bay-60-6583 and Inflammation

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Adult; Aminopyridines; Animals; Apyrase; Cells, Cultured; Cytokines; GPI-Linked Proteins; Humans; Infertility, Male; Inflammation; Male; Mice; Mice, Inbred C57BL; Middle Aged; Receptor, Adenosine A2B; Receptors, Purinergic P1; RNA Interference; RNA, Small Interfering; Testis

Inflammation is responsible for secondary organ failure after trauma and hemorrhagic shock (T/HS). Adenosine, acting through four G protein-coupled cell surface receptors, A1, A2A, A2B, and A3, exerts a number of tissue protective and anti-inflammatory effects. The goal of the present study was to test the effect of A2B adenosine receptor stimulation on T/HS-induced organ injury and inflammation in rats. Rats after T/HS were resuscitated with Ringer's lactate containing the A2B receptor agonist BAY 60-6583 or its vehicle. We found that BAY 60-6583 decreased T/HS-induced lung permeability and plasma creatine kinase levels but failed to affect T/HS-induced lung neutrophil infiltration and IκBα expression and plasma alanine aminotransferase levels. Thus, we conclude that stimulation of A2B receptors protects against T/HS-induced lung and muscle injury.

Topics: Acute Lung Injury; Aminopyridines; Animals; Blotting, Western; Disease Models, Animal; Inflammation; Male; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2B; Shock, Hemorrhagic; Wounds and Injuries

Chronic obstructive pulmonary disease (COPD) is a neutrophilic inflammatory disorder that is weakly responsive to glucocorticoids. Identification of ways to enhance the anti-inflammatory activity of glucocorticoids is, therefore, a major research objective. Adenosine receptor agonists that target the A2B-receptor subtype are efficacious in several cell-based assays and preclinical models of inflammation. Accordingly, the present study was designed to determine if a selective A2B-receptor agonist, 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulphanyl]acetamide (Bay 60-6583), and a glucocorticoid, dexamethasone, in combination display putative anti-inflammatory activity that is superior to either drug alone. In BEAS-2B human airway epithelial cells stably transfected with cAMP-response element (CRE) and glucocorticoid response element (GRE) reporter constructs, Bay 60-6583 promoted CRE-dependent transcription and enhanced GRE-dependent transcription by an adenosine A2B-receptor-mediated mechanism that was associated with cAMP formation and abolished by an inhibitor of cAMP-dependent protein kinase. Analysis of the concentration-response relationship that described the enhancement of GRE-dependent transcription showed that Bay 60-6583 increased the magnitude of response without affecting the potency of dexamethasone. Bay 60-6583 and dexamethasone also induced a panel of genes that, collectively, could have benefit in COPD. These were categorized into genes that were induced in a positive cooperative manner (RGS2, p57(kip2)), an additive manner (TTP, BRL-1), or by Bay 60-6583 (CD200, CRISPLD2, SOCS3) or dexamethasone (GILZ) only. Thus, the gene induction "fingerprints" produced by Bay 60-6583 and dexamethasone, alone and in combination, were distinct. Collectively, through their actions on gene expression, an adenosine A2B-receptor agonist and a glucocorticoid administered together may have utility in the treatment of inflammatory disorders that respond suboptimally to glucocorticoids as a monotherapy.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Algorithms; Aminopyridines; Calcium; Cell Line; Cyclic AMP; Cyclic AMP Response Element Modulator; Cytosol; Dexamethasone; Epithelial Cells; Humans; Inflammation; Phenethylamines; Pulmonary Disease, Chronic Obstructive; Real-Time Polymerase Chain Reaction; Receptor, Adenosine A2B; Receptors, Glucocorticoid; Respiratory Mucosa; RNA, Messenger; Transfection

High fat diet and its induced changes in glucose homeostasis, inflammation and obesity continue to be an epidemic in developed countries. The A2b adenosine receptor (A2bAR) is known to regulate inflammation. We used a diet-induced obesity murine knockout model to investigate the role of this receptor in mediating metabolic homeostasis, and correlated our findings in obese patient samples.. Administration of high fat, high cholesterol diet (HFD) for sixteen weeks vastly upregulated the expression of the A2bAR in control mice, while A2bAR knockout (KO) mice under this diet developed greater obesity and hallmarks of type 2 diabetes (T2D), assessed by delayed glucose clearance and augmented insulin levels compared to matching control mice. We identified a novel link between the expression of A2bAR, insulin receptor substrate 2 (IRS-2), and insulin signaling, determined by Western blotting for IRS-2 and tissue Akt phosphorylation. The latter is impaired in tissues of A2bAR KO mice, along with a greater inflammatory state. Additional mechanisms involved include A2bAR regulation of SREBP-1 expression, a repressor of IRS-2. Importantly, pharmacological activation of the A2bAR by injection of the A2bAR ligand BAY 60-6583 for four weeks post HFD restores IRS-2 levels, and ameliorates T2D. Finally, in obese human subjects A2bAR expression correlates strongly with IRS-2 expression.. Our study identified the A2bAR as a significant regulator of HFD-induced hallmarks of T2D, thereby pointing to its therapeutic potential.

Topics: Adenosine A2 Receptor Agonists; Adult; Aminopyridines; Animals; Cholesterol; Diabetes Mellitus, Type 2; Dietary Fats; Female; Gene Expression Regulation; Glucose; Homeostasis; Humans; Inflammation; Insulin; Insulin Receptor Substrate Proteins; Male; Mice; Mice, Knockout; Middle Aged; Obesity; Receptor, Adenosine A2B; Sterol Regulatory Element Binding Protein 1

Gastrointestinal ischemia/reperfusion (IR) injury significantly contributes to the morbidity and mortality of critical illness. In this study, we hypothesized a protective role for extracellular adenosine signaling in intestinal IR injury. Initial profiling studies of mucosal scrapings following murine IR demonstrated selective induction of the A2B adenosine receptor (A2BAR) transcript. Moreover, gene-targeted mice for the A2BAR showed more profound intestinal IR injury compared with controls. In contrast, A2AAR(-/-) mice exhibited no differences in intestinal injury compared with littermate controls. In addition, selective inhibition of the A2BAR resulted in enhanced intestinal inflammation and injury during IR. Furthermore, A2BAR agonist treatment (BAY 60-6583) protected from intestinal injury, inflammation, and permeability dysfunction in wild-type mice, whereas the therapeutic effects of BAY 60-6583 were abolished following targeted A2BAR gene deletion. Taken together, these studies demonstrate the A2BAR as a novel therapeutic target for protection during gastrointestinal IR injury.

Topics: Aminopyridines; Animals; Blotting, Western; Gene Expression Profiling; Inflammation; Intestinal Mucosa; Intestines; Mice; Mice, Knockout; Receptor, Adenosine A2B; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

Acute renal failure from ischemia significantly contributes to morbidity and mortality in clinical settings, and strategies to improve renal resistance to ischemia are urgently needed. Here, we identified a novel pathway of renal protection from ischemia using ischemic preconditioning (IP).. For this purpose, we utilized a recently developed model of renal ischemia and IP via a hanging weight system that allows repeated and atraumatic occlusion of the renal artery in mice, followed by measurements of specific parameters or renal functions. Studies in gene-targeted mice for each individual adenosine receptor (AR) confirmed renal protection by IP in A1(-/-), A2A(-/-), or A3AR(-/-) mice. In contrast, protection from ischemia was abolished in A2BAR(-/-) mice. This protection was associated with corresponding changes in tissue inflammation and nitric oxide production. In accordance, the A2BAR-antagonist PSB1115 blocked renal protection by IP, while treatment with the selective A2BAR-agonist BAY 60-6583 dramatically improved renal function and histology following ischemia alone. Using an A2BAR-reporter model, we found exclusive expression of A2BARs within the reno-vasculature. Studies using A2BAR bone-marrow chimera conferred kidney protection selectively to renal A2BARs.. These results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia.

Topics: Adenosine; Adenosine A2 Receptor Antagonists; Aminopyridines; Animals; Blood Vessels; Cytoprotection; Extracellular Fluid; Female; Inflammation; Ischemia; Kidney; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Receptor, Adenosine A2B; Signal Transduction; Xanthines

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

Topics: 5'-Nucleotidase; Adenosine; Adenosine A2 Receptor Agonists; Adenosine Monophosphate; Aminopyridines; Animals; Cell Movement; Dendritic Cells; Endothelium, Vascular; Inflammation; L-Selectin; Lipopolysaccharides; Lymph Nodes; Mice; Mice, Knockout; Receptor, Adenosine A2B; T-Lymphocytes; Tumor Necrosis Factor-alpha; Up-Regulation; Venules