cAMP may be a potential method to ameliorate PAH.

Limonene‐induced Activation of A2A Adenosine Receptors Reduces Airway Inflammation and Reactivity in a Mouse Model of Asthma

Animal models of asthma have shown that limonene (terpene in citrus fruits) reduces inflammation and airway reactivity. However, the mechanism of these effects is unknown. We hypothesized that limonene activates A2A adenosine receptors (A2AAR) to produce its effects in asthma, after comparing Glide scores for limonene (−5.19) and A2AAR agonist CGS 21680 (−8.43). We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) as well NECA (nonselective adenosine analog) 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 20 μg 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). Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Airway responsiveness to MCh (48 mg/ml) in WT SEN group was significantly lowered with limonene treatment (420±92.57% in SEN vs. 56.23±25% in SEN+LIM). However, limonene did not have an effect in A2AKO (553±85% in SEN vs. 588±34% in SEN+LIM). NECA‐induced increased airway responsiveness was attenuated in limonene‐treated WT SEN mice (1002±161 in SEN vs. 250±226%, p&lt;0.05) while no effect was observed in A2AKO groups (541±44% in SEN vs. 528±147% in SEN+LIM). 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&lt;0.05) while no difference was observed in A2AKO (76.33±1.35% in SEN vs 79±0.69% in SEN+LIM). These data suggest that limonene‐induced reduction in airway inflammation and airway reactivity occurs via activation of A2AAR.Support or Funding InformationLong Island University start‐up funding (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of adenosine A2A or A2B receptors causes hypothermia in mice

Extracellular adenosine is a danger/injury signal that initiates protective physiology, such as hypothermia. Adenosine has been shown to trigger hypothermia via agonism at A1 and A3 adenosine receptors (A1AR, A3AR). Here, we find that adenosine continues to elicit hypothermia in mice null for A1AR and A3AR and investigated the effect of agonism at A2AAR or A2BAR. The poorly brain penetrant A2AAR agonists CGS-21680 and PSB-0777 caused hypothermia, which was not seen in mice lacking A2AAR. MRS7352, a likely non-brain penetrant A2AAR antagonist, inhibited PSB-0777 hypothermia. While vasodilation is probably a contributory mechanism, A2AAR agonism also caused hypometabolism, indicating that vasodilation is not the sole mechanism. The A2BAR agonist BAY60-6583 elicited hypothermia, which was lost in mice null for A2BAR. Low intracerebroventricular doses of BAY60-6583 also caused hypothermia, indicating a brain site of action, with neuronal activation in the preoptic area and paraventricular nucleus of the hypothalamus. Thus, agonism at any one of the canonical adenosine receptors, A1AR, A2AAR, A2BAR, or A3AR, can cause hypothermia. This four-fold redundancy in adenosine-mediated initiation of hypothermia may reflect the centrality of hypothermia as a protective response.

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

Although adenosine 2A (A2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A2A receptor siRNA injections. A2A receptor siRNA injections selectively knocked down A2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced phrenic motor facilitation. Upregulation of A2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A2A receptors for pMF are unknown. This is an important question since the physiological outcome of A2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89±2%). Intrapleural siRNA injections were used to selectively knock down A2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A2A receptor knock-down was verified by a ∼45% decrease in A2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P<0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A2A receptor agonist CGS21680 was greatly attenuated in siA2A (21%) versus siNT treated rats (147%; P<0.01). There were no significant effects of siA2A on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced pMF.

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.

Activation of A1 and A2a adenosine receptors promotes neural progenitor cell proliferation

Neural progenitor cells (NPCs) play a key role not only in the maintenance of the adult central nervous system (CNS) but also in the ability to recover from injury and disease. In this study, we established a 96-well-based screening system to screen small molecules modulating the proliferation of NPCs. A compound library composed of 1280 compounds was screened. We found that the A1 adenosine receptor agonist cyclopentyladenosine (CPA) and the A2a adenosine receptor agonist CGS-21680 increased proliferation of NPCs. The A1 adenosine receptor agonist-induced cell proliferation was attenuated by A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPA). Accordingly, the A2a adenosine receptor agonist-induced cell proliferation was attenuated by A2a adenosine receptor antagonist SCH-58261. Further study indicated that CPA and CGS-21680 treatment induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and CPA-induced or CGS-21680-induced cell proliferation was inhibited by ERK and Akt inhibitors. These results suggested that the activation of A1 and A2a adenosine receptor stimulated the proliferation of NPCs via the ERK and Akt signaling pathways.

Disruption of A2AR-D2R Heteroreceptor Complexes After A2AR Transmembrane 5 Peptide Administration Enhances Cocaine Self-Administration in Rats

Antagonistic allosteric A2AR-D2R receptor-receptor interactions in heteroreceptor complexes counteract cocaine self-administration and cocaine seeking in rats as seen in biochemical and behavioral experiments. It was shown that the human A2AR transmembrane five (TM5) was part of the interface of the human A2AR-D2R receptor heteromer. In the current paper, the rat A2AR synthetic TM5 (synthTM5) peptide disrupts the A2AR-D2R heteroreceptor complex in HEK293 cells as shown by the bioluminescence resonance energy transfer method. Rat A2AR synthTM5 peptide, microinjected into the nucleus accumbens, produced a complete counteraction of the inhibitory effects of the A2AR agonist CGS21680 on cocaine self-administration. It was linked to a disappearance of the accumbal A2AR-D2R heteroreceptor complexes and the A2AR agonist induced inhibition of D2R recognition using proximity ligation assay and biochemical binding techniques. However, possible effects of the A2AR synthTM5 peptide on accumbal A2AR-D3R and A2AR-D4R heteroreceptor complexes remain to be excluded. Evidence is provided that accumbal A2AR-D2R-like heteroreceptor complexes with their antagonistic receptor-receptor interactions can be major targets for treatment of cocaine use disorder.