A2B Adenosine Receptor Activation Research Articles

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.

Adenosine A2B receptor agonist improves epidermal barrier integrity in a murine model of epidermal hyperplasia

Adenosine regulates multiple physiological processes through the activation of four receptor subtypes, of which the A2B adenosine receptor (A2BAR) has the lowest affinity for adenosine. Being the adenosine receptor subtype most prominently expressed in epidermis, we recently described the antiproliferative and anti-inflammatory effect of the selective A2BAR agonist BAY60–6583 (BAY) in human keratinocytes stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), so we sought to establish the effect of topical application of BAY in a model of murine epidermal hyperplasia.Topical application of BAY (1 or 10 μg/site) prevented the inflammatory reaction and skin lesions induced by TPA, minimizing hyperproliferation and acanthosis, as well as the expression of specific markers of proliferative keratinocytes. On the other hand, pre-treatment with the selective A2BAR antagonist, PSB-1115 (PSB, 5 or 50 μg/site) reversed these beneficial effects. Additionally, BAY application normalized the expression of epidermal barrier proteins, whose integrity is altered in inflammatory skin diseases, while treatment with the antagonist alone worsened it.Our results, besides confirming the anti-inflammatory and antiproliferative effects of the A2BAR agonist, further demonstrate a role of A2BAR activation to preserve the epidermal barrier. Therefore, the activation of A2BAR may constitute a possible new pharmacological target for the treatment of skin inflammatory diseases such as psoriasis.

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.

A2BR facilitates the pathogenesis of H. pylori-associated GU by inducing oxidative stress through p38 MAPK phosphorylation

Gastric ulcers significantly impact the quality of life of patients, the pathogenesis of which is closely associated with Helicobacter pylori (HP) infection. Oxidative stress is involved in the pathological mechanism of gastric ulcers. Recently, adenosine A2B Receptor (A2BR) was reported to activate the p38MAPK pathway. However, the role of A2BR in gastric ulcers remains unknown. In the present study, the biological function of A2BR in HP-induced gastric ulcers was investigated to explore novel targets for gastric ulcers. GES-1 cells were infected with HP, followed by incubation with 10 μM BAY60-6583 (A2BR agonist) and 25 nM PSB1115 (A2BR antagonist). In HP-infected GES-1 cells, an increased apoptotic rate, enhanced migration ability, excessive release of reactive oxygen species (ROS), increased malondialdehyde (MDA) levels, and decreased superoxide dismutase (SOD) activity were observed, accompanied by the activation of p38MAPK signaling, which were dramatically aggravated by BAY60-6583 and alleviated by PSB1115. In animal experiments, rats were treated with 2 mg/kg BAY60-6583 and 10 mg/kg PSB1115, followed by gastric ulcer modeling 30 min later. In HP-infected rats, increased ulcer area, elevated pepsin activity, increased hematoxylin and eosin (HE) pathological scores, increased MDA levels, and decreased SOD activity were observed, which were further aggravated by BAY60-6583 and ameliorated by PSB1115. Finally, the effects of A2BR activation on apoptosis, migration, oxidative stress, and p38MAPK signaling in HP-infected GES-1 cells were reversed by an inhibitor of the p38MAPK pathway. Collectively, A2BR facilitated the pathogenesis of HP-induced gastric ulcers by inducing oxidative stress through p38MAPK activation.

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.

A2B adenosine receptor activation and modulation by protein kinase C

Protein kinase C (PKC) isoforms regulate many important signaling pathways. Here, we report that PKC activation by phorbol 12-myristate 13-acetate (PMA) enhanced A2B adenosine receptor (AR)-mediated, but not β2-adrenergic receptor-mediated, cAMP accumulation, in H9C2 cardiomyocyte-like and HEK293 cells. In addition to enhancement, PKC (PMA-treatment) also activated A2BAR with low Emax (H9C2 and NIH3T3 cells endogenously expressing A2BAR), or with high Emax (A2BAR-overexpressing HEK293 cells) to induce cAMP accumulation. A2BAR activation induced by PKC was inhibited by A2BAR and PKC inhibitors but enhanced by A2BAR overexpression. Gαi isoforms and PKCγ isoform were found to be involved in both enhancement of A2BAR function and A2BAR activation. Thus,we establish PKC as an endogenous modulator and activator of A2BAR, involving Giα and PKCγ. Depending on signaling pathway, PKC could activate and enhance,or alternatively inhibit A2BAR activity. These findings are relevant to common functions of A2BAR and PKC, e.g. cardioprotection and cancer progression/treatment.

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.

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.

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.

Activation of A2B adenosine receptor protects against demyelination in a mouse model of schizophrenia.

The purpose of the present study was to explore the effects of A2B adenosine receptor (A2BAR) on learning, memory and demyelination in a dizocilpine maleate (MK-801)-induced mouse model of schizophrenia (SCZ). BAY 60-6583, an agonist of A2BAR, or PSB 603, an antagonist of A2BAR, was used to treat SCZ in this model. The Morris Water Maze (MWM) was utilized to determine changes in cognitive function. Moreover, western blotting, immunohistochemistry and immunofluorescence were conducted to investigate the myelination and oligodendrocyte (OL) alterations at differentiation and maturation stages. The MWM results showed that learning and memory were impaired in SCZ mice, while subsequent treatment with BAY 60-6583 alleviated these impairments. In addition, western blot analysis revealed that myelin basic protein (MBP) and chondroitin sulphate proteoglycan 4 (NG2) expression levels were significantly decreased in MK-801-induced mice, while the expression of G protein-coupled receptor 17 (GPR17) was increased. Additionally, the number of anti-adenomatous polyposis coli clone CC-1/OL transcription factor 2 (CC-1+/Olig2+) cells was also decreased. Notably, BAY 60-6583 administration could reverse these changes, resulting in a significant increase in MBP and NG2 protein expression, and in the number of CC-1+/Olig2+ cells, while GPR17 protein expression levels were decreased. The present study indicated that the selective activation of A2BAR using BAY 60-6583 could improve the impaired learning and memory of SCZ mice, as well as protect the myelin sheath from degeneration by regulating the survival and maturation of OLs.

A2B adenosine receptor inhibition ameliorates hypoxic-ischemic injury in neonatal mice via PKC/Erk/Creb/HIF-1α signaling pathway

Periventricular leukomalacia (PVL), the dominant cerebral white matter injury disease, is induced by hypoxia–ischemia and inflammation in premature infants. The activation of A2B adenosine receptor (A2BAR) is shown to involve into inflammation, ischemia, and other typical stress reactions, but its exact function in PVL has not been clarified. We gained initial insight from PVL mouse model (P9) by the induction of hypoxia–ischemia with right carotid ligation followed by exposure to hypoxia and intraperitoneal (i.p.) injection of Lipopolysaccharide (LPS). The results showed that treatment of PSB-603, an A2BAR selective antagonist, greatly ameliorated cerebral ischemic injury by increasing bodyweights, reducing infarct volume, brain injury,inflammation andcontributing to long-term learning memory functionalrecoveryof the PVL mice. Meanwhile, PSB-603 treatment suppressed neurons apoptosis as characterized byreducing of Caspase-3 level, inhibited microglia activation and attenuated hypomyelination through promoting MBP expression and oligodendrocytes differentiation. A2BAR inhibition also augmented PKC expression, the activity of PKC downstream signaling molecules were then explored. Erk expression and Creb phosphorylation exhibited upregulation in PSB-603 treatment group compared with the control group. Hypoxia Inducible Factor-1α (HIF-1α), a direct target of hypoxia, which is a key regulator of adenosine signaling by binding to the A2BAR promoter to induce expression of A2BAR, was shown to be decreased by PSB-603. Taken together, A2BAR inhibition can ameliorate hypoxic-ischemic injury in PVL mice maybe through PKC/Erk/Creb/HIF-1α signaling pathway.

100 Activation of A2B adenosine receptor normalizes filaggrin and caspase-14 levels in a murine model of epidermal hyperplasia

A2B adenosine receptor (AR) is the subtype mainly expressed within the epidermis, regulating keratinocyte proliferation and inflammatory responses. In the murine model of epidermal hyperplasia induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), we recently demonstrated the protective role of A2B AR activation, reducing macroscopic and microscopic lesions and restoring the epidermal barrier. Caspase-14 is a crucial protease in the activation and catabolism of filaggrin, a protein with a pivotal role in the development of the cornified envelope.

Simvastatin attenuates acute lung injury by activation of A2B adenosine receptor

To explore the protective mechanism of simvastatin in acute lung injury (ALI), the lipopolysaccharide (LPS) induced (5 mg/kg) ALI rat model was used to examine the effects of simvastatin. Following simvastatin treatment, the histopathological evaluation of lung tissues was made using hematoxylin and eosin (H&E) staining. Also, myeloperoxidase (MPO) activity and the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined by ELISA. Blood gas analyses of arterial blood samples were performed to assess the pulmonary gas exchange. Moreover, the neutrophil count and total protein content were determined in the bronchoalveolar lavage (BAL) fluid. The ratio of wet lung to dry lung (W/D) and the alveolar fluid clearance (AFC) were calculated to estimate the severity of edema. Lastly, the levels of A2BAR, CFTR, claudin4, and claudin18 were also measured by qRT-PCR and Western blotting. Simvastatin treatment, in a dose-related manner, markedly improved the lung histological injury and decreased the levels of TNF-α, IL-1β, and increased IL-10 in LPS induced ALI. Also, pulmonary neutrophil count was alleviated. Besides, a decreased ratio of W/D lung also confirmed the simvastatin intervention. Notably, simvastatin reduced the levels of A2BAR, CFTR, and claudin18 but upregulated claudin4 in lung tissues. Additionally, treatment with PSB1115, an antagonist of A2BAR, countered the protective effect of simvastatin in ALI. Our study demonstrates that simvastatin has a protective effect against LPS-induced ALI by activating A2BAR and should be exploited as a novel therapeutic target for the treatment of ALI.

Activation of central adenosine A2B receptors mediate brain ghrelin-induced improvement of intestinal barrier function through the vagus nerve in rats

Leaky gut that is a condition reflecting intestinal barrier dysfunction has been attracting attention for its relations with many diseases such as irritable bowel syndrome or Alzheimer dementia. We have recently demonstrated that ghrelin acts in the brain to improve leaky gut via the vagus nerve. In the present study, we tried to clarify the precise central mechanisms by which ghrelin improves intestinal barrier function through the vagus nerve. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue in rats. Adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), blocked the intracisternal ghrelin-induced improvement of intestinal hyperpermeability while dopamine, cannabinoid or opioid receptor antagonist failed to prevent it. Since DPCPX can block adenosine A1 and adenosine A2B receptors, we examined which subtype is involved in the mechanism. Intracisternal injection of adenosine A2B agonist but not adenosine A1 agonist improved colonic hyperpermeability, while peripheral injection of adenosine A2B agonist failed to improve it. Intracisternal adenosine A2B agonist-induced improvement of colonic hyperpermeability was blocked by vagotomy. Adenosine A2B specific antagonist, alloxazine blocked the ghrelin- or central vagal stimulation by 2-deoxy-d-glucose-induced improvement of intestinal hyperpermeability. These results suggest that activation of adenosine A2B receptors in the central nervous system is capable of improving intestinal barrier function through the vagal pathway, and the adenosine A2B receptors may mediate the ghrelin-induced improvement of leaky gut in a vagal dependent fashion. These findings may help us understand the pathophysiology in not only gastrointestinal diseases but also non-gastrointestinal diseases associated with the altered intestinal permeability.

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 IFNβ 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.

Insulin requires A2B adenosine receptors to modulate the L-arginine/nitric oxide signalling in the human fetoplacental vascular endothelium from late-onset preeclampsia

Late-onset preeclampsia (LOPE) associates with reduced umbilical vein reactivity and endothelial nitric oxide synthase (eNOS) activity but increased human cationic amino acid (hCAT-1)–mediated L-arginine transport involving A2A adenosine receptor in the fetoplacental unit. This study addresses the A2B adenosine receptor (A2BAR)-mediated response to insulin in the fetoplacental vasculature from LOPE. Umbilical veins and HUVECs were obtained from women with normal (n = 37) or LOPE (n = 35) pregnancies. Umbilical vein rings reactivity to insulin was assayed in the absence or presence of adenosine and MRS-1754 (A2BAR antagonist) in a wire myograph. HUVECs were exposed to insulin, MRS-1754, BAY60–6583 (A2BAR agonist), NECA (general adenosine receptors agonist) or NG-nitro-L-arginine methyl ester (NOS inhibitor). A2BAR, hCAT-1, total and phosphorylated eNOS, Akt and p44/42mapk protein abundance were determined by Western blotting. Insulin receptors A (IR-A) and B (IR-B), eNOS and hCAT-1 mRNA were determined by qPCR. Firefly/Renilla luciferase assay was used to determine −1606 bp SLC7A1 (hCAT-1) promoter activity. L-Citrulline content was measured by HPLC, L-[3H]citrulline formation from L-[3H]arginine by the Citrulline assay, and intracellular cGMP by radioimmunoassay. LOPE-reduced dilation of vein rings to insulin was restored by MRS-1754. HUVECs from LOPE showed higher A2BAR, hCAT-1, and IR-A expression, Akt and p44/42mapk activation, and lower NOS activity. MRS-1754 reversed the LOPE effect on A2BAR, hCAT-1, Akt, and eNOS inhibitory phosphorylation. Insulin reversed the LOPE effect on A2BAR, IR-A and eNOS, but increased hCAT-1–mediated transport. Thus, LOPE alters endothelial function, causing an imbalance in the L-arginine/NO signalling pathway to reduce the umbilical vein dilation to insulin requiring A2BAR activation in HUVECs.

The Detrimental Action of Adenosine on Glutamate-Induced Cytotoxicity in PC12 Cells Can Be Shifted towards a Neuroprotective Role through A1AR Positive Allosteric Modulation.

Glutamate cytotoxicity is implicated in neuronal death in different neurological disorders including stroke, traumatic brain injury, and neurodegenerative diseases. Adenosine is a nucleoside that plays an important role in modulating neuronal activity and its receptors have been identified as promising therapeutic targets for glutamate cytotoxicity. The purpose of this study is to elucidate the role of adenosine and its receptors on glutamate-induced injury in PC12 cells and to verify the protective effect of the novel A1 adenosine receptor positive allosteric modulator, TRR469. Flow cytometry experiments to detect apoptosis revealed that adenosine has a dual role in glutamate cytotoxicity, with A2A and A2B adenosine receptor (AR) activation exacerbating and A1 AR activation improving glutamate-induced cell injury. The overall effect of endogenous adenosine in PC12 cells resulted in a facilitating action on glutamate cytotoxicity, as demonstrated by the use of adenosine deaminase and selective antagonists. However, enhancing the action of endogenous adenosine on A1ARs by TRR469 completely abrogated glutamate-mediated cell death, caspase 3/7 activation, ROS production, and mitochondrial membrane potential loss. Our results indicate a novel potential therapeutic strategy against glutamate cytotoxicity based on the positive allosteric modulation of A1ARs.

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.

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.