A2B Receptor Activation Research Articles

Adenosine A2B receptor activation regulates the balance between T helper 17 cells and regulatory T cells, and inhibits regulatory T cells exhaustion in experimental autoimmune myositis.

Idiopathic inflammatory myopathy (IIM) is a systemic autoimmune disease characterized by skeletal muscle involvement. This study aimed to investigate the role of adenosine receptor signalling pathways in the development of experimental autoimmune myositis (EAM). An ecto-5'-nucleotidase (CD73) inhibitor, adenosine receptor agonists, a hypoxia-inducible factor-1α (HIF-1α) inhibitor or a vehicle were administered to control and EAM mice. Murine splenic CD4+ or regulatory T cells (Tregs) were isolated using magnetic beads and subsequently stimulated with an adenosine A2B receptor agonist, a HIF-1α inhibitor, or vehicle in vitro. In cross-sectional studies, we collected 64 serum samples (69% female, 49±9years), 63 peripheral blood samples (70% female, 50±11years), and 34 skeletal muscle samples (71% female, 63±6years) from patients with IIM. Additionally, 35 serum samples and 30 peripheral blood samples were obtained from age- and sex-matched healthy controls, and six quadriceps muscle samples were collected from patients with osteoarthritis to serve as the normal group. Patients with IIM exhibited increased CD73 [dermatomyositis (DM), polymyositis (PM): P<0.01; immune-mediated necrotizing myopathy (IMNM): P<0.0001] and adenosine deaminase (ADA) expression (DM: P<0.001; PM, IMNM: P<0.0001) in the skeletal muscles, and serum ADA levels [56.7 (95% CI: 53.7, 58.7) vs. 198.8 (95% CI: 186.2, 237.3) ng/μL, P<0.0001]. Intervention with a CD73 inhibitor exacerbated (P=0.0461), whereas adenosine receptor agonists (A1: P=0.0009; A2B: P<0.0001; A3: P=0.0001) and the HIF-1α inhibitor (P=0.0044) alleviated skeletal muscle injury in EAM mice. Elevated expression of programmed cell death protein-1 (PD1: P=0.0023) and T-cell immunoglobulin and mucin-domain containing-3 (TIM3: P<0.0001) in skeletal muscles of patients with IIM were correlated with creatine kinase levels (PD1, r=0.7072, P<0.0001; TIM3, r=0.4808, P=0.0046). PD1+CD4+ (r=0.3243, P=0.0115) and PD1+CD8+ (r=0.3959, P=0.0017) T cells were correlated with Myositis Disease Activity Assessment Visual Analogue Scale scores (muscle) in IIM. The exhausted Tregs were identified in the skeletal muscles of patients with IIM. Activation of the A2B adenosine receptor downregulated HIF-1α (protein or mRNA level, P<0.01), resulting in decreased T helper cell 17 (Th17) (13.58% vs. 5.43%, P=0.0201) and phosphorylated-signal transducer and activator of transcription 3 (p-STAT3)+ Th17 (16.32% vs. 6.73%, P=0.0029), decreased exhausted Tregs (PD1+ Tregs: 53.55% vs. 40.28%, P=0.0005; TIM3+ Tregs: 3.93% vs. 3.11%, P=0.0029), and increased Tregs (0.45% vs. 2.89%, P=0.0006) in EAM mice. The exhausted T cells may be pathogenic in IIM,andthe activation of adenosine A2B receptor signalling pathway can regulate Th17/Treg balance and inhibit Tregsexhaustion, thereby slowing EAM disease progression.

Sustained adenosine release: Revealing its impact on osteogenic signalling pathways of human mesenchymal stromal cells

Non-healing fractures, a global health concern arising from trauma, osteoporosis, and tumours, can lead to severe disabilities. Adenosine, integral to cellular energy metabolism, gains prominence in bone regeneration via adenosine A2B receptor activation. This study introduces a controlled-release system for localized adenosine delivery, fostering human mesenchymal stromal cell (hMSC) differentiation into functional bone cells. The study investigates how the ratio of lactic acid to glycolic acid in microparticles can influence adenosine release and explores the downstream effects on gene expression and metabolic profiles of osteogenic differentiation in hMSCs cultured in growth and osteoinductive media. Insights into adenosine-modulated signalling pathways during MSC differentiation, with osteogenic factors, provide a comprehensive understanding of the pathways involved. Analysing gene expression and metabolic profiles unravels adenosine's regulatory mechanisms in MSC differentiation. Sustained adenosine release from microparticles induces mineralization, synergizing with osteogenic media supplements, showcasing the potential of adenosine for treating critical bone defects and metabolic disorders. This study highlights the efficacy of a polymeric microparticle-based delivery system, offering novel strategies for bone repair. Unveiling adenosine's roles and associated signalling pathways advances our comprehension of molecular mechanisms steering bone regeneration, propelling innovative biomaterial, combined with metabolites, approaches for clinical use.

Involvement of Cannabinoid Receptors and Adenosine A2B Receptor in Enhanced Migration of Lung Cancer A549 Cells Induced by γ-Ray Irradiation.

Residual cancer cells after radiation therapy may acquire malignant phenotypes such as enhanced motility and migration ability, and therefore it is important to identify targets for preventing radiation-induced malignancy in order to increase the effectiveness of radiotherapy. G-Protein-coupled receptors (GPCRs) such as adenosine A2B receptor and cannabinoid receptors (CB1, CB2, and GPR55) may be involved, as they are known to have roles in proliferation, invasion, migration and tumor growth. In this study, we investigated the involvement of A2B and cannabinoid receptors in γ-radiation-induced enhancement of cell migration and actin remodeling, as well as the involvement of cannabinoid receptors in cell migration enhancement via activation of A2B receptor in human lung cancer A549 cells. Antagonists or knockdown of A2B, CB1, CB2, or GPR55 receptor suppressed γ-radiation-induced cell migration and actin remodeling. Furthermore, BAY60-6583 (an A2B receptor-specific agonist) enhanced cell migration and actin remodeling in A549 cells, and this enhancement was suppressed by antagonists or knockdown of CB2 or GPR55, though not CB1 receptor. Our results indicate that A2B receptors and cannabinoid CB1, CB2, and GPR55 receptors all contribute to γ-radiation-induced acquisition of malignant phenotypes, and in particular that interactions of A2B receptor and cannabinoid CB2 and GPR55 receptors play a role in promoting cell migration and actin remodeling. A2B receptor-cannabinoid receptor pathways may be promising targets for blocking the appearance of malignant phenotypes during radiotherapy of lung cancer.

8-Aminopurines in the Cardiovascular and Renal Systems and Beyond.

Screening of compounds comprising 8-substituted guanine revealed that 8-aminoguanosine and 8-aminoguanine cause diuresis/natriuresis/glucosuria, yet decrease potassium excretion. Subsequent investigations demonstrated that 8-aminoguanosine's effects are mediated by its metabolite 8-aminoguanine. The mechanism by which 8-aminoguanine causes diuresis/natriuresis/glucosuria involves inhibition of PNPase (purine nucleoside phosphorylase), which increases renal interstitial inosine levels. Additional evidence suggests that inosine, via indirect or direct adenosine A2B receptor activation, increases renal medullary blood flow which enhances renal excretory function. Likely, 8-aminoguanine has pleiotropic actions that also alter renal excretory function. Indeed, the antikaliuretic effects of 8-aminoguanine are independent of PNPase inhibition. 8-Aminoguanine is an endogenous molecule; nitrosative stress leads to production of biomolecules containing 8-nitroguanine moieties. Degradation of these biomolecules releases 8-nitroguanosine and 8-nitro-2'-deoxyguanosine which are converted to 8-aminoguanine. Also, guanosine and guanine per se may contribute to 8-aminoguanine formation. 8-Aminoinosine, 8-aminohypoxanthine, and 8-aminoxanthine likewise induce diuresis/natriuresis/glucosuria, yet do not reduce potassium excretion. Thus, there are several pharmacologically active 8-aminopurines with nuanced effects on renal excretory function. Chronic treatment with 8-aminoguanine attenuates hypertension in deoxycorticosterone/salt rats, prevents strokes, and increases lifespan in Dahl salt-sensitive rats on a high salt diet and attenuates the metabolic syndrome in rats; 8-aminoguanosine retards progression of pulmonary hypertension in rats and anemia and organ damage in sickle cell mice. 8-Aminoguanine reverses age-associated lower urinary tract dysfunction and retinal degeneration. 8-Aminopurines represent a new class of agents (and potentially endogenous factors) that have beneficial effects on the cardiovascular system and kidneys and may turn back the clock in age-associated diseases.

FUNDC1: a key mediator of adenosine A2BR activation-induced inhibition of cardiac mitophagy under ischemia/reperfusion conditions.

Mitophagy is an essential factor in mitochondrial quality control and myocardial ischaemia/reperfusion (I/R) injury protection. Because adenosine A2B receptor (A2BR) activation exerts a major role in reducing myocardial I/R injury, the effects of adenosine A2BR activation on cardiac mitophagy under reperfusion conditions were investigated. 110 adult Wistar rats (7-10 w), weighing 250-350 grams, were cultured in specific-pathogen-free (SPF) conditions before experiments. All hearts were removed and reperfused by Langendorff device. Six hearts with coronary flow (CF) values >28 or <10 mL/min were excluded. Others were arbitrarily divided into the following groups: sham operation group, I/R group, BAY60-6583 (BAY) (1-1,000 nM) + I/R group, PP2 + BAY + I/R group. After ischemia in rats, reperfusion was performed. H9c2 cells were placed in an imitated ischemic environment followed by Tyrode's solution to stimulate hypoxia/reoxygenation (H/R) injury. The mitochondrial fluorescence indicator MitoTracker Green and lysosomal fluorescence indicator LysoTracker Red were used to examine mitochondria and lysosomes, respectively. Colocalization of mitochondrial and autophagy marker proteins was determined by immunofluorescence. Autophagic flow currents were tested by Ad-mCherry-GFP-LC3B. Protein-protein interactions were predicted using a database and analyzed by co-immunoprecipitation. Autophagy marker protein, mitophagy marker protein, and mitophagy protein FUNDC1 were detected by immunoblotting. Compared with those in the I/R group, myocardial autophagy and mitophagy were suppressed by the selective adenosine A2BR agonist BAY, and this effect was inhibited by the selective Src tyrosine kinase inhibitor PP2, indicating that adenosine A2BR activation could inhibit myocardial autophagy and mitophagy by activating Src tyrosine kinase. In support, in H9c2 cells, the selective Src tyrosine kinase inhibitor PP2 inhibited the effect of BAY on TOM20 with LC3 or mitochondria with lysosomes colocalization and autophagy flow. Here, we showed that mitochondrial FUNDC1 co-precipitated with Src tyrosine kinase after BAY was added. Consistently, the immunofluorescence and western blotting results demonstrated that compared to that in the H/R group, the expression of mitochondrial FUNDC1 was reduced by BAY, but this effect was reversed by PP2. Adenosine A2BR activation may inhibit myocardial mitophagy by downregulating expression of the mitochondrial FUNDC1 by activating Src tyrosine kinase under I/R conditions and could increase the interaction between Src tyrosine kinase and FUNDC1.

8-Aminoguanine and Its Actions on Renal Excretory Function.

The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Normoxic induction of HIF-1α by adenosine-A2B R signaling in epicardial stromal cells formed after myocardial infarction.

Myocardial infarction (MI) activates the epicardium to form epicardial stromal cells (EpiSC) that reside in the epicardial hypoxic microenvironment. Paracrine factors secreted by EpiSC were shown to modulate the injury response of the post-MI heart and improve cardiac function. We have previously reported that the expression of the angiogenic cytokines vascular endothelial growth factor A (VEGFA) and IL-6 is strongly upregulated in EpiSC by adenosine acting via the A2B receptor (A2B R). Since tissue hypoxia is well known to be a potent stimulus for the generation of extracellular adenosine, the present study explored the crosstalk of A2B R activation and hypoxia-hypoxia-inducible factor 1 alpha (HIF-1α) signaling in cultured EpiSC, isolated from rat hearts 5days after MI. We found substantial nuclear accumulation of HIF-1α after A2B R activation even in the absence of hypoxia. This normoxic HIF-1α induction was PKC-dependent and involved upregulation of HIF-1α mRNA expression. While the influence of hypoxia on adenosine generation and A2B R signaling was only minor, hypoxia and A2B R activation cumulatively increased VEGFA expression. Normoxic A2B R activation triggered an HIF-1α-associated cell-protective metabolic switch and reduced oxygen consumption. HIF-1α targets and negative regulators PHD2 and PHD3 were only weakly induced by A2B R signaling, which may result in a sustained HIF-1α activity. The A2B R-mediated normoxic HIF-1α induction was also observed in cardiac fibroblasts from healthy mouse hearts, suggesting that this mechanism is also functional in other A2B R-expressing cell types. Altogether, we identified A2B R-mediated HIF-1α induction as novel aspect in the HIF-1α-adenosine crosstalk, which modulates EpiSC activity and can amplify HIF-1α-mediated cardioprotection.

Hyperinflammation and airway surface liquid dehydration in cystic fibrosis: purinergic system as therapeutic target.

The exacerbate inflammatory response contributes to the progressive loss of lung function in cystic fibrosis (CF), a genetic disease that affects the osmotic balance of mucus and mucociliary clearance, resulting in a microenvironment that favors infection and inflammation. The purinergic system, an extracellular signaling pathway characterized by nucleotides, enzymes and receptors, may have a protective role in the disease, through its action in airway surface liquid (ASL) and anti-inflammatory response. To make up this review, studies covering topics of CF, inflammation, ASL and purinergic system were selected from the main medical databases, such as Pubmed and ScienceDirect. We propose several ways to modulate the purinergic system as a potential therapy for CF, like inhibition of P2X7, activation of P2Y2, A2A and A2B receptors and blocking of adenosine deaminase. Among them, we postulate that the most suitable strategy is to block the action of adenosine deaminase, which culminates in the increase of Ado levels that presents anti-inflammatory actions and improves mucociliary clearance. Furthermore, it is possible to maintain the physiological levels of ATP to control the hydration of ASL. These therapies could correct the main mechanisms that contribute to the progression of CF.

Involvement of CD73 and A2B Receptor in Radiation-Induced DNA Damage Response and Cell Migration in Human Glioblastoma A172 Cells.

Glioblastoma is the most common malignant tumor of the central nervous system and is treated with a combination of surgery, radiation and chemotherapy. However, the tumor often acquires radiation resistance, which is characterized by an increased DNA damage response (DDR). Here, we show that CD73, which generates extracellular adenosine from ATP, and A2B receptor, which is activated by adenosine, are involved in the γ-radiation-induced DDR and the enhanced migration ability of human glioblastoma cell line A172. To investigate DDR, we evaluated ataxia telangiectasia mutated (ATM) activation and focus formation of histone H2A isoform γ (γH2AX) and p53-binding protein 1 (53BP1) in the nucleus of A172 cells after γ-irradiation. Antagonists of A2B receptor and CD73, or knockdown with small interfering RNA (siRNA), suppressed γ-radiation-induced DDR and promoted γ-radiation-induced cell death, as well as suppressing γ-radiation-induced cell migration and actin remodeling. These results suggest that activation of A2B receptor by extracellular adenosine generated via CD73 promotes γ-radiation-induced DDR, leading to recovery from DNA damage, and also enhances cell migration and actin remodeling. The CD73-A2B receptor pathway may be a promising target for overcoming radiation resistance and the acquisition of malignant phenotypes during radiotherapy of glioblastoma.

Extracellular Adenosine Induces IL-6 Production through Activation of A2B Receptor and Epidermal Growth Factor Receptor in Human Keratinocyte HaCaT Cells

Extracellular Adenosine Induces IL-6 Production through Activation of A2B Receptor and Epidermal Growth Factor Receptor in Human Keratinocyte HaCaT Cells

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, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A2A and A2B receptors. The pharmacological studies were carried out with A2A adenosine receptor knock-out (A2AKO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A2AKO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A2AKO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A2AKO. However, limonene reduced neutrophils in sensitized A2AKO mice, suggesting that it may activate A2B receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A2AAR but A2B receptors may also play a supporting role.

Exogenous Adenosine Antagonizes Excitatory Amino Acid Toxicity in Primary Astrocytes.

Excitatory toxicity is still a hot topic in the study of ischemic stroke, and related research has focused mainly on neurons. Adenosine is an important neuromodulator that is known as a "biosignature" in the central nervous system (CNS). The protective effect of exogenous adenosine on neurons has been confirmed, but its mechanism remains elusive. In this study, astrocytes were pretreated with adenosine, and the effects of an A2a receptor (A2aR) inhibitor (SCH58261) and A2b receptor (A2bR) inhibitor (PSB1115) on excitatory glutamate were investigated. An oxygenglucose deprivation/reoxygenation (OGD/R) and glutamate model was generated invitro. Post-model assessment includedexpression levels of glutamate transporters (glt-1), gap junction protein (Cx43) and glutamate receptor (AMPAR), Na+-K+-ATPase activity, and diffusion distance of dyes. Glutamate and glutamine contents were determined at different time points. The results showed that (1) adenosine could improve the function of Na+-K+-ATPase, upregulate the expression of glt-1, and enhance the synthesis of glutamine in astrocytes. This effect was associated with A2aR activation but not with A2bR activation. (2) Adenosine could inhibit the expression of gap junction protein (Cx43) and reduce glutamate diffusion. Inhibition of A2aR attenuated adenosine inhibition of gap junction intercellular communication (GJIC) in the OGD/R model, while it enhanced adenosine inhibition of GJIC in the glutamate model, depending on the glutamate concentration. (3) Adenosine could cause AMPAR gradually entered the nucleus from the cytoplasm, thereby reducing the expression of AMPAR on the cell membrane. Taken together, the results indicate that adenosine plays a role of anti-excitatory toxicity effect in protection against neuronal death and the functional recovery of ischemic stroke mainly by targeting astrocytes, which are closely related to A2aR. The present study provided a scientific basis for adenosine prevention and ischemic stroke treatment, thereby providing a new approach for alleviating ischemic stroke.

Adenosine A2B receptor activation stimulates alveolar fluid clearance through alveolar epithelial sodium channel via cAMP pathway in endotoxin-induced lung injury.

Clinical studies have established that the capacity of removing excess fluid from alveoli is impaired in most patients with acute respiratory distress syndrome. Impaired alveolar fluid clearance (AFC) correlates with poor outcomes. Adenosine A2B receptor (A2BAR) has the lowest affinity with adenosine among four adenosine receptors. It is documented that A2BAR can activate adenylyl cyclase (AC) resulting in elevated cAMP. Based on the understanding that cAMP is a key regulator of epithelial sodium channel (ENaC), which is the limited step in sodium transport, we hypothesized that A2BAR signaling may affect AFC in acute lung injury (ALI) through regulating ENaC via cAMP, thus attenuating pulmonary edema. To address this, we utilized pharmacological approaches to determine the role of A2BAR in AFC in rats with endotoxin-induced lung injury and further focused on the mechanisms in vitro. We observed elevated pulmonary A2BAR level in rats with ALI and the similar upregulation in alveolar epithelial cells exposed to LPS. A2BAR stimulation significantly attenuated pulmonary edema during ALI, an effect that was associated with enhanced AFC and increased ENaC expression. The regulatory effects of A2BAR on ENaC-α expression were further verified in cultured alveolar epithelial type II (ATII) cells. More importantly, activation of A2BAR dramatically increased amiloride-sensitive Na+ currents in ATII cells. Moreover, we observed that A2BAR activation stimulated cAMP accumulation, whereas the cAMP inhibitor abolished the regulatory effect of A2BAR on ENaC-α expression, suggesting that A2BAR activation regulates ENaC-α expression via cAMP-dependent mechanism. Together, these findings suggest that signaling through alveolar epithelial A2BAR promotes alveolar fluid balance during endotoxin-induced ALI by regulating ENaC via cAMP pathway, raising the hopes for treatment of pulmonary edema due to ALI.

Activation of adenosine A2A but not A2B receptors is involved in uridine adenosine tetraphosphate-induced porcine coronary smooth muscle relaxation

Activation of both adenosine A2A and A2B receptors (A2BR) contributes to coronary vasodilation. We previously demonstrated that uridine adenosine tetraphosphate (Up4A) is a novel vasodilator in the porcine coronary microcirculation, acting mainly on A2AR in smooth muscle cells (SMC). We further investigated whether activation of A2BR is involved in Up4A-mediated coronary SMC relaxation. Both A2AR and A2BR may stimulate H2O2 production leading to activation of KATP channels in SMCs, we also studied the involvement of H2O2 and KATP channels in Up4A-mediated effect. Coronary small arteries dissected from the apex of porcine hearts were mounted on wire myograph for Up4A concentration responses. Up4A-induced coronary SMC relaxation was attenuated by A2AR but not A2BR antagonism or non-selective P2R antagonism, despite greater endogenous A2BR expression vs. A2AR in both coronary small arteries and primary cultured coronary SMCs. Moreover, Up4A-induced coronary SMC relaxation was blunted by H2O2 catabolism. This effect was not altered by KATP channel blockade. Combination of H2O2 catabolism and A2AR antagonism attenuated Up4A-induced coronary SMC relaxation to the similar extent as A2AR antagonism alone. Collectively, Up4A-induced porcine coronary SMC relaxation is mediated by activation of A2AR-H2O2 pathway. This process does not involve A2BR, P2R or KATP channels.

Agonists activate different A2B adenosine receptor signaling pathways in MBA-MD-231 breast cancer cells with distinct potencies.

It is well established that some receptor types including G protein-coupled receptors may transduce effects through more than one signaling pathway. This holds also true for A2B adenosine receptors which were shown to trigger an increase in intracellular Ca2+ levels in addition to the canonical stimulation of adenylyl cyclase. We have recently shown that activation of A2B receptors in the breast cancer cell line MBA-MD-231 elicits a reduction in ERK1/2 phosphorylation, an effect that might be exploited in treatment of cancer cell growth and proliferation. In this study, we investigate whether structurally divers agonists show functional selectivity for any of the signaling pathways leading to an increase of intracellular cAMP or Ca2+, or the reduction of ERK1/2 phosphorylation. As agonists, adenosine derivatives were used bearing different substitutions in 2- and 6-position and, in addition, a ligand with a non-nucleoside structure was tested. It was found that all the tested ligands showed similar pharmacological profiles for the three responses investigated in MBA-MD-231 cells. However, the reduction of ERK1/2 phosphorylation occurred with 40-500-fold higher potency compared to stimulation of adenylyl cyclase or increasing intracellular Ca2+ levels. Based on these observations, it seems possible to utilize activation of A2B adenosine receptors expressed in certain cancers to limit cell growth and proliferation due to reduction of MAPK activity without activation of other signaling pathways potentially responsible for side effects.

Elevated cyclic-AMP represses expression of exchange protein activated by cAMP (EPAC1) by inhibiting YAP-TEAD activity and HDAC-mediated histone deacetylation

Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts.Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP.Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.

Activation of adenosine A2b receptor attenuates high glucose-induced apoptosis in H9C2 cells via PI3K/Akt signaling.

High glucose plays a vital role in apoptosis in H9C2 cells. However, the exact molecular mechanism remains unclear. In this study, we aimed to evaluate the cardio-protective role of A2b receptor in high glucose-induced cardiomyocyte apoptosis via PI3K/Akt pathway. Adenosine A2b receptor agonist (Bay506583), antagonist (MRS1754), and Akt inhibitor (LY294002) were applied respectively to H9C2 cells before exposed to high glucose for 12h. Apoptosis of H9C2 cells was determined by TUNEL assay and the apoptosis rate by flow cytometry. The protein level of adenosine A2b receptor, p-Akt, total Akt, cleaved capase-3, cleaved capase-9, bax, and bcl-2 was measured by western blotting. The results demonstrated that apoptosis of H9C2 cardiomyocytes triggered by high-glucose treatment was time-dependent. The protein level of A2b receptor and activated Akt was both decreased in cardiomyocyte with high-glucose treatment. Moreover, we found that high glucose-induced apoptosis in H9C2 cells could be attenuated by administration of adenosine A2b receptor agonist Bay606583. This effect could be reversed by Akt inhibitor LY294002. In conclusion, activation of A2b receptor could prevent high glucose-induced apoptosis of H9C2 cells in vitro to a certain extent by activating PI3K/Akt signaling. In conclusion, these results suggested that activation of A2b receptor could be a novel therapeutic approach to high glucose-induced cardiomyocyte injury.

Shockwaves Inhibit Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Association with Adenosine and A2B Receptors

Extracorporeal shockwave therapy (ESWT) has emerged as the important choice for the treatment of many orthopedic disorders. Our previous mechanistic studies suggest that ESWT promoted osteogenesis of human mesenchymal stem cells (hMSCs) through mechanisms that involve adenosine 5′-triphosphate (ATP) release. In this study, we investigated the effect of ESWT on chondrogenesis of hMSCs. We demonstrate that ESWT treatment caused a significant release of adenosine from hMSCs; ESWT treatment increased the levels of A2B receptor (A2BR) in hMSCs under 3-D culture conditions. ESWT, exogenous adenosine and specialized A2BR agonist suppressed hMSC chondrogenic differentiation through downregulating the expressions of aggrecan (ACAN), Collagen Type I alpha 2(COL1A2), Collagen Type II alpha 1(COL2A1), Sex-Determining Region YBox 9 (SOX9) and Sex-Determining Region YBox 6 (SOX6). Selective A2BR antagonists induced chondrogenic differentiation of hMSCs. This study indicated that shockwave therapy inhibits hMSC chondrogenic differentiation through or partially through regulation of adenosine release and activation of A2B receptor under 3-D culture conditions.

Inhibition of TPA-induced hemorrhagic transformation by adenosine A2b receptor activation after cerebral ischemia

Background: The thrombolytic agent tissue plasminogen activator (tPA) is underutilized in ischemic stroke, in part because of its limited therapeutic window and hemorrhagic complications. Identifying an adjuvant drug that reduces the risk of hemorrhage could increase the applicability of tPA. Studies have shown that A2b receptor (A2bR) can regulate vascular leakage reduce MMP9 expression, so A2bR is the potential target.

Inhibition of tPA-induced hemorrhagic transformation involves adenosine A2b receptor activation after cerebral ischemia

Tissue plasminogen activator (tPA) is administered after ischemic stroke to dissolve intravascular clots, but its use can lead to hemorrhagic transformation (HT). Therapeutic strategies to reduce hemorrhagic complications of tPA might be of benefit for stroke patients. Adenosine A2b receptor (A2bR) plays pivotal roles in regulating vascular protection in peripheral organs. This study explored whether A2bR agonist BAY 60-6583 reduces hemorrhage risk after tPA usage. Using a rat transient middle cerebral artery occlusion model, we showed that mRNA and protein expression of A2bR increased to a greater extent after ischemia-reperfusion than did expression of the other three adenosine receptors (A1, A2a, and A3). tPA administration reduced A2bR expression in ischemic brain microvessels. Post-treatment with BAY 60-6583 (1mg/kg) at the start of reperfusion reduced lesion volume in the absence or presence of tPA (10mg/kg) and attenuated brain swelling, blood-brain barrier disruption, and tPA-exacerbated HT at 24h. Additionally, BAY 60-6583 mitigated sensorimotor deficits in the presence of tPA. BAY 60-6583 inhibited tPA-enhanced matrix metalloprotease-9 activation, probably through elevation of tissue inhibitor of matrix metalloproteinases-1 expression, and thereby reduced degradation of tight junction proteins. These effects would likely protect cerebrovascular integrity. A2bR agonists as an adjuvant to tPA could be a promising strategy for decreasing the risk of HT during treatment for ischemic stroke.