Shenfu Administration Improves Cardiac Fibrosis in Rats With Myocardial Ischemia-Reperfusion Through Adenosine A2a Receptor Activation.

OBJECTIVE: Shenfu injection (SFI) is commonly used for cardiac dysfunction in China. Adenosine receptors have been reported to exert anti-fibrosis effects. The intent of this study was to evaluate that SFI attenuates cardiac fibrosis through activating of adenosine A2a receptor (A2aR) in rats with myocardial ischemia-reperfusion (MI/R). METHODS: Sprague Dawley male rats were randomly divided into five groups, nine rats in each group. Injections in all rat groups were carried out prior to reperfusion, and in the sham and MI/R groups, only vehicle was injected. Injections in the remaining group were as follows: 5 mL/kg in the SFI group; 15 mg/kg nicorandil in the A2R agonist group; and 5 mL/kg SFI plus 5 mg/kg MSX-3 in the SFI + A2aR antagonist group. Changes in cyclic adenosine monophosphate (cAMP) and the development of myocardial infarction and cardiac fibrosis were documented among the groups. Additionally, the levels of A2aR, collagen Ⅰ, collagen Ⅲ, fibronectin, and matrix metalloproteinase-9 (MMP-9) were measured. RESULTS: Following injection with SFI or nicorandil, the cAMP concentration, infarct area, and cardiac fibrosis induced by MI/R injury were significantly decreased (p < 0.05). Additionally, the levels of collagen Ⅰ, collagen Ⅲ, fibronectin, and MMP-9 were clearly suppressed by SFI or nicorandil when compared with the MI/R group (p<0.01). However, the protective effects of SFI were counteracted by MSX-3. A negative correlation between A2aR and collagen I and collagen III was found (p = 0.00). CONCLUSION: SFI activated the A2aR to reduce myocardial fibrosis caused by MI/R injury, which provided an underlying mechanism of action of SFI.

Heterobivalent Ligand for the Adenosine A2A-Dopamine D2 Receptor Heteromer.

A G protein-coupled receptor heteromer that fulfills the established criteria for its existence in vivo is the complex between adenosine A2A (A2AR) and dopamine D2 (D2R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A2AR-D2R heteromer with various spacer lengths. The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer has been evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the formation of the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. This combination of techniques has permitted us to identify compound 26 [KDB1 (A2AR) = 2.1 nM, KDB1 (D2R) = 0.13 nM], with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites. Moreover, bioluminescence resonance energy transfer experiments indicate that 26 favors the stabilization of the A2AR-D2R heteromer.

Blockade of adenosine A2A receptors inhibits Tremulous Jaw Movements as well as expression of zif-268 and GAD65 mRNAs in brain motor structures.

Tremor is one of the motor symptoms of Parkinson's disease (PD), present also in neuroleptic-induced parkinsonism. Tremulous Jaw Movements (TJMs) are suggested to be a well-validated rodent model of PD resting tremor. TJMs can be induced by typical antipsychotics and are known to be reduced by different drugs, including adenosine A2A receptor antagonists. The aim of the present study was to search for brain structures involved in the tremorolytic action of SCH58261, a selective A2A receptor antagonist, in TJMs induced by subchronic pimozide. Besides TJMs, we evaluated in the same animals the expression of zif-268 mRNA (neuronal responsiveness marker), and mRNA levels for glutamic acid decarboxylase 65-kDa isoform (GAD65) and vesicular glutamate transporters 1 and 2 (vGluT1/2) in selected brain structures, as markers of GABAergic and glutamatergic neurons, respectively. We found that SCH58261 reduced the pimozide-induced TJMs. Pimozide increased the zif-268 mRNA level in the striatum, nucleus accumbens (NAc) core, and substantia nigra pars reticulata (SNr). Additionally, it increased GAD65 mRNA in the striatum and SNr, and vGluT2 mRNA levels in the subthalamic nucleus (STN). A positive correlation between zif-268, GAD65 and vGluT2 mRNAs and TJMs was found. SCH58261 reversed the pimozide-increased zif-268 mRNA in the striatum and NAc core and GAD65 mRNA in the striatum and SNr. In contrast, SCH58261 did not influence vGluT2 mRNA in STN. The present study suggests an importance of the striato-subthalamo-nigro-thalamic circuit in neuroleptic-induced TJMs. The tremorolytic effect of A2A receptor blockade seems to involve this circuit bypassing, however, STN.

Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is widely known for its multiple systems damage, especially neurocognitive deficits in children. Since their discovery, adenosine A2A receptors (A2ARs) have been considered as key elements in signaling pathways mediating neurodegenerative diseases such as Huntington's and Alzheimer's, as well as cognitive function regulation. Herein, we investigated A2AR role in cognitive impairment induced by chronic intermittent hypoxia (CIH). Mice were exposed to CIH 7 h every day for 4 weeks, and intraperitoneally injected with A2AR agonist CGS21680 or A2AR antagonist SCH58261 half an hour before IH exposure daily. The 8-arm radial arm maze was utilized to assess spatial memory after CIH exposures.To validate findings using pharmacology, the impact of intermittent hypoxia was investigated in A2AR knockout mice. CIH-induced memory dysfunction was manifested by increased error rates in the radial arm maze test. The behavioral changes were associated with hippocampal pathology, neuronal apoptosis, and synaptic plasticity impairment. The stimulation of adenosine A2AR exacerbated memory impairment with more serious neuropathological damage, attenuated long-term potentiation (LTP), syntaxin down-regulation, and increased BDNF protein. Moreover, apoptosis-promoting protein cleaved caspase-3 was upregulated while anti-apoptotic protein Bcl-2 was downregulated. Consistent with these findings, A2AR inhibition with SCH58261 and A2AR deletion exhibited the opposite result. Overall, these findings suggest that A2AR plays a critical role in CIH-induced impairment of learning and memory by accelerating hippocampal neuronal apoptosis and reducing synaptic plasticity. Blockade of adenosine A2A receptor alleviates cognitive dysfunction after chronic exposure to intermittent hypoxia in mice.

Adenosine A2A receptor modulates microglia-mediated synaptic pruning of the retinogeniculate pathway during postnatal development.

Synapse pruning is essential not only for the developmental establishment of synaptic connections in the brain but also for the pathogenesis of neurodevelopmental and neurodegenerative disorders. However, there are no effective pharmacological means to regulate synaptic pruning during early development. Using the eye-specific segregation of the dorsal lateral geniculate nucleus (dLGN) as a model of synaptic pruning coupled with adenosine A2A receptor (A2AR) antagonism and knockout, we demonstrated while genetic deletion of the A2AR throughout the development attenuated eye-specific segregation with the attenuated microglial phagocytosis at postnatal day 5 (P5), selective treatment with the A2AR antagonist KW6002 at P2-P4 facilitated synaptic pruning of visual pathway with microglial activation, increased lysosomal activity in microglia and increased microglial engulfment of retinal ganglion cell (RGC) inputs in the dLGN at P5 (but not P10). Furthermore, KW6002-mediated facilitation of synaptic pruning was activity-dependent since tetrodotoxin (TTX) treatment abolished the KW6002 facilitation. Moreover, the A2AR antagonist also modulated postsynaptic proteins and synaptic density at early postnatal stages as revealed by the reduced immunoreactivity of postsynaptic proteins (Homer1 and metabotropic glutamate receptor 5) and colocalization of presynaptic VGlut2 and postsynaptic Homer1 puncta in the dLGN. These findings suggest that A2AR can control pruning by multiple actions involving the retinal wave, microglia engulfment, and postsynaptic stability. Thus, A2AR antagonists may represent a novel pharmacological strategy to modulate microglia-mediated synaptic pruning and treatment of neurodevelopmental disorders associated with dysfunctional pruning.

A2A receptor agonists and P2Y12 receptor antagonists modulate expression of thrombospondin-1 (TSP-1) and its secretion from Human Microvascular Endothelial Cells (HMEC-1).

BACKGROUNDS AND AIMS: To address the problem of resistance to standard antiplatelet therapy in some patients, our team proposed a purinoceptor-dependent dual therapy. Its efficacy is also determined by the condition of the vascular endothelium which, by secreting numerous factors, is involved in hemostasis. Among them, thrombospondin-1 is important in the context of thrombotic events. Therefore we sought to determine if the novel dual purinoceptor-dependent concept is associated with TSP-1 changes in vascular endothelial cells. METHODS AND RESULTS: TSP-1 expression in human microvascular endothelial cells was determined at transcriptional and protein level. We performed real-time PCR, the Western blot analysis and ELISA test. We found that TSP-1 mRNA and protein expression levels significantly changed in response to P1R agonists treatment. Furthermore, we have observed that co-administration of selective A2AR agonists (UK-432,097 or MRE0094) with P2Y12R antagonists altered TSP-1 expression levels, and the direction of these changes was not synergistic. MRE0094 applied with ARC69931MX or R-138727 increased mRNA expression from 39 to 56 or 57%, respectively (*P < 0.05 vs. MRE0094; ***P < 0.001 vs. control). Also, in the case of the P2Y12R antagonists used together with UK-432,097, there was an increase from 53 to 71 and 70% (*P < 0.05 vs. UK-432,097; ***P < 0.001 vs. control). The observed trends in gene expression were reflected in the protein expression and the level of its secretion from HMEC-1. CONCLUSION: The article presents evidence which proves that the purinoceptor-dependent concept is associated with TSP-1 changes in endothelial cells (EC). Moreover, Human Microvascular Endothelial Cells treatment applied together with agonists (MRE0094 or UK-432,097) and P2Y12R antagonist did not result in any synergistic effect, implicating a possible crosstalk between G proteins in GPCRs dependent signaling. Therefore, we suggest that understanding of the specific mechanism underlying TSP-1 alterations in EC in the context of the dual purinoceptor-dependent approach is essential for antiplatelet therapies and should be the subject of future research.

The adenosine A2A receptor alleviates postoperative delirium-like behaviors by restoring blood cerebrospinal barrier permeability in rats.

Postoperative delirium (POD) is a common post-operative complication in elderly patients that is associated with increased morbidity and mortality. However, the neuropathogenesis of this complication remains unknown. The blood-cerebrospinal fluid barrier (BCB) and brain-blood barrier (BBB) are composed of tight junctions between cells that form physical barriers, and BBB damage plays an important role in the neuropathogenesis of POD. Nevertheless, the role of BCB in POD remains to be elucidated. Herein, we investigated the effect of adenosine A2A receptor (A2A R), a key regulator of the permeability of barriers, on surgery-induced increased permeability of BCB and POD-like behaviors. Open field, buried food, and Y maze tests were used to evaluate behavioral changes in rats after surgery. Levels of tight junction proteins, adherens junction proteins, A2A R, GTP-RhoA, and ROCK2 in the choroid plexus were assessed by western blotting. The concentrations of NaFI and FITC-dextran in the cerebrospinal fluid (CSF) were detected by fluorescence spectrophotometry. Transmission electron microscopy was applied to observe the ultrastructure of the choroid plexus. Surgery/anesthesia decreased the levels of tight junction (e.g., ZO-1, occludin, and claudin1) proteins, increased concentrations of NaFI and FITC-dextran in CSF, damaged the ultrastructure of choroid plexus, and induced POD-like behaviors in rats. An A2A R antagonist alleviated POD-like behaviors in rats. Furthermore, the A2A R antagonist increased the levels of tight junction proteins and restored the permeability of BCB in rats with POD. Fasudil, a selective Rho-associated protein kinase 2 (ROCK2) inhibitor, ameliorated POD-like behaviors induced by A2A R activation. Moreover, fasudil also abolished the increased levels of GTP-RhoA/ROCK2, decreased levels of tight junction proteins, and increased permeability of BCB caused by A2A R activation. Our findings demonstrate that A2A R might participate in regulating BCB permeability in rats with POD via the RhoA/ROCK2 signaling pathway, which suggests the potential of A2A R as a therapeutic target for POD.

The role of peripheral adenosine receptors in glutamate-induced pain nociceptive behavior.

The role of peripheral adenosine receptors in pain is a controversial issue and seems to be quite different from the roles of spinal and central adenosine receptors. The present study is aimed at clarifying the role of these receptors in peripheral nociception. To clarify this, studies were done on Swiss mice with adenosine receptor agonists and antagonists. Nociceptive behavior was induced by subcutaneous injection of glutamate (10 µmol) into the ventral surface of the hind paw of mice. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Results showed that intraplantar (i.pl.) administration of N6-cyclohexyl-adenosine (CHA), an adenosine A1 receptor agonist, at 1 or 10 µg/paw significantly reduced glutamate-induced nociception (p<0.01 and p<0.001 vs. vehicle, respectively, n=8-10). In contrast, i.pl. injection of hydrochloride hydrate (CGS21680, an adenosine A2A receptor agonist) (1 µg/paw) induced a significant increase in glutamate-induced nociception compared to the vehicle (p<0.05, n=8), while 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385, an adenosine A2A receptor antagonist) (20 µg/paw) caused a significant reduction (p<0.05, n=7-8). There were no significant effects on i.pl. administration of four additional adenosine receptor drugs-8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A1 antagonist, 1-10 µg/paw), N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA, an A2B agonist, 1-100 µg/paw), alloxazine (an A2B antagonist, 0.1-3 µg/paw), and 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) (an A3 agonist, 1-100 µg/paw) (p>0.05 vs. vehicle for all tests). We also found that prior administration of DPCPX (3 µg/paw) significantly blocked the anti-nociceptive effect of CHA (1 µg/paw) (p<0.05, n=7-9). Similarly, ZM241385 (20 µg/paw) administered prior to CGS21680 (1 µg/paw) significantly blocked CGS21680-induced exacerbation of nociception (p<0.05, n=8). Finally, inosine (10 and 100 µg/paw), a novel endogenous adenosine A1 receptor agonist recently reported by our research group, was also able to reduce glutamate-induced nociception (p<0.001 vs. vehicle, n=7-8). Interestingly, as an A1 adenosine receptor agonist, the inosine effect was significantly blocked by the A1 antagonist DPCPX (3 µg/paw) (p<0.05, n=7-9) but not by the A2A antagonist ZM241385 (10 µg/paw, p>0.05). In summary, these results demonstrate for the first time that i.pl administration of inosine induces an anti-nociceptive effect, similar to that elicited by CHA and possibly mediated by peripheral adenosine A1 receptor activation. Moreover, our results suggest that peripheral adenosine A2A receptor activation presents a pro-nociceptive effect, exacerbating glutamate-induced nociception independent of inosine-induced anti-nociceptive effects.

Release of adenosine-induced immunosuppression: Comprehensive characterization of dual A2A/A2B receptor antagonist.

Immunosuppression is one of the main mechanisms facilitating tumor expansion. It may be driven by immune checkpoint protein expression, anti-inflammatory cytokine secretion or enhanced metabolic enzyme production, leading to the subsequent build-up of metabolites such as adenosine. Under physiological conditions, adenosine prevents the development of tissue damage resulting from a prolonged immune response; the same mechanism might be employed by tumor tissue to promote immunosuppression. Immune cells expressing A2A and A2B adenosine receptors present in an adenosine-rich environment have suppressed effector functions, such as cytotoxicity, proinflammatory cytokine release, antigen presentation and others, making them inert to cancer cells. This study was designed to investigate the dual antagonist potential of SEL330-639 to abolish adenosine-driven immunosuppression. SEL330-639 has slow dissociation kinetics. It inhibits cAMP production in human CD4+ cells, CD8+ cells and moDCs, which leads to diminished CREB phosphorylation and restoration of antitumor cytokine production (IL-2, TNFα, IL-12) in multiple primary human immune cells. The aforementioned results were additionally validated by gene expression analysis and functional assays in which NK cell line cytotoxicity was recovered by SEL330-639. Adenosine-driven immunosuppression is believed to preclude the effectiveness of immune checkpoint inhibitor therapies. Hence, there is an urgent need to develop new immuno-oncological strategies. Here, we comprehensively characterize SEL330-639, a novel dual A2A/A2B receptor antagonist effective in both lymphoid and myeloid cell populations with nanomolar potency. Due to its tight binding to the A2A and A2B receptors, this binding is sustained even at high adenosine concentrations mimicking the upper limit of the range of adenosine levels observed in the tumor microenvironment.

Effect of dopaminergic medication on adenosine 2A receptor availability in patients with Parkinson's disease.

OBJECTIVE: To assess the necessity of withdrawing dopaminergic medication in Parkinson's disease (PD) patients for accurate estimation of adenosine 2A receptor (A2AR) availability using [11C]TMSX PET imaging. This was accomplished by studying the short-term effect of the cessation of dopaminergic medication on A2AR availability in non-dyskinetic patients with PD treated with dopaminergic medication. METHODS: Eight PD patients (age 67.9 ± 5.6 years; 6 men, 2 women) without dyskinesia were enrolled in this study. A2AR availability was measured using PET imaging with a [7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([11C]TMSX) radioligand after a short term cessation of dopaminergic medication (12hrs for levodopa, 24hrs for dopamine agonists and MAO-B inhibitors). Repeated PET imaging was performed while the patients were back 'on' their regular dopaminergic medication (median 13 days after first imaging). Conventional MRI was acquired for anatomical reference. Specific binding of [11C]TMSX was quantified as distribution volume ratios (DVR) for caudate, pallidum and putamen using Logan graphical method with clustered gray matter reference region. RESULTS: No significant differences were observed for the DVRs in all three striatal regions between 'on' and 'off' medication states. Strong correlations were also observed between the two states. Statistical equivalence was found in pallidum (TOST equivalence test, p = 0.045) and putamen (TOST equivalence test, p = 0.022), but not in caudate DVR (TOST equivalence test, p = 0.201) between the two medication states. CONCLUSIONS: Our results show that dopaminergic medication has no significant short-term effect on the availability of A2A receptors in putamen and pallidum of patients with PD. However, relatively poor repeatability was demonstrated in the caudate.

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 A2A receptors (A2AR) 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 A2AR, 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 A2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A1R antagonist DPCPX, and the A2AR 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-A2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

Cannabinoid type 1 receptors in A2a neurons contribute to cocaine-environment association.

RATIONALE: Cannabinoid type 1 receptors (CB1Rs) are widely expressed within the brain's reward circuits and are implicated in regulating drug induced behavioral adaptations. Understanding how CB1R signaling in discrete circuits and cell types contributes to drug-related behavior provides further insight into the pathology of substance use disorders. OBJECTIVE AND METHODS: We sought to determine how cell type-specific expression of CB1Rs within striatal circuits contributes to cocaine-induced behavioral plasticity, hypothesizing that CB1R function in distinct striatal neuron populations would differentially impact behavioral outcomes. We crossed conditional Cnr1fl/fl mice and striatal output pathway cre lines (Drd1a -cre; D1, Adora2a -cre; A2a) to generate cell type-specific CB1R knockout mice and assessed their performance in cocaine locomotor and associative behavioral assays. RESULTS: Both knockout lines retained typical locomotor activity at baseline. D1-Cre x Cnr1fl/fl mice did not display hyperlocomotion in response to acute cocaine dosing, and both knockout lines exhibited blunted locomotor activity across repeated cocaine doses. A2a-cre Cnr1fl/fl, mice did not express a preference for cocaine paired environments in a two-choice place preference task. CONCLUSIONS: This study aids in mapping CB1R-dependent cocaine-induced behavioral adaptations onto distinct striatal neuron subtypes. A reduction of cocaine-induced locomotor activation in the D1- and A2a-Cnr1 knockout mice supports a role for CB1R function in the motor circuit. Furthermore, a lack of preference for cocaine-associated context in A2a-Cnr1 mice suggests that CB1Rs on A2a-neuron inhibitory terminals are necessary for either reward perception, memory consolidation, or recall. These results direct future investigations into CB1R-dependent adaptations underlying the development and persistence of substance use disorders.

Neuronal mechanisms of adenosine A2A receptors in the loss of consciousness induced by propofol general anesthesia with functional magnetic resonance imaging.

Propofol is the most common intravenous anesthetic agent for induction and maintenance of anesthesia, and has been used clinically for more than 30 years. However, the mechanism by which propofol induces loss of consciousness (LOC) remains largely unknown. The adenosine A2A receptor (A2A R) has been extensively proven to have an effect on physiological sleep. It is, therefore, important to investigate the role of A2A R in the induction of LOC using propofol. In the present study, the administration of the highly selective A2A R agonist (CGS21680) and antagonist (SCH58261) was utilized to investigate the function of A2A R under general anesthesia induced by propofol by means of animal behavior studies, resting-state magnetic resonance imaging and c-Fos immunofluorescence staining approaches. Our results show that CGS21680 significantly prolonged the duration of LOC induced by propofol, increased the c-Fos expression in nucleus accumbens (NAc) and suppressed the functional connectivity of NAc-dorsal raphe nucleus (DR) and NAc-cingulate cortex (CG). However, SCH58261 significantly shortened the duration of LOC induced by propofol, decreased the c-Fos expression in NAc, increased the c-Fos expression in DR, and elevated the functional connectivity of NAc-DR and NAc-CG. Collectively, our findings demonstrate the important roles played by A2A R in the LOC induced by propofol and suggest that the neural circuit between NAc-DR maybe controlled by A2A R in the mechanism of anesthesia induced by propofol.

How do adenosine A2A receptors regulate motor function?

Adenosine A2A receptor antagonism is a new therapeutic strategy in the symptomatic treatment of Parkinson's disease (PD). This review addresses how adenosine A2A receptors are involved with the control of motor function via the basal ganglia-thalamocortical circuit, and considers the anatomical localization and physiological function of the receptor, along with its ultrastructural localization in critical areas/neurons of the circuit. Based on this understanding of the functional significance of the adenosine A2A receptor in the basal ganglia, the mode of action of A2A receptor antagonists is explored in terms of the dynamic functioning of the basal ganglia and the activity of the internal circuits of the striatum in PD. Finally, the pathophysiological differences between the normal and PD states are examined to emphasize the importance of the adenosine A2A receptor.

Can adenosine A2A receptor antagonists modify motor behavior and dyskinesia in experimental models of Parkinson's disease?

Current treatment of the motor symptoms of Parkinson's disease (PD) focuses on dopamine replacement therapies. While these treatments are initially highly effective, with long-term use and disease progression, the therapeutic response is often limited by the development of motor complications, dopaminergic side effects, and residual unresponsive motor and non-motor symptoms. An alternative or additive treatment approach may be to target non-dopaminergic receptors within the motor control pathways, which function to modulate basal ganglia output. Adenosine A2A receptors are one potential non-dopaminergic target as they are selectively localized to the basal ganglia and to the indirect output pathway known to modulate the striato-thalamo-cortical loops critical to the expression of the motor symptoms of PD. This paper reviews the preclinical evidence base for the ability of adenosine A2A receptor blockade to influence motor function and modulate dyskinesia expression. There is consensus that adenosine A2A receptor antagonists - administered either as a monotherapy or in combination with l-DOPA or dopamine agonists - improve motor function in both rodent and primate models of PD, and should be effective for treating the motor symptoms of PD in humans. Importantly, the improvements in motor function were seen in the absence of dyskinesia. The introduction of a non-dopaminergic approach to modifying basal ganglia function provides a useful addition to the range of available therapies for treating PD, and there is a rational basis for a drug that focuses on modifying basal ganglia output.

Can adenosine A2A receptor antagonists be used to treat cognitive impairment, depression or excessive sleepiness in Parkinson's disease?

Treatment of non-motor symptoms of Parkinson's disease (PD) is a major unmet need. Targeting adenosine A2A receptors may address some of the neuropsychiatric components of non-motor symptoms - notably cognitive impairment, depression and excessive daytime sleepiness. A2A receptors are located primarily on the indirect gamma-aminobutyric acid (GABA)-ergic striatal output pathway but are also present to some extent in limbic areas of the brain, particularly the nucleus accumbens. Extensive studies show that adenosine antagonists are effective in reversing cognitive deficits in a range of experimental models related to the early executive and visuo-spatial deficits seen in PD. Similarly, A2A receptor antagonists can reverse depressive symptoms in experimental models of PD, including models with high predictive value of effect in humans, and to the same extent as classical antidepressants. Importantly, A2A antagonists are effective in models of the motivational symptoms of depression, which may relate to the apathetic/anhedonic expression of depression that can occur in PD. Adenosine and A2A receptors play a prominent role in regulating the sleep-wake cycle with arousal attributed to A2A receptor antagonism. In rodents, A2A receptor antagonists appear to induce arousal in the active part of the daily cycle only, and not during the inactive phase. This was suggested in small clinical studies in PD where A2A antagonism improved daytime sleepiness without impairing nocturnal sleep. In conclusion, A2A antagonists have potential to affect a range of neuropsychiatric components of PD; this clinical potential requires further investigation in humans.

Pharmacological interactions between adenosine A2A receptor antagonists and different neurotransmitter systems.

While Parkinson's disease (PD) is traditionally characterized by dopaminergic neuron degeneration, several neurotransmitters and neuromodulators besides dopamine are also involved in the onset and progression of the disease and its symptoms. The other principal neurotransmitters/neuromodulators known to control basal ganglia functions and, in particular, motor functions, are GABA, glutamate, serotonin (5-HT), noradrenaline, acetylcholine, adenosine and endocannabinoids. Among these, adenosine is the most relevant, acting through its adenosine A2A receptor. Work in experimental models of PD has established the effects of A2A receptor antagonists, including the alleviation of disrupted dopamine functions and improved efficacy of dopamine replacement therapy. Moreover, positive interactions between A2A receptor antagonists and both D2 and D1 receptor agonists have been described in vitro at the receptor-receptor level or in more complex in vivo models of PD, respectively. In addition, the interactions between A2A receptor antagonists and glutamate ionotropic GluN2B-containing N-Methyl-d-aspartic acid receptors, or metabotropic glutamate (mGlu) receptors, including both mGlu5 receptor inhibitors and mGlu4 receptor activators, have been reported in both in vitro and in vivo animal models of PD, as have positive interactions between A2A and endocannabinoid CB1 receptor antagonists. At the same time, a combination of A2A receptor antagonists and 5-HT1A-5-HT1B receptor agonists have been described to modulate the expression of dyskinesia induced by chronic dopamine replacement therapy.

Do caffeine and more selective adenosine A2A receptor antagonists protect against dopaminergic neurodegeneration in Parkinson's disease?

The adenosine A2A receptor is a major target of caffeine, the most widely used psychoactive substance worldwide. Large epidemiological studies have long shown caffeine consumption is a strong inverse predictor of Parkinson's disease (PD). In this review, we first examine the epidemiology of caffeine use vis-à-vis PD and follow this by looking at the evidence for adenosine A2A receptor antagonists as potential neuroprotective agents. There is a wealth of accumulating biological, epidemiological and clinical evidence to support the further investigation of selective adenosine A2A antagonists, as well as caffeine, as promising candidate therapeutics to fill the unmet need for disease modification of PD.

Intracellular adenosine released from THP-1 differentiated human macrophages is involved in an autocrine control of Leishmania parasitic burden, mediated by adenosine A2A and A2B receptors.

Leishmania infected macrophages have conditions to produce adenosine. Despite its known immunosuppressive effects, no studies have yet established whether adenosine alter Leishmania parasitic burden upon macrophage infection. This work aimed at investigating whether endogenous adenosine exerts an autocrine modulation of macrophage response towards Leishmania infection, identifying its origin and potential pharmacological targets for visceral leishmaniasis (VL), using THP-1 differentiated macrophages. Adenosine deaminase treatment of infected THP-1 cells reduced the parasitic burden (29.1 ± 2.2%, P < 0.05). Adenosine A2A and A2B receptor subtypes expression was confirmed by RT-qPCR and by immunocytochemistry and their blockade with selective adenosine A2A and A2B antagonists reduced the parasitic burden [14.5 ± 3.1% (P < 0.05) and 12.3 ± 3.1% (P < 0.05), respectively; and 24.9 ± 2.8% (P < 0.05), by the combination of the two antagonists)], suggesting that adenosine A2 receptors are tonically activated in infected THP-1 differentiated macrophages. The tonic activation of adenosine A2 receptors was dependent on the release of intracellular adenosine through equilibrative nucleoside transporters (ENT1/ENT2): NBTI or dipyridamole reduced (~25%) whereas, when ENTs were blocked, adenosine A2 receptor antagonists failed to reduce and A2 agonists increase parasitic burden. Effects of adenosine A2 receptors antagonists and ENT1/2 inhibitor were prevented by L-NAME, indicating that nitric oxide production inhibition prevents adenosine from increasing parasitic burden. Results suggest that intracellular adenosine, released through ENTs, elicits an autocrine increase in parasitic burden in THP-1 macrophages, through adenosine A2 receptors activation. These observations open the possibility to use well-established ENT inhibitors or adenosine A2 receptor antagonists as new therapeutic approaches in VL.

The effect of caffeine on cognitive performance is influenced by CYP1A2 but not ADORA2A genotype, yet neither genotype affects exercise performance in healthy adults.

PURPOSE: To determine the influence of two commonly occurring genetic polymorphisms on exercise, cognitive performance, and caffeine metabolism, after caffeine ingestion. METHODS: Eighteen adults received caffeine or placebo (3 mg kg-1) in a randomised crossover study, with measures of endurance exercise (15-min cycling time trial; 70-min post-supplementation) and cognitive performance (psychomotor vigilance test; PVT; pre, 50 and 95-min post-supplementation). Serum caffeine and paraxanthine were measured (pre, 30 and 120-min post-supplementation), and polymorphisms in ADORA2A (rs5751876) and CYP1A2 (rs762551) genes analysed. RESULTS: Caffeine enhanced exercise performance (P < 0.001), but effects were not different between participants with ADORA2A 'high' (n = 11) vs. 'low' (n = 7) sensitivity genotype (+ 6.4 ± 5.8 vs. + 8.2 ± 6.8%), or CYP1A2 'fast' (n = 10) vs. 'slow' (n = 8) metabolism genotype (+ 7.2 ± 5.9 vs. + 7.0 ± 6.7%, P > 0.05). Caffeine enhanced PVT performance (P < 0.01). The effect of caffeine was greater for CYP1A2 'fast' vs. 'slow' metabolisers for reaction time during exercise (- 18 ± 9 vs. - 1.0 ± 11 ms); fastest 10% reaction time at rest (- 18 ± 11 vs. - 3 ± 15 ms) and lapses at rest (- 3.8 ± 2.7 vs. + 0.4 ± 0.9) (P < 0.05). There were no PVT differences between ADORA2A genotypes (P > 0.05). Serum caffeine and paraxanthine responses were not different between genotypes (P > 0.05). CONCLUSION: Caffeine enhanced CYP1A2 'fast' metabolisers' cognitive performance more than 'slow' metabolisers. No other between-genotype differences emerged for the effect of caffeine on exercise or cognitive performance, or metabolism.