CGS-21680 defers cisplatin-induced AKI-CKD transition in C57/BL6 mice.

Acute kidney injury (AKI), with a high mortality and morbidity, is known as a risk factor for developing progressive chronic kidney disease (CKD). Targeting transition of AKI to CKD displays an excellent therapeutic potential. This study aims at investigating the role of CGS-21680, selective A2AR agonist, in deferring Cis-induced AKI-CKD transition. The AKI-CKD transition model was induced in C57/BL6 mice by repeated low doses of Cis (2.5 mg/kg i.p for 5 consecutive days in two cycles with a recovery phase of 16 days between two cycles). CGS-21680 was administered daily for 6 weeks (0.1 mg/kg, i.p). Urine, blood, and kidney were collected at three different time points to track the disease progression. CGS-21680 administration preserved kidney function and attenuated tubular damage as evidenced by hematoxylin-eosin (H&E) histopathology. CGS-21680 significantly restored oxidative status as reflected by reduced malondialdehyde (MDA) content and increased total antioxidant capacity (TAC). CGS-21680 showed anti-inflammatory effect as indicated by decreased TNF-α and iNOS. Additionally, CGS-21680 ameliorated endothelial dysfunction and enhanced renal vasodilation as evidenced by upregulation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) expression and down regulation of endothelin-1 (ET-1) and its receptor endothelin-A (ET-A) receptor expression. CGS-21680 also attenuated renal fibrosis as reflected by the reduction of percentage of fibrosis in Masson's trichome-stained renal sections and down regulation of transforming growth factor beta1 (TGF-ß1) protein expression in IHC-stained renal sections. In conclusion, CGS-21680 could defer Cis-induced AKI-CKD transition via its vasodilatory, antioxidant, anti-inflammatory, and anti-fibrotic effects.

An agonist of the adenosine A2A receptor, CGS21680, promotes corneal epithelial wound healing via the YAP signalling pathway.

BACKGROUND AND PURPOSE: The adenosine A2A receptor (A2AR) is involved in various physiological and pathological processes in the eye; however, the role of the A2AR signalling in corneal epithelial wound healing is not known. Here, the expression, therapeutic effects and signalling mechanism of A2AR in corneal epithelial wound healing were investigated using the A2AR agonist CGS21680. EXPERIMENTAL APPROACH: A2AR localization and expression during wound healing in the murine cornea were determined by immunofluorescence staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The effect of CGS21680 on corneal epithelial wound healing in the lesioned corneal and cultured human corneal epithelial cells (hCECs) by modulating cellular proliferation and migration was critically evaluated. The role of Hippo-YAP signalling in mediating the CGS21680 effect on wound healing by pharmacological inhibition of YAP signalling was explored. KEY RESULTS: A2AR expression was up-regulated after corneal epithelial injury. Topical administration of CGS21680 dose-dependently promoted corneal epithelial wound healing in the injured corneal epithelium by promoting cellular proliferation. Furthermore, CGS21680 accelerated the cellular proliferation and migration of hCECs in vitro. A2AR activation promoted early up-regulation and later down-regulation of YAP signalling molecules, and pharmacological inhibition of YAP signalling reverted CGS21680-mediated wound healing effect in vivo and in vitro. CONCLUSION AND IMPLICATIONS: A2AR activation promotes wound healing by enhancing cellular proliferation and migration through the YAP signalling pathway. A2ARs play an important role in the maintenance of corneal epithelium integrity and may represent a novel therapeutic target for facilitating corneal epithelial wound healing.

Neural and molecular investigation into the paraventricular thalamic-nucleus accumbens circuit for pain sensation and non-opioid analgesia.

The paucity of medications with novel mechanisms for pain treatment combined with the severe adverse effects of opioid analgesics has led to an imperative pursuit of non-opioid analgesia and a better understanding of pain mechanisms. Here, we identify the putative glutamatergic inputs from the paraventricular thalamic nucleus to the nucleus accumbens (PVTGlut→NAc) as a novel neural circuit for pain sensation and non-opioid analgesia. Our in vivo fiber photometry and in vitro electrophysiology experiments found that PVTGlut→NAc neuronal activity increased in response to acute thermal/mechanical stimuli and persistent inflammatory pain. Direct optogenetic activation of these neurons in the PVT or their terminals in the NAc induced pain-like behaviors. Conversely, inhibition of PVTGlut→NAc neurons or their NAc terminals exhibited a potent analgesic effect in both naïve and pathological pain mice, which could not be prevented by pretreatment of naloxone, an opioid receptor antagonist. Anterograde trans-synaptic optogenetic experiments consistently demonstrated that the PVTGlut→NAc circuit bi-directionally modulates pain behaviors. Furthermore, circuit-specific molecular profiling and pharmacological studies revealed dopamine receptor 3 as a candidate target for pain modulation and non-opioid analgesic development. Taken together, these findings provide a previously unknown neural circuit for pain sensation and non-opioid analgesia and a valuable molecular target for developing future safer medication.

Adenosine A2A receptor controls the gateway of the choroid plexus.

The choroid plexus (CP) is one of the key gateways regulating the entry of peripheral immune cells into the CNS. However, the neuromodulatory mechanisms of maintaining its gateway activity are not fully understood. Here, we identified adenosine A2A receptor (A2AR) activity as a regulatory signal for the activity of CP gateway under physiological conditions. In association with a tightly closed CP gateway, we found that A2AR was present at low density in the CP. The RNA-seq analysis revealed that the A2AR antagonist KW6002 affected the expression of the cell adhesion molecules' (CAMs) pathway and cell response to IFN-γ in the CP. Furthermore, blocking or activating A2AR signaling in the CP resulted in a decreased and an increased, respectively, expression of lymphocyte trafficking determinants and disruption of the tight junctions (TJs). Furthermore, A2AR signaling regulates the CP permeability. Thus, A2AR activity in the CP may serve as a therapeutic target for remodeling the immune homeostasis in the CNS with implications for the treatment of neuroimmunological disorders.

The role of dorsomedial striatum adenosine 2A receptors in the loss of goal-directed behaviour.

RATIONALE: Adenosine A2A receptors (A2AR) in the dorsal striatum have been implicated in goal-directed behaviour. While activation of these receptors with several methods has resulted in an insensitivity to outcome devaluation, particular explanations for how they disrupt behaviour have not been explored. We both confirm a role for A2A receptors in goal-directed responding and evaluate additional behavioural aspects of goal-directed control to more fully understand the role of A2A receptors in instrumental behaviour. OBJECTIVES: To examine the effects of the adenosine A2A agonist CGS-21680 in the DMS on response-outcome encoding, updating representations of outcome value and on the ability to inhibit behaviour when reward is not available. METHODS: Male rats were trained to lever press for food reward. The A2AR agonist CGS-21680 was infused into the dorsomedial striatum either before an outcome devaluation test, prior to training with two distinct response-outcome associations or prior to a test of discriminative stimulus control over instrumental performance. RESULTS: Intra-DMS administration of CGS-21680 impaired sensitivity to outcome devaluation. CGS-21680 treatment did not impair acquisition of specific response-outcome associations, selective control of responding based on the presence of stimuli that signaled when reward was or was not available, discrimination between stimuli or lever choices nor did it influence the effect of devaluation on the amounts of food eaten in a consumption test. CONCLUSIONS: CGS-21680 impairs the ability to modulate responding based on recent changes to outcome value, an effect that is not accounted for by impairments in behavioural inhibition, discrimination, encoding the specific outcome of a response or the effectiveness of specific satiety.

Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif.

BACKGROUND: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. METHODS: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. RESULTS AND CONCLUSIONS: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.

Adenosine A2A Receptor: A New Neuroprotective Target in Light-Induced Retinal Degeneration.

Continuous illumination induces the degeneration of photoreceptors. This animal model of light-induced retinal degeneration resembles many characteristics of human degenerative diseases of the outer retina, such as age-related macular degeneration. This work aimed to evaluate the potential neuroprotective effect of the modulation of adenosine A2A receptor in the model of light-induced retinal degeneration. Sprague-Dawley rats were intravitreally injected in the right eye with either CGS 21680, an adenosine A2A receptor agonist, or SCH 58261, an adenosine A2A receptor antagonist. Contralateral eyes were injected with respective vehicles as control. Then, rats were subjected to continuous illumination (12,000 lux) for 24 h. Retinas were processed by glial fibrillary acidic protein (GFAP) immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, Western blotting (WB), and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Another group of rats was subjected to functional studies by electroretinography. Animals treated with CGS21680 showed a significant increase of apoptotic nuclei in the outer nuclear layer and a significant increase of GFAP immunoreactive area of the retinas but did not alter WB nor electroretinography results. qRT-PCR showed that CGS 21680 significantly increased the expression of interleukin-1ß. On the opposite, SCH 58261 significantly decreased apoptotic nuclei in the outer nuclear layer and GFAP immunoreactive area of the retinas. It also significantly decreased GFAP and activated caspase-3 levels as measured by WB and preserved retinal function, as treated eyes showed significantly greater amplitudes of a- and b-waves and oscillatory potentials. qRT-PCR revealed that SCH 58261 significantly decreased the expression of tumor necrosis factor-α. These results show that the blockade of the A2A receptor before the start of the pathogenic process is neuroprotective, as it prevents light-induced retinal damage. The use of A2A receptor antagonists deserves to be evaluated in retinal degenerative diseases.

Upregulation of Src Family Tyrosine Kinases in the Rat Striatum by Adenosine A2A Receptors.

Adenosine A2A receptors are Golf-coupled receptors and are predominantly expressed in the striatum of mammalian brains. As a mostly postsynaptic receptor, A2A receptors are implicated in the regulation of a variety of intracellular signaling pathways in striatopallidal output neurons and are linked to the pathogenesis of various neuropsychiatric and neurological disorders. This study investigated the possible role of A2A receptors in the modulation of the Src family kinase (SFK) in the adult rat striatum. In acutely prepared striatal slices, adding the A2A receptor agonist PSB-0777 induced a significant increase in phosphorylation of SFKs at a conserved autophosphorylation site (Y416) in the caudate putamen (CPu). This increase was also seen in the nucleus accumbens (NAc). Another A2A agonist CGS-21680 showed the similar ability to elevate SFK Y416 phosphorylation in the striatum. Treatment with the A2A receptor antagonist KW-6002 blocked the effect of PSB-0777 on SFK Y416 phosphorylation. In addition, PSB-0777 enhanced kinase activity of two key SFK members (Src and Fyn) immunoprecipitated from the striatum. These data demonstrate a positive linkage from A2A receptors to the SFK signaling pathway in striatal neurons. Activation of A2A receptors leads to the upregulation of phosphorylation of SFKs (Src and Fyn) at an activation-associated autophosphorylation site and kinase activity of these SFK members.

Arbutin effectively ameliorates the symptoms of Parkinson's disease: the role of adenosine receptors and cyclic adenosine monophosphate.

An antagonistic communication exists between adenosinergic and dopaminergic signaling in the basal ganglia, which suggests that the suppression of adenosine A2A receptors-cyclic adenosine monophosphate pathway may be able to restore the disrupted dopamine transmission that results in motor symptoms in Parkinson's disease (PD). Arbutin is a natural glycoside that possesses antioxidant, anti-inflammatory, and neuroprotective properties. The purpose of this study was to investigate whether arbutin could ameliorate the symptoms of PD and to examine the underlying mechanism. In this study, Swiss albino mouse models of PD were established by the intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine for 4 successive days, with the concurrent intraperitoneal administration of arbutin (50 and 100 mg/kg) for 7 days. The results showed that arbutin significantly reduced lipid peroxidation, total nitrite levels, and inflammation in the substantia nigra and striatum of PD mouse models. In addition, arbutin decreased the activity of endogenous antioxidants, reduced the levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and γ-aminobutyric acid, and minimized neurodegeneration in the striatum. Arbutin also reduced the abnormal performance of PD mouse models in the open field test, bar test, pole test, and rotarod test. The therapeutic efficacy of arbutin was similar to that of madopar. The intraperitoneal injection of the A2AR agonist CGS21680 (0.5 mg/kg) attenuated the therapeutic effects of arbutin, whereas the intraperitoneal injection of forskolin (3 mg/kg) enhanced arbutin-mediated improvements. These findings suggest that arbutin can improve the performance of PD mouse models by inhibiting the function of the A2AR and enhancing the effects of cyclic adenosine monophosphate. This study was approved by the Institutional Animal Ethics Committee (1616/PO/Re/S/12/CPCSEA) on November 17, 2019 (approval No. IAEC/2019/010).

The A2A receptor agonist CGS 21680 has beneficial and adverse effects on disease development in a humanised mouse model of graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative method for blood cancers and other blood disorders, but is limited by the development of graft-versus-host disease (GVHD). GVHD results in inflammatory damage to the host liver, gastrointestinal tract and skin, resulting in high rates of morbidity and mortality in HSCT recipients. Activation of the A2A receptor has been previously demonstrated to reduce disease in allogeneic mouse models of GVHD. This study aimed to investigate the effect of A2A activation on disease development in a humanised mouse model of GVHD. Immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (IL)-2 receptor γnull (NSG) mice injected with human (h) peripheral blood mononuclear cells (hPBMCs), were treated with either the A2A agonist CGS 21680 or control vehicle. Contrary to the beneficial effect of A2A activation in allogeneic mouse models, CGS 21680 increased weight loss, and failed to reduce the clinical score or increase survival in this humanised mouse model of GVHD. Moreover, CGS 21680 reduced T regulatory cells and increased serum human IL-6 concentrations. Conversely, CGS 21680 reduced serum human tumour necrosis factor (TNF)-α concentrations and leukocyte infiltration into the liver, indicating that A2A activation can, in part, reduce molecular and histological GVHD in this model. Notably, CGS 21680 also prevented healthy weight gain in NSG mice not engrafted with hPBMCs suggesting that this compound may be suppressing appetite or metabolism. Therefore, the potential benefits of A2A activation in reducing GVHD in HSCT recipients may be limited and confounded by adverse impacts on weight, decreased T regulatory cell frequency and increased IL-6 production.

Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of ß-catenin in osteoblasts.

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A2A receptors (A2ARs) and Wingless/Integrated-ß-catenin pathways. Cytosolic ß-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular ß-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) ß-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular ß-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 µM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 ß-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular ß-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, ß-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of ß-catenin in osteoblasts.

Attenuation of adverse effects of noise induced hearing loss on adult neurogenesis and memory in rats by intervention with Adenosine A2A receptor agonist.

Hearing loss and cognitive decline are commonly associated with aging and morbidity. Present clinical interest lies in whether peripheral hearing loss promotes cognitive decline and if prophylaxis with selective adenosine receptor agonist CGS21680 effectively mitigates the adverse effects. In the current study, male Sprague Dawley rats weighing 200-250 g m were randomly allocated into three groups: Group 1) rats exposed to 100 dB SPL white noise, 2 h a day for 15 consecutive days, 2) rats supplemented with an adenosine receptor agonist, CGS21680 at 100 µg/kg/day prior to noise exposure and 3) unexposed control rats. Baseline hearing and cognition assessed by auditory brainstem response (ABR) and water maze respectively was undertaken for all the groups. Phalloidin stain and synaptic ribbons count in cochlea, and, Ki67, DCX and NeuN in hippocampus were observed by immunohistochemistry. It was inferred that noise exposed rats showed elevated thresholds of ABR and poorer performances in spatial working memory when compared with controls. On the contrary, CGS21680 administered group exhibited improved ABR and cognitive functions with shorter mean latency and path-length to reach the platform, significant reduction in the noise induced loss of synaptic ribbons count and increased number of Ki67 and doublecortin (DCX) positive cells compared to their noise exposed counterparts. Pharmacologic intervention with selective A2A receptor agonist CGS21680 provided adequate protection from noise by effectively maintaining hearing threshold levels, cell viability in cochlea and hippocampus & functional/intact reference memory.

IPP-1 controls Akt/CREB phosphorylation extension in A2a adenosine receptor signaling cascade in MIN6 pancreatic ß-cell line.

Signaling through A2a adenosine receptor specifically prevent pancreatic ß-cells (PBCs) loses under diabetogenic conditions. However, signaling mediators of this receptor in PBCs remained unidentified. Thus, we aimed to investigate the possible involvement of PKA/Akt/IPP-1/CREB pathway in MIN6 ß-cells. In addition, we investigated IPP-1 role in A2a receptor signaling pathway. The expression of A2a receptor in MIN6 cell line was evaluated by RT-PCR and its functionality confirmed by quantification of cAMP in response to the CGS 21680, an A2a receptor agonist. MTT and Brdu assays were used to evaluate cell viability and proliferation, respectively. PKA activity and insulin release were evaluated using ELISA methods. P-Akt/Akt, p-IPP-1/IPP-1, and p-CREB/CREB levels were assessed using western blotting. IPP-1 knock down assessments was performed using specific siRNA. Our result revealed that MIN6 cells express A2a receptor which actively increased cAMP levels (with EC50 = 2.41 µM) and PKA activity. Activation of this receptor increased cell viability, proliferation and insulin release. Moreover, we mentioned A2a receptor stimulation increased p-Akt, p-IPP-1, and p-CREB levels in dose (max at 10 µM of CGS 21680) and time (max at 30 min after CGS 21680 treatment) dependent manner. Interestingly, herein, we found in IPP-1 knocked down cells, A2a receptor failed to activate Akt and CREB. Altogether, we mentioned that in MIN6 cells A2a receptor increase cell viability, proliferation and insulin release through PKA/Akt/IPP-1/CREB signaling pathway. In addition, we conclude A2a receptor signaling through this pathway is dependent to activation of IPP-1.

Adenosine and Sleep.

The classic endogenous somnogen adenosine promotes sleep via A1 and A2A receptors. In this chapter, we present an overview of the current knowledge regarding the regulation of adenosine levels, adenosine receptors, and available pharmacologic and genetic tools to manipulate the adenosine system. This is followed by a summary of current knowledge of the role of adenosine and its receptors in the regulation of sleep and wakefulness. Despite strong data implicating numerous brain areas, including the basal forebrain, the tuberomammillary nucleus, the lateral hypothalamus, and the nucleus accumbens, in the adenosinergic control of sleep, the complete neural circuitry in the brain involved in the sleep-promoting effects of adenosine remains unclear. Moreover, the popular demand for natural sleep aids has led to a search for natural compounds that can promote sleep via adenosine receptor activation. Finally, we discuss the effects of caffeine in man and the possible use of more selective adenosine receptor drugs for the treatment of sleep disorders.

Reinterpreting anomalous competitive binding experiments within G protein-coupled receptor homodimers using a dimer receptor model.

An increasing number of G protein-coupled receptors (GPCRs) have been reported to be expressed in the plasma membrane as dimers. Since most ligand binding data are currently fitted by classical equations developed only for monomeric receptors, the interpretation of data could be misleading in the presence of GPCR dimers. On the other hand, the equations developed from dimer receptor models assuming the existence of two orthosteric binding sites within the dimeric molecule offer the possibility to directly calculate macroscopic equilibrium dissociation constants for the two sites, an index of cooperativity (DC) that reflects the molecular communication within the dimer and, importantly, a constant of radioligand-competitor allosteric interaction (KDAB) in competitive assays. Here, we provide a practical way to fit competitive binding data that allows the interpretation of apparently anomalous results, such as competition curves that could be either bell-shaped, monophasic or biphasic depending on the assay conditions. The consideration of a radioligand-competitor allosteric interaction allows fitting these curve patterns both under simulation conditions and in real radioligand binding experiments, obtaining competitor affinity parameters closer to the actual values. Our approach is the first that, assuming the formation of receptor homodimers, is able to explain several experimental results previously considered erroneous due to their impossibility to be fitted. We also deduce the radioligand concentration responsible for the conversion of biphasic to monophasic or to bell-shaped curves in competitive radioligand binding assays. In conclusion, bell-shaped curves in competitive binding experiments constitute evidence for GPCR homodimerization.

A2a adenosine receptor agonist improves endoplasmic reticulum stress in MIN6 cell line through protein kinase A/ protein kinase B/ Cyclic adenosine monophosphate response element-binding protein/ and Growth Arrest And DNA-Damage-Inducible 34/ eukaryotic Initiation Factor 2α pathways.

Endoplasmic reticulum (ER) stress is one of the main molecular events underlying pancreatic beta cell (PBC) failure, apoptosis, and a decrease in insulin secretion. Recent studies have highlighted the fundamental role of A2a adenosine receptor (A2aR) in potentiation of insulin secretion and proliferation of PBCs. However, possible protective effects of A2aR signaling against ER stress have not been elucidated yet. Thus, in the present study, we aimed to investigate the effects of A2aR activation in MIN6 beta cells undergoing tunicamycin (TM)-mediated ER stress. A2aR expression and activity were evaluated using real-time polymerase chain reaction and measurement of the cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), phospho-protein kinase B or Akt (p-Akt)/Akt, and phospho-Cyclic adenosine monophosphate response element-binding protein/CREB levels in response to a specific agonist (CGS 21680). Survival and proliferation in TM and CGS 21680 cotreated cells were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), annexin V-fluorescein isothiocyanate (FITC)/propidium iodide staining, colony formation, and 5-bromo-2'-deoxyuridine (Brdu) assays. In addition, the effects of A2aR stimulation on insulin secretion were evaluated using the enzyme-linked immunosorbent assay. B-cell lymphoma 2 (Bcl-2), phospho-eukaryotic Initiation Factor 2α (p-eIF2α)/eIF2α, growth arrest and DNA-damage-inducible 34 (GADD34), X-box binding protein 1 (XBP-1), spliced X-box binding protein 1 (XBP-1s), immunoglobulin heavy-chain-binding protein (BIP), and CCAAT-enhancer-binding protein homologous protein (CHOP) levels were evaluated using western blotting. Our results showed a decrease in A2aR expression and p-Akt/Akt and p-CREB/CREB levels in TM-pretreated cells. We also mentioned that CGS 21680 effectively increased cell survival, proliferation, and insulin secretion in TM-treated cells. The antiapoptotic effects were possibly mediated through Bcl-2 upregulation. Our western blotting results indicated that A2aR effectively downregulated p-eIF2α/eIF2α, XBP-1, XBP-1s, BIP, and CHOP levels, whereas GADD34 was upregulated. Altogether, the present study revealed that A2aR signaling through PKA/Akt/CREB mediators alleviated TM cytotoxicity effects in MIN6 beta cells. Thus, the stimulation of this receptor was seen as a new approach to control ER stress in the PBC cells.

Characterization of the trigeminovascular actions of several adenosine A2A receptor antagonists in an in vivo rat model of migraine.

BACKGROUND: Migraine is considered a neurovascular disorder, but its pathophysiological mechanisms are not yet fully understood. Adenosine has been shown to increase in plasma during migraine attacks and to induce vasodilation in several blood vessels; however, it remains unknown whether adenosine can interact with the trigeminovascular system. Moreover, caffeine, a non-selective adenosine receptor antagonist, is included in many over the counter anti-headache/migraine treatments. METHODS: This study used the rat closed cranial window method to investigate in vivo the effects of the adenosine A2A receptor antagonists with varying selectivity over A1 receptors; JNJ-39928122, JNJ-40529749, JNJ-41942914, JNJ-40064440 or JNJ-41501798 (0.3-10 mg/kg) on the vasodilation of the middle meningeal artery produced by either CGS21680 (an adenosine A2A receptor agonist) or endogenous CGRP (released by periarterial electrical stimulation). RESULTS: Regarding the dural meningeal vasodilation produced neurogenically or pharmacologically, all JNJ antagonists: (i) did not affect neurogenic vasodilation but (ii) blocked the vasodilation produced by CGS21680, with a blocking potency directly related to their additional affinity for the adenosine A1 receptor. CONCLUSIONS: These results suggest that vascular adenosine A2A (and, to a certain extent, also A1) receptors mediate the CGS21680-induced meningeal vasodilation. These receptors do not appear to modulate prejunctionally the sensory release of CGRP. Prevention of meningeal arterial dilation might be predictive for anti-migraine drugs, and since none of these JNJ antagonists modified per se blood pressure, selective A2A receptor antagonism may offer a novel approach to antimigraine therapy which remains to be investigated in clinical trials.

Sustained reversal of central neuropathic pain induced by a single intrathecal injection of adenosine A2A receptor agonists.

Central neuropathic pain is a debilitating outcome of spinal cord injury (SCI) and current treatments to alleviate this pain condition are ineffective. A growing body of literature suggests that activating adenosine A2A receptors (A2ARs) decreases the production of proinflammatory cytokines and increases the production of anti-inflammatory cytokines. Here, the effect of administering intrathecal A2AR agonists on central neuropathic pain was measured using hindpaw mechanical allodynia in a rat model of SCI termed spinal neuropathic avulsion pain (SNAP). Other models of SCI cause extensive damage to the spinal cord, resulting in paralysis and health problems. SNAP rats with unilateral low thoracic (T13)/high lumbar (L1) dorsal root avulsion develop below-level bilateral allodynia, without concomitant motor or health problems. A single intrathecal injection of the A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine HCl (CGS21680) reversed SCI-induced allodynia for at least 6 weeks. The reversal is likely in part mediated by interleukin (IL)-10, as intrathecally administering neutralizing IL-10 antibodies 1 week after CGS21680 abolished the anti-allodynic effect of CGS21680. Dorsal spinal cord tissue from the ipsilateral site of SCI (T13/L1) was assayed 1 and 6 weeks after CGS21680 for IL-10, CD11b, and tumor necrosis factor (TNF) gene expression. CGS21680 treatment did not change IL-10 gene expression but did significantly decrease CD11b and TNF gene expression at both timepoints. A second A2AR agonist, 4-(3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl)prop-2-ynyl)piperidine-1-carboxylic acid methyl ester (ATL313), was also able to significantly prevent and reverse SCI-induced allodynia for several weeks after a single intrathecal injection, providing converging lines of evidence of A2AR involvement. The enduring pain reversal after a single intrathecal injection of A2AR agonists suggests that A2AR agonists could be exciting new candidates for treating SCI-induced central neuropathic pain.

Critical roles of adenosine A2A receptor in regulating the balance of Treg/Th17 cells in allergic asthma.

INTRODUCTION: Deficiency of Treg cells and hyperactivity of Th17 cells together are involved in the immunological pathogenesis of asthma. The adenosine A2A receptor (A2AR) plays a critical role in the increased Foxp3 expression of Treg cells and the decreased Th17 generation. OBJECTIVE: The study aimed to investigate A2AR expression in peripheral blood and its regulatory effect on balance of Treg/Th17 cells in asthma. METHODS: Thirty-one patients with chronic persistent asthma were recruited and divided into 18 intermittent to mild asthma patients, 13 moderate to severe asthma patients. A2AR, Foxp3, and ROR-γt mRNA expression levels in peripheral blood mononuclear cells (PBMCs) were measured by quantitative polymerase chain reaction (qPCR). TGF-ß, IL-17, and IgE in plasma were detected with enzyme-linked immunosorbent assay (ELISA). Forty-two BALB/c mice were randomly, equally assigned to control group, ovalbumin (OVA) group and OVA + CGS (CGS21680, A2AR agonist) group. The infiltration of lung inflammation cells were evaluated by HE, A2AR, Foxp3, and ROR-γt mRNA in lung tissues measured by qPCR, TGF-ß, IL-17, and IgE in plasma measured with ELISA, and IL-17 and TGF-ß protein in lung tissues analyzed with immunohistochemical. RESULTS: Our results showed that expression A2AR mRNA in PBMCs was associated with asthma severity. Foxp3 mRNA, TGF-ß, and FEV1%pred positively correlated with A2AR mRNA in asthma. ROR-γt mRNA and IL-17 negatively correlated with A2AR mRNA in asthma. CGS could promote Foxp3 mRNA expression, TGF-ß, and improve lung function while inhibit ROR-γt mRNA expression, IL-17, and the infiltration of lung inflammation cells. CONCLUSION: A2AR could regulate the balance of Treg/Th17 cells in asthma.

Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603.

Endogenous adenosine A2B receptors (A2BAR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A2BAR antagonist PSB 603 in HEK 293 cells. The A2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A2AAR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA EMAX with no significant effect on EC50 values. Kinetics analysis of the effect of PSB 603 on the A2BAR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A2BAR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high.