Diminished iron concentrations increase adenosine A2A receptor levels in mouse striatum and cultured human neuroblastoma cells

Brain iron insufficiency has been implicated in several neurological disorders. The dopamine system is consistently altered in studies of iron deficiency in rodent models. Changes in striatal dopamine D₂ receptors are directly proportional to the degree of iron deficiency. In light of the unknown mechanism for the iron deficiency-dopamine connection and because of the known interplay between adenosinergic and dopaminergic systems in the striatum we examined the effects of iron deficiency on the adenosine system. We first attempted to assess whether there is a functional change in the levels of adenosine receptors in response to this low iron. Mice made iron-deficient by diet had an increase in the density of striatal adenosine A_2A (A_2AR) but not A₁ receptor (A₁R) compared to mice on a normal diet. Between two inbred murine strains, which had 2-fold differences in their striatal iron concentrations under normal dietary conditions, the strain with the lower striatal iron had the highest striatal A_2AR density. Treatment of SH-SY5Y (human neuroblastoma) cells with an iron chelator resulted in increased density of A_2AR. In these cells, A_2AR agonist-induced cyclic AMP production was enhanced in response to iron chelation, also demonstrating a functional upregulation of A_2AR. A significant correlation (r² = 0.79) was found between a primary marker of cellular iron status (transferrin receptor (TfR)) and A_2AR protein density. In conclusion, the A_2AR is increased across different iron-insufficient conditions. The relation between A_2AR and cellular iron status may be an important pathway by which adenosine may alter the function of the dopaminergic system.     

Central adenosine A2A receptors: an overview

Recent advances in molecular biology, biochemistry, cell biology and behavioral pharmacology together with the development of more selective ligands to the various adenosine receptors have increased our understanding of the functioning of central adenosine A_2A receptors. The A_2A receptor is one of four adenosine receptors found in the brain. Its expression is highest in striatum, nucleus accumbens and olfactory tubercles, although it also occurs in neurons and microglia in most other brain regions. The receptor has seven transmembrane domains and couples via Gs to adenyl cyclase stimulation. Antagonistic interactions between A_2A receptors and dopamine D₂ receptors have been described, as stimulation of the A_2A receptor leads to a reduction in the affinity of D₂ receptors for D₂ receptor agonists. The A_2A receptor is thought to play a role in a number of physiological responses and pathological conditions. Indeed, A_2A receptor antagonists may be useful for the treatment of acute and chronic neurodegenerative disorders such as cerebral ischemia or Parkinson's disease. A_2A receptor agonists may treat certain types of seizures or sleep disorders. This review discusses the characteristics, distribution, pharmacochemical properties and regulation of central A_2A receptors, as well as A_2A receptor-mediated behavioural responses and their potential role in various neuropsychiatric disorders.     

Agonists of A1 and A2A adenosine receptors attenuate methamphetamine-induced overflow of dopamine in rat striatum

The effect of adenosine receptor agonists on the release of striatal dopamine (DA), induced by repeated doses of methamphetamine (MTH), was evaluated. Rats received three injections of MTH (5 mg/kg i.p.) at 2-h intervals. The release of DA in the striatum was measured by a microdialysis in freely moving animals. The agonist of adenosine A₁ receptor, N⁶-cyclopentyladenosine (CPA) and the agonist of adenosine A_2A receptor, 2-[p-(carboxy-ethyl)phenylethylamino]-5′-N-ethylcarboxyamidoadenosine (CGS 21680), either of them being infused locally into the striatum at concentrations of 50 and 100 μM, produced decreases in the extracellular DA level during exposure to MTH, and a weaker effect on the levels of DOPAC and HVA. The above effects were reversed by the specific antagonists of adenosine A₁ and A_2A receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 3,7-dimethyl-1-propargylxanthine (DMPX), respectively. Our results indicate that both the A₁ and A_2A adenosine receptors appear to be involved in reducing the excessive release of DA in the striatum; furthermore, they suggest a neuroprotective role of adenosine in MTH neurotoxicity.     

Activation of adenosine A2A receptor protects sympathetic neurons against nerve growth factor withdrawal

Adenosine mediates a range of effects in the central nervous system (CNS), including the promotion of neuronal survival, but its actions on sympathetic neurons are less well characterized. We therefore sought to understand the role of endogenous adenosine in contributing to the survival of neurotrophin-dependent sympathetic neurons. Rat superior cervical ganglion (SCG) cultures were maintained in the continuous presence of nerve growth factor (NGF) and then exposed to adenosine deaminase (ADA), to deplete endogenous adenosine. This resulted in a marked increase in cellular apoptosis, to a level that approximated the effect of NGF withdrawal. Furthermore, the addition of exogenous adenosine to NGF-deprived SCG neurons resulted in enhanced cell survival. Analysis of adenosine receptor (AR) subtypes on these neurons, using real-time RT-PCR and receptor binding analyses, revealed that the A2A receptor was the major subtype present. Accordingly, the A2A receptor agonist CGS21680 significantly reduced both ADA-induced and NGF-withdrawal-induced neuronal apoptosis, whereas the A1 receptor agonist R-PIA had no such effect. The survival-promoting effect of CGS21680 was eliminated when cells were coincubated with a molar excess of an A2A receptor antagonist. Finally, follow-up experiments revealed that CGS21680 prevented the induction of early apoptotic events, such as changes in mitochondrial integrity and caspase activation, and that it also triggered an increase in ERK activation, which was essential for neurotrophin-independent cell survival. Taken together, these findings provide evidence that endogenous adenosine may be important in mediating protection of sympathetic neurons and that it may act via the A2A receptor subtype.     

The effects of selective A1 and A2a adenosine receptor antagonists on cerebral ischemic injury in the gerbil

Cerebral ischemia of 5 min duration was induced in unanesthetized Mongolian gerbils by bilateral occlusion of the carotid arteries. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity following ischemia and by a histopathological assessment of the extent of CA1 hippocampal pyramidal cell injury and loss 5 days after ischemia. The A_2a adenosine receptor selective antagonists 8-(3-chlorostyryl) caffeine (CSC; 0.1 mg/kg i.p.) and 4-amino-l-phenyl[1,2,4]-triazolo[4,3-a] quinoxaline (CP 66,713; 0.1 mg/kg i.p.) reduced the extent of ischemia-induced injury. An A1 selective receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1.0 mg/kg i.p.), enhanced ischemia-evoked injury. These results suggest that adenosine A_2a receptor antagonists may be useful for the prevention of cerebral injuries resulting from stroke or cardiac arrest.     

In vivo labelling of the adenosine A2A receptor in mouse brain using the selective antagonist [3H]SCH 58261

The selective A2A receptor antagonist [3H]SCH 58261 was injected intravenously in mice and the radioactivity accumulating in various brain regions was determined by tissue sampling. Radioactivity levels in regions of interest such as the striatum were highest 15 min after injection and quickly declined thereafter (30 min and 1 h postinjection) in a time-dependent manner. The amount of labelling was ranked as follows: striatum (4.6 +/- 0.3 fmol/mg protein) > cortex > hippocampus > pons = hypothalamus > cerebellum (0.5 +/- 0.05 fmol/mg protein). Specific labelling of the A2A receptor occurred in striatum and cortex because significantly less radioactivity accumulated in these areas from adenosine A2A receptor knockout mice as compared to wild-type littermates. In control outbred CD1 mice, a striatum-to-cerebellum ratio of 7.6 +/- 0.6 was found. At 30 min postinjection, the nonselective adenosine receptor antagonist caffeine reduced the radioactivity due to [3H]SCH 58261 in the striatum by 32% at 1 mg/kg i.p. and by 66% at the stimulant dose of 6.25 mg/kg i.p. Radioactivity in the striatum was lowered, respectively, by 66 and 86% 30 min after injection of 3 or 10 mg/kg i.p. doses of unlabelled SCH 58261. The present results indicate that [3H]SCH 58261 directly labels striatal A2A receptors in vivo. Thus [3H]SCH 58261 is an excellent tool for studying brain distribution and A2A receptor occupancy of various compounds ranging from xanthines, such as caffeine, to other A2A antagonists.     

Endogenous adenosine facilitates neurotransmission via A2A adenosine receptors in the rat superior colliculus in vivo

The concentration of endogenous adenosine in the cerebrospinal fluid increased 2–3-fold of the original level in the area of rat superior colliculus after the intraperitoneal administration of an adenosine deaminase inhibitor, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenosine, 10 mg/kg). Potentials evoked in the superior colliculus by optic tract stimulation were also facilitated by 120–160% of their initial amplitudes. A selective A₁ adenosine receptor antagonist, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), failed to reduce such EHNA-induced facilitation. However, a selective A_2A adenosine receptor antagonist, KF17837 (8(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) completely eliminated the facilitatory effects of EHNA. Northern blot analysis demonstrated abundant expression of A₁ adenosine receptor mRNA in the superior colliculus. RT-PCR analysis was able to detect the concomitant expression of A_2A adenosine receptor mRNA, but at levels lower than one-tenth of the striatal expression. In the superior colliculus, A_2A adenosine receptors function predominantly on the facilitatory effects of adenosine, irrespective of the ubiquitous expression of A₁ adenosine receptors.     

Single and repeated episodes of ethanol withdrawal increase adenosine A1, but not A2A, receptor density in mouse brain

A history of multiple ethanol withdrawal experiences has been shown to exacerbate the severity of future withdrawal episodes, and this sensitization of the withdrawal response has been hypothesized to represent a ‘kindling' phenomenon. Since adenosine functions as an inhibitory modulator of seizure activity and may interact with ethanol to influence neuronal excitability, the present study was conducted to examine the effects of single and repeated episodes of ethanol withdrawal on adenosine A₁ and A_2A receptors in adult C3H/He mice. Mice were chronically exposed to ethanol vapor in inhalation chambers and tested for withdrawal seizures following multiple withdrawal (MW) experience (four cycles of 16 h ethanol intoxication interrupted by 8 h periods of abstinence), single withdrawal experience following 16 h (SW) or 64 h (CE) continuous ethanol intoxication, or no ethanol exposure (controls). Separate groups of mice from each withdrawal condition were used to generate pooled cortical and striatal tissue for ligand saturation experiments using [³H]cyclohexyladenosine to label A₁ receptors and [³H]CGS 21680 to label A_2A receptors. Results indicated that withdrawal seizures were significantly more severe in mice with multiple withdrawal experience in comparison to animals that experienced only a single withdrawal episode, even when total amount of ethanol exposure was equated among groups. The density of A₁ receptors in cerebral cortex was significantly increased over controls 8 h following final ethanol withdrawal by approximately 35% in SW and CE groups, with the largest increase observed in the MW group (56%). Withdrawal treatment groups did not differ in cortical A₁ binding sites immediately upon withdrawal from ethanol, and no significant differences in binding of [³H]CGS 21680 to striatal A_2A receptors were observed following ethanol withdrawal. Ethanol exposure and withdrawal did not significantly alter ligand affinity for either adenosine receptor. These results indicate that adenosine A₁ receptors are selectively upregulated during ethanol withdrawal and that the degree of upregulation may be enhanced following multiple withdrawal episodes. Further, these observations suggest that the upregulation of brain A₁ receptors during ethanol withdrawal may represent a compensatory inhibitory response to increased seizure severity associated with repeated episodes of ethanol withdrawal.     

Preladenant, a selective A2A receptor antagonist, is active in primate models of movement disorders

Parkinson's Disease (PD) and Extrapyramidal Syndrome (EPS) are movement disorders that result from degeneration of the dopaminergic input to the striatum and chronic inhibition of striatal dopamine D₂ receptors by antipsychotics, respectively. Adenosine A_2A receptors are selectively localized in the basal ganglia, primarily in the striatopallidal (“indirect”) pathway, where they appear to operate in concert with D₂ receptors and have been suggested to drive striatopallidal output balance. In cases of dopaminergic hypofunction, A_2A receptor activation contributes to the overdrive of the indirect pathway. A_2A receptor antagonists, therefore, have the potential to restore this inhibitor imbalance. Consequently, A_2A receptor antagonists have therapeutic potential in diseases of dopaminergic hypofunction such as PD and EPS. Targeting the A_2A receptor may also be a way to avoid the issues associated with direct dopamine agonists. Recently, preladenant was identified as a potent and highly selective A_2A receptor antagonist, and has produced a significant improvement in motor function in rodent models of PD. Here we investigate the effects of preladenant in two primate movement disorder models. In MPTP-treated cynomolgus monkeys, preladenant (1 or 3 mg/kg; PO) improved motor ability and did not evoke any dopaminergic-mediated dyskinetic or motor complications. In Cebus apella monkeys with a history of chronic haloperidol treatment, preladenant (0.3–3.0 mg/kg; PO) delayed the onset of EPS symptoms evoked by an acute haloperidol challenge. Collectively, these data support the use of preladenant for the treatment of PD and antipsychotic-induced movement disorders.     

Adenosine A2A receptor agonists increase Fos-like immunoreactivity in mesolimbic areas

Expression of the early-gene c-fos is an useful method for studying potential sites of action of drugs active in the CNS. Stimulation of adenosine A_2A receptors by CGS 21680 (5 mg/kg) induced an increase in Fos-like immunoreactivity in the rat nucleus accumbens shell, while in the rostral pole and core CGS 21680 induced Fos-like immunoreactivity only after a high dose. CGS 21680 (5 mg/kg) stimulated c-fos expression also in the lateral septal nucleus and dorso-medial striatum, but not in the dorso-lateral striatum. A similar pattern of Fos-like immunoreactivity was obtained after administration of the A_2A agonist HENECA (5 mg/kg) which displays higher selectivity for A_2A receptors than CGS 21680. Administration of the selective A_2A antagonist SCH 58261 counteracted CGS 21680-induced Fos-like immunoreactivity. Lesions of the dopaminergic mesostriatal projection by 6-hydroxydopamine and stimulation of dopamine D₂/D₃ receptors by quinpirole, prevented CGS 21680-induced Fos-like immunoreactivity in the nucleus accumbens shell. The present results show that stimulation of A_2A receptors induces a profile of c-fos expression similar to that of atypical neuroleptics. A_2A receptor stimulation has been reported to have dopamine antagonistic actions, it is therefore suggested that A_2A agonists might have antipsychotic activity without producing extrapyramidal side effects.     

A1 and A2A adenosine receptors and A1 mRNA in mouse brain: effect of long-term caffeine treatment

The effect of oral treatment with caffeine, in doses that are known to produce marked adaptive effects, was investigated on A₁ and A_2A receptors in the mouse brain. Caffeine (0.1, 0.3 or 1 g/l) was added to the drinking water and the animals were sacrificed after a 14-day treatment period. Ligand binding to A₁ receptors was studied, using quantitative autoradiography, with the agonist [³H]cyclohexyladenosine (CHA) and the antagonist [³H]1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Caffeine did not remain in the sections during the autoradiography experiments. Caffeine treatment (1 g/l, but not 0.1 or 0.3 g/l) tended to increase [³H]CHA binding to the CA3 subfield of the hippocampus, but in no other region studied. There was no change in the number of A₁ receptors since [³H]DPCPX binding to the CA3, cerebral and cerebellar cortex was not influenced by caffeine treatment. There was similarly no change in the ability of CHA to displace [³H]DPCPX binding, suggesting that there are no major changes in the proportion of A₁ receptors that are coupled to G-proteins. mRNA for the A₁ receptor, measured by in situ hybridization, did not differ significantly between caffeine-treated and control mice in the structures examined. Thus, higher doses of caffeine can cause an increase in A₁ agonist binding without a corresponding change in A₁ mRNA or in A₁ antagonist binding, suggesting that the adaptive changes seen upon prolonged caffeine treatment may be in sites different from A₁ receptors. Caffeine (1 g/l) increased A_2A receptors in the striatum measured as binding of the agonist [³H]CGS 21680 suggesting that up-regulation of A_2A receptors may be an adaptive effect of caffeine intake.     

Electrophysiological and behavioural evidence for an antagonistic modulatory role of adenosine A2A receptors in dopamine D2 receptor regulation in the rat dopamine-denervated striatum

It has been shown that striatal adenosine A2A receptors can antagonistically interact with dopamine D2 receptors at the membrane level leading to a decrease in the affinity and efficacy of D2 receptors. Extracellular recordings and rotational behaviour were employed to obtain a correlate to these findings in an animal model of Parkinson's disease (PD). The recordings were performed in rats with unilateral 6-hydroxydopamine (6-OHDA)-induced catecholamine depletion. While recording in the dopamine-depleted striatum, local applications of the dopamine D2 agonist quinpirole reduced neuronal activity. However, when the adenosine A2A antagonist MSX-3 was applied simultaneously with quinpirole, the inhibition of neuronal firing seen after quinpirole alone was significantly potentiated (P< 0.001, n = 11). In contrast, local application of CGS 21680 attenuated the effect of quinpirole. The doses of MSX-3 and CGS 21680 used to achieve the modulation of quinpirole action had no effect per se on striatal neuronal firing. Furthermore, rotational behaviour revealed that MSX-3 dose-dependently increased the number of turns when administrated together with a threshold dose of quinpirole while no enhancement was achieved when MSX-3 was combined with SKF 38393. MSX-3 alone did not induce rotational behaviour. In conclusion, this study shows that low ineffective doses of MSX-3 enhance the effect of quinpirole on striatal firing rate, while the A2A agonist exerts the opposite action. This mechanism gives a therapeutic potential to A2A antagonists in the treatment of PD by enhancing D2 receptor function.     

A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase-2 in the hippocampus after kainate lesions

The systemic administration of kainate (10 mg/ml) into adult Wistar rats produces seizures and neurodegeneration. We have studied the effect of kainate administration on cPLA₂ and COX-2 immunoreactivities after 3 days and 1, 2, 4 and 11 weeks. The cPLA₂ immunoreactivity was increased in hippocampal neurons at 1 and 3 days after kainate injection suggesting that PLA₂ may be involved in neurodegeneration. Increased cPLA₂ and COX-2 immunoreactivities in astrocytes at 1, 2, 4 and 11 weeks after kainate injection indicate an adaptive astrocytic response that may be associated with gliosis.     

Effects of chronic administration of caffeine on adenosine A1 and A2 receptors in rat brain

Chronic administration of caffeine (75 mg/kg/day) to rats for 12 days increased [3H]R-PIA binding in the cerebral cortex and cerebellum and [3H]NECA binding to high affinity receptor sites in the striatum. The results indicate that both adenosine A1 and A2 receptor subtypes possess mechanisms of adaptation to chronic caffeine treatment. In addition, adenosine A1 receptor binding shows heterogenous neuroanatomical pattern indicating that the A1 response to caffeine treatment presents regional variation in the rat brain.     

Up-regulation of striatal adenosine A2A receptors with iron deficiency in rats: Effects on locomotion and cortico-striatal neurotransmission

Brain iron deficiency leads to altered dopaminergic function in experimental animals, which can provide a mechanistic explanation for iron deficiency-related human sensory-motor disorders, such as Restless Legs Syndrome (RLS). However, mechanisms linking both conditions have not been determined. Considering the strong modulation exerted by adenosine on dopamine signaling, one connection could involve changes in adenosine receptor expression or function. In the striatum, presynaptic A_2A receptors are localized in glutamatergic terminals contacting GABAergic dynorphinergic neurons and their function can be analyzed by the ability of A_2A receptor antagonists to block the motor output induced by cortical electrical stimulation. Postsynaptic A_2A receptors are localized in the dendritic field of GABAergic enkephalinergic neurons and their function can be analyzed by studying the ability of A_2A receptor antagonists to produce locomotor activity and to counteract striatal ERK1/2 phosphorylation induced by cortical electrical stimulation. Increased density of striatal A_2A receptors was found in rats fed during 3 weeks with an iron-deficient diet during the post-weaning period. In iron-deficient rats, the selective A_2A receptor antagonist MSX-3, at doses of 1 and 3 mg/kg, was more effective at blocking motor output induced by cortical electrical stimulation (presynaptic A_2A receptor-mediated effect) and at enhancing locomotor activation and blocking striatal ERK phosphorylation induced by cortical electrical stimulation (postsynaptic A_2A receptor-mediated effects). These results indicate that brain iron deficiency induces a functional up-regulation of both striatal pre- and postsynaptic A_2A receptor, which could be involved in sensory-motor disorders associated with iron deficiency such as RLS.     

Immunohistochemical detection of A1 adenosine receptors in rat brain with emphasis on localization in the hippocampal formation, cerebral cortex, cerebellum, and basal ganglia

Polyclonal antisera were generated against two identical regions of rat and human A₁ adenosine receptors using synthetic multiple-antigenic-peptides as immunogens. Western blotting showed that the antisera recognized a single protein in brain of the expected size for A₁ receptors. Immunohistochemistry of CHO cells transfected with the rat or human A₁ adenosine receptor cDNAs showed robust labeling of the cell surface. In contrast, labeling was not apparent over non-transfected CHO cells, nor over CHO cells expressing A_2a receptors. The pattern of immunoreactivity in rat brain was similar to that expected for A₁ adenosine receptors. In contrast to receptor autoradiography or in situ hybridization methods, immunohistochemistry allowed identification of individually labeled cells and processes. Heavy labeling was apparent in many brain regions. In the hippocampal formation, strong labeling was present on granule cell bodies and dendrites, mossy fibers, and pyramidal neurons. In cerebellum, basket cells were the most heavily labeled cell type. Less intense staining was present over granule cells. In cerebral cortex, pyramidal cells were the most heavily labeled cell type, and some interneurons were also labeled. In the basal ganglia, 43% of neurons in the globus pallidus were labeled. In the caudate-putamen region, 38% of neurons were labeled. Heavy labeling was present in most thalamic nuclei, and moderate to heavy labeling was seen in many brainstem nuclei. These data identify specific cellular sites of A₁ receptor expression and support the concept of cellular specificity of A₁ adenosine receptor action.     

Endogenous adenosine delays the onset of hypoxic depolarization in the rat hippocampus in vitro via an action at A1 receptors

The effect of endogenous adenosine on the delay to hypoxic depolarization (HD) was examined utilizing in vitro slices of gerbil hippocampus. Adenosine receptor antagonists were used to block the actions of adenosine during hypoxia, and the delay to HD was measured in the CA1 region. Both a broad spectrum antagonist (theophylline) and an A₁ receptor-specific antagonist (8-cyclopentyl-1,3-dimethylxanthine; CPT) shortened the delay to HD. These findings indicate that endogenous adenosine working through A₁ receptors prolongs the delay to HD. This effect may contribute to the neuroprotective influence of adenosine and its analogs.     

Adenosine A2A receptor gene: Evidence for association of risk variants with panic disorder and anxious personality

Adenosine A_2A receptors are suggested to play an important role in different brain circuits and pathways involved in anxiety reactions. A variant within the corresponding ADORA2A gene (rs5751876) increased the risk for panic disorder (PD), for elevated anxiety during challenge tests in healthy probands and for anxiety-related arousal in blood-injury phobia. These multiple effects may mirror a more general effect of the SNP on basic personality traits. In the present study we therefore aimed to replicate the original finding in a large PD sample and extend it by investigating an additional proband sample characterized for different anxiety-related personality scores. In addition, as rs5751876 is assumed not to be the disease variant itself but to be in linkage disequilibrium (LD) with the true functional polymorphism other SNPs of potentially functional relevance were identified by re-sequencing the whole gene including several newly identified regions of putative regulatory potential and analysed for their impact on PD and anxious personality. We were indeed able to replicate rs5751876 as risk factor for PD, particularly PD with agoraphobia. Rs5751876 and several other variants in high LD (rs5751862, rs2298383 and rs3761422) as well as the corresponding haplotypes were also associated with different anxiety-related personality scores (Bonferroni corrected P_all < 0.05). Of these variants, rs2298383 shows functional potential based on in silico analyses and might therefore represent the true underlying causal variant. Our data provide further support for an important role of ADORA2A variants in the pathogenesis of anxiety disorders and anxious personality reflecting their potential as basic susceptibility factors.