Enhanced survival of lethally irradiated adenosine A3 receptor knockout mice. A role for hematopoietic growth factors?

Adenosine A₃ receptor knockout (A₃AR KO) mice and their wild-type (WT) counterparts were compared from the point of view of their abilities to survive exposures to lethal doses of γ-radiation belonging to the range of radiation doses inducing the bone marrow acute radiation syndrome. Parameters of cumulative 30-day survival (experiment using a midlethal radiation dose) or cumulative 11-day survival (experiment using an absolutely lethal radiation dose), and of mean survival time were evaluated. The values of A₃AR KO mice always reflected their higher survival in comparison with WT ones, the P values being above the limit for statistical significance after the midlethal radiation dose and standing for statistical significance after the absolutely lethal radiation dose. This finding was considered surprising, taking into account the previously obtained findings on defects in numbers and functional properties of peripheral blood cells in A₃AR KO mice. Therefore, previous hematological analyses of A₃AR KO mice were supplemented in the present studies with determination of serum levels of the granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Though distinct differences in these parameters were observed between A₃AR KO and WT mice, none of them could explain the relatively high postirradiation survival of A₃AR KO mice. Further studies on these mice comprising also those on other than hemopoietic tissues and organs can help to clarify their relative radioresistance.     

Adenosine A<Subscript>1</Subscript> receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury

Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A₁ receptor deficiency (A₁R^?/?) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction size evaluated. Flow cytometry was performed on brain-infiltrating cells, and semi-automated analysis of flow cytometric data was applied. A₁R^?/? mice displayed larger infarctions (+33 %, p?<?0.05) and performed worse in beam walking tests (44 % more mistakes, p?<?0.05) than wild-type (WT) mice. Myeloid cell activation after injury was enhanced in A₁R^?/? versus WT brains. Activated B lymphocytes expressing IL-10 infiltrated the brain after HI in WT, but were less activated and did not increase in relative frequency in A₁R^?/?. Also, A₁R^?/? B lymphocytes expressed less IL-10 than their WT counterparts, the A₁R antagonist DPCPX decreased IL-10 expression whereas the A₁R agonist CPA increased it. CD4⁺ T lymphocytes including FoxP3⁺ T regulatory cells, were unaffected by genotype, whereas CD8⁺ T lymphocyte responses were smaller in A₁R^?/? mice. Using PCA to characterize the immune profile, we could discriminate the A₁R^?/? and WT genotypes as well as sham operated from HI-subjected animals. We conclude that A₁R signaling modulates IL-10 expression by immune cells, influences the activation of these cells in vivo, and affects outcome after HI.     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Adenosine A2A receptor deficiency prevents p38MAPK activation and apoptosis of mouse hippocampal cells in the chronic hypoxic-hypercapnia model

ABSTRACT

This study aims to study the effects of adenosine A_2A receptor (A_2AR) on hippocampal cell apoptosis and the putative mechanisms in a mouse model of chronic hypoxic-hypercapnia. Wild-type (WT) or A_2AR knockout (A_2AR KO) mice were randomly divided into normal control (NC) groups and chronic hypoxic-hypercapnia (4HH) groups. Compared with their corresponding NC groups (WT-NC and KO-NC), the apoptosis index (AI), caspase-3 activity, Bax mRNA and P-p38 protein expression in the hippocampus of 4HH groups (WT-4HH and KO-4HH) were significantly increased, while Bcl2 mRNA expression was significantly decreased (P < 0.05). Moreover, A_2AR deficiency significantly rescued the effect of chronic hypoxic-hypercapnia on apoptosis when compared with the WT-4HH group (P < 0.05). A_2AR deficiency inhibits hippocampal cell apoptosis in mice exposed to chronic hypoxic-hypercapnia, which might be associated with dampened p38 MAPK activation and Bax mRNA expression, and augmented Bcl-2 mRNA expression.     

The role of activated adenosine receptors in degranulation of human LAD2 mast cells

Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A_2A, A_2B, and A₃, but not A₁, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA''s enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A_2A antagonist separately inhibited NECA''s enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334"}}CGS15943. BMMCs also expressed A_2A, A_2B, and A₃ ARs. but not A₁AR detectably. We conclude that (a) A₁AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A_2A, A_2B, and A₃ ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.     

Adenosine A(2A) receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91^phox deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A_2A receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A_2A receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91^phox deficient mice is attenuated by CGS21680. These data suggest that treatment with A_2AR agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91^phox deficiency.     

Restraint stress fails to modulate cutaneous hypersensitivity responses in mice lacking the adenosine A1 receptor

Psychological stress has long been associated with effects on immune function and disease. In particular, differential effects of acute and chronic stress on skin immunity occur in the rodent restraint stress model, with acute stress enhancing and chronic stress suppressing cutaneous hypersensitivity. Extracellular levels of adenosine are known to modulate diverse biological activities in the CNS and peripheral tissues and serve an important protective function against physiological stressors such as inflammation and ischemia. In this study, we utilized the restraint stress model and the skin sensitizer dinitrofluorobezene to test the hypothesis that perceived stress influences contact hypersensitivity through an adenosine A₁ receptor-mediated mechanism. We subjected hapten-sensitized A₁ receptor knockout (A1 KO) mice and their wild-type (WT) littermates to either acute (2.5 h) or chronic (5 h daily × 4 weeks) restraint stress, followed by hapten re-challenge of the pinna. Daily measurements of the resulting pinna swellings from each group were compared to reactions in non-stressed controls. In WT mice, pinna swelling was augmented in acutely stressed mice and suppressed in the chronically stressed group. In contrast, contact hypersensitivity responses in the A1 KO mice failed to be affected by either acute or chronic stress. Absence of the adenosine A₁ receptor did not affect levels of plasma corticosterone or urine catecholamines under these stressful conditions but did lead to reduced numbers of circulating neutrophil granulocytes compared to stressed WT animals. These results suggest that the adenosine A₁ receptor pathway plays a role in the process by which perceived psychological stress influences the contact hypersensitivity response.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

Synthesis of 3′-Acetamidoadenosine Derivatives as Potential A3 Adenosine Receptor Agonists

On the basis of high binding affinity of 3′-aminoadenosine derivatives 2b at the human A₃ adenosine receptor (AR), 3′-acetamidoadenosine derivatives 3a–e were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A₃AR, our results revealed that 3′-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A₃AR.     

Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells

Angiogenesis is critical to wound repair due to its role in providing oxygen and nutrients that are required to support the growth and function of reparative cells in damaged tissues. Adenosine receptors are claimed to be of paramount importance in driving wound angiogenesis by inducing VEGF. However, the underlying mechanisms for the regulation of adenosine receptors in VEGF as well as eNOS remain poorly understood. In the present study, we found that adenosine and the non-selective adenosine receptor agonists (NECA) induced tube formation in HMEC-1 in a dose-dependent manner. Adenosine or NECA (10 µmol/L) significantly augmented the number and length of the segments in comparison with the control. Simultaneously, VEGF and eNOS were significantly upregulated following the administration of 10 µmol/L NECA, while they were suppressed after A_2B AR genetic silencing and pharmacological inhibition by MRS1754. In addition, VEGF expression and eNOS bioavailability elimination significantly reduced the formation of capillary-like structures. Furthermore, the activation of A_2B AR by NECA significantly increased the intracellular cAMP levels and concomitant CREB phosphorylation, eventually leading to the production of VEGF in HMEC-1. However, the activated PKA-CREB pathway seemed to be invalidated in the induction of eNOS. Moreover, we found that the elicited PI3K/AKT signaling in response to the induction of NECA assisted in regulating eNOS but failed to impact on VEGF generation. In conclusion, the A_2B AR activation-driven angiogenesis via cAMP-PKA-CREB mediated VEGF production and PI3K/AKT-dependent upregulation of eNOS in HMEC-1.     

Role of A2A adenosine receptors in regulation of opsonized E. coli-induced macrophage function

Adenosine is a biologically active molecule that is formed at sites of metabolic stress associated with trauma and inflammation, and its systemic level reaches high concentrations in sepsis. We have recently shown that inactivation of A_2A adenosine receptors decreases bacterial burden as well as IL-10, IL-6, and MIP-2 production in mice that were made septic by cecal ligation and puncture (CLP). Macrophages are important in both elimination of pathogens and cytokine production in sepsis. Therefore, in the present study, we questioned whether macrophages are responsible for the decreased bacterial load and cytokine production in A_2A receptor-inactivated septic mice. We showed that A_2A KO and WT peritoneal macrophages obtained from septic animals were equally effective in phagocytosing opsonized E. coli. IL-10 production induced by opsonized E. coli was decreased in macrophages obtained from septic A_2A KO mice as compared to WT counterparts. In contrast, the release of IL-6 and MIP-2 induced by opsonized E. coli was higher in septic A_2A KO macrophages than WT macrophages. These results suggest that peritoneal macrophages are not responsible for the decreased bacterial load and diminished MIP-2 and IL-6 production that are observed in septic A_2A KO mice. In contrast, peritoneal macrophages may contribute to the suppressive effect of A_2A receptor inactivation on IL-10 production during sepsis.     

Adenosine A2A receptors modulate BDNF both in normal conditions and in experimental models of Huntington’s disease

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Functional interactions between adenosine A_2A receptors (A_2ARs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A_2ARs regulate both BDNF functions and levels in the brain. Whereas BDNF (10 ng/ml) increased the slope of excitatory postsynaptic field potentials (fEPSPs) in hippocampal slices from wild-type (WT) mice, it was completely ineffective in slices taken from A_2AR knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A_2AR KO vs. WT mice. Having found an even marked reduction in the striatum of A_2AR KO mice, and as both BDNF and A_2ARs have been implicated in the pathogenesis of Huntington’s disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A_2ARs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration (quinolinic acid-lesioned rats) and in a transgenic mice model of HD (R6/2 mice). In both QA-lesioned rats and early symptomatic R6/2 mice (8 weeks), the systemic administration of the A_2AR antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A_2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone. The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation.     

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Potent and selective adenosine A₁ receptor (A₁AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a K_i value of 0.96 nM for PQ-69 in cloned hA₁ receptor, which was 217-fold more selective compared with hA_2A receptors and >1,000-fold selectivity for hA₁ over hA₃ receptor. The results obtained from [³⁵S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A₁AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A₁AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t_1/2) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A₁AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A₁AR function, and it could be developed as a potential therapeutic agent.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9424-5) contains supplementary material, which is available to authorized users.     

Potent and selective adenosine A(2A) receptor antagonists: [1,2,4]-triazolo[4,3-c]pyrimidin-3-ones

Antagonism of the adenosine A_2A receptor affords a possible treatment of Parkinson’s disease. In the course of investigating pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine A_2A antagonists, we prepared [1,2,4]-triazolo[4,3-c]pyrimidin-3-ones with potent and selective (vs A₁) A_2A antagonist activity. Structure-activity relationships are described for this series.     

Molecular dynamics simulations reveal initial structural and dynamic features for the A2AR as a result of ligand binding

G-protein-coupled receptors (GPCRs) are membrane proteins that have a wide variety of physiological roles. Adenosine receptors belong to the GPCR family. Adenosine receptors are implicated in many physiological disorders, such as Parkinson's disease, Huntington's disease, inflammatory and immune's disease and many others. Interestingly, crystal structures of the active and inactive conformations of the A₂-subtype adenosine receptor (A₂AR) have been solved. These two structures could be used to get insights about the conformational changes that occur during the process of activation/inactivation processes of this receptor. Therefore, two ligand-free simulations of the native active (PDB code: 3QAK) and inactive (PDB code: 3EML) conformations of the A₂AR and two halo-simulations were carried out to observe the initial conformational changes induced by coupling adenosine to the inactive conformation and caffeine to the active conformation. Furthermore, we constructed an A₂AR model that contained four thermostabilising mutations, L48A, T65A, Q89A and A54L, which had previously been determined to stabilise the bound conformation of the agonist, and we ran molecular dynamics simulations of this mutant to investigate how these point mutations might affect the inactive conformation of this receptor. This study provides insights about the initial structural and dynamic features that occur as a result of the binding of caffeine and adenosine in the active and inactive A₂AR structures, respectively, as well as the introduction of some mutations on the inactive structure of the A₂AR. Moreover, we provide useful and detailed information regarding structural features such as toggle switch and ionic lock during the activation/inactivation processes of this receptor.     

A new D₂ dopamine receptor agonist allosterically modulates A(2A) adenosine receptor signalling by interacting with the A(2A)/D₂ receptor heteromer

The structural and functional interaction between D₂ dopamine receptor (DR) and A_2A adenosine receptor (AR) has suggested these two receptors as a pharmacological target in pathologies associated with dopamine dysfunction, such as Parkinson's disease. In transfected cell lines it has been demonstrated the activation of D₂DR induces a significant negative regulation of A_2AAR-mediated responses, whereas few data are at now available about the regulation of A_2AAR by D₂DR agonists at receptor recognition site. In this work we confirmed that in A_2AAR/D₂DR co-transfected cells, these receptors exist as homo- and hetero-dimers. The classical D₂DR agonists were able to negatively modulate both A_2AAR affinity and functionality. These effects occurred even if any significant changes in A_2AAR/D₂DR energy transfer interaction could be detected in BRET experiments.Since the development of new molecules able to target A_2A/D₂ dimers may represent an attractive tool for innovative pharmacological therapy, we also identified a new small molecule, 3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine (compound 1), full agonist of D₂DR and modulator of A_2A-D₂ receptor dimer. This compound was able to negatively modulate A_2AAR binding properties and functional responsiveness in a manner comparable to classical D₂R agonists. In contrast to classical agonists, compound 1 led to conformational changes in the quaternary structure in D₂DR homomers and heteromers and induced A_2AAR/D₂DR co-internalization. These results suggest that compound 1 exerts a high control of the function of heteromers and could represent a starting point for the development of new drugs targeting A_2AAR/D₂ DR heteromers.     

Deletion of CD73 increases exercise power in mice

Ecto-5′-nucleotidase or CD73 is the main source of extracellular adenosine involved in the activation of adenosine A_2A receptors, responsible for the ergogenic effects of caffeine. We now investigated the role of CD73 in exercise by comparing female wild-type (WT) and CD73 knockout (KO) mice in a treadmill-graded test to evaluate running power, oxygen uptake (V̇O₂), and respiratory exchange ratio (RER) — the gold standards characterizing physical performance. Spontaneous locomotion in the open field and submaximal running power and V̇O₂ in the treadmill were similar between CD73-KO and WT mice; V̇O₂max also demonstrated equivalent aerobic power, but CD73-KO mice displayed a 43.7 ± 4.2% larger critical power (large effect size, P < 0.05) and 3.8 ± 0.4% increase of maximum RER (small effect size, P < 0.05). Thus, KO of CD73 was ergogenic; i.e., it increased physical performance.     

Pharmacophore mapping and in silico screening to identify new potent leads for A2A adenosine receptor as antagonists

A_2A adenosine receptor (AR) antagonists play an important role in neurodegenerative diseases like Parkinson’s disease. A 3D-QSAR study of A_2A AR antagonists, was taken up to design best pharmacophore model. The pharmacophoric features (ADHRR) containing a hydrogen bond acceptor (A), a hydrogen bond donor (D), a hydrophobic group (H) and two aromatic rings (R), is projected as the best predictive pharmacophore model. The QSAR model was further treated as a template for in silico search of databases to identify new scaffolds. The binding patterns of the leads with A_2A AR are analysed using docking studies and novel potent ligands of A_2A AR are projected.