Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.     

The A2A adenosine receptor rescues neuritogenesis impaired by p53 blockage via KIF2A,a kinesin family member

The A_2A adenosine receptor (A_2AR) is a G‐protein–coupled receptor. We previously reported that the C terminus of the A_2AR binds to translin‐associated protein X (TRAX) and modulates nerve growth factor (NGF)‐evoked neurite outgrowth in PC12 cells. Herein, we show that neuritogenesis of primary hippocampal neurons requires p53 because blockage of p53 suppressed neurite outgrowth. The impaired neuritogenesis caused by p53 blockage was rescued by activation of the A_2AR (designated the A_2A rescue effect) in a TRAX‐dependent manner. Importantly, suppression of a TRAX‐interacting protein (kinesin heavy chain member 2A, KIF2A) inhibited the A_2A rescue effect, whereas overexpression of KIF2A caused a rescue effect. Expression of a KIF2A fragment (KIF2A₅₁₄), which disturbed the interaction between KIF2A and TRAX, blocked the rescue effect. Transient colocalization of TRAX and KIF2A was detected in the nucleus of PC12 cells upon NGF treatment. These data suggest that functional interaction between KIF2A and TRAX is critical for the A_2A rescue effect. Moreover, p53 blockage during NGF treatment prevented the redistribution of KIF2A from the nucleus to the cytoplasmic region. Expression of a nuclear‐retained KIF2A variant (NLS‐KIF2A) did not rescue the impaired neurite outgrowth as did the wild‐type KIF2A. Therefore, redistribution of KIF2A to the cytoplasmic fraction is a prerequisite for neurite outgrowth. Collectively, we demonstrate that KIF2A functions downstream of p53 to mediate neuritogenesis of primary hippocampal neurons and PC12 cells. Stimulation of the A_2AR rescued neuritogenesis impaired by p53 blockage via an interaction between TRAX and KIF2A. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 604–621, 2010     

Genetic and pharmacological inactivation of the adenosine A2A receptor attenuates 3-nitropropionic acid-induced striatal damage

Adenosine A2A receptor (A2AR) antagonism attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and quinolinic acid-induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3-nitropriopionic acid (3-NP). 3-NP was administered to A2AR knockout (KO) and wild-type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3-NP-treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3-NP-treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3-NP-induced striatal damage was observed in A2AR KO mice in a 129-Steel background. In addition, the effect of pharmacological antagonism on 3-NP-induced striatal neurotoxicity was tested by pre-treatment of C57Bl/6 mice with the A2AR antagonist 8-(3-chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3-NP alone or 3-NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3-NP and in five of six mice treated with CSC (20 mg/kg) plus 3-NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3-NP. Since the clinical and neuropathological features of 3-NP-induced striatal damage resemble those observed in Huntington's disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntington's disease patients.     

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.     

Balance of purines may determine life or death of retinal ganglion cells as A3 adenosine receptors prevent loss following P2X7 receptor stimulation

The purines ATP and adenosine can act as a coordinated team of transmitters. As extracellular adenosine is frequently derived from the enzymatic dephosphorylation of released ATP, the distinct actions of the two purines can be synchronized. In retinal ganglion cells (RGCs), stimulation of the P2X7 receptor for ATP leads to increased intracellular Ca2+ and death. Here we define the contrasting effects of adenosine and identify protective actions mediated by the A3 receptor. Adenosine attenuated the rise in Ca2+ produced by the P2X7 agonist 3'-O-(4-benzoylbenzoyl)ATP (BzATP). Adenosine was also neuroprotective, increasing the survival of ganglion cells exposed to BzATP. The A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronimide (Cl-IB-MECA) mimicked the inhibition of the Ca2+ rise, whereas the A3 antagonist 3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191) reduced the protective effects of adenosine. Both Cl-IB-MECA and a second A3 receptor agonist IB-MECA reduced the cell loss triggered by BzATP. The actions of BzATP were mimicked by ATPgammaS, but not by ATP. In summary, adenosine can stop the rise in Ca2+ and cell death resulting from stimulation of the P2X7 receptor on RGCs, with the A3 adenosine receptor contributing to this protection. Hydrolysis of ATP into adenosine and perhaps inosine shifts the balance of purinergic action from that of death to the preservation of life.     

Caffeine inhibition of rat carotid body chemoreceptors is mediated by A2A and A2B adenosine receptors

Caffeine, an unspecific antagonist of adenosine receptors, is commonly used to treat the apnea of prematurity. We have defined the effects of caffeine on the carotid body (CB) chemoreceptors, the main peripheral controllers of breathing, and identified the adenosine receptors involved. Caffeine inhibited basal (IC50, 210 microm) and low intensity (PO2 approximately 66 mm Hg/30 mm K+) stimulation-induced release of catecholamines from chemoreceptor cells in intact preparations of rat CB in vitro. Opposite to caffeine, 5'-(N-ethylcarboxamido)adenosine (NECA; an A2 agonist) augmented basal and low-intensity hypoxia-induced release. 2-p-(2-Carboxyethyl)phenethyl-amino-5'-N-ethylcaboxamido-adenosine hydrochloride (CGS21680), 2-hexynyl-NECA (HE-NECA) and SCH58621 (A2A receptors agents) neither affected catecholamine release nor altered the caffeine effects. The 8-cycle-1,3-dipropylxanthine (DPCPX; an A1/A2B antagonist) and 8-(4-{[(4-cyanophenyl)carbamoylmethyl]-oxy}phenyl)-1,3-di(n-propyl)xanthine (MRS1754; an A2B antagonist) mimicking of caffeine indicated that caffeine effects are mediated by A2B receptors. Immunocytochemical A2B receptors were located in tyrosine hydroxylase positive chemoreceptor cells. Caffeine reduced by 52% the chemosensory discharges elicited by hypoxia in the carotid sinus nerve. Inhibition had two components with pharmacological analysis indicating that A2A and A2B receptors mediate, respectively, the low (17 x 10(-9) m) and high (160 x 10(-6) m) IC50 effects. It is concluded that endogenous adenosine, via presynaptic A2B and postsynaptic A2A receptors, can exert excitatory effects on the overall output of the rat CB chemoreceptors.     

Behavioral control by striatal adenosine A2A‐dopamine D2 receptor heteromers

G protein‐coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A_2A receptors (A_2AR) and dopamine D₂ receptors (D₂R) predominantly form A_2AR‐D₂R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A_2AR and D₂R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain‐related differences, a new D₂R‐deficient mouse with the same genetic background (CD‐1) than the A_2AR knock‐out mouse was generated. Locomotor activity, pre‐pulse inhibition (PPI) and drug‐induced catalepsy were then evaluated in wild‐type, A_2AR and D₂R knock‐out mice, with and without the concomitant administration of either the D₂R agonist sumanirole or the A_2AR antagonist SCH442416. SCH442416‐mediated locomotor effects were demonstrated to be dependent on D₂R signaling. Similarly, a significant dependence on A_2AR signaling was observed for PPI and for haloperidol‐induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A_2AR‐D₂R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders.     

Alpha1-adrenergic receptor antagonist tamsulosin ameliorates aging-induced memory impairment by enhancing neurogenesis and suppressing apoptosis in the hippocampus of old-aged rats

Age-related memory decline is closely associated with decreased neurogenesis and increased apoptosis in the hippocampus. Noradrenaline exerts its effect by selectively binding to and activating adrenergic receptors (ARs). Tamsulosin, α₁-AR antagonist, is reported to have access to the brain and interact with α₁-AR. In this study, the effects of tamsulosin on short-term and spatial learning memory in terms of neurogenesis and apoptosis were investigated using rats. Step-down avoidance test for short-term memory and radial 8-arm maze test for spatial learning memory were conducted. Neurogenesis was detected by 5-bromo-2’-deoxyuridine (BrdU) immunohistochemistry and apoptosis was evaluated by caspase-3 immunohistochemisty and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNE) staining. Western blot for protein kinase C (PKC), cAMP-responsive element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB), phosphatidylinositol 3-kinase (PI 3-kinase), Akt, Bcl-2, and Bax was conducted. In the aged rats, short-term and spatial learning memory was declined. Hippocampal nerogenesis was suppressed and hippocampal apoptosis was enhanced in the aged rats. In addition, phosphorylation of PKCα, CREB, PI-3 kinase, and Akt was decreased in the hippocampus of old-aged rats. Tamsulosin activated PKC/CREB and PI-3 kinase/Akt pathways. With these pathways, BDNF-TrkB signaling enhanced hippocampal neurogenesis and suppressed apoptosis in the old-aged rats. As the results, tamsulosin improved performance of short-term and spatial learning memory in the aged rats.     

In vivo effects of adenosine A(2) receptor agonist and antagonist on neuronal and astrocytic intermediary metabolism studied with ex vivo (13)C MR spectroscopy.

The effect of adenosine A(2) receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-ethylcarboxamidoadenosine (CGS 21680) and antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) on [1-(13)C]glucose and [1,2-(13)C]acetate metabolism was studied in rats by (13)C magnetic resonance (MR) spectroscopy and HPLC. In the cortex a significant reduction was observed in the amounts of [2-(13)C]GABA and [3-(13)C]aspartate from [1-(13)C]glucose in CGS 21680. In the subcortex the concentration of labelled [4-(13)C]glutamate was increased in both treatment groups. The amounts of [2 + 3-(13)C]succinate and [3-(13)C]lactate were increased in the CGS 21680 group compared to control, and the DMPX group showed an increase in the total amount of [6-(13)C]N-acetyl aspartate compared to control in the subcortex. Astrocyte metabolism was only affected in the cortex as shown by a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in glutamate and glutamine in the treatment groups. Labelling from [1,2-(13)C]acetate was not much affected by CGS 21680 or DMPX. However, the amount of [1,2-(13)C]acetate in cortex and subcortex was reduced in the DMPX group. In the cortex a reduction in the labelling of [3-(13)C]GABA in the DMPX group compared to control and an increase in the total amount of taurine in both treatment groups was detected. The present study shows that A(2) receptor agonist and antagonist have similar effects; however, in cortex GABAergic neurones and astrocytes were affected in contrast to subcortex, where glutamatergic neurones showed the greatest changes.     

Therapeutic potential of A2 adenosine receptor pharmacological regulators in the treatment of cardiovascular diseases,recent progress,and prospective

Adenosine and its analogs are of particular interest as potential therapeutic agents for treatment of cardiovascular diseases (CVDs). A2 adenosine receptor subtypes (A2a and A2b) are extensively expressed in cardiovascular system, and modulation of these receptors using A2 adenosine receptor agonists or antagonists regulates heart rate, blood pressure, heart rate variability, and cardiovascular toxicity during both normoxia and hypoxia conditions. Regulation of A2 adenosine receptor signaling via specific and novel pharmacological regulators is a potentially novel therapeutic approach for a better understanding and hence a better management of CVDs. This review summarizes the role of pharmacological A2 adenosine receptor regulators in the pathogenesis of CVDs.     

Extracellular vesicles with ubiquitinated adenosine A2A receptor in plasma of patients with coronary artery disease

Extracellular vesicles (EV) can transfer cellular molecules for specific intercellular communication with potential relevance in pathological conditions. We searched for the presence in plasma from coronary artery disease (CAD) patients of EV containing the adenosine A_2A receptor (A_2AR), a signalling receptor associated with myocardial ischaemia and whose expression is related to homocysteine (HCy) metabolism. Using protein organic solvent precipitation for plasma EV preparation and Western blotting for protein identification, we found that plasma from CAD patients contained various amounts of EV with ubiquitin bound to A_2AR. Interestingly, the presence of ubiquitinated A_2AR in EV from patients was dependent on hyperhomocysteinemia, the amount being inversely proportional to A_2AR expression in peripheral mononuclear cells in patients with the highest levels of HCy. CEM, a human T cell line, was also found to released EV containing various amounts of ubiquitinated A_2AR in stimulated conditions depending on the hypoxic status and HCy level of culture medium. Together, these data show that ubiquitinated A_2AR‐containing EV circulate in the plasma of CAD patients and that this presence is related to hyperhomocysteinemia. A_2AR in plasma EV could be a useful tool for diagnosis and a promising drug for the treatment of CAD.     

大鼠侧脑室注射腺苷A1受体反义寡聚脱氧核苷酸抑制脑缺血耐受的诱导

目的:观察侧脑室注射腺苷A1受体(ARA1)反义寡聚脱氧核苷酸(As-ODN)对脑缺血预处理(CIP)脑保护作用的影响,进一步探讨腺苷A1受体在CIP脑保护作用中的作用。方法:将54只凝闭双侧椎动脉的Wistar大鼠分为Sham组、CIP组、损伤性脑缺血组、CIP 损伤性脑缺血组、双蒸水 CIP 损伤性脑缺血组、ARA1As-ODN组、ARA1As-ODN CIP组、和ARA1As-ODN CIP 损伤性脑缺血组。ARA1As-ODN的剂量分为10nmol/5μl和20nmol/5μl,溶于双蒸水中,侧脑室注射。所有动物均在Sham手术后或末次全脑缺血/再灌注后7d断头取脑,硫堇染色观察海马CA1区锥体神经元迟发性死亡(DND)情况。结果:Sham组和CIP组均未见DND。与Sham、CIP组相比,损伤性脑缺血组出现了明显的DND,表现为组织学分级(HG)升高和锥体神经元密度(ND)下降(P<0.05)。CIP可显著抑制损伤性脑缺血引起的DND。与CIP 损伤性缺血组相比,ARA1As-ODN CIP 损伤性脑缺血组出现了显著的DND,表现为HG升高、ND降低(P<0.05),这种变化与ARA1As-ODN的剂量呈明显正相关。结论:腺苷A1受体As-ODN可阻断CIP诱导的脑缺血耐受,进一步证实了腺苷A1受体表达上调参与CIP诱导的脑缺血耐受。     

Neuroprotection by adenosine A2A receptor blockade in experimental models of Parkinson's disease

Adenosine A2A receptors are abundant in the caudate-putamen and involved in the motor control in several species. In MPTP-treated monkeys, A2A receptor-blockade with an antagonist alleviates parkinsonian symptoms without provoking dyskinesia, suggesting this receptor may offer a new target for the antisymptomatic therapy of Parkinson's disease. In the present study, a significant neuroprotective effect of A2A receptor antagonists is shown in experimental models of Parkinson's disease. Oral administration of A2A receptor antagonists protected against the loss of nigral dopaminergic neuronal cells induced by 6-hydroxydopamine in rats. A2A antagonists also prevented the functional loss of dopaminergic nerve terminals in the striatum and the ensuing gliosis caused by MPTP in mice. The neuroprotective property of A2A receptor antagonists may be exerted by altering the packaging of these neurotoxins into vesicles, thus reducing their effective intracellular concentration. We therefore conclude that the adenosine A2A receptor may provide a novel target for the long-term medication of Parkinson's disease, because blockade of this receptor exerts both acutely antisymptomatic and chronically neuroprotective activities.     

Short-term TNF-Alpha treatment induced A2B adenosine receptor desensitization in human astroglial cells

Long-term glial cell treatment with the proinflammatory cytokine TNF-alpha has been demonstrated to increase the functional responsiveness of A(2B) adenosine receptors (A(2B) ARs), which in turn synergize with the cytokine inducing chronic astrogliosis. In the present study, we investigated the short-term effects of TNF-alpha on A(2B) AR functional responses in human astroglial cells (ADF), thus simulating the acute phase of cerebral damage which is characterized by both cytokine and adenosine high level release. Short-term TNF-alpha cell treatment caused A(2B) AR phosphorylation inducing, in turn, impairment in A(2B) AR-G protein coupling and cAMP production. These effects occurred in a time-dependent manner with a maximum following 3-h cell exposure. Moreover, we showed PKC intracellular kinase is mainly involved in the TNF-alpha-mediated regulation of A(2B) AR functional responses. The results may indicate the A(2B) AR functional impairment as a cell defense mechanism to counteract the A(2B) receptor-mediated effects during the acute phase of brain damage, underlying A(2B) AR as a target to modulate early inflammatory responses.     

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions

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Differential glutamate-dependent and glutamate-independent adenosine A1 receptor-mediated modulation of dopamine release in different striatal compartments

Adenosine and dopamine are two important modulators of glutamatergic neurotransmission in the striatum. However, conflicting reports exist about the role of adenosine and adenosine receptors in the modulation of striatal dopamine release. It has been previously suggested that adenosine A(1) receptors localized in glutamatergic nerve terminals indirectly modulate dopamine release, by their ability to modulate glutamate release. In the present study, using in vivo microdialysis, we provide evidence for the existence of a significant glutamate-independent tonic modulation of dopamine release in most of the analyzed striatal compartments. In the dorsal, but not in the ventral, part of the shell of the nucleus accumbens (NAc), blockade of A(1) receptors by local perfusion with the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine or by systemic administration of the non-selective adenosine antagonist caffeine induced a glutamate-dependent release of dopamine. On the contrary, A(1) receptor blockade induced a glutamate-independent dopamine release in the core of the NAc and the nucleus caudate-putamen. Furthermore, using immunocytochemical and functional studies in rat striatal synaptosomes, we demonstrate that a fraction of striatal dopaminergic terminals contains adenosine A(1) receptors, which directly inhibit dopamine release independently of glutamatergic transmission.