Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson’s disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 μM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A₁ (A₁R) and/or A_2A receptors (A_2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A₁R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A₁R agonist, did not alter GUO effects. Regarding A_2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A_2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A₁R antagonist DPCPX, and the A_2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A₁R-A_2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

     

Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 μM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti‐inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A₁ receptor, phosphatidylinositol‐3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin‐sensitive G‐protein‐coupled signaling, blockade of adenosine A_2A receptors (A_2AR), but not via A₁ receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.     

Dopamine D2 receptor-mediated modulation of adenosine A2A receptor agonist binding within the A2AR/D2R oligomer framework

The molecular interaction between adenosine A_2A and dopamine D₂ receptors (A_2ARs and D₂Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine–dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson’s disease). While the effects of A_2AR challenge on D₂R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D₂R-mediated allosteric modulation of A_2AR binding when an A_2AR/D₂R oligomer is established. Thus, we synthesized fluorescent A_2AR agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A_2AR binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D₂R-mediated negative allosteric modulation on A_2AR agonist binding that was oligomer-formation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A_2AR agonist binding. Overall, the here-developed methods were found valid to explore the ability of drugs acting on D₂Rs to modulate A_2AR binding, thus serving to facilitate the preliminary selection of D₂R-like candidate drugs in the management of Parkinson’s disease.     

Dual blockade of the A1 and A2A adenosine receptor prevents amyloid beta toxicity in neuroblastoma cells exposed to aluminum chloride

In a previous work we have shown that exposure to aluminum (Al) chloride (AlCl₃) enhanced the neurotoxicity of the amyloid beta_25-35 fragment (Abeta_25-35) in neuroblastoma cells and affected the expression of Alzheimer's disease (AD)-related genes. Caffein, a compound endowed with beneficial effects against AD, exerts neuroprotection primarily through its antagonist activity on A_2A adenosine receptors (A_2AR), although it also inhibits A₁Rs with similar potency. Still, studies on the specific involvement of these receptors in neuroprotection in a model of combined neurotoxicity (Abeta_25-35 + AlCl₃) are missing. To address this issue, cultured SH-SY5Y cells exposed to Abeta_25-35 + AlCl₃ were assessed for cell viability, morphology, intracellular ROS activity and expression of apoptosis-, stress- and AD-related proteins. To define the role of A₁R and A_2ARs, pretreatment with caffein, specific receptor antagonists (DPCPX or SCH58261) or siRNA-mediated gene knockdown were delivered. Results indicate that AlCl₃ treatment exacerbated Abeta_25-35 toxicity, increased ROS production, lipid peroxidation, β-secretase-1 (BACE1) and amyloid precursor protein (APP). Interestingly, SCH58261 successfully prevented toxicity associated to Abeta_25-35 only, whereas pretreatment with both DPCPX and SCH58261 was required to fully avert Abeta_25-35 + AlCl₃-induced damage, suggesting that A₁Rs might also be critically involved in protection during combined toxicity. The effects of caffein were mimicked by both N-acetyl cysteine, an antioxidant, and desferrioxamine, likely acting through distinct mechanisms. Altogether, our data establish a novel protective function associated with A₁R inhibition in the setting of combined Abeta_25-35 + AlCl₃ neurotoxicity, and expand our current knowledge on the potential beneficial role of caffein to prevent AD progression in subjects environmentally exposed to aluminum.     

Homocysteine concentration and adenosine A2A receptor production by peripheral blood mononuclear cells in coronary artery disease patients

Hyperhomocysteinemia is associated with coronary artery disease (CAD). The mechanistic aspects of this relationship are unclear. In CAD patients, homocysteine (HCy) concentration correlates with plasma level of adenosine that controls the coronary circulation via the activation of adenosine A_2A receptors (A_2AR). We addressed in CAD patients the relationship between HCy and A_2AR production, and in cellulo the effect of HCy on A_2AR function. 46 patients with CAD and 20 control healthy subjects were included. We evaluated A_2AR production by peripheral blood mononuclear cells using Western blotting. We studied in cellulo (CEM human T cells) the effect of HCy on A_2A R production as well as on basal and stimulated cAMP production following A_2A R activation by an agonist‐like monoclonal antibody. HCy concentration was higher in CAD patients vs controls (median, range: 16.6 [7‐45] vs 8 [5‐12] µM, P < 0.001). A_2A R production was lower in patients vs controls (1.1[0.62‐1.6] vs 1.53[0.7‐1.9] arbitrary units, P < 0.001). We observed a negative correlation between HCy concentration and A_2A R production (r = ?0.43; P < 0.0001), with decreased A_2A R production above 25 µM HCy. In cellulo, HCy inhibited A_2AR production, as well as basal and stimulated cAMP production. In conclusion, HCy is negatively associated with A_2A R production in CAD patients, as well as with A_2A R and cAMP production in cellulo. The decrease in A_2A R production and function, which is known to hamper coronary blood flow and promote inflammation, may support CAD pathogenesis.     

Enhancement of inosine-mediated A2AR signaling through positive allosteric modulation

Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15 h) than adenosine (half-life < 10 s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A_2A receptor (A_2AR). Relative to adenosine inosine exhibits a lower affinity towards the A_2AR. Therefore, it is generally believed that inosine is incapable of activating the A_2AR through direct engagement, but indirectly activates the A_2AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A_2AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A_2AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A_2AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A_2AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A_2AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A_2AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.     

Guanosine promotes cytotoxicity via adenosine receptors and induces apoptosis in temozolomide-treated A172 glioma cells

Gliomas are a malignant tumor group whose patients have survival rates around 12 months. Among the treatments are the alkylating agents as temozolomide (TMZ), although gliomas have shown multiple resistance mechanisms for chemotherapy. Guanosine (GUO) is an endogenous nucleoside involved in extracellular signaling that presents neuroprotective effects and also shows the effect of inducing differentiation in cancer cells. The chemotherapy allied to adjuvant drugs are being suggested as a novel approach in gliomas treatment. In this way, this study evaluated whether GUO presented cytotoxic effects on human glioma cells as well as GUO effects in association with a classical chemotherapeutic compound, TMZ. Classical parameters of tumor aggressiveness, as alterations on cell viability, type of cell death, migration, and parameters of glutamatergic transmission, were evaluated. GUO (500 and 1000 μM) decreases the A172 glioma cell viability after 24, 48, or 72 h of treatment. TMZ alone or GUO plus TMZ also reduced glioma cell viability similarly. GUO combined with TMZ showed a potentiation effect of increasing apoptosis in A172 glioma cells, and a similar pattern was observed in reducing mitochondrial membrane potential. GUO per se did not elevate the acidic vesicular organelles occurrence, but TMZ or GUO plus TMZ increased this autophagy hallmark. GUO did not alter glutamate transport per se, but it prevented TMZ-induced glutamate release. GUO or TMZ did not alter glutamine synthetase activity. Pharmacological blockade of glutamate receptors did not change GUO effect on glioma viability. GUO cytotoxicity was partially prevented by adenosine receptor (A₁R and A_2AR) ligands. These results point to a cytotoxic effect of GUO on A172 glioma cells and suggest an anticancer effect of GUO as a putative adjuvant treatment, whose mechanism needs to be unraveled.     

Neuroprotection afforded by adenosine A2A receptor blockade is modulated by corticotrophin‐releasing factor (CRF) in glutamate injured cortical neurons

In situations of hypoxia, glutamate excitotoxicity induces neuronal death. The release of extracellular adenosine is also triggered and is accompanied by an increase of the stress mediator, corticotrophin‐releasing factor (CRF). Adenosine A_2A receptors contribute to glutamate excitoxicity and their blockade is effective in stress‐induced neuronal deficits, but the involvement of CRF on this effect was never explored. We now evaluated the interaction between A_2A and CRF receptors (CRFR) function, upon glutamate insult. Primary rat cortical neuronal cultures (9 days in vitro) expressing both CRF₁R and CRF₂R were challenged with glutamate (20–1000 μM, 24 h). CRF₁R was found to co‐localize with neuronal markers and CRF₂R to be present in both neuronal and glial cells. The effects of the CRF and A_2A receptors ligands on cell viability were measured using propidium iodide and Syto‐13 fluorescence staining. Glutamate decreased cell viability in a concentration‐dependent manner. Urocortin (10 pM), an agonist of CRF receptors, increased cell survival in the presence of glutamate. This neuroprotective effect was abolished by blocking either CRF₁R or CRF₂R with antalarmin (10 nM) or anti‐Sauvagine‐30 (10 nM), respectively. The blockade of A_2A receptors with a selective antagonist SCH 58261 (50 nM) improved cell viability against the glutamate insult. This effect was dependent on CRF₂R, but not on CRF₁R activation. Overall, these data show a protective role of CRF in cortical neurons, against glutamate‐induced death. The neuroprotection achieved by A_2A receptors blockade requires CRF₂R activation. This interaction between the adenosine and CRF receptors can explain the beneficial effects of using A_2A receptor antagonists against stress‐induced noxious effects.     

Targeting the upregulation of reactive oxygen species subsequent to hyperglycemia prevents type 1 diabetic cardiomyopathy in mice

Cardiac oxidative stress is an early event associated with diabetic cardiomyopathy, triggered by hyperglycemia. We tested the hypothesis that targeting left-ventricular (LV) reactive oxygen species (ROS) upregulation subsequent to hyperglycemia attenuates type 1 diabetes-induced LV remodeling and dysfunction, accompanied by attenuated proinflammatory markers and cardiomyocyte apoptosis. Male 6-week-old mice received either streptozotocin (55 mg/kg/day for 5 days), to induce type 1 diabetes, or citrate buffer vehicle. After 4 weeks of hyperglycemia, the mice were allocated to coenzyme Q₁₀ supplementation (10 mg/kg/day), treatment with the angiotensin-converting-enzyme inhibitor (ACE-I) ramipril (3 mg/kg/day), treatment with olive oil vehicle, or no treatment for 8 weeks. Type 1 diabetes upregulated LV NADPH oxidase (Nox2, p22^phox, p47^phox and superoxide production), LV uncoupling protein UCP3 expression, and both LV and systemic oxidative stress (LV 3-nitrotyrosine and plasma lipid peroxidation). All of these were significantly attenuated by coenzyme Q₁₀. Coenzyme Q₁₀ substantially limited type 1 diabetes-induced impairments in LV diastolic function (E:A ratio and deceleration time by echocardiography, LV end-diastolic pressure, and LV −dP/dt by micromanometry), LV remodeling (cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis), and LV expression of proinflammatory mediators (tumor necrosis factor-α, with a similar trend for interleukin IL-1β). Coenzyme Q_10's actions were independent of glycemic control, body mass, and blood pressure. Coenzyme Q₁₀ compared favorably to improvements observed with ramipril. In summary, these data suggest that coenzyme Q₁₀ effectively targets LV ROS upregulation to limit type 1 diabetic cardiomyopathy. Coenzyme Q₁₀ supplementation may thus represent an effective alternative to ACE-Is for the treatment of cardiac complications in type 1 diabetic patients.     

BDNF prevents NMDA‐induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved

NMDA receptor‐mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain‐derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A_2ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA‐induced effects in HD models, and the possible involvement of A_2ARs. In corticostriatal slices from wild‐type mice and age‐matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild‐type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A_2AR blockade. The protective effect of BDNF against NMDA‐induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A_2AR ligands in HD.     

Inactivation of neuronal forebrain A2A receptors protects dopaminergic neurons in a mouse model of Parkinson’s disease

Adenosine A_2A receptors antagonists produce neuroprotective effects in animal models of Parkinson’s disease (PD). As neuroinflammation is involved in PD pathogenesis, both neuronal and glial A_2A receptors might participate to neuroprotection. We employed complementary pharmacologic and genetic approaches to A_2A receptor inactivation, in a multiple MPTP mouse model of PD, to investigate the cellular basis of neuroprotection by A_2A antagonism. MPTP·HCl (20 mg/kg daily for 4 days) was administered in mice treated with the A_2A antagonist SCH58261, or in conditional knockout mice lacking A_2A receptors on forebrain neurons (fbnA_2AKO mice). MPTP‐induced partial loss of dopamine neurons in substantia nigra pars compacta (SNc) and striatum (Str), associated with increased astroglial and microglial immunoreactivity in these areas. Astroglia was similarly activated 1, 3, and 7 days after MPTP administration, whereas maximal microglial reactivity was detected on day 1, returning to baseline 7 days after MPTP administration. SCH58261 attenuated dopamine cell loss and gliosis in SNc and Str. Selective depletion of A_2A receptors in fbnA_2AKO mice completely prevented MPTP‐induced dopamine neuron degeneration and gliosis in SNc, and partially counteracted gliosis in Str. Results provide evidence of a primary role played by neuronal A_2A receptors in neuroprotective effects of A_2A antagonists in a multiple MPTP injections model of PD. With the symptomatic antiparkinsonian potential of several A_2A receptor antagonists being pursued in clinical trials, this study adds to the rationale for broader clinical benefit and use of these drugs early in the treatment of PD.     

Caffeine has a dual influence on NMDA receptor–mediated glutamatergic transmission at the hippocampus

Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A₁R and A₂R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca²⁺ homeostasis. We found that caffeine (30–200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A₁Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A_2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A_2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca²⁺ transients in neuronal cell culture, an action mimicked by the A₁R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca²⁺ homeostasis.

     

Synthesis of novel 7-imino-2-thioxo-3,7-dihydro-2H-thiazolo [4,5-d] pyrimidine derivatives as adenosine A2A receptor antagonists

Novel bicyclic thiazolopyrimidine compounds (15–26) were synthesized to develop adenosine A_2A receptor (A_2AR) antagonist for the treatment of Parkinson’s disease (PD). The binding affinity of the compounds (15–26) with A_2AR was evaluated using radioligand binding assay on isolated membranes from stably transfected HEK293 cells. Selectivity of the compounds towards A_2AR was assessed by comparing their binding affinities with A₁ receptors (A₁R). cAMP concentrations were measured from HEK293 cells treated with compounds (15–26) as compared to NECA (A_2AR agonist). The compound (16) possessed strongest A_2AR binding affinity (K_i value = 0.0038 nM) and selectivity (737-fold) versus A₁R. Decrease in A_2AR-coupled release of endogenous cAMP from HEK293 cells treated with compounds (15–26) is evocative of their potential as A_2AR antagonist.     

Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation

Adenosine A_2A receptor (A_2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A_2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A_2AR expression are still obscure. Here, we demonstrated that 2 weeks of SAM treatment produced a significant reduction in the rat striatal A_2AR messenger RNA (mRNA) and protein content as well as A_2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5′UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A_2AR expression could be established. Overall, we concluded that striatal A_2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A_2AR expression.     

Adenosine receptors regulate exosome production

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A₁Rs, A_2ARs, and A_2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A₁R^?/?, A_2AR^?/?, and A_2BR^?/? rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A₁Rs, A_2ARs, and A_2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A₁R and A_2AR constrained exosome production under normal conditions (p?=?0.0297; p?=?0.0409, respectively), and A₁R, A_2AR, and A_2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p?=?0.0028) by the selective A_2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A₁R and A_2BR agonists (p?=?0.0474). The selective A_2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A_2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A_2ARs suppress exosome release in all cell types examined, whereas effects of A₁Rs and A_2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A_2AR antagonists may inadvertently increase exosome production from tumor cells.

     

Receptor desensitization and blockade of the suppressive effects of prostaglandin E<Subscript>2</Subscript> and adenosine on the cytotoxic activity of human melanoma-infiltrating T lymphocytes

Previous studies document that PGE₂ and adenosine suppress production of inflammatory cytokines. The present study demonstrates for the first time that (1) PGE₂ and 2-chloroadenosine (CADO; a stable analog of adenosine) directly inhibit the cytolytic function of human tumor-infiltrating lymphocytes (TILs); (2) the combination PGE₂ and CADO have additive suppressive effects; and (3) the cooperative immunosuppressive actions of PGE₂ and CADO are mediated via EP2 receptors (EP2Rs) and A_2A receptors (A_2ARs) and are due to amplification of cAMP production, activation of protein kinase A (PKA) and T cell receptor (TCR) inhibitor Csk leading to inhibition of Lck, ZAP-70 and Akt phosphorylation. (4) During ex vivo expansion, TILs undergo three stages of differentiation converting from TILs with high cytotoxic activity and relative resistance to combined EP2R/A_2AR suppression (stage I) to TILs retaining high cytotoxicity and gaining sensitivity to combined suppression (stage II) and then to TILS that are less cytotoxic and very sensitive to combined suppression (stage III). (5) Finally, we find that pretreatment of TILs with non-inhibitory concentrations of EP2R agonists (such as PGE₂ or butaprost) or A_2AR agonists (such as CADO or CGS21680) increases their cytotoxic activity and induces resistance to EP2R and A_2AR inhibitory signaling (cross-resistance) due to homologous and heterologous desensitization and internalization of EP2Rs and A_2ARs, thus preventing their inhibitory signaling. We conclude that inducing resistance of TILs to the suppressive effects of PGE₂ and adenosine in the tumor microenvironment could represent a novel strategy for improving the efficacy of adoptive immunotherapy.     

Anti-inflammatory effects of purine nucleosides, adenosine and inosine, in a mouse model of pleurisy: evidence for the role of adenosine A2 receptors

Adenosine and its metabolite, inosine, have been described as molecules that participate in regulation of inflammatory response. The aim of this study was to investigate the effect of adenosine and inosine in a mouse model of carrageenan-induced pleurisy as well as the participation of adenosine receptors in this response. Injection of carrageenan into the pleural cavity induced an acute inflammatory response characterized by leukocyte migration, pleural exudation, and increased release of interleukin-1β and tumor necrosis factor-α in pleural exudates. The treatment with adenosine (0.3–100 mg/kg, i.p.) and inosine (0.1–300 mg/kg, i.p.) 30 min before carrageenan injection reduced significantly all these parameters analyzed. Our results also demonstrated that A_2A and A_2B receptors seem to mediate the adenosine and inosine effects observed, since pretreatment with selective antagonists of adenosine A_2A (ZM241385) and A_2B (alloxazine) receptors, reverted the inhibitory effects of adenosine and inosine in pleural inflammation. The involvement of A₂ receptors was reinforced with adenosine receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment, since its anti-inflammatory effects were reversed completely and partially with ZM241385 and alloxazine injection, respectively. Moreover, the combined treatment with subeffective dose of adenosine (0.3 mg/kg) and inosine (1.0 mg/kg) induced a synergistic anti-inflammatory effect. Thus, based on these findings, we propose that inosine contributes with adenosine to exert anti-inflammatory effects in pleural inflammation, reinforcing the notion that endogenous nucleosides play an important role in controlling inflammatory diseases. This effect is likely mediated by the activation of adenosine A₂ subtype receptors and inhibition of production or release of pro-inflammatory cytokines.     

Modification of Cytokine Milieu by A2A Adenosine Receptor Signaling–Possible Application for Inflammatory Diseases

Pro‐inflammatory cytokine TNF‐alpha (TNF) production from in vitro lipopolysaccharide (LPS)‐stimulated human peripheral blood CD14⁺ cells (PB‐CD14) was inhibited by A_2A adenosine receptor (AdoR) (A_2AR) or ß₂ adrenergic receptor (ADR) (ß₂R) signaling in a concentration‐dependent manner. These inhibitory effects were presumably mediated by the increase in intracellular cAMP. Furthermore A_2AR agonist and ß₂R agonist synergistically inhibited the TNF production of LPS‐stimulated PB‐CD14 cells. These results suggest that the anti‐inflammatory effect of extracellular adenosine is, at least in part, due to the modification of the cytokine milieu via A_2A signaling, and that the targeting of both A_2AR and ß₂R may have strong therapeutic potential for the inflammatory diseases.     

Anticonvulsant and analgesic in neuropathic pain activity in a group of new aminoalkanol derivatives

As part of the presented research, thirteen new aminoalkanol derivatives were designed and obtained by chemical synthesis. In vivo studies (mice, i.p.) showed anticonvulsant activity (MES) of nine compounds, and in the case of one compound (R,S-trans-2-((2-(2,3,5-trimethylphenoxy)ethyl)amino)cyclohexan-1-ol, 4) both anticonvulsant (ED₅₀ MES = 15.67 mg/kg, TD₅₀ rotarod = 78.30 mg.kg, PI = 5.00) and analgesic activity (OXA-induced neuropathic pain, active at 15 mg/kg). For selected active compounds additional in vitro studies have been performed, including receptor studies (5-HT_1A), evaluation of antioxidant activity (DPPH assay), metabolism studies as well as safety panel (mutagenicity, safety in relation to the gastrointestinal flora, cytotoxicity towards astrocytes as well as impact on their proliferation and cell cycle).     

Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N ⁶-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A₃ receptor (hA₃R) ligands with affinities in the high nanomolar range (K _i values of 159 and 649 nM, respectively). These values were comparable to the observed K _i value of adenosine on hA₃R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A₃R. In a functional assay in Chinese hamster ovary cells transfected with hA₃R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A₃R antagonist VUF5574. Both IPA and reference A₃R agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A₃R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A₃R-independent mechanism, as was previously reported for other A₃R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A₃R.