Hypoxia-inducible factor 1-dependent expression of adenosine receptor 2B promotes breast cancer stem cell enrichment.

Breast cancer stem cells (BCSCs), which are characterized by a capacity for unlimited self-renewal and for generation of the bulk cancer cell population, play a critical role in cancer relapse and metastasis. Hypoxia is a common feature of the cancer microenvironment that stimulates the specification and maintenance of BCSCs. In this study, we found that hypoxia increased expression of adenosine receptor 2B (A2BR) in human breast cancer cells through the transcriptional activity of hypoxia-inducible factor 1. The binding of adenosine to A2BR promoted BCSC enrichment by activating protein kinase C-δ, which phosphorylated and activated the transcription factor STAT3, leading to increased expression of interleukin 6 and NANOG, two key mediators of the BCSC phenotype. Genetic or pharmacological inhibition of A2BR expression or activity decreased hypoxia- or adenosine-induced BCSC enrichment in vitro, and dramatically impaired tumor initiation and lung metastasis after implantation of MDA-MB-231 human breast cancer cells into the mammary fat pad of immunodeficient mice. These data provide evidence that targeting A2BR might be an effective strategy to eradicate BCSCs.

Adenosine receptor 2B activity promotes autonomous growth, migration as well as vascularization of head and neck squamous cell carcinoma cells.

Adenosine is a signaling molecule that exerts dual effects on tumor growth: while it inhibits immune cell function and thereby prevents surveillance by the immune system, it influences tumorigenesis directly via activation of adenosine receptors on tumor cells at the same time. However, the adenosine-mediated mechanisms affecting oncogenic processes particularly in head and neck squamous cell carcinomas (HNSCC) are not fully understood. Here, we investigated the role of adenosine receptor activity on HNSCC-derived cell lines. Targeting the adenosine receptor A2B (ADORA2B) on these cells with the inverse agonist/antagonist PSB-603 leads to inhibition of cell proliferation, transmigration as well as VEGFA secretion in vitro. At the molecular level, these effects were associated with cell cycle arrest as well as the induction of the apoptotic pathway. In addition, shRNA-mediated downmodulation of ADORA2B expression caused decreased proliferation. Moreover, in in vivo xenograft experiments, chemical and genetic abrogation of ADORA2B activity impaired tumor growth associated with decreased tumor vascularization. Together, our findings characterize ADORA2B as a crucial player in the maintenance of HNSCC and, therefore, as a potential therapeutic target for HNSCC treatment.

HIF1A up-regulates the ADORA2B receptor on alternatively activated macrophages and contributes to pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a deadly chronic lung disease. Extracellular accumulation of adenosine and subsequent activation of the ADORA2B receptor play important roles in regulating inflammation and fibrosis in IPF. Additionally, alternatively activated macrophages (AAMs) expressing ADORA2B have been implicated in mediating adenosine's effects in IPF. Although hypoxic conditions are present in IPF, hypoxia's role as a direct modulator of macrophage phenotype and identification of factors that regulate ADORA2B expression on AAMs in IPF is not well understood. In this study, an experimental mouse model of pulmonary fibrosis and lung samples from patients with IPF were used to examine the effects and interactions of macrophage differentiation and hypoxia on fibrosis. We demonstrate that hypoxia-inducible factor 1-α (HIF1A) inhibition in late stages of bleomycin-induced injury attenuates pulmonary fibrosis in association, with reductions in ADORA2B expression in AAMs. Additionally, ADORA2B deletion or pharmacological antagonism along with HIF1A inhibition disrupts AAM differentiation and subsequent IL-6 production in cultured macrophages. These findings suggest that hypoxia, through HIF1A, contributes to the development and progression of pulmonary fibrosis through its regulation of ADORA2B expression on AAMs, cell differentiation, and production of profibrotic mediators. These studies support a potential role for HIF1A or ADORA2B antagonists in the treatment of IPF.-Philip, K., Mills, T. W., Davies, J., Chen, N.-Y., Karmouty-Quintana, H., Luo, F., Molina, J. G., Amione-Guerra, J., Sinha, N., Guha, A., Eltzschig, H. K., Blackburn, M. R. HIF1A up-regulates the ADORA2B receptor on alternatively activated macrophages and contributes to pulmonary fibrosis.

Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani.

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.

Identification of hypoxia-inducible factor HIF-1A as transcriptional regulator of the A2B adenosine receptor during acute lung injury.

Although acute lung injury (ALI) contributes significantly to critical illness, resolution often occurs spontaneously through endogenous pathways. We recently found that mechanical ventilation increases levels of pulmonary adenosine, a signaling molecule known to attenuate lung inflammation. In this study, we hypothesized a contribution of transcriptionally controlled pathways to pulmonary adenosine receptor (ADOR) signaling during ALI. We gained initial insight from microarray analysis of pulmonary epithelia exposed to conditions of cyclic mechanical stretch, a mimic for ventilation-induced lung disease. Surprisingly, these studies revealed a selective induction of the ADORA2B. Using real-time RT-PCR and Western blotting, we confirmed an up to 9-fold induction of the ADORA2B following cyclic mechanical stretch (A549, Calu-3, or human primary alveolar epithelial cells). Studies using ADORA2B promoter constructs identified a prominent region within the ADORA2B promoter conveying stretch responsiveness. This region of the promoter contained a binding site for the transcription factor hypoxia-inducible factor (HIF)-1. Additional studies using site-directed mutagenesis or transcription factor binding assays demonstrated a functional role for HIF-1 in stretch-induced increases of ADORA2B expression. Moreover, studies of ventilator-induced lung injury revealed induction of the ADORA2B during ALI in vivo that was abolished following HIF inhibition or genetic deletion of Hif1a. Together, these studies implicate HIF in the transcriptional control of pulmonary adenosine signaling during ALI.

Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells.

Men with sickle cell disease (SCD) risk developing priapism. Recognizing that SCD is a disease of hypoxia, we investigated the effect of hypoxia on gene expression in corporal smooth muscle (CSM) cells. Rat CSM cells in vitro were treated with CoCl2 or low oxygen tension to mimic hypoxia. Hypoxic conditions increased expression of genes previously associated with priapism in animal models. Variable coding sequence a1 (Vcsa1; the rat opiorphin homologue, sialorphin), hypoxia-inducible factor 1a (Hif-1a), and A2B adenosine receptor (a2br) were increased by 10-, 4-, and 6-fold, respectively, by treatment with CoCl2, whereas low oxygen tension caused increases in expression of 3-, 4-, and 1.5-fold, respectively. Sialorphin-treated CSM cells increased expression of Hif-1a and a2br by 4-fold, and vcsa1-siRNA treatment reduced expression by ∼50%. Using a Hif-1a inhibitor, we demonstrated up-regulation of a2br by sialorphin is dependent on Hif-1a, and knockdown of vcsa1 expression with vcsa1-siRNA demonstrated that hypoxic-up-regulation of Hif-1a is dependent on vcsa1. In CSM from a SCD mouse, there was 15-fold up-regulation of opiorphin at a life stage prior to priapism. We conclude that in CSM, opiorphins are master regulators of the hypoxic response. Opiorphin up-regulation in response to SCD-associated hypoxia activates CSM "relaxant" pathways; excessive activation of these pathways results in priapism.

A2B Adenosine Receptor Agonist Improves Erectile Function in Diabetic Rats.

Diabetes is an important risk factor for erectile dysfunction (ED). Recent studies have indicated that A2B adenosine receptor (ADORA2B) signaling is essential for penile erection. Thus, we hypothesize that diabetic ED may be attributed to impaired A2B adenosine signaling. To test this hypothesis, we generated diabetic rats by injecting streptozocin as animal model. After 12 weeks, immunohistochemistry staining was used to localize the expression of ADORA2B. Western Blot and quantitative PCR were employed to determine ADORA2B expression level. Intracavernosal pressure (ICP) measurement was used to evaluate erectile function. Diabetic rats received a single intravenous injection of BAY 60-6583, an ADORA2B agonist, or vehicle solution, at 60 min before the ICP measurement. The results showed that ADORA2B expressed in the nerve bundle, smooth muscle, and endothelium in penile tissue of control mice. Western Blot and quantitative PCR results indicated that the expression levels of ADORA2B protein and mRNA were significantly reduced in penile tissues of diabetic rats. Functional studies showed that the erectile response induced by electrical stimulation was remarkably decreased in diabetic rats, compared with age-matched control rats. However, at 60 min after BAY 60-6583 treatment, the erectile function was improved in diabetic rats, suggesting that enhancement of ADORA2B signaling may improve erectile function in diabetic ED. This preclinical study has revealed a previously unrecognized therapeutic possibility of BAY 60-6583 as an effective and mechanism-based drug to treat diabetic ED. In conclusion, we propose that impaired A2B adenosine signaling is one of the pathological mechanisms of diabetic ED.

Expression of adenosine A2b receptor in rat type II and III taste cells.

We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 µM adenosine or 10 µM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats.

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy.

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.

Adenosine 2B receptor expression is post-transcriptionally regulated by microRNA.

We have reported that epithelial adenosine 2B receptor (A(2B)AR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor alpha (TNF-alpha). Here, we examined the mechanism that governs A(2B)AR expression during colitis. A 1.4-kb sequence of the A(2B)AR promoter was cloned into the pFRL7 luciferase vector. Anti-microRNA (miRNA) was custom-synthesized based on specific miRNA binding sites. The binding of miRNA to the 3'-untranslated region (UTR) of A(2B)AR mRNA was examined by cloning this 3'-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-alpha induced a 35-fold increase in A(2B)AR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A(2B)AR mRNA following TNF-alpha treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3'-UTR of the A(2B)AR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-alpha reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A(2B)AR levels by >60%. Blockade of miR27b increased A(2B)AR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A(2B)AR mRNA. Collectively, these data demonstrate that TNF-alpha-induced A(2B)AR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A(2B)AR expression in murine colitis.

Adenosine A2A receptor is involved in cell surface expression of A2B receptor.

The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological processes. However, the mechanisms of their intracellular maturation and trafficking are poorly understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that the levels of cell surface expression of A2BR were significantly lower than those of A2AR. A large portion of the A2BR was degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules in transfectants suggested that A2BR does not have the dominant forward transport signal for export from the endoplasmic reticulum to the cell surface. A2BR surface expression was increased in A2BR chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with the A2AR CT. Co-transfection of A2AR with A2BR enhanced surface expression of A2BR though the F(X)(6)LL motif in the A2AR CT. The requirements of A2AR expression for better A2BR cell surface expression was not only established in transfectants but also confirmed by observations of much lower levels of A2BR-induced intracellular cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR(-/-) mice than in wild type mice. The results of mechanistic studies suggested that poor A2BR expression at the cell surface might be accounted for mainly by the lack of a dominant forward transport signal from the endoplasmic reticulum to the plasma membrane; it is likely that A2BR forms a hetero-oligomer complex for better function.

Oxidative/nitrosative stress selectively altered A(2B) adenosine receptors in chronic obstructive pulmonary disease.

The primary aim of this study was to investigate adenosine receptors (ARs) in bronchoalveolar lavage (BAL) macrophages from patients with chronic obstructive pulmonary disease (COPD) and age-matched healthy smokers. A(2B)ARs were significantly decreased in BAL macrophages from patients with COPD when compared with healthy smokers. The effect of proinflammatory cytokines and oxidative/nitrosative stress on AR expression and function in U937 cells before and after PMA treatment was evaluated. IL-1beta and TNF-alpha treatment up-regulated A(2A)- and A(3)ARs but not A(1)- or A(2B)ARs, whereas IL-6 did not modify AR expression. In contrast, oxidative/nitrosative stress selectively decreased A(2B)AR expression, which was associated with a reduction in the potency of the adenosine agonist 5'-N-ethylcarboxamideadenosine (NECA) to induce cAMP. Further, the ability of NECA to enhance cell proliferation was increased after oxidative/nitrosative stress. The specific involvement of A(2B)ARs was investigated by using potent and selective A(2B)AR antagonist and by A(2B)AR knockdown using siRNA and demonstrated responses similar to those obtained with oxidative/nitrosative stress. N-acetylcysteine (NAC), an antioxidant agent, counteracted the decrease in A(2B)AR expression, as well as the altered NECA effects on cAMP and cell proliferation. These findings highlight the central role of A(2B)ARs in alveolar macrophages, suggesting that their modulation could represent an innovative pharmacological strategy to manage COPD.-Varani, K., Caramori, G., Vincenzi, F., Tosi, A., Barczyk, A., Contoli, M., Casolari, P., Triggiani, M., Hansel, T., Leung, E., MacLennan, S., Barnes, P. J., Fan Chung, K., Adcock, I., Papi, A., Borea, P. A. Oxidative/nitrosative stress selectively altered A(2B) adenosine receptors in chronic obstructive pulmonary disease.

Effects of thoracic epidural anesthesia on flow and endothelium of internal thoracic artery in coronary artery bypass graft surgery.

OBJECTIVES: The internal thoracic artery (ITA) is the preferred conduit for coronary artery bypass graft (CABG) surgery. The authors investigated whether thoracic epidural anesthesia (TEA) as an adjunct to general anesthesia (GA) can increase the blood flow of the ITA. DESIGN: A prospective randomized study. SETTING: A university hospital. PARTICIPANTS: Patients with ischemic heart disease. INTERVENTIONS: Thirty patients scheduled for elective CABG surgery were randomized to receive either GA (n = 15) or GA + TEA (n = 15) after receiving institutional review board approval. Demographics showed similarity between the groups. The epidural catheter was inserted in the thoracic region between T1 and T5 levels. In the GA + TEA group, the patients received a 20-mg bolus of 0.25% bupivacaine through epidural catheters 1 hour before surgery, and this was followed by the infusion (20 mg/h) of 0.25% bupivacaine. In all patients, ITA free blood flow was measured before cardiopulmonary bypass and without the administration of any vasodilatory agent. A short segment of ITA was excised for histologic examination; immunocytochemistry analysis was performed using antirabbit polyclonal VEGF antibody, rabbit polyclonal inducible nitric oxide synthase (i-NOS) antibody, and adenosine anti-A2B receptor antibody. The immunoreactivity rates then were evaluated. MAIN RESULTS: The mean ITA free flow in the GA + TEA group was significantly higher than in the GA group (56.0 ± 9.0 mL/min v 39.6 ± 14 mL/min, p = 0.001). Immunostaining intensity in the sections after incubation with each primary antibody increased in the GA + TEA group compared with the GA group. CONCLUSIONS: The results of this study indicated that TEA increased ITA free blood flow significantly via increased VEGF, i-NOS, and adenosine-A2B receptor expressions. Therefore, the use of TEA as an adjunct to GA might be considered as an alternative to vasoactive agents for increasing ITA flow in CABG surgery.

Extracellular 3',5'-cAMP-adenosine pathway inhibits glomerular mesangial cell growth.

Abnormal growth of glomerular mesangial cells (GMCs) contributes to the pathophysiology of many types of nephropathy. Because adenosine is an autocrine/paracrine factor that potentially could regulate GMC proliferation and because the extracellular 3',5'-cAMP-adenosine pathway (i.e., the conversion of extracellular 3',5'-cAMP to 5'-AMP and adenosine on the cell surface) could generate adenosine in the biophase of GMC receptors, we investigated the role of the 3',5'-cAMP-adenosine pathway in modulating growth [cell proliferation, DNA synthesis ([(3)H]thymidine incorporation), collagen synthesis ([(3)H]proline incorporation), and mitogen-activated protein kinase activity] of GMCs. The addition of exogenous 3',5'-cAMP to human GMCs increased extracellular levels of 5'-AMP, adenosine, and inosine, and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), 1,3-dipropyl-8-p-sulfophenylxanthine (ecto-phosphodiesterase inhibitor), and alpha,beta-methylene-adenosine-5'-diphosphate (ecto-5'-nucleotidase inhibitor) attenuated the increases in adenosine and inosine. Forskolin augmented extracellular 3',5'-cAMP and adenosine concentrations, and 2',5'-dideoxyadenosine (adenylyl cyclase inhibitor) blocked these increases. Exogenous 3',5'-cAMP and forskolin inhibited all indices of cell growth, and antagonism of A(2) [(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine, KF17837] or A(1)/A(2) (1,3-dipropyl-8-p-sulfophenylxanthine, DPSPX), but not A(1) (8-cyclopentyl-1,3-dipropylxanthine), or A(3){N-(2-methoxyphenyl)-N'-[2-(3-pyridinyl)-4-quinazolinyl]-urea, VUF5574}, adenosine receptors blocked the growth-inhibitory actions of exogenous 3',5'-cAMP, but not the effects of 8-bromo-3',5'-cAMP (stable 3',5'-cAMP analog). Erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor) plus 5-iodotubercidin (adenosine kinase inhibitor) enhanced the growth inhibition by exogenous 3',5'-cAMP and forskolin, and A(2) receptor antagonism blocked this effect. In rat GMCs, down-regulation of A(2B) receptors with antisense, but not sense or scrambled, oligonucleotides abrogated the inhibitory effects of 3',5'-cAMP and forskolin on cell growth. The extracellular 3',5'-cAMP-adenosine pathway exists in GMCs and attenuates cell growth via A(2B) receptors. Pharmacological augmentation of this pathway could abate pathological glomerular remodeling.

Adenosine receptor-mediated adhesion of endothelial progenitors to cardiac microvascular endothelial cells.

Intracoronary delivery of endothelial progenitor cells (EPCs) is an emerging concept for the treatment of cardiovascular disease. Enhancement of EPC adhesion to vascular endothelium could improve cell retention within targeted organs. Because extracellular adenosine is elevated at sites of ischemia and stimulates neovascularization, we examined the potential role of adenosine in augmenting EPC retention to cardiac microvascular endothelium. Stimulation of adenosine receptors in murine embryonic EPCs (eEPCs) and cardiac endothelial cells (cECs) rapidly, within minutes, increased eEPC adhesion to cECs under static and flow conditions. Similarly, adhesion of human adult culture-expanded EPCs to human cECs was increased by stimulation of adenosine receptors. Furthermore, adenosine increased eEPC retention in isolated mouse hearts perfused with eEPCs. We determined that eEPCs and cECs preferentially express functional A1 and A2B adenosine receptor subtypes, respectively, and that both subtypes are involved in the regulation of eEPC adhesion to cECs. We documented that the interaction between P-selectin and its ligand (P-selectin glycoprotein ligand-1) plays a role in adenosine-dependent eEPC adhesion to cECs and that stimulation of adenosine receptors in cECs induces rapid cell surface expression of P-selectin. Our results suggest a role for adenosine in vasculogenesis and its potential use to stimulate engraftment in cell-based therapies.

Identification of zebrafish A2 adenosine receptors and expression in developing embryos.

The A2A adenosine receptor (AdR) subtype has emerged as an attractive target in the pursuit of improved therapy for Parkinson's disease (PD). This report focuses on characterization of zebrafish a2 AdRs. By mining the zebrafish EST and genomic sequence databases, we identified two zebrafish a2a (adora2a.1 and adora2a.2) genes and one a2b (adora2b) AdR gene. Sequence comparisons indicate that the predicted zebrafish A2 AdR polypeptides share 62-74% amino acid identity to mammalian A2 AdRs. We mapped the adora2a.1 gene to chromosome 8, the adora2a.2 gene to chromosome 21, and the adora2b gene to chromosome 5. Whole mount in situ hybridization analysis indicates zebrafish a2 AdR genes are expressed primarily within the central nervous system (CNS). Zebrafish are known to be sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that causes selective loss of dopaminergic neurons and PD-like symptoms in humans as well as in animal models. Here we show that caffeine, an A2A AdR antagonist, is neuroprotective against the adverse effects of MPTP in zebrafish embryos. These results suggest that zebrafish AdRs may serve as useful targets for testing novel therapeutic strategies for the treatment of PD.

A2B adenosine receptors regulate the mucus clearance component of the lung's innate defense system.

Adenosine (ADO) signaling is altered in both asthma and chronic obstructive pulmonary disease, and the A(2B) adenosine receptor (A(2B)-R) may drive pulmonary inflammation. Accordingly, it has been proposed that specific inhibition of the A(2B)-R could treat inflammatory lung diseases. However, stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by ADO may be crucial in permitting the superficial epithelium to maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Our goal was to determine which ADO receptor (ADO-R) underlies ASL volume regulation in bronchial epithelia. We used PCR techniques to determine ADO-R expression in bronchial epithelia and used nasal potential difference measurements, Ussing chambers studies, and XZ-confocal microscopy to look at Cl- secretion and ASL volume regulation. The A(2B)-R was the most highly expressed ADO-R in donor specimens of human bronchial epithelia, and inhibition of ADO-R in vivo prevented activation of CFTR. A(2B)-R was the only ADO-R detected in cultured human bronchial epithelial cells and inhibition of this receptor with specific A(2B)-R antagonists resulted in ASL height collapse and a failure to effect ASL height homeostasis. Removal of ADO with ADO deaminase and replacement with 5'N-ethylcarboxamide adenosine resulted in dose-dependent changes in ASL height, and suggested that the cell surface (ADO) may be in excess of 1 microM, which is sufficient to activate A(2B)-R. A(2B)-R are required for ASL volume homeostasis in human airways, and therapies directed at inhibiting A(2B)-R may lead to a cystic fibrosis-like phenotype with depleted ASL volume and mucus stasis.

Cardioprotection by ecto-5'-nucleotidase (CD73) and A2B adenosine receptors.

BACKGROUND: Ecto-5'-nucleotidase (CD73)-dependent adenosine generation has been implicated in tissue protection during acute injury. Once generated, adenosine can activate cell-surface adenosine receptors (A1 AR, A2A AR, A2B AR, A3 AR). In the present study, we define the contribution of adenosine to cardioprotection by ischemic preconditioning. METHODS AND RESULTS: On the basis of observations of CD73 induction by ischemic preconditioning, we found that inhibition or targeted gene deletion of cd73 abolished infarct size-limiting effects. Moreover, 5'-nucleotidase treatment reconstituted cd73-/- mice and attenuated infarct sizes in wild-type mice. Transcriptional profiling of adenosine receptors suggested a contribution of A2B AR because it was selectively induced by ischemic preconditioning. Specifically, in situ ischemic preconditioning conferred cardioprotection in A1 AR-/-, A2A AR-/-, or A3 AR-/- mice but not in A2B AR-/- mice or in wild-type mice after inhibition of the A2B AR. Moreover, A2B AR agonist treatment significantly reduced infarct sizes after ischemia. CONCLUSIONS: Taken together, pharmacological and genetic evidence demonstrate the importance of CD73-dependent adenosine generation and signaling through A2B AR for cardioprotection by ischemic preconditioning and suggests 5'-nucleotidase or A2B AR agonists as therapy for myocardial ischemia.

B-Myb regulates the A(2B) adenosine receptor in vascular smooth muscle cells.

The A(2B) adenosine receptor (A(2B)AR) has been described to control various vascular functions, including inhibition of smooth muscle cell proliferation. Here, we sought to understand the regulation of A(2B)AR gene expression in aortic vascular smooth muscle cells (VSMCs), with a focus on the proliferation phase. Assays with A(2B)AR-beta-gal aortic VSMCs, in which the endogenous A(2B)AR gene promoter drives the expression of prokaryotic beta-galactosidase (beta-gal) instead of the endogenous A(2B)AR gene, show that beta-gal expression is upregulated when the cells are induced to exit from cell cycle arrest. Similarly, the level of A(2B)AR mRNA is upregulated in proliferating primary aortic VSMCs. In search of related mechanisms, it was noted that the A(2B)AR gene promoter contains several putative binding sites for the proliferation-inducing transcription factor, B-Myb. Using a clone of the 5' region upstream of the mouse A(2B)AR gene linked to a reporter gene, B-Myb site deletion mutants were generated. It was determined that B-Myb upregulates the A(2B)AR gene promoter, and specific promoter binding sites were identified as functional. In accordance, B-Myb also elevates endogenous A(2B)AR mRNA and receptor activity, and this activity decreases cell proliferation. Our data are novel in that they show that this proliferation-inhibiting A(2B)AR is itself an inducible receptor regulated by B-Myb.

Effect of A2B adenosine receptor gene ablation on adenosine-dependent regulation of proinflammatory cytokines.

Pharmacological studies suggest that A(2B) adenosine receptors mediate proinflammatory effects of adenosine. This concept was recently challenged by the finding that A(2B) adenosine receptor knockout (A(2B)KO) mice had moderate inflammation due to elevated basal plasma tumor necrosis factor (TNF)-alpha and an exaggerated response to lipopolysaccharide (LPS) challenge. However, it is unclear whether this phenomenon actually reflects the loss of putative taming of proinflammatory cytokine production via activation of A(2B) receptors by endogenous adenosine. In this report, we examined adenosine receptor-dependent regulation of interleukin (IL)-6 and TNF-alpha blood plasma levels in A(2B)KO and wild-type mice in vivo and their release from peritoneal macrophages ex vivo. Stimulation of adenosine receptors with 5'-N-ethylcarboxamidoadenosine (NECA) up-regulated IL-6 and suppressed LPS-induced TNF-alpha in wild-type mice. The selective A(2B) antagonists 3-isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine (MRS 1754) inhibited NECA-induced IL-6 release but not the suppression of LPS-induced TNF-alpha secretion from macrophages. Genetic ablation of A(2B) receptors abrogated NECA-induced increases in IL-6 release from mouse peritoneal macrophages and dramatically reduced the ability of NECA to raise IL-6 plasma levels in vivo. In contrast, the absence of A(2B) adenosine receptors did not affect NECA-induced suppression of LPS-activated TNF-alpha release in macrophages, nor did it reduce the ability of NECA to suppress LPS-induced increase in TNF-alpha plasma levels in vivo. Thus, our results indicate that stimulation of A(2B) receptors up-regulates the proinflammatory cytokine IL-6 and argue against the recently suggested anti-inflammatory role of A(2B) receptors in suppression of LPS-stimulated TNF-alpha production by adenosine.