Adenosine signalling to astrocytes coordinates brain metabolism and function.

Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.

Klotho Null Mutation Involvement in Adenosine A2B Receptor-Related Skeletal Muscle Degeneration.

Klotho is known for its age-suppressing function and has been implicated in sarcopenia pathology. It has recently been proposed that the adenosine A2B receptor plays a crucial role in skeletal muscle energy expenditure. However, the association between Klotho and A2B remains elusive. In this study, Klotho knockout mice, aged 10 weeks, and wild-type mice, aged 10 and 64 weeks, were used for comparison in indicators of sarcopenia (n = 6 for each group). PCR was performed to confirm the mice genotypes. Skeletal muscle sections were analyzed using hematoxylin and eosin staining as well as immunohistochemistry staining. The skeletal muscle cross-sectional area was significantly reduced in Klotho knockout mice and wild-type mice, aged 64 weeks, when compared with wild-type mice, aged 10 weeks, with a decreased percentage of type IIa and IIb myofibers. Likely impaired regenerative capacity, as reflected by the reduction of paired box 7 (Pax7)- and myogenic differentiation protein 1 (MyoD)-positive cells, was also observed in Klotho knockout mice and aged wild-type mice. 8-Hydroxy-2-deoxyguanosine expression was enhanced with Klotho knockout and aging, indicating higher oxidative stress. Adenosine A2B signaling was impaired, with a lower expression of the A2B receptor and the cAMP-response element binding protein in Klotho knockout and aged mice. The present study provides the novel finding that sarcopenia involves adenosine signaling under the influence of Klotho knockout.

Activation of adenosine A2B receptor alleviates myocardial ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress and restoring autophagy flux.

Myocardial ischemia-reperfusion injury (MIRI) poses a significant threat to patients with coronary heart disease. Adenosine A2A receptors have been known as a protective role in MIRI by regulating autophagy, so we assumed that activation of adenosine A2B receptor (A2BAR) might exert a similar effect during MIRI and underlying mechanism be related to proteostasis maintenance as well. In situ hearts were subjected to 30 min of ischemia and 120 min of reperfusion (IR), while invitro cardiomyocytes from neonatal rats experienced 6 h of oxygen-glucose deprivation followed by 12 h of reoxygenation (OGDR). Initially, we observed that post-ischemia-reperfusion induced autophagy flux blockade and ERS both in vivo and in vitro, evident through the increased expression of p62, LC3II, and BIP, which indicated the deteriorated proteostasis. We used a selective A2BAR agonist, Bay 60-6583, to explore the positive effects of A2BAR on cardiomyocytes and found that A2BAR activation rescued damaged cardiac function and morphological changes in the IR group and improved frail cell viability in the OGDR group. The A2BAR agonist also alleviated the blockage of autophagic flux, coupled with augmented ERS in the IR/OGDR group, which was reassured by using an autophagy inhibitor chloroquine (CQ) and ERS inhibitor (4-PBA) in vitro. Additionally, considering cAMP/PKA as a well-known downstream effector of A2BAR, we utilized H89, a selective PKA inhibitor. We observed that the positive efficacy of Bay 60-6583 was inhibited by H89. Collectively, our findings demonstrate that the A2BAR/cAMP/PKA signaling pathway exerts a protective role in MIRI by mitigating impaired autophagic flux and excessive ERS.

Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming.

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline.

Blockage of the adenosine A2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension.

Sustained pressure overload and fibrosis of the right ventricle (RV) are the leading causes of mortality in pulmonary arterial hypertension (PAH). Although the role of adenosine in PAH has been attributed to the control of pulmonary vascular tone, cardiac reserve, and inflammatory processes, the involvement of the nucleoside in RV remodelling remains poorly understood. Conflicting results exist on targeting the low-affinity adenosine A2B receptor (A2BAR) for the treatment of PAH mostly because it displays dual roles in acute vs. chronic lung diseases. Herein, we investigated the role of the A2BAR in the viability/proliferation and collagen production by cardiac fibroblasts (CFs) isolated from RVs of rats with monocrotaline (MCT)-induced PAH. CFs from MCT-treated rats display higher cell viability/proliferation capacity and overexpress A2BAR compared to the cells from healthy littermates. The enzymatically stable adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA, 1-30 µM), concentration-dependently increased growth, and type I collagen production by CFs originated from control and PAH rats, but its effects were more prominent in cells from rats with PAH. Blockage of the A2BAR with PSB603 (100 nM), but not of the A2AAR with SCH442416 (100 nM), attenuated the proliferative effect of NECA in CFs from PAH rats. The A2AAR agonist, CGS21680 (3 and 10 nM), was virtually devoid of effect. Overall, data suggest that adenosine signalling via A2BAR may contribute to RV overgrowth secondary to PAH. Therefore, blockage of the A2AAR may be a valuable therapeutic alternative to mitigate cardiac remodelling and prevent right heart failure in PAH patients.

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.

Adenosine Stimulates Beating of Neonatal Brain-Derived Cilia through Adenosine A2B Receptor on the Cilia and Activation of Protein Kinase A Pathway.

Motile cilia in the ependymal cells that line the brain ventricles play pivotal roles in cerebrospinal fluid (CSF) flow in well-defined directions. However, the substances and pathways which regulate their beating have not been well studied. Here, we used primary cultured cells derived from neonatal mouse brain that possess motile cilia and found that adenosine (ADO) stimulates ciliary beating by increasing the ciliary beat frequency (CBF) in a concentration-dependent manner, with the ED50 value being 5 µM. Ciliary beating stimulated by ADO was inhibited by A2B receptor (A2BR) antagonist MRS1754 without any inhibition by antagonists of other ADO receptor subtypes. The expression of A2BR on the cilia was also confirmed by immunofluorescence. The values of CBF were also increased by forskolin, which is an activator of adenylate cyclase, whereas they were not further increased by the addition of ADO. Furthermore, ciliary beating was not stimulated by ADO in the presence of a protein kinase A (PKA) inhibitors. These results altogether suggest that ADO stimulates ciliary beating through A2BR on the cilia, and activation of PKA.

Dexmedetomidine alleviates pulmonary fibrosis through the ADORA2B-Mediated MAPK signaling pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronically progressive fibrotic pulmonary disease characterized by an uncertain etiology, a poor prognosis, and a paucity of efficacious treatment options. Dexmedetomidine (Dex), an anesthetic-sparing alpha-2 adrenoceptor (α2AR) agonist, plays a crucial role in organ injury and fibrosis. However, the underlying mechanisms of IPF remain unknown. METHODS: In our study, the role of Dex in murine pulmonary fibrosis models was determined by Dex injection intraperitoneally in vivo. Fibroblast activation and myofibroblast differentiation were assessed after Dex treatment in vitro. The activation of MAPK pathway and the expression of Adenosine A2B receptor (ADORA2B) were examined in lung myofibroblasts. Moreover, the role of ADORA2B in Dex suppressing myofibroblast differentiation and pulmonary fibrosis was determined using the ADORA2B agonist BAY60-6583. RESULTS: The results revealed that Dex could inhibit Bleo-induced pulmonary fibrosis in mice. In vitro studies revealed that Dex suppressed TGF-ß-mediated MAPK pathway activation and myofibroblast differentiation. Furthermore, Dex inhibits myofibroblast differentiation and pulmonary fibrosis via downregulating ADORA2B expression. CONCLUSIONS: Our findings suggest Dex as a potential therapeutic agent for pulmonary fibrosis. Dex may alleviate lung fibrosis and myofibroblast differentiation through the ADORA2B-mediated MAPK signaling pathway.

Regionally selective cardiovascular responses to adenosine A2A and A2B receptor activation.

Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein-coupled receptors (A1 , A2A , A2B , A3 ). Here, we have investigated the effect of A2A and A2B -selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. NanoBRET ligand binding studies were used to confirm receptor selectivity. The regional hemodynamic effects of adenosine A2A and A2B selective agonists were investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery, and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of the A2A -selective agonist CGS 21680 (0.1, 0.3, 1 µg kg-1 min-1 ) or the A2B -selective agonist BAY 60-6583 (4,13.3, 40 µg kg-1 min-1 ) following predosing with the A2A -selective antagonist SCH 58261 (0.1 or 1 mg kg-1 min-1 ), the A2B /A2A antagonist PSB 1115 (10 mg kg-1 min-1 ) or vehicle. The A2A -selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A2B -selective agonist BAY 60-6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Taken together, these data indicate that A2A and A2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury.

Extracellular cyclic dinucleotides induce polarized responses in barrier epithelial cells by adenosine signaling.

Cyclic dinucleotides (CDNs) are secondary messengers used by prokaryotic and eukaryotic cells. In mammalian cells, cytosolic CDNs bind STING (stimulator of IFN gene), resulting in the production of type I IFN. Extracellular CDNs can enter the cytosol through several pathways but how CDNs work from outside eukaryotic cells remains poorly understood. Here, we elucidate a mechanism of action on intestinal epithelial cells for extracellular CDNs. We found that CDNs containing adenosine induced a robust CFTR-mediated chloride secretory response together with cAMP-mediated inhibition of Poly I:C-stimulated IFNß expression. Signal transduction was strictly polarized to the serosal side of the epithelium, dependent on the extracellular and sequential hydrolysis of CDNs to adenosine by the ectonucleosidases ENPP1 and CD73, and occurred via activation of A2B adenosine receptors. These studies highlight a pathway by which microbial and host produced extracellular CDNs can regulate the innate immune response of barrier epithelial cells lining mucosal surfaces.

A2BAR Antagonism Decreases the Glomerular Expression and Secretion of Chemoattractants for Monocytes and the Pro-Fibrotic M2 Macrophages Polarization during Diabetic Nephropathy.

Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.

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 5 days 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.

Inosine monophosphate and inosine differentially regulate endotoxemia and bacterial sepsis.

Inosine monophosphate (IMP) is the intracellular precursor for both adenosine monophosphate and guanosine monophosphate and thus plays a central role in intracellular purine metabolism. IMP can also serve as an extracellular signaling molecule, and can regulate diverse processes such as taste sensation, neutrophil function, and ischemia-reperfusion injury. How IMP regulates inflammation induced by bacterial products or bacteria is unknown. In this study, we demonstrate that IMP suppressed tumor necrosis factor (TNF)-α production and augmented IL-10 production in endotoxemic mice. IMP exerted its effects through metabolism to inosine, as IMP only suppressed TNF-α following its CD73-mediated degradation to inosine in lipopolysaccharide-activated macrophages. Studies with gene targeted mice and pharmacological antagonism indicated that A2A , A2B, and A3 adenosine receptors are not required for the inosine suppression of TNF-α production. The inosine suppression of TNF-α production did not require its metabolism to hypoxanthine through purine nucleoside phosphorylase or its uptake into cells through concentrative nucleoside transporters indicating a role for alternative metabolic/uptake pathways. Inosine augmented IL-ß production by macrophages in which inflammasome was activated by lipopolysaccharide and ATP. In contrast to its effects in endotoxemia, IMP failed to affect the inflammatory response to abdominal sepsis and pneumonia. We conclude that extracellular IMP and inosine differentially regulate the inflammatory response.

Clustering of adenosine A2B receptors with ectonucleotidases in caveolin-rich lipid rafts underlies immunomodulation by Leishmania amazonensis.

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

Physiology and effects of nucleosides in mice lacking all four adenosine receptors.

Adenosine is a constituent of many molecules of life; increased free extracellular adenosine indicates cell damage or metabolic stress. The importance of adenosine signaling in basal physiology, as opposed to adaptive responses to danger/damage situations, is unclear. We generated mice lacking all four adenosine receptors (ARs), Adora1-/-;Adora2a-/-;Adora2b-/-;Adora3-/- (quad knockout [QKO]), to enable investigation of the AR dependence of physiologic processes, focusing on body temperature. The QKO mice demonstrate that ARs are not required for growth, metabolism, breeding, and body temperature regulation (diurnal variation, response to stress, and torpor). However, the mice showed decreased survival starting at about 15 weeks of age. While adenosine agonists cause profound hypothermia via each AR, adenosine did not cause hypothermia (or bradycardia or hypotension) in QKO mice, indicating that AR-independent signals do not contribute to adenosine-induced hypothermia. The hypothermia elicited by adenosine kinase inhibition (with A134974), inosine, or uridine also required ARs, as each was abolished in the QKO mice. The proposed mechanism for uridine-induced hypothermia is inhibition of adenosine transport by uridine, increasing local extracellular adenosine levels. In contrast, adenosine 5'-monophosphate (AMP)-induced hypothermia was attenuated in QKO mice, demonstrating roles for both AR-dependent and AR-independent mechanisms in this process. The physiology of the QKO mice appears to be the sum of the individual knockout mice, without clear evidence for synergy, indicating that the actions of the four ARs are generally complementary. The phenotype of the QKO mice suggests that, while extracellular adenosine is a signal of stress, damage, and/or danger, it is less important for baseline regulation of body temperature.

Design, synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A2A/A2B adenosine receptor antagonists.

A series of novel dual A2A/A2B AR antagonists based on the triazole-pyrimidine-methylbenzonitrile core were designed and synthesised. The A2A AR antagonist cAMP functional assay results were encouraging for most target compounds containing quinoline or its open-ring bioisosteres. In addition, compound 7i displayed better inhibitory activity on A2B AR (IC50 14.12 nM) and higher potency in IL-2 production than AB928. Moreover, molecular docking studies were carried out to explain the rationality of molecular design and the activity of compound 7i. Further studies on 7f and 7i revealed good liver microsomes stabilities and acceptable in vivo PK profiles. This study provides insight into the future development of dual A2A/A2B AR antagonists for cancer immunotherapy.

Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells.

The purinergic system is fundamental in the tumor microenvironment, since it regulates tumor cell interactions with the immune system, as well as growth and differentiation in autocrine-paracrine responses. Here, we investigated the role of the adenosine A2B receptor (A2BR) in ovarian carcinoma-derived cells' (OCDC) properties. From public databases, we documented that high A2BR expression is associated with a better prognostic outcome in ovarian cancer patients. In vitro experiments were performed on SKOV-3 cell line to understand how A2BR regulates the carcinoma cell phenotype associated with cell migration. RT-PCR and Western blotting revealed that the ADORA2B transcript (coding for A2BR) and A2BR were expressed in SKOV-3 cells. Stimulation with BAY-606583, an A2BR agonist, induced ERK1/2 phosphorylation, which was abolished by the antagonist PSB-603. Pharmacological activation of A2BR reduced cell migration and actin stress fibers; in agreement, A2BR knockdown increased migration and enhanced actin stress fiber expression. Furthermore, the expression of E-cadherin, an epithelial marker, increased in BAY-606583-treated cells. Finally, cDNA microarrays revealed the pathways mediating the effects of A2BR activation on SKOV-3 cells. Our results showed that A2BR contributed to maintaining an epithelial-like phenotype in OCDC and highlighted this purinergic receptor as a potential biomarker.

Preliminary Evidence of the Potent and Selective Adenosine A2B Receptor Antagonist PSB-603 in Reducing Obesity and Some of Its Associated Metabolic Disorders in Mice.

The adenosine A2A and A2B receptors are promising therapeutic targets in the treatment of obesity and diabetes since the agonists and antagonists of these receptors have the potential to positively affect metabolic disorders. The present study investigated the link between body weight reduction, glucose homeostasis, and anti-inflammatory activity induced by a highly potent and specific adenosine A2B receptor antagonist, compound PSB-603. Mice were fed a high-fat diet for 14 weeks, and after 12 weeks, they were treated for 14 days intraperitoneally with the test compound. The A1/A2A/A2B receptor antagonist theophylline was used as a reference. Following two weeks of treatment, different biochemical parameters were determined, including total cholesterol, triglycerides, glucose, TNF-α, and IL-6 blood levels, as well as glucose and insulin tolerance. To avoid false positive results, mouse locomotor and spontaneous activities were assessed. Both theophylline and PSB-603 significantly reduced body weight in obese mice. Both compounds had no effects on glucose levels in the obese state; however, PSB-603, contrary to theophylline, significantly reduced triglycerides and total cholesterol blood levels. Thus, our observations showed that selective A2B adenosine receptor blockade has a more favourable effect on the lipid profile than nonselective inhibition.

A2B Adenosine Receptor Enhances Chemoresistance of Glioblastoma Stem-Like Cells under Hypoxia: New Insights into MRP3 Transporter Function.

Glioblastoma is the most common and aggressive primary brain tumor, characterized by its high chemoresistance and the presence of a cell subpopulation that persists under hypoxic niches, called glioblastoma stem-like cells (GSCs). The chemoresistance of GSCs is mediated in part by adenosine signaling and ABC transporters, which extrude drugs outside the cell, such as the multidrug resistance-associated proteins (MRPs) subfamily. Adenosine promotes MRP1-dependent chemoresistance under normoxia. However, adenosine/MRPs-dependent chemoresistance under hypoxia has not been studied until now. Transcript and protein levels were determined by RT-qPCR and Western blot, respectively. MRP extrusion capacity was determined by intracellular 5 (6)-Carboxyfluorescein diacetate (CFDA) accumulation. Cell viability was measured by MTS assays. Cell cycle and apoptosis were determined by flow cytometry. Here, we show for the first time that MRP3 expression is induced under hypoxia through the A2B adenosine receptor. Hypoxia enhances MRP-dependent extrusion capacity and the chemoresistance of GSCs. Meanwhile, MRP3 knockdown decreases GSC viability under hypoxia. Downregulation of the A2B receptor decreases MRP3 expression and chemosensibilizes GSCs treated with teniposide under hypoxia. These data suggest that hypoxia-dependent activation of A2B adenosine receptor promotes survival of GSCs through MRP3 induction.

Irreversible Antagonists for the Adenosine A2B Receptor.

Blockade of the adenosine A2B receptor (A2BAR) represents a potential novel strategy for the immunotherapy of cancer. In the present study, we designed, synthesized, and characterized irreversible A2BAR antagonists based on an 8-p-sulfophenylxanthine scaffold. Irreversible binding was confirmed in radioligand binding and bioluminescence resonance energy transfer(BRET)-based Gα15 protein activation assays by performing ligand wash-out and kinetic experiments. p-(1-Propylxanthin-8-yl)benzene sulfonyl fluoride (6a, PSB-21500) was the most potent and selective irreversible A2BAR antagonist of the present series with an apparent Ki value of 10.6 nM at the human A2BAR and >38-fold selectivity versus the other AR subtypes. The corresponding 3-cyclopropyl-substituted xanthine derivative 6c (PSB-21502) was similarly potent, but was non-selective versus A1- and A2AARs. Attachment of a reactive sulfonyl fluoride group to an elongated xanthine 8-substituent (12, Ki 7.37 nM) resulted in a potent, selective, reversibly binding antagonist. Based on previous docking studies, the lysine residue K2697.32 was proposed to react with the covalent antagonists. However, the mutant K269L behaved similarly to the wildtype A2BAR, indicating that 6a and related irreversible A2BAR antagonists do not interact with K2697.32. The new irreversible A2BAR antagonists will be useful tools and have the potential to be further developed as therapeutic drugs.