Brain adenosine concentrations can reach micromolar concentrations in stressful situations such as stroke, neurodegenerative diseases or hypoxic regions of brain tumours. Adenosine can act by receptor-independent mechanism by reversing the reaction catalysed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation and inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases. Astrocytes are essential in maintaining brain homeostasis but their pathological activation and uncontrolled proliferation plays a role in neurodegeneration and glioma. Adenosine can affect cell proliferation, but the effect of increased adenosine concentration on proliferation of astrocytes is not clarified and was addressed in present work. Human astrocytes (HA) were treated for 3 days with test drugs. Cell proliferation/viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay and by cell counting. Cell death was evaluated by assessing lactate dehydrogenase release and by western blot analysis of αII-Spectrin cleavage. 30 µM-Adenosine caused a 40% ± 3% (p < .05, n = 5) reduction in cell proliferation/viability, an effect reversed by 2U/ml-adenosine deaminase, but unchanged in the presence of antagonists of any of the adenosine receptors. Adenosine alone did not induce cell death. 100 µM-Homocysteine alone caused 16% ± 3% (p < .05) decrease in HA proliferation. Combined action of adenosine and homocysteine decreased HA proliferation by 76% ± 4%, an effect higher (p < .05) than the sum of the effects of adenosine and homocysteine alone (56% ± 5%). The inhibitory effect of adenosine on HA proliferation/viability was mimicked by two adenosine kinase inhibitors and attenuated in the presence of folate (100 µM) or SAM (50-100 µM). The results suggest that adenosine reduces HA proliferation by a receptor-independent mechanism probably involving reversal of SAH hydrolase-catalysed reaction.

Osteoarthritis (OA) is characterized by cartilage destruction and chondrocytes have a central role in this process. With age and inflammation chondrocytes have reduced capacity to synthesize and maintain ATP, a molecule important for cartilage homeostasis. Here we show that concentrations of ATP and adenosine, its metabolite, fall after treatment of mouse chondrocytes and rat tibia explants with IL-1β, an inflammatory mediator thought to participate in OA pathogenesis. Mice lacking A2A adenosine receptor (A2AR) or ecto-5'nucleotidase (an enzyme that converts extracellular AMP to adenosine) develop spontaneous OA and chondrocytes lacking A2AR develop an 'OA phenotype' with increased expression of Mmp13 and Col10a1. Adenosine replacement by intra-articular injection of liposomal suspensions containing adenosine prevents development of OA in rats. These results support the hypothesis that maintaining extracellular adenosine levels is an important homeostatic mechanism, loss of which contributes to the development of OA; targeting adenosine A2A receptors might treat or prevent OA.

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5'-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a 'calm down' signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001.

A recent study (American Journal of Physiology-Cell Physiology 304: C406-C421, 2013) suggests that extracellular guanosine increases extracellular adenosine by modifying the disposition of extracellular adenosine ("guanosine-adenosine mechanism") and that the guanosine-adenosine mechanism is not mediated by classical adenosine transport systems (SLC28 and SLC29 families) nor by classical adenosine-metabolizing enzymes. The present investigation had two aims: 1) to test the hypothesis that the "guanosine-adenosine mechanism" affects cell proliferation; and 2) to determine whether the transporters SLC19A1, SLC19A2, SLC19A3 or SLC22A2 (known to carrier guanosine analogues) might be responsible for the guanosine-adenosine mechanism. In the absence of added adenosine, guanosine had little effect on the proliferation of coronary artery vascular smooth muscle cells (vascular conduit cells) or preglomerular vascular smooth muscle cells (vascular resistance cells). However, in the presence of added adenosine (3 or 10 μmol/L), guanosine (10 to 100 μmol/L) decreased proliferation of both cell types, thus resulting in a highly significant (p<0.000001) interaction between guanosine and adenosine on cell proliferation. The guanosine-adenosine interaction on cell proliferation was abolished by 1,3-dipropyl-8-(p-sulfophenyl)xanthine (adenosine receptor antagonist). Guanosine (30 μmol/L) increased extracellular levels of adenosine when adenosine (3 μmol/L) was added to the medium. This effect was not reproduced by high concentrations of methotrexate (100 μmol/L), thiamine (1000 μmol/L), chloroquine (1000 μmol/L) or acyclovir (10,000 μmol/L), archetypal substrates for SLC19A1, SLC19A2, SLC19A3, and SLC22A2, respectively; and guanosine still increased adenosine levels in the presence of these compounds.

We investigated the role of adenosine in citalopram-induced cardiotoxicity.

Previous studies evaluated the adenosine receptor antagonists alone to determine their effects on oxidative stress, but little is known about adenosine's protective efficacy when oxidative injury occurs in vivo. Adenosine is a crucial signaling molecule recognized by four distinct G-protein-coupled receptors (GPCRs) (i.e., A1R, A2AR, A2BR, and A3R) and protects cells against pathological conditions. The present study was performed to evaluate the role of antagonist modulation in the setting of paraquat toxicity with adenosine pretreatment. First, PC12 cells were exposed to paraquat (850 μM) and adenosine (30 μM) to develop an in vitro model for the antagonist effect assay. Second, we found that the A1R antagonist DPCPX enhanced the viability of paraquat-induced PC12 cells that underwent adenosine pretreatment. Moreover, the A2AR antagonist ZM241385 decreased the viability of paraquat-induced PC12 cells that underwent adenosine pretreatment. Our findings indicate that adenosine protection requires a dual blockade of A1R and activation of A2AR to work at its full potential, and the A2B and A3 adenosine receptor antagonists increased paraquat-induced oxidative damage. This represents a novel pharmacological strategy based on A1/A2A interactions and can assist in clarifying the role played by AR antagonists in the treatment of neurodegenerative diseases.

Adenosine A(2A) receptor antagonists have been proposed as an effective therapy in the treatment of Parkinson's disease. To explore the possibility that dopamine denervation may produce modifications in adenosine A(2A) transmission, we measured the extracellular concentration of adenosine and adenosine A(2A) receptor mRNA in the striatum of rats infused unilaterally with 6-hydroxydopamine in the medial forebrain bundle. Fifteen days after 6-hydroxydopamine infusion, extracellular adenosine levels, measured by in vivo microdialysis, were significantly lower (-35%) in the dopamine-denervated striatum. At the time of the decrease in adenosine levels, an increase in striatal adenosine A(2A) receptor mRNA levels (+20%), measured by in situ hybridization, was observed. Modifications in adenosine A(2A) transmission, following nigrostriatal dopamine neuron degeneration, establish a potential neural basis for the effectiveness of adenosine A(2A) receptor antagonists in the treatment of Parkinson's disease.

The neurochemical effects of adenosine signaling in small-fiber neuropathy leading to neuropathic pain are yet to be explored in a direct manner. This study examined this system at the level of ligand (through the ectonucleotidase activity of prostatic acid phosphatase [PAP]) and adenosine A1 receptors (A1Rs) in resiniferatoxin (RTX) neuropathy, a peripheral neurodegenerative disorder that specifically affects nociceptive nerves expressing transient receptor potential vanilloid type 1 (TRPV1). We conducted immunohistochemistry on dorsal root ganglion (DRG) neurons, high-performance liquid chromatography for functional assays, and pharmacological interventions to alter PAP and A1Rs in mice with RTX neuropathy. In DRG of RTX neuropathy, PAP(+) neurons were reduced compared with vehicle-treated mice (P = 0.002). Functionally, PAP ectonucleotidase activity was consequently reduced (ie, the content of adenosine in DRG, P = 0.012). PAP(+) neuronal density was correlated with the degree of mechanical allodynia, which was reversed by intrathecal (i.t.) lumbar puncture injection of recombinant PAP with a dose-dependent effect. Furthermore, A1Rs were downregulated (P = 0.002), and this downregulation was colocalized with the TRPV1 receptor (31.0% ± 2.8%). Mechanical allodynia was attenuated in a dose-dependent response by i.t. injection of the A1R ligand, adenosine; however, no analgesia was evident when an exogenous adenosine was blocked by A1R antagonist. This study demonstrated dual mechanisms of neuropathic pain in TRPV1-induced neuropathy, involving a reduced adenosine system at both the ligand (adenosine) and receptor (A1Rs) levels.

Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A2ARs. While blockade of the A2AARs subtype effectively rescues lymphocyte activity, with four A2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A2AAR/A2BAR dimers in tissues that coexpress the two receptor subtypes, where the A2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy.

Intraperitoneal adenosine reduces abdominal adhesions. However, because of the ultra-short half-life and low solubility of adenosine, optimal efficacy requires multiple dosing.

Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds.

Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by 2-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain.

The activity of a cell-surface ecto-adenosine deaminase (eADA) is markedly increased in the endothelial activation and vascular inflammation leading to decreased adenosine concentration and alterations in adenosine signalling. Depending on the specific pathway activated, extracellular purines mediate host cell response or regulate growth and cytotoxicity on tumour cells. The aim of this study was to test the effects of adenosine deaminase inhibition by 2'deoxycoformycin (dCF) on the breast cancer development. dCF treatment decreased a tumour growth and a final tumour mass in female BALB/c mice injected orthotopically with 4T1 cancer cells. dCF also counteracted cancer-induced endothelial dysfunction in orthotopic and intravenous 4T1 mouse breast cancer models. In turn, this low dCF dose had a minor effect on immune stimulation exerted by 4T1 cell implantation. In vitro studies revealed that dCF suppressed migration and invasion of 4T1 cells via A2a and A3 adenosine receptor activation as well as 4T1 cell adhesion and transmigration through the endothelial cell layer via A2a receptor stimulation. Similar effects of dCF were observed in human breast cancer cells. Moreover, dCF improved a barrier function of endothelial cells decreasing its permeability. This study highlights beneficial effects of adenosine deaminase inhibition on breast cancer development. The inhibition of adenosine deaminase activity by dCF reduced tumour size that was closely related to the decreased aggressiveness of tumour cells by adenosine receptor-dependent mechanisms and endothelial protection.

A series of 12 novel polyethylene-glycol(PEG)-alkynyl C2-adenosine(ADN) conjugates were synthesized using a robust Sonogashira coupling protocol and characterized by NMR spectroscopy and mass spectrometry analysis. The ADN-PEG conjugates showed null to moderate toxicity in murine macrophages and 12c was active against Mycobacterium aurum growth (MIC = 62.5 mg/L). The conjugates were not active against Mycobacterium bovis BCG. Conjugates 10b and 11b exhibited high water solubility with solubility values of 1.22 and 1.18 mg/ml, respectively, in phosphate buffer solutions at pH 6.8. Further, 10b and 11b induced a significant increase in cAMP accumulation in RAW264.7 cells comparable with that induced by adenosine. Analogues 10c, 11c and 12c were docked to the A1 , A2A , A2B and A3 adenosine receptors (ARs) using crystal-structures and homology models. ADN-PEG-conjugates bearing chains with up to five ethyleneoxy units could be well accommodated within the binding sites of A1 , A2A and A3 ARs. Docking studies showed that compound 10b and 11b were the best A2A receptor binders of the series, whereas 12c was the best binder for A1 AR. In summary, introduction of hydrophilic PEG substituents at the C2 of adenine ring significantly improved water solubility and did not affect AR binding properties of the ADN-PEG conjugates.

The early release of adenosine following traumatic brain injury (TBI) suppresses seizures and brain inflammation; thus, it is important to elucidate the cellular sources of adenosine following injurious stimuli triggered by TBI so that therapeutics for enhancing the early adenosine-release response can be optimized. Using mass spectrometry with 13 C-labeled standards, we investigated in cultured rat neurons, astrocytes, and microglia the effects of oxygen-glucose deprivation (OGD; models energy failure), H2 O2 (produces oxidative stress), and glutamate (induces excitotoxicity) on intracellular and extracellular levels of 5'-AMP (adenosine precursor), adenosine, and inosine and hypoxanthine (adenosine metabolites). In neurons, OGD triggered increases in intracellular 5'-AMP (2.8-fold), adenosine (2.6-fold), inosine (2.2-fold), and hypoxanthine (5.3-fold) and extracellular 5'-AMP (2.2-fold), adenosine (2.4-fold), and hypoxanthine (2.5-fold). In neurons, H2 O2 did not affect intracellular or extracellular purines; yet, glutamate increased intracellular adenosine, inosine, and hypoxanthine (1.7-fold, 1.7-fold, and 1.6-fold, respectively) and extracellular adenosine, inosine, and hypoxanthine (2.9-fold, 2.1-fold, and 1.6-fold, respectively). In astrocytes, neither H2 O2 nor glutamate affected intracellular or extracellular purines, and OGD only slightly increased intracellular and extracellular hypoxanthine. Microglia were unresponsive to OGD and glutamate, but were remarkably responsive to H2 O2 , which increased intracellular 5'-AMP (1.6-fold), adenosine (1.6-fold), inosine (2.1-fold), and hypoxanthine (1.6-fold) and extracellular 5'-AMP (5.9-fold), adenosine (4.0-fold), inosine (4.3-fold), and hypoxanthine (1.9-fold).

The lymphatic system controls tissue homeostasis by draining protein-rich lymph to the vascular system. Lymphangiogenesis, the formation of lymphatic vessels, is a normal event in childhood but promotes tumor spread and metastasis during adulthood. Blocking lymphangiogenesis may therefore be of therapeutic interest. Production of adenosine is enhanced in the tumor environment and contributes to tumor progression through stimulation of angiogenesis. In this study, we determined whether adenosine affects lymphangiogenesis.

Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression.

Analysis of the Cryptosporidium parvum genome demonstrates that the parasite cannot synthesize purines de novo and reveals that the sole route for purine salvage by the parasite is via adenosine kinase (CpAK). In order to initiate a biochemical characterization of CpAK and ultimately validate this apparently essential enzyme as a therapeutic target, the CpAK gene was redesigned for optimum codon usage, overexpressed in Escherichia coli, and the recombinant protein purified to homogeneity and characterized. CpAK appears to be specific for adenosine among the naturally occurring nucleosides but can utilize ATP, GTP, UTP and CTP as the phosphate donor. The enzyme exhibits K(m) values of 1.4microM for adenosine and 41microM for ATP, has a pH optimum approximately 7.0, and is dependent upon the presence of a divalent cation. Structure-activity data intimate that catalysis requires contacts between residues on CpAK with the six-position of the purine ring and the O2' and O3' hydroxyls of the ribose sugar. Additionally, 4-nitro-6-benzylthioinosine, a compound that demonstrates therapeutic promise against the related parasite Toxoplasma gondii, also inhibits adenosine phosphorylation by CpAK. The overproduction and purification of CpAK now enables a thorough evaluation of its potential as a drug target.