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.

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.

A2bR-dependent signaling alters immune cell composition and enhances IL-6 formation in the ischemic heart.

Although the cardioprotective effect of adenosine is undisputed, the role of the adenosine A2b receptor (A2bR) in ischemic cardiac remodeling is not defined. In this study we aimed to unravel the role A2bR plays in modulating the immune response and the healing mechanisms after myocardial infarction. Genetic and pharmacological (PSB603) inactivation of A2bR as well as activation of A2bR with BAY60-6583 does not alter cardiac remodeling of the infarcted (50-min left anterior descending artery occlusion/reperfusion) murine heart. Flow cytometry of immune cell subsets identified a significant increase in B cells, NK cells, CD8 and CD4 T cells, as well as FoxP3-expressing regulatory T cells in the injured heart in A2bR-deficient mice. Analysis of T-cell function revealed that expression and secretion of interleukin (IL)-2, interferon (IFN)γ, and tumor necrosis factor (TNF)α by T cells is under A2bR control. In addition, we found substantial cellular heterogeneity in the response of immune cells and cardiomyocytes to A2bR deficiency: while in the absence of A2bR, expression of IL-6 was greatly reduced in cardiomyocytes and immune cells except T cells, and expression of IL-1ß was strongly reduced in cardiomyocytes, granulocytes, and B cells as determined by quantitative PCR. Our findings indicate that A2bR signaling in the ischemic heart triggers substantial changes in cardiac immune cell composition of the lymphoid lineage and induces a profound cell type-specific downregulation of IL-6 and IL-1ß. This suggests the presence of a targetable adenosine-A2bR-IL-6-axis triggered by adenosine formed by the ischemic heart. NEW & NOTEWORTHY Genetic deletion and pharmacological inactivation/activation of A2bR does not alter cardiac remodeling after MI but is associated by compensatory upregulation of various pro- and anti-inflammatory immune cell subsets (B cells, NK cells, CD8 and CD4 T cells, regulatory T cells). In the inflamed heart, A2bR modulates the expression of IL-2, IFNγ, TNFα in T cells and of IL-6 in cardiomyocytes, monocytes, granulocytes and B cells. This suggests an important adenosine-IL-6 axis, which is controlled by A2bR via local adenosine.

Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity.

Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a lethal, fibroproliferative disease. Pulmonary hypertension (PH) can develop secondary to IPF and increase mortality. Alternatively, activated macrophages (AAMs) contribute to the pathogenesis of both IPF and PH. Here we hypothesized that adenosine signaling through the ADORA2B on AAMs impacts the progression of these disorders and that conditional deletion of ADORA2B on myeloid cells would have a beneficial effect in a model of these diseases. Conditional knockout mice lacking ADORA2B on myeloid cells (Adora2B(f/f)-LysM(Cre)) were exposed to the fibrotic agent bleomycin (BLM; 0.035 U/g body weight, i.p.). At 14, 17, 21, 25, or 33 d after exposure, SpO2, bronchoalveolar lavage fluid (BALF), and histologic analyses were performed. On day 33, lung function and cardiovascular analyses were determined. Markers for AAM and mediators of fibrosis and PH were assessed. Adora2B(f/f)-LysM(Cre) mice presented with attenuated fibrosis, improved lung function, and no evidence of PH compared with control mice exposed to BLM. These findings were accompanied by reduced expression of CD206 and arginase-1, markers for AAMs. A 10-fold reduction in IL-6 and a 5-fold decrease in hyaluronan, both linked to lung fibrosis and PH, were also observed. These data suggest that activation of the ADORA2B on macrophages plays an active role in the pathogenesis of lung fibrosis and PH.

Blocking A2B adenosine receptor alleviates pathogenesis of experimental autoimmune encephalomyelitis via inhibition of IL-6 production and Th17 differentiation.

Adenosine is a key endogenous signaling molecule that regulates immune responses. A(2B) adenosine receptor (AR) is a relatively low-affinity receptor for adenosine, and the activation of A(2B)AR is believed to require pathological level of adenosine that is associated with ischemia, inflammation, trauma, or other types of stress. The role of A(2B)AR in the pathogenesis of multiple sclerosis (MS) is still unclear. In this study, we discovered that A(2B)AR was upregulated both in the peripheral blood leukocytes of MS patients and the peripheral lymphoid tissues of experimental autoimmune encephalomyelitis (EAE) mice. A(2B)AR-specific antagonists, CVT-6883 and MRS-1754, alleviated the clinical symptoms of EAE and protected the CNS from immune damage. A(2B)AR-knockout mice also developed less severe EAE. Further study indicated that blocking or deleting A(2B)AR inhibited Th17 cell differentiation by blocking IL-6 production from APCs such as dendritic cells. In dendritic cells, A(2B)AR was also upregulated during the development of EAE. CVT-6883 and genetic deletion of A(2B)AR significantly reduced adenosine-mediated IL-6 production. The phospholipase Cß-protein kinase C and p38 MAPK pathways were found to be involved in the A(2B)AR-mediated IL-6 production. Our findings not only revealed the pathological role of A(2B)AR in EAE, but also suggested that this receptor might be a new therapeutic target for the development of anti-MS drugs.

A novel mechanism of control of NFκB activation and inflammation involving A2B adenosine receptors.

The nuclear factor kappa B (NFκB) pathway controls a variety of processes, including inflammation, and thus, the regulation of NFκB has been a continued focus of study. Here, we report a newly identified regulation of this pathway, involving direct binding of the transcription factor NFκB1 (the p105 subunit of NFκB) to the C-terminus of the A(2B) adenosine receptor (A(2B)AR), independent of ligand activation. Intriguingly, binding of A(2B)AR to specific sites on p105 prevents polyubiquitylation and degradation of p105 protein. Ectopic expression of the A(2B)AR increases p105 levels and inhibits NFκB activation, whereas p105 protein levels are reduced in cells from A(2B)AR-knockout mice. In accordance with the known regulation of expression of anti- and pro-inflammatory cytokines by p105, A(2B)AR-null mice generate less interleukin (IL)-10, and more IL-12 and tumor necrosis factor (TNF-α). Taken together, our results show that the A(2B)AR inhibits NFκB activation by physically interacting with p105, thereby blocking its polyubiquitylation and degradation. Our findings unveil a surprising function for the A(2B)AR, and provide a novel mechanistic insight into the control of the NFκB pathway and inflammation.

A2B adenosine receptor expression by myeloid cells is proinflammatory in murine allergic-airway inflammation.

Asthma is a chronic condition with high morbidity and healthcare costs, and cockroach allergens are an established cause of urban pediatric asthma. A better understanding of cell types involved in promoting lung inflammation could provide new targets for the treatment of chronic pulmonary disease. Because of its role in regulating myeloid cell-dependent inflammatory processes, we examined A(2B) R expression by myeloid cells in a cockroach allergen model of murine asthma-like pulmonary inflammation. Both systemic and myeloid tissue-specific A(2B) R deletion significantly decreased pulmonary inflammatory cell recruitment, airway mucin production, and proinflammatory cytokine secretion after final allergen challenge in sensitized mice. A(2B) R deficiency resulted in a dramatic reduction on Th2-type airways responses with decreased pulmonary eosinophilia without augmenting neutrophilia, and decreased lung IL-4, IL-5, and IL-13 production. Chemokine analysis demonstrated that eotaxin 1 and 2 secretion in response to repeated allergen challenge is myeloid cell A(2B) R dependent. In contrast, there were no differences in the levels of the CXC chemokines keratinocyte-derived chemokine and MIP-2 in the myeloid cell A(2B) R-deficient mice, strengthening A(2B) R involvement in the development of Th2-type airways inflammation. Proinflammatory TNF-α, IFN-γ, and IL-17 secretion were also reduced in systemic and myeloid tissue-specific A(2B) R deletion mouse lines. Our results demonstrate Th2-type predominance for A(2B) R expression by myeloid cells as a mechanism of development of asthma-like pulmonary inflammation.

Adora2b adenosine receptor signaling protects during acute kidney injury via inhibition of neutrophil-dependent TNF-α release.

Renal ischemia is among the leading causes of acute kidney injury (AKI). Previous studies have shown that extracellular adenosine is a prominent tissue-protective cue elicited during ischemia, including signaling events through the adenosine receptor 2b (Adora2b). To investigate the functional role of Adora2b signaling in cytokine-mediated inflammatory pathways, we screened wild-type and Adora2b-deficient mice undergoing renal ischemia for expression of a range of inflammatory cytokines. These studies demonstrated a selective and robust increase of TNF-α levels in Adora2b-deficient mice following renal ischemia and reperfusion. Based on these findings, we next sought to understand the contribution of TNF-α on ischemic AKI through a combination of loss- and gain-of-function studies. Loss of TNF-α, through either Ab blockade or study of Tnf-α-deficient animals, resulted in significantly attenuated tissue injury and improved kidney function following renal ischemia. Conversely, transgenic mice with overexpression of TNF-α had significantly pronounced susceptibility to AKI. Furthermore, neutrophil depletion or reconstitution of Adora2b(-/-) mice with Tnf-α-deficient neutrophils rescued their phenotype. In total, these data demonstrate a critical role of adenosine signaling in constraining neutrophil-dependent production of TNF-α and implicate therapies targeting TNF-α in the treatment of ischemic AKI.

Enhanced mast cell activation in mice deficient in the A2b adenosine receptor.

Antigen-mediated cross-linking of IgE bound to mast cells via the high affinity receptor for IgE triggers a signaling cascade that results in the release of intracellular calcium stores, followed by an influx of extracellular calcium. The collective increase in intracellular calcium is critical to the release of the granular contents of the mast cell, which include the mediators of acute anaphylaxis. We show that the sensitivity of the mast cell to antigen-mediated degranulation through this pathway can be dramatically influenced by the A2b adenosine receptor. Loss of this Gs-coupled receptor on mouse bone marrow-derived mast cells results in decreased basal levels of cyclic AMP and an excessive influx of extracellular calcium through store-operated calcium channels following antigen activation. Mice lacking the A2b receptor display increased sensitivity to IgE-mediated anaphylaxis. Collectively, these findings show that the A2b adenosine receptor functions as a critical regulator of signaling pathways within the mast cell, which act in concert to limit the magnitude of mast cell responsiveness when antigen is encountered.

Netrin-1 signaling dampens inflammatory peritonitis.

Previous studies implicated the anti-inflammatory potential of the adenosine 2B receptor (A2BAR). A2BAR activation is achieved through adenosine, but this is limited by its very short t(1/2). To further define alternative adenosine signaling, we examined the role of netrin-1 during acute inflammatory peritonitis. In this article, we report that animals with endogenous repression of netrin-1 (Ntn1(+/-)) demonstrated increased cell count, increased peritoneal cytokine concentration, and pronounced histological changes compared with controls in a model of zymosan A peritonitis. Exogenous netrin-1 significantly decreased i.p. inflammatory changes. This effect was not present in animals with deletion of A2BAR (A2BAR(-/-)). A2BAR(-/-) animals demonstrated no change in cell count, i.p. cytokine concentration, or histology in response to netrin-1 injection. These data strengthen the role of netrin-1 as an immunomodulatory protein exerting its function in dependence of the A2BAR and further define alternative adenosine receptor signaling.

A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice.

Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.

Distinct roles for the A2B adenosine receptor in acute and chronic stages of bleomycin-induced lung injury.

Adenosine is an extracellular signaling molecule that is generated in response to cell injury where it orchestrates tissue protection and repair. Whereas adenosine is best known for promoting anti-inflammatory activities during acute injury responses, prolonged elevations can enhance destructive tissue remodeling processes associated with chronic disease states. The generation of adenosine and the subsequent activation of the adenosine 2B receptor (A(2B)R) is an important processes in the regulation of both acute and chronic lung disease. The goal of this study was to examine the contribution of the A(2B)R in models of bleomycin-induced lung injury that exhibit varying degrees of acute and chronic injury. Intratracheal bleomycin exposure results in substantial acute lung injury followed by progressive fibrosis. In this model, genetic removal of the A(2B)R resulted in enhanced loss of barrier function and increased pulmonary inflammation, with few differences in indexes of pulmonary fibrosis. These results support an anti-inflammatory role for this receptor in this model of acute lung injury. In contrast, systemic exposure of mice to bleomycin resulted in modest acute lung injury together with progressive pulmonary fibrosis. In this model, the effects of A(2B)R removal on acute lung injury were negligible; however, there were substantial reductions in pulmonary fibrosis, supporting a profibrotic role for this receptor. A(2B)R-dependent regulation of IL-6 production was identified as a potential mechanism involved in the diminished pulmonary fibrosis seen in A(2B)R knockout mice exposed to i.p. bleomycin. These studies highlight the distinct roles of A(2B)R signaling during acute and chronic stages of lung injury.

Adenosine A(2B) receptor deficiency promotes host defenses against gram-negative bacterial pneumonia.

RATIONALE: Activation of the adenosine A(2B) receptor (A(2B)R) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. OBJECTIVES: To test the hypothesis that absence of adenosine A(2B) receptor signaling promotes host defense against bacterial pneumonia. METHODS: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A(2B)R. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. MEASUREMENTS AND MAIN RESULTS: A(2B)R(-/-) mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow-derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A(2B)R(-/-) and wild-type mice, but A(2B)R(-/-) neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A(2B)R(-/-) mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. CONCLUSIONS: These data suggest that the absence of A(2B)R signaling enhances antimicrobial activity in gram-negative bacterial pneumonia.

A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation.

Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5'-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A(2B) receptor (A(2B)R) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A(2B)R signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A(2B)R in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A(2B)R activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.

A2B adenosine receptors protect against sepsis-induced mortality by dampening excessive inflammation.

Despite intensive research, efforts to reduce the mortality of septic patients have failed. Adenosine is a potent extracellular signaling molecule, and its levels are elevated in sepsis. Adenosine signals through G-protein-coupled receptors and can regulate the host's response to sepsis. In this study, we studied the role of A(2B) adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A(2B) receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A(2B) knockout mice was associated with increased levels of inflammatory cytokines and chemokines and augmented NF-kappaB and p38 activation in the spleen, heart, and plasma in comparison with wild-type animals. In addition, A(2B) receptor knockout mice showed increased splenic apoptosis and phosphatase and tensin homolog activation and decreased Akt activation. Experiments using bone-marrow chimeras revealed that it is the lack of A(2B) receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A(2B) receptor-deficient mice. These results indicate that A(2B) receptor activation may offer a new therapeutic approach for the management of sepsis.

Signaling through the A2B adenosine receptor dampens endotoxin-induced acute lung injury.

Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia, or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (AR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9 + or - 3.4-fold, p < 0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS exposure. Studies utilizing A2BAR promoter constructs and RNA protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury.

Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice.

Adenosine plays a role in physiological and pathological conditions, and A(2) adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A(2B)AR and its relationship to the A(2A)AR in coronary flow (CF) changes using A(2B) single-knockout (KO) and A(2A/2B) double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A(2A)KO and A(2B)KO and A(2A/2B)DKO mice. BAY 60-6583 (a selective A(2B) agonist) had no effect on CF in A(2B)KO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min(-1)·g(-1)). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A(2B)KO mice (maximum of 34.6 ± 4.7 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Also, CGS-21680 (a selective A(2A) agonist) increased CF in A(2B)KO mice (maximum of 29 ± 1.9 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min(-1)·g(-1)). SCH-58261 (an A(2A)-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A(2B)KO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min(-1)·g(-1)) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min(-1)·g(-1)). NECA did not induce any increase in CF in A(2A/2B)DKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Furthermore, the mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker 5-hydroxydecanoate had no effect on the NECA-induced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min(-1)·g(-1)), A(2A)KO (12.5 ± 2.3 ml·min(-1)·g(-1)), and A(2B)KO (16.2 ± 0.8 ml·min(-1)·g(-1)) mice was significantly blunted by the K(ATP) channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min(-1)·g(-1), respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min(-1)·g(-1)) and BAY 60-6583-induced (16.4 ± 1.60 ml·min(-1)·g(-1)) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min(-1)·g(-1), respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A(2A)ARs in A(2B)KO mice and demonstrates that both A(2A)ARs and A(2B)ARs induce CF changes through K(ATP) channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders.

Adenosine A2A and A2B receptors are both required for adenosine A1 receptor-mediated cardioprotection.

All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A(2A) and/or A(2B) receptors modulate adenosine A(1) receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A(2A) knockout (KO), and A(2B)KO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A(2A) receptor agonist CGS-21680 (200 nM) and the A(2B) agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A(2A) or A(2B) receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A(2A) and A(2B) receptors are required for adenosine A(1) receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes.

Contribution of adenosine A2B receptors to inflammatory parameters of experimental colitis.

Inflammatory diseases influence tissue metabolism, significantly altering the profile of extracellular adenine nucleotides. A number of studies have suggested that adenosine (Ado) may function as an endogenously generated anti-inflammatory molecule. Given the central role of intestinal epithelial cells to the development of colitis, we hypothesized that specific Ado receptors would contribute to disease resolution in mucosal inflammation as modeled by dextran sodium sulfate (DSS) colitis. Initial profiling studies revealed that murine intestinal epithelial cells express predominantly the Ado A2B receptor (AA2BR) and to a lesser extent AA2AR. Guided by these results, we examined the contribution of AA2BR to colitis. Initial studies indicated that the severity of colitis was increased in Aa2br(-/-) mice relative to Aa2br(+/+) controls, as reflected by increased weight loss, colonic shortening, and disease activity indices. Likewise, enteral administration of the selective AA2BR inhibitor PSB1115 to Aa2br(+/+) mice resulted in a similar increase in severity of DSS colitis. Cytokine profiling of colonic tissue revealed specific deficiencies in IL-10 in Aa2br(-/-) mice relative to controls. Extensions of these findings in cultured human intestinal epithelial cells revealed that stable Ado analogs induce IL-10 mRNA and protein and that such increases can be blocked with PSB1115. Taken together, these studies indicate a central regulatory role for AA2BR-modulated IL-10 in the acute inflammatory phase of DSS colitis, thereby implicating AA2BR as an endogenously protective molecule expressed on intestinal epithelial cells.