Adenosine A1 Receptor Agonist Research Articles

Structure-Activity Studies of 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as A3 Adenosine Receptor Positive Allosteric Modulators.

We previously reported 1H-imidazo[4,5-c]quinolin-4-amines as A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs). A3AR agonists, but not PAMs, are in clinical trials for inflammatory diseases and liver conditions. We synthesized new analogues to distinguish 2-cyclopropyl antagonist 17 (orthosteric interaction demonstrated by binding and predicted computationally) from PAMs (derivatives with large 2-alkyl/cycloalkyl/bicycloalkyl groups). We predicted PAM binding at a hydrophobic site on the A3AR cytosolic interface. Although having low Caco-2 permeability and high plasma protein binding, hydrophobic 2-cyclohept-4-enyl-N-3,4-dichlorophenyl, MRS7788 18, and 2-heptan-4-yl-N-4-iodophenyl, MRS8054 39, derivatives were orally bioavailable in rat. 2-Heptan-4-yl-N-3,4-dichlorophenyl 14 and 2-cyclononyl-N-3,4-dichlorophenyl 20 derivatives and 39 greatly enhanced Cl-IB-MECA-stimulated [35S]GTPγS binding Emax, with only 12b trending toward decreasing the agonist EC50. A feasible route for radio-iodination at the p-position of a 4-phenylamino substituent suggests a potential radioligand for allosteric site binding. Herein, we advanced an allosteric approach to developing A3AR-activating drugs that are potentially event- and site-specific in action.

Adenosine A3 Receptor (A3AR) Agonist for the Treatment of Bleomycin-Induced Lung Fibrosis in Mice.

Adenosine receptors (ARs) are involved in the suppression and development of inflammatory and fibrotic conditions. Specifically, AR activation promotes differentiation of lung fibroblasts into myofibroblasts, typical of a fibrotic event. Pulmonary fibrosis is a severe disease characterized by inflammation and fibrosis of unknown etiology and lacking an effective treatment. The present investigation explored the action of MRS5980, a new, highly potent and selective A3AR agonist, in an established murine model of lung fibrosis. The effects of either vehicle or MRS5980 were studied in mice following intratracheal bleomycin administration. We evaluated the role of the A3AR agonist on lung stiffness, studying the airway resistance to inflation, oxidative stress (8-OHdG and MDA), inflammation, pro- and anti-inflammatory marker levels (IL-1β, IL-6, TNF-α, IL-10 and IL-17A) and fibrosis establishment, evaluating transforming growth factor (TGF)-β expression and α-smooth muscle actin (α-SMA) deposition in lungs. Bleomycin administration increased lung stiffness, TGF-β levels, α-SMA deposition, and inflammatory and oxidative stress markers. The treatment with MRS5980 attenuated all the analyzed functional, biochemical and histopathological markers in a dose-dependent manner. Our findings support the therapeutic potential of A3AR agonists in lung fibrosis by demonstrating reduced disease progression, as indicated by decreased inflammation, TGF-β expression and fibrotic remodeling.

The antidepressant-like effect of guanosine involves the modulation of adenosine A1 and A2A receptors.

Guanosine has been considered a promising candidate for antidepressant responses, but if this nucleoside could modulate adenosine A1 (A1R) and A2A (A2AR) receptors to exert antidepressant-like actions remains to be elucidated. This study investigated the role of A1R and A2AR in the antidepressant-like response of guanosine in the mouse tail suspension test and molecular interactions between guanosine and A1R and A2AR by docking analysis. The acute (60min) administration of guanosine (0.05mg/kg, p.o.) significantly decreased the immobility time in the tail suspension test, without affecting the locomotor performance in the open-field test, suggesting an antidepressant-like effect. This behavioral response was paralleled with increased A1R and reduced A2AR immunocontent in the hippocampus, but not in the prefrontal cortex, of mice. Guanosine-mediated antidepressant-like effect was not altered by the pretreatment with caffeine (3mg/kg, i.p., a non-selective adenosine A1R/A2AR antagonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX - 2mg/kg, i.p., a selective adenosine A1R antagonist), or 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)-phenol (ZM241385 - 1mg/kg, i.p., a selective adenosine A2AR antagonist). However, the antidepressant-like response of guanosine was completely abolished by adenosine (0.5mg/kg, i.p., a non-selective adenosine A1R/A2AR agonist), N-6-cyclohexyladenosine (CHA - 0.05mg/kg, i.p., a selective adenosine A1 receptor agonist), and N-6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)ethyl]adenosine (DPMA - 0.1mg/kg, i.p., a selective adenosine A2A receptor agonist). Finally, docking analysis also indicated that guanosine might interact with A1R and A2AR at the adenosine binding site. Overall, this study reinforces the antidepressant-like of guanosine and unveils a previously unexplored modulation of the modulation of A1R and A2AR in its antidepressant-like effect.

A1 Adenosine Receptor Activation Inhibits P2X3 Receptor-Mediated ATP Currents in Rat Dorsal Root Ganglion Neurons.

Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) concentration dependently suppressed P2X3 receptor-mediated and α,β-methylene-ATP (α,β-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons. CPA significantly decreased the maximal current response to α,β-meATP, as shown a downward shift of the concentration-response curve for α,β-meATP. CPA suppressed ATP currents in a voltage-independent manner. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. Moreover, CPA suppressed the membrane potential depolarization and action potential bursts, which were induced by α,β-meATP in DRG neurons. Finally, CPA relieved α,β-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors. These results indicated that CPA inhibited the activity of P2X3 receptors in rat primary sensory neurons by activating A1 adenosine receptors and its downstream cAMP signaling pathway, revealing a novel peripheral mechanism underlying its analgesic effect.

Ex Vivo Feedback Control of Neurotransmission Using a Photocaged Adenosine A1 Receptor Agonist.

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

Role of A1 Adenosinergic System in Multiple Sclerosis and Possible Therapeutic Strategy

Multiple sclerosis (MS) is one of the most affecting autoimmune neurodegenerative disease characterized by chronic neuroinflammation, demyelination and impaired neuronal conduction. The oligodendrocytes toxicity by inflammatory cytokines and oxy-radicals are considered to be the most important factor in demyelination of motor neurons. The dysfunction of neuronal A1 adenosine receptor (A1AR) contributes to the demyelination of neurons by triggering the pro-inflammatory cytokines, oxy-radicals and neuroinflammatory cascades. In MS pathogenesis, Antigen presenting cells, MHC protein, CD4+T-cells, GM-CSF along with effector cells enhance the activation of macrophages in adenosinergic declined conditions, where it shows cumulative effects which leads to oligodendrocytes toxicity and demyelination of motor neurons. In general, A1AR is mainly expressed in macrophage lineage cells in central nervous system which could control the macrophage activation upon stimulation by its agonists. In this review, we have mainly emphasized on the pathogenesis of MS and highlighted the importance of adenosinergic system in reversing the molecular events in MS. In addition, we have discussed about the beneficial role of A1AR agonists in MS management.

Stimulation of adenosine A1 receptor prevents oxidative injury in H9c2 cardiomyoblasts: Role of Gβγ-mediated Akt and ERK1/2 signaling

Oxidative stress causes cellular injury and damage in the heart primarily through apoptosis resulting in cardiac abnormalities such as heart failure and cardiomyopathy. During oxidative stress, stimulation of adenosine receptor (AR) has been shown to protect against oxidative damage due to their cytoprotective properties. However, the subtype specificity and signal transductions of adenosine A1 receptor (A1R) on cardiac protection during oxidative stress have remained elusive. In this study, we found that stimulation of A1Rs with N6-cyclopentyladenosine (CPA), a specific A1R agonist, attenuated the H2O2-induced intracellular and mitochondrial reactive oxygen species (ROS) production and apoptosis. In addition, A1R stimulation upregulated the synthesis of antioxidant enzymes (catalase and GPx-1), antiapoptotic proteins (Bcl-2 and Bcl-xL), and mitochondria-related markers (UCP2 and UCP3). Blockades of Gβγ subunit of heterotrimeric Gαi protein antagonized A1R-mediated antioxidant and antiapoptotic effects, confirming the potential role of Gβγ subunit-mediated A1R signaling. Additionally, cardioprotective effects of CPA mediated through PI3K/Akt- and ERK1/2-dependent signaling pathways. Thus, we propose that A1R represents a promising therapeutic target for prevention of oxidative injury in the heart.

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.

Modulation of adenosine signaling reverses 3-nitropropionic acid-induced bradykinesia and memory impairment in adult zebrafish

Huntington's disease (HD) is a neurodegenerative disorder, characterized by motor dysfunction, psychiatric disturbance, and cognitive decline. In the early stage of HD, occurs a decrease in dopamine D2 receptors and adenosine A2A receptors (A2AR), while in the late stage also occurs a decrease in dopamine D1 receptors and adenosine A1 receptors (A1R). Adenosine exhibits neuromodulatory and neuroprotective effects in the brain and is involved in motor control and memory function. 3-Nitropropionic acid (3-NPA), a toxin derived from plants and fungi, may reproduce HD behavioral phenotypes and biochemical characteristics. This study investigated the effects of acute exposure to CPA (A1R agonist), CGS 21680 (A2AR agonist), caffeine (non-selective of A1R and A2AR antagonist), ZM 241385 (A2AR antagonist), DPCPX (A1R antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in an HD pharmacological model induced by 3-NPA in adult zebrafish. CPA, CGS 21680, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered via i.p. in zebrafish after 3-NPA (at dose 60 mg/kg) chronic treatment. Caffeine and ZM 241385 reversed the bradykinesia induced by 3-NPA, while CGS 21680 potentiated the bradykinesia caused by 3-NPA. Moreover, CPA, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA reversed the 3-NPA-induced memory impairment. Together, these data support the hypothesis that A2AR antagonists have an essential role in modulating locomotor function, whereas the activation of A1R and blockade of A2AR and A1R and modulation of adenosine levels may reduce the memory impairment, which could be a potential pharmacological strategy against late-stage symptoms HD.

Upregulated adenosine 2A receptor accelerates post-infectious irritable bowel syndrome by promoting CD4+ T cells' T helper 17 polarization.

BACKGROUNDPost-infectious irritable bowel syndrome (PI-IBS) is generally regarded as a functional disease. Several recent studies have reported the involvement of low-grade inflammation and immunological dysfunction in PI-IBS. T helper 17 (Th17) polarization occurs in IBS. Adenosine and its receptors participate in intestinal inflammation and immune regulation.AIMTo investigate the role of Th17 polarization of CD4+ T cells regulated by adenosine 2A receptor (A2AR) in PI-IBS.METHODSA PI-IBS model was established by infecting mice with Trichinella spiralis. The intestinal A2AR and CD4+ T lymphocytes were detected by immunohistochemistry, and the inflammatory cytokines were detected by enzyme-linked immunoassay. CD4+ T lymphocytes present in the animal’s spleen were separated and cultured with or without A2AR agonist and antagonist. Western blotting and real-time quantitative polymerase chain reaction were performed to determine the effect of A2AR on the cells and intestinal tissue. Cytokine production was determined. The protein and mRNA levels of A2AR associated signaling pathway molecules were also evaluated. Furthermore, A2AR agonist and antagonist were injected into the mouse model and the clinical features were observed.RESULTSThe PI-IBS mouse model showed increased expression of ATP and A2AR (P < 0.05), and inhibition of A2AR improved the clinical features in PI-IBS, including the abdominal withdrawal reflex and colon transportation test (P < 0.05). The number of intestinal CD4+ T cells and interleukin-17 (IL-17) protein levels increased during PI-IBS, which was reversed by administration of the A2AR antagonist (P < 0.05). CD4+ T cells expressed A2AR and produced IL-17 in vitro, which was regulated by the A2AR agonist and antagonist. The A2AR antagonist increased the production of IL-17 by CD4+ T cells via the Janus kinase-signal transducer and activator of transcription-receptor-related orphan receptor γ signaling pathway.CONCLUSIONThe results of the present study suggested that the upregulation of A2AR increases PI-IBS by promoting the Th17 polarization of CD4+ T cells.

Adenosine is released during thalamic oscillations to provide negative feedback control

Physiological oscillations in the cortico-thalamo-cortical loop occur during processes such as sleep, but these can become dysfunctional in pathological conditions such as absence epilepsy. The purine neuromodulator adenosine can act as an endogenous anticonvulsant: it is released into the extracellular space during convulsive seizures to activate A1 receptors suppressing on-going activity and delaying the occurrence of the next seizure. However, the role of adenosine in thalamic physiological and epileptiform oscillations is less clear. Here we have combined immunohistochemistry, electrophysiology, and fixed potential amperometry (FPA) biosensor measurements to characterise the release and actions of adenosine in thalamic oscillations measured in rodent slices. In the thalamus, A1 receptors are highly expressed particularly in the ventral basal (VB) thalamus and reticular thalamic nucleus (nRT) supporting a role for adenosine signalling in controlling oscillations. In agreement with previous studies, both adenosine and adenosine A1 receptor agonists inhibited thalamic oscillations in control (spindle-like) and in epileptic conditions. Here we have shown for the first time that both control and epileptiform oscillations are enhanced (i.e., increased number of oscillatory cycles) by blocking A1 receptors consistent with adenosine release occurring during oscillations. Although increases in extracellular adenosine could not be directly detected during control oscillations, clear increases in adenosine concentration could be detected with a biosensor during epileptiform oscillation activity. Thus, adenosine is released during thalamic oscillations and acts via A1 receptors to feedback and reduce thalamic oscillatory activity.

Activation of adenosine A3 receptor attenuates progression of osteoarthritis through inhibiting the NLRP3/caspase‐1/GSDMD induced signalling

The specific adenosine A3 receptor (A3AR) agonist (CF101) has potential for inflammation and pain in various disease, such as arthritis, cancer and neuropathic pain, while the role of A3AR in post‐traumatic OA and the underlying mechanism is largely unknown. CF101 was orally administrated in OA rats induced by anterior cruciate ligament transection (ACLT) surgery, and the rat primary chondrocytes were stimulated by hydrogen peroxide (H2O2, 300 μM). Histologic grading system was performed for detecting cartilage degeneration and immunohistochemistry for determining pyroptosis. The moleculars associated with cartilage homeostasis and inflammatory cytokines were analysed; moreover, the activation of NLRP3 inflammasome was determined. CF101 treatment significantly attenuated OA cartilage damage, OA‐related pain and cartilage pyroptosis. Chondrocytes stimulated by H2O2 evoked ROS release, thereby promoting the activation of NLRP3 inflammasome and facilitating the cleavage of GSDMD, which ultimately resulted in the mass release of pro‐inflammatory cytokines including IL‐1β and IL‐18, and production of matrix hydrolase. The pre‐treatment with CF101 powerfully inhibited the above process both in vivo and in vitro. Our findings demonstrated that activation of A3AR attenuates OA progression and relieves pain perception through suppression of cartilage degradation and inhibition of ROS/NLRP3/GSDMD signalling, indicating pyroptosis is a potential candidate for OA treatment.

Drugs Targeting the A3 Adenosine Receptor: Human Clinical Study Data.

The A3 adenosine receptor (A3AR) is overexpressed in pathological human cells. Piclidenoson and namodenoson are A3AR agonists with high affinity and selectivity to A3AR. Both induce apoptosis of cancer and inflammatory cells via a molecular mechanism entailing deregulation of the Wnt and the NF-κB signaling pathways. Our company conducted phase I studies showing the safety of these 2 molecules. In the phase II studies in psoriasis patients, piclidenoson was safe and demonstrated efficacy manifested in significant improvements in skin lesions. Namodenoson is currently being developed to treat liver cancer, where prolonged overall survival was observed in patients with advanced liver disease and a Child–Pugh B score of 7. A pivotal phase III study in this patient population has been approved by the FDA and the EMA and is currently underway. Namodenoson is also being developed to treat non-alcoholic steatohepatitis (NASH). A Phase IIa study has been successfully concluded and showed that namodenoson has anti-inflammatory, anti-fibrosis, and anti-steatosis effects. A phase IIb study in NASH is currently enrolling patients. In conclusion, A3AR agonists are promising drug candidates in advanced stages of clinical development and demonstrate safety and efficacy in their targeted indications.

Synthesis of 2-chloropurine ribosides with chiral amino acid amides at C6 and their evaluation as A1 adenosine receptor agonists

A series of purine ribonucleosides bearing chiral amino acid amides at the C6 position of 2-chloropurine was synthesized. Molecular docking of the synthesized analogs of 2-chloroadenosine by their affinity for A1 adenosine receptors (A1ARs) was conducted. The investigation of A1AR stimulating activity of synthesized nucleosides was carried out in a model of an isolated mouse atrium. We have shown that derivatives with tyrosine, valine, and serine residues exhibit the properties of A1AR partial agonists. Animal experiments in the open field test have shown that these compounds have different profiles of psychoactive action. These nucleosides have an ophthalmic hypotensive effect and reduce intraocular pressure in a manner slightly inferior to that of timolol and brimonidine. The synthesized nucleosides can be the basis for further design and synthesis of new A1AR agonists.

Adenosine receptor signaling regulates effector T cell activation through the limitation of aerobic glucose metabolism.

Abstract Background In a mouse model of colitis, we previously reported the protective role of the adenosine axis on effector T (Teff) cell responses and disease progression. Inflammatory reactions are paired with cell metabolic reprogramming from oxidative phosphorylation (OXPHOS) to aerobic glycolysis which is also a hallmark of T cell activation. Thus, we sought to evaluate the role of the adenosine 2A receptor (A2AR) signaling in the regulation of T cell metabolism. Methods Teff cells were reactivated in the presence or absence of A2AR agonists. Gene expression of mTOR, HIF-1α and cMyc (transcriptional factors of glycolysis-related genes) were quantified by qPCR. T cell metabolic phenotype was evaluated by transcriptomic profiling (Nanostring nCounter® Metabolic Pathway Panel), as well as real-time extracellular flux analysis, glucose uptake, and ATP production assays. Results mTOR, HIF-1α and cMyc gene expression increased as soon as 2hr post stimulation. At this time point, transcriptomic analysis revealed an A2AR agonist-induced downregulation of pathways related to T cell activation (i.e. mTOR, MAPK, PI3K, NFκB and cMyc pathways) and energy metabolism (i.e glycolysis, autophagy, fatty acid synthesis) whereas oxidative metabolism pathways (OXPHOS, fatty acid oxidation, glutamine metabolism) were upregulated (Pathway Score q-value &amp;lt;0.05). Functional assays showed a decreased de novo glucose uptake and glycolysis rate after A2AR agonist treatment while OXPHOS capacity and ATP production were not modulated. Conclusion A2AR signaling provokes a rewiring of the activated T cell metabolism towards an oxidative state making adenosine a potential T cell metabolic regulator that can be exploited in the treatment of IBD. Supported by NIH R01AI079145

Therapeutic Potential of Highly Selective A3 Adenosine Receptor Ligands in the Central and Peripheral Nervous System.

Ligands of the Gi protein-coupled adenosine A3 receptor (A3R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A3R. The present review summarizes information on the effect of latest-generation A3R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A3R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed.

A3 adenosine receptor agonist IB-MECA reverses chronic cerebral ischemia-induced inhibitory avoidance memory deficit

BackgroundChronic cerebral ischemia (CCI) is a major cause of subcortical ischemic vascular dementia. Here, we examined the neuroprotective action of the A3 adenosine receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA) on white matter lesions following CCI. MethodsA CCI mouse model was established using unilateral common carotid artery occlusion. IB-MECA and 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS1523), an antagonist of the A3 adenosine receptor, were administered by intraperitoneal injection. The inhibitory avoidance test was used to examine changes in memory performance. Microtubule-associated protein 2 (MAP-2) and neurofilament were assessed by immunohistochemistry, while expressions of phosphorylated extracellular signal-regulated kinase (ERK), ERK, interferon-beta (IFN-β), and glial fibrillary acidic protein (GFAP) were assessed by western blot assay. ResultsThe memory retention score was reduced in vehicle-treated mice compared with IB-MECA-treated (p < 0.05) mice. Compared to sham-operated mice, p-ERK, GFAP and IFN-β were increased, while MAP-2 and neurofilament were reduced in vehicle-treated mice (p < 0.01 for each). IB-MECA reduced ERK phosphorylation (p < 0.01) and GFAP expression (p < 0.05), but upregulated MAP-2 and IFN-β (p < 0.01 for both), compared with vehicle. MRS1523 suppressed the effects of IB-MECA on the memory deficit, ERK phosphorylation, and on MAP-2, neurofilament, GFAP and IFN-β levels. ConclusionOur results suggest that A3 adenosine receptor stimulation ameliorates CCI-induced memory deficits, modulates the ERK signaling pathway, preserves MAP-2 and neurofilament expression, regulates GFAP expression, and upregulates the anti-inflammatory cytokine IFN-β. Thus, the A3 adenosine receptor may be a therapeutic target for treatment of cognitive disorders and cerebral inflammatory diseases.

Analgesia with 5' extracellular nucleotidase-mediated electroacupuncture for neuropathic pain.

Acupuncture is a treatment for neuropathic pain, but its mechanism remains unclear. Previous studies showed that analgesia was induced in rats with neuropathic pain when their spinal cord adenosine content increased after electroacupuncture (EA); however, the mechanism behind this electroacupuncture-induced increase has not been clarified. This study aimed to determine the role that ecto-5'-nucleotidase plays in EA-induced analgesia for neuropathic pain. We performed electroacupuncture at the Zusanli acupoint on the seventh day after establishing a rat model of neuropathic pain induced through chronic constriction injuries. We observed the mechanical withdrawal threshold and thermal pain threshold and detected the expression of ecto-5'-nucleotidase in the spinal cord using Western blot. Chronic constriction injury rat models were intraperitoneally injected with α,β-methyleneadenosine 5'-diphosphate, an ecto-5'-nucleotidase inhibitor, 30 min before electroacupuncture. The adenosine content of the spinal cord was detected using high-performance liquid chromatography. Lastly, the adenosine A1 receptor agonist N6-cyclopentyladenosine was intrathecally injected into the lumbar swelling of the rats, and the mechanical withdrawal and thermal pain thresholds were reevaluated. Analgesia and increased ecto-5'-nucleotidase expression and adenosine content in the spinal cord were observed 1 h after electroacupuncture. α,β-methyleneadenosine 5'-diphosphate was able to inhibit upregulation of adenosine content and electroacupuncture-induced analgesia. After administration of N6-cyclopentyladenosine, electroacupuncture-induced analgesia was restored. Our results suggest that electroacupuncture at Zusanli can produce analgesia in chronic constriction injury rat models, possibly via the increased ecto-5'-nucleotidase expression induced through electroacupuncture, thus leading to increased adenosine expression in the spinal cord.

Adenosine A1 receptor ligands bind to \u03b1-synuclein: implications for \u03b1-synuclein misfolding and \u03b1-synucleinopathy in Parkinson\u2019s disease

BackgroundAccumulating α-synuclein (α-syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson’s disease (PD). Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) could directly interact with α-syn to modulate α-syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN).MethodsNanopore analysis and molecular docking were used to test the binding properties of CPA and DPCPX with α-syn in vitro. Sprague–Dawley rats were administered with 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-syn aggregation and neurodegeneration were examined in the SN pars compacta and hippocampal regions using confocal imaging and Western blotting.ResultsUsing nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-syn, similar to 2-aminoindan, which is expected to promote a “knot” conformation and α-syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-syn, similar to 1-aminoindan, which is expected to promote a “loop” conformation that prevents α-syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Confocal imaging and Western blot analyses revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-syn expression/aggregation and neurodegeneration in both SN pars compacta and hippocampus. In contrast, DPCPX and 1-aminoindan attenuated the CPA-induced α-syn expression/aggregation and neurodegeneration in SN and hippocampus.ConclusionsThe results indicate that A1R agonists and drugs promoting a “knot” conformation of α-syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a “loop” conformation of α-syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in PD.

Intraocular implants loaded with A3R agonist rescue retinal ganglion cells from ischemic damage

Retinal ganglion cell (RGC) loss underlies several conditions which give rise to significant visual compromise, including glaucoma and ischaemic optic neuropathies. Neuroprotection of RGCs is a clinical well-defined unmet need in these diseases, and adenosine A3 receptor (A3R) activation emerges as a therapeutic pharmacological approach to protect RGCs.A porous biodegradable intraocular implant loaded with 2-Cl-IB-MECA (selective A3R agonist) was used as a strategy to protect RGCs.Drug-loaded PCL implants released 2-Cl-IB-MECA for an extended period and the released 2-Cl-IB-MECA limited glutamate-evoked calcium (Ca2+) rise in RGCs. Retinal thinning due to transient ischemia was not prevented by 2-Cl-IB-MECA-PCL implant. However, 2-Cl-IB-MECA-PCL implants decreased retinal cell death, promoted the survival of RGCs, preserved optic nerve structure and anterograde axonal transport. We further demonstrated that 2-Cl-IB-MECA-loaded PCL implants were able to enhance RGC function that was compromised by transient ischemia. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA released from the PCL implant, this can be envisaged a good therapeutic strategy to protect RGCs.