Extracellular Adenosine Formation Research Articles

Increased Synaptic ATP Release and CD73-Mediated Formation of Extracellular Adenosine in the Control of Behavioral and Electrophysiological Modifications Caused by Chronic Stress.

Increased ATP release and its extracellular catabolism through CD73 (ecto-5'-nucleotidase) lead to the overactivation of adenosine A2A receptors (A2AR), which occurs in different brain disorders. A2AR blockade blunts mood and memory dysfunction caused by repeated stress, but it is unknown if increased ATP release coupled to CD73-mediated formation of extracellular adenosine is responsible for A2AR overactivation upon repeated stress. This was now investigated in adult rats subject to repeated stress for 14 consecutive days. Frontocortical and hippocampal synaptosomes from stressed rats displayed an increased release of ATP upon depolarization, coupled to an increased density of vesicular nucleotide transporters and of CD73. The continuous intracerebroventricular delivery of the CD73 inhibitor α,β-methylene ADP (AOPCP, 100 μM) during restraint stress attenuated mood and memory dysfunction. Slice electrophysiological recordings showed that restraint stress decreased long-term potentiation both in prefrontocortical layer II/III-layer V synapses and in hippocampal Schaffer fibers-CA1 pyramid synapses, which was prevented by AOPCP, an effect occluded by adenosine deaminase and by the A2AR antagonist SCH58261. These results indicate that increased synaptic ATP release coupled to CD73-mediated formation of extracellular adenosine contributes to mood and memory dysfunction triggered by repeated restraint stress. This prompts considering interventions decreasing ATP release and CD73 activity as novel strategies to mitigate the burden of repeated stress.

CD73, a Promising Therapeutic Target of Diclofenac, Promotes Metastasis of Pancreatic Cancer through a Nucleotidase Independent Mechanism

CD73, a cell surface-bound nucleotidase, facilitates extracellular adenosine formation by hydrolyzing 5'-AMP to adenosine. Several studies have shown that CD73 plays an essential role in immune escape, cell proliferation and tumor angiogenesis, making it an attractive target for cancer therapies. However, there are limited clinical benefits associated with the mainstream enzymatic inhibitors of CD73, suggesting that the mechanism underlying the role of CD73 in tumor progression is more complex than anticipated, and further investigation is necessary. In this study, CD73 is found to overexpress in the cytoplasm of pancreatic ductal adenocarcinoma (PDAC) cells and promotes metastasis in a nucleotidase-independent manner, which cannot be restrained by the CD73 monoclonal antibodies or small-molecule enzymatic inhibitors. Furthermore, CD73 promotes the metastasis of PDAC by binding to the E3 ligase TRIM21, competing with the Snail for its binding site. Additionally, a CD73 transcriptional inhibitor, diclofenac, a non-steroidal anti-inflammatory drug, is more effective than the CD73 blocking antibody for the treatment of PDAC metastasis. Diclofenac also enhances the therapeutic efficacy of gemcitabine in the spontaneous KPC (LSL-KrasG12D/+ , LSL-Trp53R172H/+ , and Pdx-1-Cre) pancreatic cancer model. Therefore, diclofenac may be an effective anti-CD73 therapy, when used alone or in combination with gemcitabine-based chemotherapy regimen, for metastatic PDAC.

CD73-Mediated Formation of Extracellular Adenosine Is Responsible for Adenosine A2A Receptor-Mediated Control of Fear Memory and Amygdala Plasticity.

Adenosine A2A receptors (A2AR) control fear memory and the underlying processes of synaptic plasticity in the amygdala. In other brain regions, A2AR activation is ensured by ATP-derived extracellular adenosine formed by ecto-5'-nucleotidase or CD73. We now tested whether CD73 is also responsible to provide for the activation of A2AR in controlling fear memory and amygdala long-term potentiation (LTP). The bilateral intracerebroventricular injection of the CD73 inhibitor αβ-methylene ADP (AOPCP, 1 nmol/ventricle/day) phenocopied the effect of the A2AR blockade by decreasing the expression of fear memory, an effect disappearing in CD73-knockout (KO) mice and in forebrain neuronal A2AR-KO mice. In the presence of PPADS (20 μM) to eliminate any modification of ATP/ADP-mediated P2 receptor effects, both AOPCP (100 μM) and the A2AR antagonist, SCH58261 (50 nM), decreased LTP magnitude in synapses of projection from the external capsula into the lateral amygdala, an effect eliminated in slices from both forebrain neuronal A2AR-KO mice and CD73-KO mice. These data indicate a key role of CD73 in the process of A2AR-mediated control of fear memory and underlying synaptic plasticity processes in the amygdala, paving the way to envisage CD73 as a new therapeutic target to interfere with abnormal fear-like emotional processing.

Association Between Adenosine A2A Receptors and Connexin 43 Regulates Hemichannels Activity and ATP Release in Astrocytes Exposed to Amyloid-β Peptides.

Increasing evidence implicates astrocytes and the associated purinergic modulation in Alzheimer's disease (AD), characterized by cognitive deficits involving the extracellular amyloid-β peptides (Aβ) accumulation. Aβ can affect astrocytic gliotransmitters release, namely ATP, which is rapidly metabolized into adenosine by ecto-5'-nucleotidase, CD73, resulting in adenosine A2A receptors (A2AR) activation that bolsters neurodegeneration. AD's brains exhibit an upregulation of A2AR and of connexin 43 (Cx43), which in astrocytes forms hemichannels that can mediate ATP release. However, a coupling between astrocytic A2AR and Cx43 remains to be established. This was now investigated using astrocytic primary cultures exposed to Aβ1-42 peptides. Aβ triggered ATP release through Cx43 hemichannels, a process blocked by A2AR antagonists and mimicked by selective A2AR activation. A2AR directly regulated hemichannels activity and prevented Cx43 upregulation and phosphorylation observed in Aβ1-42-exposed astrocytes. Moreover, a proximity ligand assay revealed a physical association between astrocytic A2AR and Cx43. Finally, the blockade of CD73-mediated extracellular formation of ATP-derived adenosine prevented the Aβ-induced increase of Cx43 hemichannel activity and of ATP release. Overall, the data identify a feed-forward loop involving astrocytic A2AR and Cx43 hemichannels, whereby A2AR increase Cx43 hemichannel activity leading to increased ATP release, which is converted into adenosine by CD73, sustaining the increased astrocytic A2AR activity in AD-like conditions.

Plasma levels of the cardiovascular protective endogenous nucleoside adenosine are reduced in patients with primary aldosteronism without affecting ischaemia-reperfusion injury: A prospective case-control study.

BackgroundPatients with primary aldosteronism (PA) experience more cardiovascular events compared to patients with essential hypertension (EHT), independent from blood pressure levels. In animals, mineralocorticoid receptor antagonists limit ischaemia‐reperfusion (IR) injury by increasing extracellular adenosine formation and adenosine receptor stimulation. Adenosine is an endogenous compound with profound cardiovascular protective effects. Firstly, we hypothesized that patients with PA have lower circulating adenosine levels which might contribute to the observed increased cardiovascular risk. Secondly, we hypothesized that by this mechanism, patients with PA are more susceptible to IR compared to patients with EHT.DesignIn our prospective study in 20 patients with PA and 20 patients with EHT, circulating adenosine was measured using a pharmacological blocker solution that halts adenosine metabolism after blood drawing. Brachial artery flow‐mediated dilation (FMD) before and after forearm IR was used as a well‐established method to study IR injury.ResultsPatients with PA had a 33% lower adenosine level compared to patients with EHT (15.3 [13.3‐20.4] vs 22.7 [19.4‐36.8] nmol/L, respectively, P < .01). The reduction in FMD after IR, however, did not differ between patients with PA and patients with EHT (−1.0 ± 2.9% vs −1.6 ± 1.6%, respectively, P = .52).ConclusionsAs adenosine receptor stimulation induces various powerful protective cardiovascular effects, its lower concentration in patients with PA might be an important novel mechanism that contributes to their increased cardiovascular risk. We suggest that modulation of the adenosine metabolism is an exciting novel pharmacological opportunity to limit cardiovascular risk in patients with PA that needs further exploration.

Mast Cells Are Directly Activated by Cancer Cell–Derived Extracellular Vesicles by a CD73- and Adenosine-Dependent Mechanism

We have recently shown that mast cells (MCs), which constitute an important part of the tumor microenvironment (TME), can be directly activated by cancer cells under conditions that recapitulate cell to cell contact. However, MCs are often detected in the tumor periphery rather than intratumorally. Therefore, we investigated the possibility of MC activation by cancer cell–derived extracellular vesicles (EVs). Here we show that exposure of MCs to EVs derived from pancreatic cancer cells or non–small cell lung carcinoma results in MC activation, evident by the increased phosphorylation of the ERK1/2 MAP kinases. Further, we show that, similarly to activation by cancer cell contact, activation by EVs is dependent on the ecto enzyme CD73 that mediates extracellular formation of adenosine and on signaling by the A3 adenosine receptor. Finally, we show that activation by either cell contact or EVs upregulates expression of angiogenic and tissue remodeling genes, including IL8, IL6, VEGF, and amphiregulin. Collectively, our findings indicate that both intratumorally localized MCs and peripheral MCs are activated and reprogrammed in the TME either by contact with the cancer cells or by their released EVs.

Synaptic and memory dysfunction in a β-amyloid model of early Alzheimer's disease depends on increased formation of ATP-derived extracellular adenosine

Adenosine A2A receptors (A2AR) overfunction causes synaptic and memory dysfunction in early Alzheimer's disease (AD). In a β-amyloid (Aβ1-42)-based model of early AD, we now unraveled that this involves an increased synaptic release of ATP coupled to an increased density and activity of ecto-5′-nucleotidase (CD73)-mediated formation of adenosine selectively activating A2AR. Thus, CD73 inhibition with α,β-methylene-ADP impaired long-term potentiation (LTP) in mouse hippocampal slices, which is occluded upon previous superfusion with the A2AR antagonist SCH58261. Furthermore, α,β-methylene-ADP did not alter LTP amplitude in global A2AR knockout (KO) and in forebrain neuron-selective A2AR-KO mice, but inhibited LTP amplitude in astrocyte-selective A2AR-KO mice; this shows that CD73-derived adenosine solely acts on neuronal A2AR. In agreement with the concept that ATP is a danger signal in the brain, ATP release from nerve terminals is increased after intracerebroventricular Aβ1-42 administration, together with CD73 and A2AR upregulation in hippocampal synapses. Importantly, this increased CD73 activity is critically required for Aβ1-42 to impair synaptic plasticity and memory since Aβ1-42-induced synaptic and memory deficits were eliminated in CD73-KO mice. These observations establish a key regulatory role of CD73 activity over neuronal A2AR and imply CD73 as a novel target for modulation of early AD.

Abstract 146: Investigating and Inhibiting Neutrophil Extracellular Trap Formation in the Heart

Neutrophil extracellular traps (NETs) have been observed in multiple diseases of the cardiovascular system, such as myocardial infarction and atherosclerosis. We have recently shown that adenosine (ADO) inhibits NET formation and may be an important endogenous regulator. CD73 is expressed on many cell types and catalyzes the extracellular formation of ADO from AMP. Here, we first attempt to implicate NETs in cardiac injury, and we posit that human NETs negatively impact cardiac function by influencing native leukocyte recruitment and activation. Second, we prototype a CD73-functionalized hydrogel delivery vehicle for adenosine and test its function in vitro. We introduced NETs obtained from stimulated neutrophils into healthy hearts by intramyocardial injection in two-month-old rats. We used echo to assess cardiac function at days 0, 1 and 3 of injection. We measured the abundance of cardiac leukocyte subpopulations by flow cytometry at days 1 and 3 after injection, with immunostaining for CD45 (all leukocytes), myeloperoxidase (MPO, neutrophils), CD68 (macrophages), CD3 (T-cells), B220 (B-cells) and citrullinated histone 3 (citH3, NETs). To deliver adenosine as a NET antagonist, we designed a polyethylene (PEG) hydrogel composed of a 4-armed PEG incorporating a VPM protease-degradable crosslinker for cargo release, and an RGD peptide to enhance gel-tissue attachment. We tested gel polymerization by measuring storage and loss moduli at 4% and 6% PEG content (w/v) and 0, 1 uM and 1mM RGD concentrations. We functionalized the gel with CD73 to test its adenosine production capability. We found a significant decreased in cardiac function assessed by global longitudinal strain at day 1 in the NET group compared to saline. By flow cytometry, we found an increase in neutrophils, macrophages and NET formation at day 1, while B- and T-cells were increased at day 3. We found that increasing the PEG content of our hydrogel, but not RGD, increases gel stiffness. We also found that the CD73 functionalized hydrogel successfully catalyzes the formation of adenosine in vitro. In conclusion, we found that NETs negatively impact cardiac function, and successfully tested an adenosine hydrogel source in vitro. Next, we will study the effect of our gel on NETs in vivo.

Enhanced ATP release and CD73-mediated adenosine formation sustain adenosine A2A receptor over-activation in a rat model of Parkinson's disease.

Parkinson's disease (PD) involves an initial loss of striatal dopamine terminals evolving into degeneration of dopamine neurons in substantia nigra (SN), which can be modelled by 6-hydroxydopamine (6-OHDA) administration. Adenosine A2A receptor blockade attenuates PD features in animal models, but the source of the adenosine causing A2A receptor over-activation is unknown. As ATP is a stress signal, we have tested if extracellular catabolism of adenine nucleotides into adenosine (through ecto-5'-nucleotidase or CD73) leads to A2A receptor over-activation in PD. Effects of blocking CD73 with α,β-methylene ADP (AOPCP) were assayed in 6-OHDA-treated rats and dopamine-differentiated neuroblastoma SH-SY5Y cells. 6-OHDA increased ATP release and extracellular conversion into adenosine through CD73 up-regulation in SH-SY5Y cells. Removing extracellular adenosine with adenosine deaminase, blocking CD73 with AOPCP, or blocking A2A receptors with SCH58261 were equi-effective in preventing 6-OHDA-induced damage in SH-SY5Y cells. In vivo striatal exposure to 6-OHDA increased ATP release and extracellular formation of adenosine from adenosine nucleotides and up-regulated CD73 and A2A receptors in striatal synaptosomes. Intracerebroventricular administration of AOPCP phenocopied effects of SCH58261, attenuating 6-OHDA-induced (a) increase of contralateral rotations after apomorphine, (b) reduction of dopamine content in striatum and SN, (c) loss of TH staining in striatum and SN, (d) motor dysfunction in the cylinder test, and (e) short-term memory impairment in the object recognition test. Our data indicate that increased ATP-derived adenosine formation is responsible for A2A receptor over-activation in PD, suggesting CD73 as a new target to manage PD.

Patients with primary aldosteronism have lower circulating adenosine levels but similar susceptibility to ischemia-reperfusion compared to patients with essential hypertension

Aim: Patients with primary aldosteronism (PA) experience more cardiovascular events compared to patients with essential hypertension (EHT), independent from blood pressure levels. In animal models, aldosterone increases myocardial ischemia-reperfusion (IR) injury. Mineralocorticoid receptor antagonists limit IR-injury by increasing extracellular adenosine formation and adenosine receptor stimulation.

Post-inflammatory Ileitis Induces Non-neuronal Purinergic Signaling Adjustments of Cholinergic Neurotransmission in the Myenteric Plexus.

Uncoupling between ATP overflow and extracellular adenosine formation changes purinergic signaling in post-inflammatory ileitis. Adenosine neuromodulation deficits were ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular adenine nucleotides in the inflamed ileum. Here, we hypothesized that inflammation-induced changes in cellular density may also account to unbalance the release of purines and their influence on [3H]acetylcholine release from longitudinal muscle-myenteric plexus preparations of the ileum of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-treated rats. The population of S100β-positive glial cells increase, whereas Ano-1-positive interstitial cells of Cajal (ICCs) diminished, in the ileum 7-days after the inflammatory insult. In the absence of changes in the density of VAChT-positive cholinergic nerves detected by immunofluorescence confocal microscopy, the inflamed myenteric plexus released smaller amounts of [3H]acetylcholine which also became less sensitive to neuronal blockade by tetrodotoxin (1 μM). Instead, [3H]acetylcholine release was attenuated by sodium fluoroacetate (5 mM), carbenoxolone (10 μM) and A438079 (3 μM), which prevent activation of glial cells, pannexin-1 hemichannels and P2X7 receptors, respectively. Sodium fluoroacetate also decreased ATP overflow without significantly affecting the extracellular adenosine levels, thus indicating that surplus ATP release parallels reactive gliosis in post-inflammatory ileitis. Conversely, loss of ICCs may explain the lower amounts of adenosine detected in TNBS-treated preparations, since blockade of Cav3 (T-type) channels existing in ICCs with mibefradil (3 μM) or inhibition of the equilibrative nucleoside transporter 1 with dipyridamole (0.5 μM), both decreased extracellular adenosine. Data indicate that post-inflammatory ileitis operates a shift on purinergic neuromodulation reflecting the upregulation of ATP-releasing enteric glial cells and the depletion of ICCs accounting for decreased adenosine overflow via equilibrative nucleoside transporters.

Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5′-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5′-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5′-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.

Extracellular Adenosine Formation by Ecto-5'-Nucleotidase (CD73) Is No Essential Trigger for Early Phase Ischemic Preconditioning.

BackgroundAdenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5’-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5’-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73-/- and wild type (WT) mice.Methods and Results3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.ConclusionThe infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5’-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine’s well known cardioprotective effect in early phase ischemic preconditioning.

The effect of eplerenone on adenosine formation in humans in vivo: a double-blinded randomised controlled study.

BackgroundIt has been suggested that mineralocorticoid receptor antagonists have direct cardioprotective properties, because these drugs reduce mortality in patients with heart failure. In murine models of myocardial infarction, mineralocorticoid receptor antagonists reduce infarct size. Using gene deletion and pharmacological approaches, it has been shown that extracellular formation of the endogenous nucleoside adenosine is crucial for this protective effect. We now aim to translate this finding to humans, by investigating the effects of the selective mineralocorticoid receptor antagonist eplerenone on the vasodilator effect of the adenosine uptake inhibitor dipyridamole, which is a well-validated surrogate marker for extracellular adenosine formation.Methods and ResultsIn a randomised, double-blinded, placebo-controlled, cross-over study we measured the forearm blood flow response to the intrabrachial administration of dipyridamole in 14 healthy male subjects before and after treatment with placebo or eplerenone (50 mg bid for 8 days). The forearm blood flow during administration of dipyridamole (10, 30 and 100 µg·min−1·dl−1) was 1.63 (0.60), 2.13 (1.51) and 2.71 (1.32) ml·dl−1·min−1 during placebo use, versus 2.00 (1.45), 2.68 (1.87) and 3.22 (1.94) ml·dl−1·min−1 during eplerenone treatment (median (interquartile range); P = 0.51). Concomitant administration of the adenosine receptor antagonist caffeine attenuated dipyridamole-induced vasodilation to a similar extent in both groups. The forearm blood flow response to forearm ischemia, as a stimulus for increased formation of adenosine, was similar during both conditions.ConclusionIn a dosage of 50 mg bid, eplerenone does not augment extracellular adenosine formation in healthy human subjects. Therefore, it is unlikely that an increased extracellular adenosine formation contributes to the cardioprotective effect of mineralocorticoid receptor antagonists.Trial RegistrationClinicalTrials.gov, NCT01837108

Ecto-5'-Nucleotidase (CD73)-Mediated Formation of Adenosine Is Critical for the Striatal Adenosine A2A Receptor Functions

Adenosine is a neuromodulator acting through inhibitory A1 receptors (A1Rs) and facilitatory A2ARs, which have similar affinities for adenosine. It has been shown that the activity of intracellular adenosine kinase preferentially controls the activation of A1Rs, but the source of the adenosine activating A2ARs is unknown. We now show that ecto-5'-nucleotidase (CD73), the major enzyme able to convert extracellular AMP into adenosine, colocalizes with A2ARs in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 coimmunoprecipitated with A2ARs and proximity ligation assays confirmed the close proximity of CD73 and A2ARs in the striatum. Accordingly, the cAMP formation in synaptosomes as well as the hypolocomotion induced by a novel A2AR prodrug that requires CD73 metabolization to activate A2ARs were observed in wild-type mice, but not in CD73 knock-out (KO) mice or A2AR KO mice. Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamine-induced sensitization, mimicking the phenotype of global or forebrain-A2AR KO mice, as well as upon pharmacological A2AR blockade. These results show that CD73-mediated formation of extracellular adenosine is responsible for the activation of striatal A2AR function. This study points to CD73 as a new target that can fine-tune A2AR activity, and a novel therapeutic target to manipulate A2AR-mediated control of striatal function and neurodegeneration.

Cortical spreading depression-induced preconditioning in mouse neocortex is lamina specific

Cortical spreading depression (CSD) is able to confer neuroprotection when delivered at least 1 day in advance of an ischemic event. However, its ability to confer neuroprotection in a more immediate time frame has not previously been investigated. Here we have used mouse neocortical brain slices to study the effects of repeated episodes of CSD in layer V and layer II/III pyramidal neurons. In layer V, CSD evoked at 15-min intervals caused successively smaller membrane depolarizations and increases in intracellular calcium compared with the response to the first CSD. With an inter-CSD interval of 30 min this preconditioning effect was much less marked, indicating that preconditioning lasts between 15 and 30 min. A single episode of CSD also provided a degree of protection in oxygen-glucose deprivation (OGD) by significantly lengthening the time a cell could withstand OGD before anoxic depolarization occurred. In layer II/III pyramidal neurons no preconditioning by CSD on subsequent episodes of CSD was observed, demonstrating that the response of pyramidal neurons to repeated CSD is lamina specific. The A1 receptor antagonist 8-cyclopentyl theophylline (8-CPT) reduced the layer V preconditioning in a concentration-related manner. Inhibition of extracellular formation of adenosine by blocking ecto-5'-nucleotidase with α,β-methyleneadenosine 5'-diphosphate prevented preconditioning in most but not all cells. Block of equilibrative nucleoside transporters 1 and 2 with dipyramidole alone or in combination with 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine also prevented preconditioning in some but not all cells. These data provide evidence that rapid preconditioning of one CSD by another is primarily mediated by adenosine.

Deletion of CD73 promotes dyslipidemia and intramyocellular lipid accumulation in muscle of mice

Context: CD73 converts extracellular AMP to adenosine which is well known to inhibit lipolysis. It is unknown, however, whether adenosine formed directly by CD73 is functionally relevant in this process. Objective: We therefore explored the effect of CD73-derived adenosine on body fat of aged mice. Results: In lean mice, extracellular adenosine formation by adipocytes is dependent on CD73. High fat diet down-regulates the expression of CD73 in wildtype mice similar to ob/ob mice. Transgenic mice chronically lacking CD73 (CD73−/−) gain significantly less body weight and show decreased superficial white fat content as well as increased serum free fatty acids and triglycerides. In addition, intramyocellular lipid levels are significantly increased. This phenotype is accompanied by an increase in blood glucose and serum insulin levels although insulin secretion and the level of insulin degrading enzyme are unaltered. Additionally, insulin-induced Akt phosphorylation is reduced in skeletal muscle of CD73−/− mice. Conclusion: CD73-derived adenosine is functionally involved in body fat homeostasis.

Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

Ecto-5′-nucleotidase (CD73; EC 3.1.3.5, e-5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15-day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e-5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, K m and V max, suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency ( V max/ K m). Finally, double immunofluorescence staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that e-5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e-5NT in the brain.

Upregulation of Ecto-5′-Nucleotidase by Rosuvastatin Increases the Vasodilator Response to Ischemia

3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) are effective in the primary and secondary prevention of cardiovascular events. Although originally developed to improve lipid profile, statins have demonstrated a surplus of beneficial pleiotropic effects, including improved endothelial function, reduced inflammation, and increased tolerance to ischemia-reperfusion injury. In preclinical studies, increased ecto-5'-nucleotidase activity, the key enzyme in extracellular adenosine formation, plays an important role in these effects. Because human data are absent, we explored the effects of rosuvastatin on ecto-5'-nucleotidase activity and the clinical relevance of increased extracellular adenosine during ischemia in humans in vivo. The forearm vasodilator responses to 3 increasing periods of forearm ischemia (2, 5, and 13 minutes) were determined during placebo and caffeine (an adenosine receptor antagonist) infusion into the brachial artery. At the end of an 8-day treatment period with rosuvastatin (20 mg per day), this whole procedure was repeated. During both experiments, ecto-5'-nucleotidase activity was determined. Vasodilator responses are expressed as the percentage increase in forearm blood flow ratio from baseline. Rosuvastatin increased ecto-5'-nucleotidase activity by 49±17% and enhanced the vasodilator response after 2, 5, and 13 minutes of ischemia in the absence (146±19, 330±26, and 987±133 to 312±77, 566±107, and 1533±267) but not in the presence of caffeine (98±25, 264±54, and 727±111 versus 95±19, 205±34, and 530±62). Rosuvastatin increases extracellular formation of adenosine in humans in vivo probably by enhancing ecto-5'-nucleotidase activity. This action results in the improvement of reactive hyperemia and may further enhance the clinical benefit of statins, in particular in conditions of ischemia.

Extracellular adenosine triggers lymphocyte entry into the central nervous system during experimental autoimmune encephalomyelitis by regulating chemokine and adhesion molecule expression in the brain (44.12)

Abstract Lymphocyte entry into the central nervous system (CNS) is a highly regulated process which serves to promote tissue repair while protecting against potentially fatal inflammation. As the formation of extracellular adenosine (catalyzed from ATP by CD39 and CD73) during inflammation serves as a negative-feedback signal to prevent excessive cell damage, our results suggest that adenosine receptor (AR) signaling also regulates CNS lymphocyte infiltration. We have shown that extracellular adenosine is required for the progression experimental autoimmune encephalomyelitis (EAE), the animal model of autoimmune inflammatory disease Multiple Sclerosis. We now show that AR signaling induces the CNS expression of the chemokine CX3CL1 (fractalkine), which acts as a chemoattractant for monocytes, lymphocytes, and NK cells. In addition, CX3CL1 expression is up-regulated in the CNS immediately prior to EAE onset only in wild type, but not CD73-/- mice, which cannot produce extracellular adenosine, are protected from EAE, and have minimal CNS lymphocyte infiltration following EAE induction. Moreover, CD39, CD73, and ARs are found clustered in the brain only at the choroid plexus, a structure which is known to regulate lymphocyte migration into the CNS. As AR signaling can induce CX3CL1 and ICAM-1 expression and lymphocyte transmigration across an in vitro choroid plexus transwell barrier, we conclude that AR signaling regulates lymphocyte entry into the CNS.