Activation Of Adenosine A1 Receptors Research Articles

A3 adenosine receptor activation mechanisms: molecular dynamics analysis of inactive, active, and fully active states.

We investigated the Gi-coupled A3 adenosine receptor (A3AR) activation mechanism by running 7.2µs of molecular dynamics (MD) simulations. Based on homology to G protein-coupled receptor (GPCR) structures, three constitutively active mutant (CAM) and the wild-type (WT) A3ARs in the apo form were modeled. Conformational signatures associated with three different receptor states (inactive R, activeR*, and bound to Gi protein mimic) were predicted by analyzing and comparing the CAMs with WT receptor and by considering site-directed mutagenesis data. Detected signatures that were correlated with receptor state included: Persistent salt-bridges involving key charged residues for activation (including a novel, putative ionic lock), rotameric state of conserved W6.48, and Na+ ions and water molecules present. Active-coupled state signatures similar to the X-ray structures of β2 adrenergic receptor-Gs protein and A2AAR-mini-Gs and the recently solved cryo-EM A1AR-Gi complexes were found. Our MD analysis suggests that constitutive activation might arise from the D1073.49-R1083.50 ionic lock destabilization in R and the D1073.49-R1113.53 ionic lock stabilization in R* that presumably lowers the energy barrier associated with an R to R* transition. This study provides new opportunities to understand the underlying interactions of different receptor states of other Gi protein-coupled GPCRs.

Long-term adenosine A1 receptor activation-induced sortilin expression promotes α-synuclein upregulation in dopaminergic neurons.

Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases. However, the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear. In this study, rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) for five weeks. The mobility of rats was evaluated by forced swimming test, while their cognitive capabilities were evaluated by Y-maze test. Expression of sortilin, α-synuclein, p-JUN, and c-JUN proteins in the substantia nigra were detected by western blot analysis. In addition, immunofluorescence staining of sortilin and α-synuclein was performed to detect expression in the substantia nigra. The results showed that, compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (5 mg/kg) + CPA co-treated rats, motor and memory abilities were reduced, surface expression of sortin and α-synuclein in dopaminergic neurons was reduced, and total sortilin and total α-synuclein were increased in CPA-treated rats. MN9D cells were incubated with 500 nM CPA alone or in combination with 10 μM SP600125 (JNK inhibitor) for 48 hours. Quantitative real-time polymerase chain reaction analysis of sortilin and α-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone, but the combination of CPA and SP600125 could inhibit this expression. Predictions made using Jasper, PROMO, and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents. A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction. After sortilin expression was inhibited by sh-SORT1, expression of p-JUN and c-JUN was detected by western blot analysis. Long-term adenosine A1 receptor activation levels upregulated α-synuclein expression at the post-transcriptional level by affecting sortilin expression. The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind to α-synuclein. Co-immunoprecipitation revealed an interaction between sortilin and α-synuclein in MN9D cells. Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression and α-synuclein accumulation, and dramatically improved host cognition and kineticism. This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan (approval No. AUP#20070090) in March 2007 and the Animals Ethics Committee of University of South China (approval No. LL0387-USC) in June 2017.

Medicinal Chemistry and Therapeutic Potential of Agonists, Antagonists and Allosteric Modulators of A1 Adenosine Receptor: Current Status and Perspectives.

Adenosine is a purine nucleoside, responsible for the regulation of a wide range of physiological and pathophysiological conditions by binding with four G-protein-coupled receptors (GPCRs), namely A1, A2A, A2B and A3 adenosine receptors (ARs). In particular, A1 AR is ubiquitously present, mediating a variety of physiological processes throughout the body, thus represents a promising drug target for the management of various pathological conditions. Agonists of A1 AR are found to be useful for the treatment of atrial arrhythmia, angina, type-2 diabetes, glaucoma, neuropathic pain, epilepsy, depression and Huntington's disease, whereas antagonists are being investigated for the treatment of diuresis, congestive heart failure, asthma, COPD, anxiety and dementia. However, treatment with full A1 AR agonists has been associated with numerous challenges like cardiovascular side effects, off-target activation as well as desensitization of A1 AR leading to tachyphylaxis. In this regard, partial agonists of A1 AR have been found to be beneficial in enhancing insulin sensitivity and subsequently reducing blood glucose level, while avoiding severe CVS side effects and tachyphylaxis. Allosteric enhancer of A1 AR is found to be potent for the treatment of neuropathic pain, culminating the side effects related to off-target tissue activation of A1 AR. This review provides an overview of the medicinal chemistry and therapeutic potential of various agonists/partial agonists, antagonists and allosteric modulators of A1 AR, with a particular emphasis on their current status and future perspectives in clinical settings.

The Role of Altered BDNF/TrkB Signaling in Amyotrophic Lateral Sclerosis

Brain derived neurotrophic factor (BDNF) is well recognized for its neuroprotective functions, via activation of its high affinity receptor, tropomysin related kinase B (TrkB). In addition, BDNF/TrkB neuroprotective functions can also be elicited indirectly via activation of adenosine 2A receptors (A2aRs), which in turn transactivates TrkB. Evidence suggests that alterations in BDNF/TrkB, including TrkB transactivation by A2aRs, can occur in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Although enhancing BDNF has been a major goal for protection of dying motor neurons (MNs), this has not been successful. Indeed, there is emerging in vitro and in vivo evidence suggesting that an upregulation of BDNF/TrkB can cause detrimental effects on MNs, making them more vulnerable to pathophysiological insults. For example, in ALS, early synaptic hyper-excitability of MNs is thought to enhance BDNF-mediated signaling, thereby causing glutamate excitotoxicity, and ultimately MN death. Moreover, direct inhibition of TrkB and A2aRs has been shown to protect MNs from these pathophysiological insults, suggesting that modulation of BDNF/TrkB and/or A2aRs receptors may be important in early disease pathogenesis in ALS. This review highlights the relevance of pathophysiological actions of BDNF/TrkB under certain circumstances, so that manipulation of BDNF/TrkB and A2aRs may give rise to alternate neuroprotective therapeutic strategies in the treatment of neural diseases such as ALS.

Indirect Medium Spiny Neurons in the Dorsomedial Striatum Regulate Ethanol-Containing Conditioned Reward Seeking.

Adenosine 2A receptor (A2AR)-containing indirect medium spiny neurons (iMSNs) in the dorsomedial striatum (DMS) contribute to reward-seeking behaviors. However, those roles for ethanol-seeking behaviors remain unknown. To investigate ethanol-seeking behaviors, we used an ethanol-containing reward (10% ethanol and 10% sucrose solution; 10E10S). Upon conditioning with 10E10S, mice that initially only preferred 10% sucrose, not 10E10S, showed a stronger preference for 10E10S. Then, we investigated whether the manipulation of the DMS-external globus pallidus (GPe) iMSNs circuit alters the ethanol-containing reward (10E10S) seeking behaviors using the combination of pharmacologic and optogenetic approaches. DMS A2AR activation dampened operant conditioning-induced ethanol-containing reward, whereas A2AR antagonist abolished the effects of the A2AR agonist and restored ethanol-containing reward-seeking. Moreover, pre-ethanol exposure potentiated the A2AR-dependent reward-seeking. Interestingly, mice exhibiting ethanol-containing reward-seeking showed the reduction of the DMS iMSNs activity, suggesting that disinhibiting iMSNs decreases reward-seeking behaviors. In addition, we found that A2AR activation reversed iMSNs neural activity in the DMS. Similarly, optogenetic stimulation of the DMS-GPe iMSNs reduced ethanol-containing reward-seeking, whereas optogenetic inhibition of the DMS-GPe iMSNs reversed this change. Together, our study demonstrates that DMS A2AR and iMSNs regulate ethanol-containing reward-seeking behaviors.SIGNIFICANCE STATEMENT Our findings highlight the mechanisms of how operant conditioning develops the preference of ethanol-containing conditioned reward. Mice exhibiting ethanol-containing reward-seeking showed a reduction of the indirect medium spiny neuronal activity in the dorsomedial striatum. Pharmacological activation of adenosine A2A receptor (A2AR) or optogenetic activation of indirect medium spiny neurons dampened operant conditioned ethanol-containing reward-seeking, whereas inhibiting this neuronal activity restored ethanol-containing reward-seeking. Furthermore, repeated intermittent ethanol exposure potentiated A2AR-dependent reward-seeking. Therefore, our finding suggests that A2AR-containing indirect medium spiny neuronal activation reduces ethanol-containing reward-seeking, which may provide a potential therapeutic target for alcohol use disorder.

Astrocytic Endocannabinoids Mediate Hippocampal Transient Heterosynaptic Depression.

Astrocytes are highly dynamic cells that modulate synaptic transmission within a temporal domain of seconds to minutes in physiological contexts such as Long-Term Potentiation (LTP) and Heterosynaptic Depression (HSD). Recent studies have revealed that astrocytes also modulate a faster form of synaptic activity (milliseconds to seconds) known as Transient Heterosynaptic Depression (tHSD). However, the mechanism underlying astrocytic modulation of tHSD is not fully understood. Are the traditional gliotransmitters ATP or glutamate released via hemichannels/vesicles or are other, yet, unexplored pathways involved? Using various approaches to manipulate astrocytes, including the Krebs cycle inhibitor fluoroacetate, connexin 43/30 double knockout mice (hemichannels), and inositol triphosphate type-2 receptor knockout mice, we confirmed early reports demonstrating that astrocytes are critical for tHSD. We also confirmed the importance of group II metabotropic glutamate receptors (mGluRs) in astrocytic modulation of tHSD using a group II agonist. Using dominant negative SNARE mice, which have disrupted glial vesicle function, we also found that vesicular release of gliotransmitters and activation of adenosine A1 receptors are not required for tHSD. As astrocytes can release lipids upon receptor stimulation, we asked if astrocyte-derived endocannabinoids are involved in tHSD. Interestingly, a cannabinoid receptor 1 (CB1R) antagonist blocked and an inhibitor of the endogenous endocannabinoid 2-arachidonyl glycerol (2-AG) degradation potentiates tHSD in hippocampal slices. Taken together, this study provides the first evidence for group II mGluR-mediated astrocytic endocannabinoids in transiently suppressing presynaptic neurotransmitter release associated with the phenomenon of tHSD.

Su1831 – Activation of Adenosine A3 Receptor Inhibits Nlrp3 Inflammasome and Pyroptosis of Colonic Epithelial Cells of Patients with Ulcerative Colitis

Su1831 – Activation of Adenosine A3 Receptor Inhibits Nlrp3 Inflammasome and Pyroptosis of Colonic Epithelial Cells of Patients with Ulcerative Colitis

Effects of CF102, an Adenosine A3 Receptors Agonist, on Memory Deficits and Oxidative Stress in a Mouse Model of Streptozotocin‐Induced Dementia of Alzheimer's Type

Alzheimer's disease (AD) is an age‐related neurodegenerative disease characterized by a progressive decline in cognitive functions. Adenosine receptors play a pivotal role in disease progression, however, adenosine A3 receptors are understudied and their effects vastly unknown. We now investigated the effects of the activation of adenosine A3 receptors on memory deficits and oxidative damage in a mouse model of streptozotocin‐induced experimental dementia of Alzheimer's type. Male Swiss mice received bilateral intracerebroventricular injections of streptozotocin (STZ, 3 mg/kg) dissolved in artificial cerebrospinal fluid. Two days after the first STZ administration, injections were repeated, and the animals were treated with the A3 receptor agonist, CF102 (200 μg/kg intraperitoneally), or vehicle (2% DMSO in saline), 1 h after surgery and once a day until the last day of behavioral evaluation (18th after the first STZ injection). The animals subjected to STZ administration showed significant recognition memory deficits evaluated by the object recognition task, spatial memory deficits evaluated by water maze test, and early and late memory deficits in the passive avoidance test. No differences of locomotor activity in the open field test were observed between groups. STZ also increased malonaldehyde and nitrite contents in the hippocampus. The treatment with CF102 significantly prevented the early and late memory deficits and object recognition memory deficits. CF102 treatment also reduced the increase of nitrite production in 37% in the hippocampus when compared to STZ group. These data highlight the therapeutic potential of A3 agonists in AD, however the role of these receptors on STZ‐induced AD model requires more investigation.Support or Funding InformationSupported by Brazilian National Research and Development Council (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES) ‐ Brazil, and the Research Support Foundation of Ceará (FUNCAP) – Brazil.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurochemical Plasticity of Phrenic Motor Neuron Adenosine 2A Receptors: Effects of Cervical Spinal Injury and Intermittent Hypoxia

Cervical spinal adenosine 2A receptor activation is necessary for certain forms of spinal respiratory motor plasticity, such as phrenic long‐term facilitation elicited by severe acute intermittent hypoxia (AIH; Nichols et al., 2012) or moderate AIH with acute cervical spinal injury (cSCI; Navarette‐Opazo et al., 2016). Whereas cervical spinal A2A receptor activation is sufficient to elicit phrenic motor facilitation (pMF; Golder et al., 2008), it undermines other, serotonin‐dependent forms of pMF via cross‐talk inhibition (Hoffman et al., 2010; Navarette‐Opazo et al., 2017). Thus, A2A receptors exert complex effects on phrenic motor plasticity. However, it is not known if conditions such as cSCI and/or repetitive exposure to intermittent hypoxia elicit neurochemical plasticity in A2A receptor expression (and associated phrenic motor plasticity). Thus, we characterized A2A receptor expression in phrenic motor neurons after chronic cSCI and prolonged exposure to different protocols of intermittent hypoxia. A2A receptor expression was determined in male Sprague Dawley rats with and without C2 spinal hemisection (C2Hx; 12 wks post‐injury) that were exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min 10.5% O2 episodes with 5‐min intervals per day; 1.5 hrs/day); 3) moderate CIH (CIH 5/5: 5‐min 10.5% O2 episodes, 5‐min intervals; 8 hrs/day); and 4) high dose CIH (CIH 2/2: 2‐min 10.5% O2 episodes, 2 min intervals; 8 hrs/day). Rats were then perfused, and cervical spinal cords sectioned (40μm). C3 to C5 sections were processed for A2A receptor and Cholera toxin B subunit (CtB, injected 14 days before C2Hx). Using a custom MATLAB algorithm, optical density in CtB‐positive phrenic motor neurons was quantified. Statistical analysis was performed using MANOVA (SAS JMP). C2Hx decreased A2A receptor expression in phrenic motor neurons (p<0.05). Intermittent hypoxia exerted protocol‐specific effects on A2A receptor expression (CIH 2/2 > CIH 5/5 > daily AIH; p=0.014). Although intermittent hypoxia appeared to increase A2A receptor expression more after C2Hx, this effect was only marginally significant (injury/treatment interaction; p=0.061). The dual effects of injury and intermittent hypoxia on A2A receptor expression suggest that adenosine receptor plasticity may be important in the development of new therapeutic strategies to improve breathing after spinal cord injury.Support or Funding InformationSupported by: NIH T32 HD043730 (LLA) OT2OD023854 (SPARC), NIH K12 HD055929 (EGR), McKnight Brain Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spinal Adenosine 2A Receptor Activation is Necessary for Phrenic Long‐Term Facilitation in Carotid‐Denervated Rats

Moderate acute intermittent hypoxia (AIH) elicits a long‐lasting increase in phrenic nerve activity, known as phrenic long‐term facilitation (pLTF). pLTF was originally demonstrated by intermittent electrical carotid sinus nerve stimulation, highlighting the pivotal role of carotid chemoafferent neurons in this form of respiratory motor plasticity. Surprisingly, there is still some residual pLTF after carotid body denervation (CBX) through unknown mechanisms. Carotid body‐dependent, serotonin 2 receptor‐induced pLTF is not expected after CBX; thus, a distinct mechanism must account for residual pLTF. Greater hypoxia‐induced hypotension after CBX might further reduce spinal tissue PO2 more during each hypoxic episode, potentially inducing pLTF via an alternate adenosine‐dependent mechanism. Thus, we tested the hypothesis that adenosine 2A (A2A) receptor activation underlies residual pLTF after CBX. Phrenic nerve activity and spinal tissue PO2 were measured in vivo during our standard pLTF protocol in urethane anesthetized, male Sprague Dawley rats. The contribution of severe hypoxic hypotension to residual pLTF after CBX was evaluated by preventing the arterial pressure drop during hypoxic episodes via systemic phenylephrine administration. We demonstrate that severe hypoxia‐induced hypotension diminishes spinal tissue oxygenation (at the same arterial PO2) during hypoxic episodes to an extent sufficient to trigger residual pLTF. Preventing hypoxia‐induced hypotension minimizes tissue hypoxia, and residual pLTF is no longer observed. Spinal A2A receptor (MSX‐3; 12 μL, 10 μM), but not a serotonin 2A receptor (Ketanserin, 500 μM, 15 μL), antagonist abolishes residual pLTF after CBX. We conclude that differences in arterial blood pressure regulation impact expression and mechanisms of phrenic motor plasticity, a finding of importance for conditions characterized by hypotension such as anemia or spinal injury, where sympathetic outflow is disrupted by injury.Support or Funding InformationSupported by: NIH Grants HL69064, SPARC and University of Florida McKnight Brain InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adenosine 2A Receptor Antagonism in Acute Cervical Contusion/Compression Injury Preserves Serotonin‐Dependent Phrenic Motor Plasticity

Cervical spinal cord injury (cSCI) disrupts descending respiratory neural pathways, causing respiratory muscle paralysis and decreased breathing capacity. Immediately following acute trauma, a suite of pathogenic mechanisms contributes to secondary degeneration and respiratory failure including spinal ischemia, excitotoxicity and inflammation. These events are accompanied by increased extracellular adenosine that activates adenosine receptor signaling cascades. Adenosine 2A receptor (A2AR) antagonism is shown to preserve neurons in neurodegenerative diseases and ischemic injury, likely due to reduced excitotoxicity and inflammation. However, little is known concerning how A2AR activation immediately following cSCI impacts the respiratory control system and its neuromodulatory inputs. Thus, the fundamental hypothesis guiding this study is that A2ARs exert pathogenic effects on respiratory function and plasticity immediately following cSCI. Serotonergic plasticity plays a significant role in the respiratory control network. For example, the phrenic motor system exhibits phrenic long‐term facilitation (pLTF) following moderate acute intermittent hypoxia (AIH), a form of phrenic motor plasticity that requires raphe serotonergic neuron activation. Spinal trauma induces neurodegeneration in descending raphe serotonergic axons, undermining the capacity for pLTF following cSCI. Here, we tested the hypothesis that A2AR antagonism in acute cSCI preserves serotonin‐dependent, AIH‐induced pLTF in adult male Sprague‐Dawley rats with mid‐cervical spinal contusion with compression (cSCC). Addition of a brief compression with dwell time exacerbated the impact of C4/5 contusion as demonstrated by: 1) high death rates (~50% vs <10% without compression); 2) severe paralysis in all limbs for up to 5 days; 3) delays in regaining of the righting reflex; 4) ventilatory deficits when challenged with 7% CO2 + 10.5% O2; and 5) profound neurodegeneration extending rostrally to the brainstem. One week post‐cSCC, pLTF is abolished, consistent with degeneration in raphe serotonergic neurons. However, even a single administration of istradefylline 1‐hour post‐cSCC preserved and even augmented pLTF 1‐week post‐cSCC. Respiratory and locomotor functional studies are in progress. These exciting preliminary data suggest that plasticity outcomes are improved by A2AR antagonism in acute cSCC.Support or Funding InformationNIH OT2OD023854 (SPARC), HL69064, and K12 HD055929 (EGR), and the McKnight Brain Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adenosine A1 receptors modulate the Na+-Hypertonicity induced glutamate release in hypothalamic glial cells

Glutamate release in response to a hypertonic stimulus is a well described phenomenon in the hypothalamus. Evidence suggests that hypothalamic glial cells release glutamate into the extracellular environment in hypertonic conditions. In the current study, we described autocrine regulation of adenosine on glutamate release induced by Na+hypertonicity in hypothalamic glial cell cultures. We showed that glial cells cultured from the cerebral cortex did not release glutamate or adenosine under hypertonic conditions. The findings suggest that the hypothalamus has specialized glial cells, which are responsive to osmotic variations. Stimulation or inhibition of adenosine A1 receptors modulates extracellular glutamate levels in hypothalamic glial cell cultures under hypertonic stimulation. Our results extend previous observations regarding the role of glial cells in the control of hypothalamic physiology. They further demonstrate for the first time that hypothalamic glial cells regulate Na+-hypertonicity-induced glutamate release by activation of adenosine A1 receptors via adenosine release.

Neuroprotective potential of adenosine A1 receptor partial agonists in experimental models of cerebral ischemia

Cerebral ischemia is the second most common cause of death and a major cause of disability worldwide. Available therapies are based only on anticoagulants or recombinant tissue plasminogen activator. Extracellular adenosine increases during ischemia and acts as a neuroprotective endogenous agent mainly by activating adenosine A1 receptors (A1 Rs) which control calcium influx, glutamate release, membrane potential, and metabolism. Accordingly, in many experimental paradigms it has been already demonstrated that the stimulation of A1 R with full agonists is able to reduce ischemia-related structural and functional brain damage; unfortunately, cardiovascular side effects and desensitization of A1 R induced by these compounds have strongly limited their exploitation in stroke therapy so far. Among the newly emerging compounds, A1 R partial agonists could be almost free of side effects and equally effective. Therefore, we decided to evaluate the neuroprotective potential of two A1 R partial agonists, namely 2'-dCCPA and 3'-dCCPA, in invitro and exvivo experimental models of cerebral ischemia. Within the experimental paradigm of oxygen-glucose deprivation invitro in human neuroblastoma (SH-SY5Y) cells both A1 R partial agonists increased cell viability. Considering the high level of expression of A1 Rs in the hippocampus and the susceptibility of CA1 region to hypoxia, we performed electrophysiological experiments in this subfield. The application of 7min of oxygen-glucose deprivation constantly produces an irreversible synaptic failure in all the C57Bl/6 mice hippocampal slices evaluated; both tested compounds allowed a significant recovery of synaptic transmission. These findings demonstrate that A1 R and its partial agonists are still of interest for cerebral ischemia therapy. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Adenosine A1 receptors and mitochondria: targets of remote ischemic preconditioning.

Adenosine is involved in classic preconditioning in most species and acts especially through adenosine A1 and A3 receptors. The aim of the present study was to evaluate whether remote ischemic preconditioning (rIPC) activates adenosine A1 receptors and improves mitochondrial function, thereby reducing myocardial infarct size. Isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion [ischemia-reperfusion (I/R)]. In a second group, before isolation of the heart, a rIPC protocol (3 cycles of hindlimb I/R) was performed. Infarct size was measured with tetrazolium staining, and Akt/endothelial nitric oxide (NO) synthase (eNOS) expression/phosphorylation and mitochondrial function were evaluated after ischemia at 10 and 60 min of reperfusion. As expected, rIPC significantly decreased infarct size. This beneficial effect was abolished only when 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor blocker) and NG-nitro-l-arginine methyl ester (NO synthesis inhibitor) were administered during the reperfusion phase. At the early reperfusion phase, rIPC induced significant Akt and eNOS phosphorylation, which was abolished by the perfusion with an adenosine A1 receptor blocker. I/R led to impaired mitochondrial function, which was attenuated by rIPC and mediated by adenosine A1 receptors. In conclusion, we demonstrated that rIPC limits myocardial infarct by activation of adenosine A1 receptors at early reperfusion in the isolated rat heart. Interestingly, rIPC appears to reduce myocardial infarct size by the Akt/eNOS pathway and improves mitochondrial function during myocardial reperfusion. NEW & NOTEWORTHY Adenosine is involved in classic preconditioning and acts especially through adenosine A1 and A3 receptors. However, its role in the mechanism of remote ischemic preconditioning is controversial. In this study, we demonstrated that remote ischemic preconditioning activates adenosine A1 receptors during early reperfusion, inducing Akt/endothelial nitric oxide synthase phosphorylation and improving mitochondrial function, thereby reducing myocardial infarct size.

Adenosine A3 receptor activation inhibits pronociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.

Extracellular adenosine promotes cell migration/invasion of Glioblastoma Stem-like Cells through A3 Adenosine Receptor activation under hypoxia

Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A3 Adenosine Receptor (A3AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A3AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A3AR on cell migration/invasion was evaluated using the A3AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A3AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAP-dependent activation of A3AR under hypoxia.

Adenosine A2A receptor blocks the A 1 receptor inhibition of renal Na + transport and oxygen consumption.

A high renal oxygen (O2 ) need is primarily associated with the renal tubular O2 consumption (VO2 ) necessary for a high rate of sodium (Na+ ) transport. Limited O2 availability leads to increased levels of adenosine, which regulates the kidney via activation of both A1 and A2A adenosine receptors (A1R and A2AR, respectively). The relative contributions of A1R and A2AR to the regulation of renal Na+ transport and VO2 have not been determined. We demonstrated that A1R activation has a dose-dependent biphasic effect on both renal Na+ /H+ exchanger-3 (NHE3), a major player in Na+ transport, and VO2 . Here, we report concentration-dependent effects of adenosine: less than 5 × 10-7 M adenosine-stimulated NHE3 activity; between 5 × 10-7 M and 10-5 M adenosine-inhibited NHE3 activity; and greater than 10-5 M adenosine reversed the change in NHE3 activity (returned to baseline). A1R activation mediated the activation and inhibition of NHE3 activity, whereas 10-4 M adenosine had no effect on the NHE3 activity due to A2AR activation. The following occurred when A1R and A2AR were activated: (a) Blockade of the A2AR receptor restored the NHE3 inhibition mediated by A1R activation, (b) the NHE-dependent effect on VO2 mediated by A1R activation became NHE independent, and (c) A2AR bound to A1R. In summary, A1R affects VO2 via NHE-dependent mechanisms, whereas A2AR acts via NHE-independent mechanisms. When both A1R and A2AR are activated, the A2AR effect on NHE3 and VO2 predominates, possibly via an A1R-A2AR protein interaction. A2AR-A1R heterodimerization is proposed as the molecular mechanism enabling the NHE-independent control of renal VO2 .

2–Benzylidene–1–Indanone Analogues as Dual Adenosine A1/A2a Receptor Antagonists for the Potential Treatment of Neurological Conditions

Previous studies explored 2-benzylidine-1-tetralone derivatives as innovative adenosine A1 and A2A receptor antagonists for alternative non-dopaminergic treatment of Parkinson's disease. This study's aim is to investigate structurally related 2-benzylidene-1-indanones with substitutions on ring A and B as novel, potent and selective adenosine A1 and A2A receptor blockers. 2-Benzylidene-1-indanone derivatives were synthesised via acid catalysed aldol condensation reactions and evaluated via radioligand binding assays to ascertain structure activity relationships to govern A1 and A2A AR affinity. The results indicated that hydroxy substitution at C4 of ring A and meta (3'), or para (4') substitution on ring B of the 2-benzylidene-1-indanone scaffold (2C: ) is preferred over substitution at C5 (2D: ) or C6 (2E: ) of ring A for adenosine A1 receptor activity and selectivity in the micromolar range. Furthermore, substitution at the meta (3') position of ring B with chlorine lead to the highly potent and selective adenosine A2A receptor antagonist, compound 2 H: . Compound 2C: and the 2Q: behaved as adenosine A1 receptor antagonists in the performed GTP shift assays. In view of these findings, compounds 2C: , 2 H: , 2Q: and 2P: are potent and selective adenosine A1 and A2A receptor antagonists for the potential treatment of neurological conditions.

The effect of dimerization on the activation and conformational dynamics of adenosine A1 receptor.

The adenosine A1 receptor (A1R) is one of four adenosine receptors in humans, which are involved in the function of the cardiovascular, respiratory and central nervous systems. Experimental results indicate that A1R can form a homodimer and that the protomer-protomer interaction in the A1R dimer is related to certain pharmacological characteristics of A1R activation. In this work, we performed docking, metadynamics simulation, conventional molecular dynamics simulations, Gaussian-accelerated molecular dynamics simulations, potential of mean force calculations, dynamic cross-correlation motions analysis and community network analysis to study the binding mode of 5'-N-ethylcarboxamidoadenosine (NECA) to A1R and the effect of dimerization on the activation of A1R. Our results show that NECA binds to A1R in a similar mode to adenosine in the A1R crystal structure and NECA in the A2AR crystal structure. The A1R homodimer can be activated by one or two agonists with NECA occupying its orthosteric pockets in one (which we call the NECA-A1R system) or both protomers (which we call the dNECA-A1R system). In the NECA-A1R system, activation is predicated in the protomer without NECA bound. In the dNECA-A1R system, only one protomer achieves the active state. These findings suggest an asymmetrical activation mechanism of the homodimer and a negative cooperativity between the two protomers. We envision that our results may further facilitate the drug development of A1R.

Evaluation of Biological Activity Exerted by an Aza-bicyclocarboxylic acid Derivative using Anischemia-Reperfusion Injury Model

The main objective of this study was to evaluate the biological activity of a new compound (derived from aza-bicyclo-carboxylic acid) against heart failure caused by the ischemia- reperfusion phenomenon. In addition, to characterize de molecular mechanism involved in the effect exerted by aza-bicyclo-carboxylic acid against infarction area, some drugs such as prazosin, metoprolol, propanolol, tamoxifen, flutamide, finasteride, nifedipine, levosimedan, adenosine, rolofylline, isoproterenol and the compound ZM-241385 were used as pharmacological tools. The data found indicated that biological activity induced by compound 3 on infarction area only was similar at effect exerted by adenosine; however, the effect produced by compound 3 was blocked with of rolofylline. Other data showed that the biological activity of compound 3 decreases the cAMP levels in a time-dependent manner. In conclusion, the results indicate that compound 3 can produce a cardioprotective effect against myocardial ischemia-reperfusion injury translated as a decrease on infarction area; this phenomenon involves A1-adenosine receptor activation and, as a result may cause changes in cAMP levels.