Adenosine Receptor Subtypes Research Articles

Mitochondrial A3 adenosine receptor as a mechanism for the protective effects of A3AR agonists on chemotherapy-induced neuropathic pain.

Alterations in mitochondrial function are the linchpin in numerous disease states including in the development of chemotherapy-induced neuropathic pain (CIPN), a major dose-limiting toxicity of widely used chemotherapeutic cytotoxins. In CIPN, mitochondrial dysfunction is characterized by deficits in mitochondrial bioenergetics (e.g., decreased ATP production) that are thought to drive the degeneration of the peripheral nerve sensory axon terminal sensory arbors in the skin (the intraepidermal nerve fibers; IENFs) and induce abnormal spontaneous discharge in peripheral nerve sensory axons. Preserving mitochondrial function is anticipated to prevent CIPN. We have now discovered that the G protein-coupled receptor, A3 adenosine receptor subtype (A3AR), is expressed on the mitochondrial outer membrane. Ex vivo application of a highly selective A3AR agonist, MRS5980, to saphenous nerve microfilaments harvested from male oxaliplatin-treated rats reversed the loss in ATP production underscoring mitoprotective effects resulting from A3AR activation on mitochondria. Moreover, in vivo administration of A3AR agonists to rats during oxaliplatin treatment was associated with reduced IENF loss and a lower incidence of spontaneous discharge in peripheral afferent axons. These effects are accompanied by improved mitochondrial ATP production in primary afferent sensory axons and overall inhibition of the development of neuropathic pain. These data identify for the first time mitochondrial A3AR and indicate that activation of A3AR protects mitochondrial function in primary afferent sensory axons against chemotherapy-induced neurotoxicity. Repurposing A3AR agonists that are already in clinical trials as anticancer agents as adjunct to chemotherapeutics will address a major unmet medical need for which there are no FDA approved drugs.Significance statement Alterations in mitochondrial function are central to the development of chemotherapy-induced neuropathy and ensuing pain syndromes. Here we show that the adenosine A3 receptor (A3AR) is expressed on mitochondria and its activation can reverse deficits in mitochondrial ATP production in peripheral sensory nerves from animals with chemotherapy-induced neuropathic pain. Moreover, administration of A3AR agonists during chemotherapy in animals prevented the development of pain and pathophysiological signs of neuropathy in sensory nerves such as the loss of intraepidermal nerve fibers and increased spontaneous firing. These findings identify for the first time mitochondrial A3AR and show that activation of A3AR protects mitochondrial function in primary afferent sensory axons against chemotherapy-induced neurotoxicity.

Cordycepin improves hyperactivation and acrosome reaction through adenosine receptors during human sperm capacitation in vitro

BackgroundSperm capacitation is a prerequisite for natural or in vitro fertilization. After capacitation, sperm become hyperactivated and undergo an acrosome reaction, which helps them penetrate the oocyte. Cordycepin, a bioactive compound first isolated from Cordyceps militaris, is an adenosine analog with numerous physiological activities. However, its effects on sperm capacitation remain unclear. This study aims to elucidate the effects and mechanisms of cordycepin on human sperm capacitation.MethodsDuring in vitro capacitation culture, healthy human sperm were treated with cordycepin (20, 100, 500 µM). Sperm motility and hyperactivation were detected using a computer-assisted sperm analyzer. Sperm acrosome reaction was measured using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin. Sperm protein kinase A (PKA) activity was analyzed using an ELISA kit. The levels of sperm protein tyrosine phosphorylation were detected by western blotting. Sperm DNA damage was detected by a sperm chromatin dispersion assay. Reactive oxygen species (ROS) were measured using the fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate. The expression and localization of adenosine receptors were analyzed by western blotting and immunofluorescence. The specific inhibitors of adenosine receptors were used to confirm their effects on cordycepin-induced sperm capacitation. Finally, molecular docking was performed to analyze the interaction between cordycepin and adenosine receptors.ResultsCordycepin improved hyperactivated sperm motility, acrosome reaction, PKA activity, and protein tyrosine phosphorylation during capacitation while having no obvious effects on sperm ROS or DNA damage. Four adenosine receptor subtypes were expressed in human sperm, but their localizations differed. Inhibition of adenosine receptors significantly decreased cordycepin-induced sperm hyperactivation and the acrosome reaction. Molecular docking showed that cordycepin can bind to the four subtypes of adenosine receptors.ConclusionCordycepin may promote human sperm capacitation through adenosine receptor-mediated signaling pathways. These findings may be useful for assisted reproductive technology and animal breeding.

Novel Agonists of Adenosine Receptors in Animal Model of Acute Myocardial Infarction.

Current treatments for acute myocardial infarction (AMI) include pain relief and attempts to improve survival. This study investigated the effects of two new ligands of the adenosine receptor, LASSBio-1027 and LASSBio-1860, on cardiac function in an experimental model of AMI. AMI was induced in Wistar rats by ligating the anterior descending coronary arteries. Infarcted animals were treated orally with vehicle (DMSO), LASSBio-1027 (30 and 70μmol/kg), or LASSBio-1860 (70μmol/kg) for seven days. Hemodynamic parameters were observed using echocardiography, whereas inflammation and fibrosis were detected using histological analysis. MI increased the filling pressure from 23.0 ± 1.6 and 14.0 ± 2.0 to 37.0 ± 3.7 and 33.2 ± 8.0, respectively indicating diastolic dysfunction. However, treatment with LASSBio-1027 (70μmol/kg) and LASSBio-1860 (70μmol/kg) reduced this parameter to 23.9 ± 5.4 and 17.1 ± 6.7. An impairment in ejection fraction from 57.1 ± 3.2 to 36.6 ± 2.0% was observed after MI, partially recovered to 47.0 ± 7.4% by LASSBio-1027 and fully restored to 61.8 ± 4.3% after 7 days of treatment with LASSBio-1860. After MI, collagen deposition in LV free wall was increased to 31.4 ± 11.0% and treatment with LASSBio-1027 reduced to 23.4 ± 6.0 and 19.7 ± 8.0% at 30 and 70μmol/kg, respectively. Similarly, LASSBio-1860 reduced collagen levels to 63.1 ± 2.0%. Fibrosis and inflammatory components of MI reduced following treatment with agonist of adenosine receptor subtype A2A. Cardiac remodeling induced by LASSBio-1027 and LASSBio-1860 may be responsible for the improvement in cardiac function in AMI through the activation of A2A adenosine receptors.

Current Understanding of the Role of Adenosine Receptors in Cancer.

Cancer, a complex array of diseases, involves the unbridled proliferation and dissemination of aberrant cells in the body, forming tumors that can infiltrate neighboring tissues and metastasize to distant sites. With over 200 types, each cancer has unique attributes, risks, and treatment avenues. Therapeutic options encompass surgery, chemotherapy, radiation therapy, hormone therapy, immunotherapy, targeted therapy, or a blend of these methods. Yet, these treatments face challenges like late-stage diagnoses, tumor diversity, severe side effects, drug resistance, targeted drug delivery hurdles, and cost barriers. Despite these hurdles, advancements in cancer research, encompassing biology, genetics, and treatment, have enhanced early detection methods, treatment options, and survival rates. Adenosine receptors (ARs), including A1, A2A, A2B, and A3 subtypes, exhibit diverse roles in cancer progression, sometimes promoting or inhibiting tumor growth depending on the receptor subtype, cancer type, and tumor microenvironment. Research on AR ligands has revealed promising anticancer effects in lab studies and animal models, hinting at their potential as cancer therapeutics. Understanding the intricate signaling pathways and interactions of adenosine receptors in cancer is pivotal for crafting targeted therapies that optimize benefits while mitigating drawbacks. This review delves into each adenosine receptor subtype's distinct roles and signaling pathways in cancer, shedding light on their potential as targets for improving cancer treatment outcomes.

A novel and selective fluorescent ligand for the study of adenosine A2B receptors.

Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

Targeting Adenosine A2b Receptor Promotes Penile Rehabilitation of Refractory Erectile Dysfunction.

The mechanisms of adenosine and specific adenosine receptor subtypes in promoting penile rehabilitation remain unclear. Single-cell RNA sequencing of human corpus cavernosum, adenosine deaminase (ADA) and adenosine receptors knock-out mice (ADA-/-, A1-/-, A2a-/-, A2b-/-, and A3-/-), and primary corpus cavernosum smooth muscle cellsare used to determine receptor subtypes responsible for adenosine-induced erection. Three rat models are established to characterize refractory erectile dysfunction (ED): age-related ED, bilateral cavernous nerve crush related ED (BCNC), and diabetes mellitus-induced ED. In single-cell RNA sequencing data, the corpus cavernosum of ED patients show a decrease in adenosine A1, A2a and A2b receptors. In vivo, A2b receptor knock-out abolishes adenosine-induced erection but not that of A1, A2a, or A3 receptor. Under hypoxic conditions in vitro, activating the A2b receptor increases HIF-1α and decreases PDE5 expression. In refractory ED models, activating the A2b receptor with Bay 60-6583 improves erectile function and down-regulates HIF-1α and TGF-β. Administering Dipyridamole (40mgKg-1) to BCNC rats improve penile adenosine levels and erectile function. Our study reveals that the A2b receptor mediates adenosine-induced penile erection. Activating the A2b receptor promotes penile rehabilitation of refractory ED by alleviating hypoxia and fibrosis.

Adenosine Stimulates Beating of Neonatal Brain-Derived Cilia through Adenosine A2B Receptor on the Cilia and Activation of Protein Kinase A Pathway.

Motile cilia in the ependymal cells that line the brain ventricles play pivotal roles in cerebrospinal fluid (CSF) flow in well-defined directions. However, the substances and pathways which regulate their beating have not been well studied. Here, we used primary cultured cells derived from neonatal mouse brain that possess motile cilia and found that adenosine (ADO) stimulates ciliary beating by increasing the ciliary beat frequency (CBF) in a concentration-dependent manner, with the ED50 value being 5 µM. Ciliary beating stimulated by ADO was inhibited by A2B receptor (A2BR) antagonist MRS1754 without any inhibition by antagonists of other ADO receptor subtypes. The expression of A2BR on the cilia was also confirmed by immunofluorescence. The values of CBF were also increased by forskolin, which is an activator of adenylate cyclase, whereas they were not further increased by the addition of ADO. Furthermore, ciliary beating was not stimulated by ADO in the presence of a protein kinase A (PKA) inhibitors. These results altogether suggest that ADO stimulates ciliary beating through A2BR on the cilia, and activation of PKA.

2-Aryladenine Derivatives as a Potent Scaffold for Adenosine Receptor Antagonists: The 6-Morpholino Derivatives.

A set of 2-aryl-9-H or methyl-6-morpholinopurine derivatives were synthesized and assayed through radioligand binding tests at human A1, A2A, A2B, and A3 adenosine receptor subtypes. Eleven purines showed potent antagonism at A1, A3, dual A1/A2A, A1/A2B, or A1/A3 adenosine receptors. Additionally, three compounds showed high affinity without selectivity for any specific adenosine receptor. The structure-activity relationships were made for this group of new compounds. The 9-methylpurine derivatives were generally less potent but more selective, and the 9H-purine derivatives were more potent but less selective. These compounds can be an important source of new biochemical tools and/or pharmacological drugs.

Adenosine A3 Receptor: From Molecular Signaling to Therapeutic Strategies for Heart Diseases.

Cardiovascular diseases (CVDs), particularly heart failure, are major contributors to early mortality globally. Heart failure poses a significant public health problem, with persistently poor long-term outcomes and an overall unsatisfactory prognosis for patients. Conventionally, treatments for heart failure have focused on lowering blood pressure; however, the development of more potent therapies targeting hemodynamic parameters presents challenges, including tolerability and safety risks, which could potentially restrict their clinical effectiveness. Adenosine has emerged as a key mediator in CVDs, acting as a retaliatory metabolite produced during cellular stress via ATP metabolism, and works as a signaling molecule regulating various physiological processes. Adenosine functions by interacting with different adenosine receptor (AR) subtypes expressed in cardiac cells, including A1AR, A2AAR, A2BAR, and A3AR. In addition to A1AR, A3AR has a multifaceted role in the cardiovascular system, since its activation contributes to reducing the damage to the heart in various pathological states, particularly ischemic heart disease, heart failure, and hypertension, although its role is not as well documented compared to other AR subtypes. Research on A3AR signaling has focused on identifying the intricate molecular mechanisms involved in CVDs through various pathways, including Gi or Gq protein-dependent signaling, ATP-sensitive potassium channels, MAPKs, and G protein-independent signaling. Several A3AR-specific agonists, such as piclidenoson and namodenoson, exert cardioprotective impacts during ischemia in the diverse animal models of heart disease. Thus, modulating A3ARs serves as a potential therapeutic approach, fueling considerable interest in developing compounds that target A3ARs as potential treatments for heart diseases.

Intrinsic ecto-5'-Nucleotidase/A1R Coupling may Confer Neuroprotection to the Cerebellum in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is widely used animal model of multiple sclerosis (MS). The disease is characterized by demyelination and neurodegeneration triggered by infiltrated autoimmune cells and their interaction with astrocytes and microglia. While neuroinflammation is most common in the spinal cord and brainstem, it is less prevalent in the cerebellum, where it predisposes to rapid disease progression. Because the induction and progression of EAE are tightly regulated by adenosinergic signaling, in the present study we compared the adenosine-producing and -degrading enzymes, ecto-5'-nucleotidase (eN/CD73) and adenosine deaminase (ADA), as well as the expression levels of adenosine receptors A1R and A2AR subtypes in nearby areas around the fourth cerebral ventricle-the pontine tegmentum, the choroid plexus (CP), and the cerebellum. Significant differences in histopathological findings were observed between pontine tegmentum and cerebellum on the same horizontal section level. Reactive astrogliosis and massive infiltration of CD4 + cells and macrophages in CP and pontine tegmentum resulted in local demyelination. In cerebellum, there was no evidence of infiltrates, microgliosis and neuroinflammation at the same sectional level. In addition, Bergman glia showed no signs of reactive gliosis. As for adenosinergic signaling, significant upregulation of eN/CD73 was observed in all areas studied, but in association with different adenosine receptor subtypes. In CP and pons, overexpression of eN/CD73 was coupled with induction of A2AR, whereas in cerebellum, a modest increase in eN/CD73 in resident Bergman glia was accompanied by a strong induction of A1R in the same type of astrocytes. Thus, the presence of specialized astroglia and intrinsic differences in adenosinergic signaling may play a critical role in the differential regional susceptibility to EAE inflammation.

Antihypertensive Effect of New Agonist of Adenosine Receptor in Spontaneously Hypertensive Rats.

Systemic arterial hypertension is a risk factor for cardiac, renal, and metabolic dysfunction. The search for new strategies to prevent and treat cardiovascular diseases led to the synthesis of new N-acylhydrazones to produce antihypertensive effect. Adenosine receptors are an alternative target to reduce blood pressure because of their vasodilatory action and antioxidant properties, which may reduce oxidative stress characteristic of systemic arterial hypertension. To evaluate the antihypertensive profile of novel selenium-containing compounds designed to improve their interaction with adenosine receptors. Vascular reactivity was evaluated by recording the isometric tension of pre-contracted thoracic aorta of male Wistar rats after exposure to increasing concentrations of each derivative (0.1 to 100 μM). To investigate the antihypertensive effect in spontaneously hypertensive rats, systolic, diastolic, and mean arterial pressure and heart rate were determined after intravenous administration of 10 and 30 μmol/kg of the selected compound LASSBio-2062. Compounds named LASSBio-2062, LASSBio-2063, LASSBio-2075, LASSBio-2076, LASSBio-2084, LASSBio-430, LASSBio-2092, and LASSBio-2093 promoted vasodilation with mean effective concentrations of 15.5 ± 6.5; 14.6 ± 2.9; 18.7 ± 9.6; 6.7 ± 4.1; > 100; 6.0 ± 3.6; 37.8 ± 11.8; and 15.9 ± 5.7 μM, respectively. LASSBio-2062 (30 μmol/kg) reduced mean arterial pressure in spontaneously hypertensive rats from 124.6 ± 8.6 to 72.0 ± 12.3 mmHg (p < 0.05). Activation of adenosine receptor subtype A3 and potassium channels seem to be involved in the antihypertensive effect of LASSBio-2062. The new agonist of adenosine receptor and activator of potassium channels is a potential therapeutic agent to treat systemic arterial hypertension.

Adenosine A2B receptor agonist improves epidermal barrier integrity in a murine model of epidermal hyperplasia

Adenosine regulates multiple physiological processes through the activation of four receptor subtypes, of which the A2B adenosine receptor (A2BAR) has the lowest affinity for adenosine. Being the adenosine receptor subtype most prominently expressed in epidermis, we recently described the antiproliferative and anti-inflammatory effect of the selective A2BAR agonist BAY60–6583 (BAY) in human keratinocytes stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), so we sought to establish the effect of topical application of BAY in a model of murine epidermal hyperplasia.Topical application of BAY (1 or 10 μg/site) prevented the inflammatory reaction and skin lesions induced by TPA, minimizing hyperproliferation and acanthosis, as well as the expression of specific markers of proliferative keratinocytes. On the other hand, pre-treatment with the selective A2BAR antagonist, PSB-1115 (PSB, 5 or 50 μg/site) reversed these beneficial effects. Additionally, BAY application normalized the expression of epidermal barrier proteins, whose integrity is altered in inflammatory skin diseases, while treatment with the antagonist alone worsened it.Our results, besides confirming the anti-inflammatory and antiproliferative effects of the A2BAR agonist, further demonstrate a role of A2BAR activation to preserve the epidermal barrier. Therefore, the activation of A2BAR may constitute a possible new pharmacological target for the treatment of skin inflammatory diseases such as psoriasis.

Impairment of adenosine signaling disrupts early embryo development: unveiling the underlying mechanisms.

Purinergic signaling has been implicated in many biological functions, including development. In this study, we investigate the functions of extracellular adenosine and adenosine receptors using a mouse embryonic stem cell (ESC) line and morula stages isolated from mouse embryos. Feeder-free mouse ESC was investigated in the absence and presence of the leukemia inhibitory factor (LIF), configuring undifferentiated cells and cells undergoing spontaneous differentiation. High alkaline phosphatase (ALPL) and low CD73 levels resulting in low adenosine (eADO) levels were characteristic for pluripotent cells in the presence of the LIF, while LIF deprivation resulted in augmented adenosine levels and reduced pluripotency marker expression, which indicated differentiation. Tracing ESC proliferation by BrdU labeling revealed that the inhibition of ALPL by levamisole resulted in a decrease in proliferation due to less eADO accumulation. Furthermore, caffeine and levamisole treatment, inhibiting adenosine receptor and eADO accumulation, respectively, reduced ESC migration, similar to that observed in the absence of the LIF. Pharmacological approaches of selective adenosine receptor subtype inhibition triggered specific adenosine receptor activities, thus triggering calcium or MAP kinase pathways leading to differentiation. In line with the in vitro data, mouse embryos at the morula stage were sensitive to treatments with A1 and A3 receptor antagonists, leading to the conclusion that A1 receptor and A3 receptor inhibition impairs proliferation and self-renewal and triggers inappropriate differentiation, respectively. The findings herein define the functions of eADO signaling in early development with implications for developmental disorders, in which adenosine receptors or ectonucleotidase dysfunctions are involved, and which could lead to malformations and miscarriages, due to exposure to caffeine.

Multi-Functional Small Molecule Alleviates Fracture Pain and Promotes Bone Healing.

Bone injuries such as fractures are one major cause of morbidities worldwide. A considerable number of fractures suffer from delayed healing, and the unresolved acute pain may transition to chronic and maladaptive pain. Current management of pain involves treatment with NSAIDs and opioids with substantial adverse effects. Herein, we tested the hypothesis that the purine molecule, adenosine, can simultaneously alleviate pain and promote healing in a mouse model of tibial fracture by targeting distinctive adenosine receptor subtypes in different cell populations. To achieve this, a biomaterial-assisted delivery of adenosine is utilized to localize and prolong its therapeutic effect at the injury site. The results demonstrate that local delivery of adenosine inhibited the nociceptive activity of peripheral neurons through activation of adenosine A1 receptor (ADORA1) and mitigated pain as demonstrated by weight bearing and open field movement tests. Concurrently, local delivery of adenosine at the fracture site promoted osteogenic differentiation of mesenchymal stromal cells through adenosine A2B receptor (ADORA2B) resulting in improved bone healing as shown by histological analyses and microCT imaging. This study demonstrates the dual role of adenosine and its material-assisted local delivery as a feasible therapeutic approach to treat bone trauma and associated pain.

1,2,4]Triazolo[1,5-c]pyrimidines as Tools to Investigate A3 Adenosine Receptors in Cancer Cell Lines.

The A3 adenosine receptor is an interesting target whose role in cancer is controversial. In this work, a structural investigation at the 2-position of the [1,2,4]triazolo[1,5-c]pyrimidine nucleus was performed, finding new potent and selective A3 adenosine receptor antagonists such as the ethyl 2-(4-methoxyphenyl)-5-(methylamino)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylate (20, DZ123) that showed a Ki value of 0.47 nM and an exceptional selectivity profile over the other adenosine receptor subtypes. Computational studies were performed to rationalize the affinity and the selectivity profile of the tested compounds at the A3 adenosine receptor and the A1 and A2A adenosine receptors. Compound 20 was tested on both A3 adenosine receptor positive cell lines (CHO-A3 AR transfected, THP1 and HCT16) and on A3 negative cancer cell lines, showing no effect in the latter and a pro-proliferative effect at a low concentration in the former. These interesting results pave the way to further investigation on both the mechanism involved and potential therapeutic applications.

Anti-Inflammatory Activities of 8-Benzylaminoxanthines Showing High Adenosine A2A and Dual A1/A2A Receptor Affinity.

Chronic inflammation plays an important role in the development of neurodegenerative diseases, such as Parkinson's disease (PD). In the present study, we synthesized 25 novel xanthine derivatives with variable substituents at the N1-, N3- and C8-position as adenosine receptor antagonists with potential anti-inflammatory activity. The compounds were investigated in radioligand binding studies at all four human adenosine receptor subtypes, A1, A2A, A2B and A3. Compounds showing nanomolar A2A and dual A1/A2A affinities were obtained. Three compounds, 19, 22 and 24, were selected for further studies. Docking and molecular dynamics simulation studies indicated binding poses and interactions within the orthosteric site of adenosine A1 and A2A receptors. In vitro studies confirmed the high metabolic stability of the compounds, and the absence of toxicity at concentrations of up to 12.5 µM in various cell lines (SH-SY5Y, HepG2 and BV2). Compounds 19 and 22 showed anti-inflammatory activity in vitro. In vivo studies in mice investigating carrageenan- and formalin-induced inflammation identified compound 24 as the most potent anti-inflammatory derivative. Future studies are warranted to further optimize the compounds and to explore their therapeutic potential in neurodegenerative diseases.

Ivy Leaf Dry Extract EA 575® Has an Inhibitory Effect on the Signalling Cascade of Adenosine Receptor A2B.

Ivy leaf dry extract EA 575® is used to improve complaints of chronic inflammatory bronchial diseases and acute inflammation of the respiratory tract accompanied by coughing. Its mechanism of action has so far been explained by influencing β2-adrenergic signal transduction. In the present study, we investigated a possible influence on adenosine receptor A2B (A2BAR) signalling, as it has been described to play a significant and detrimental role in chronic inflammatory airway diseases. The influence of EA 575® on A2BAR signalling was assessed with measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, β-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via the Lumit™ Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified, and A2BAR was found to be responsible. The present study demonstrates an inhibitory influence of EA 575® on A2BAR-mediated general cellular response, cAMP levels, β-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occur within a time frame of several hours of incubation, its mode of action can be described as indirect. The present data are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. This may offer an explanation for the beneficial clinical effects of the extract in adjuvant asthma therapy.

Curcumin Stereoisomer, Cis-Trans Curcumin, as a Novel Ligand to A1 and A3 Adenosine Receptors.

Adenosine receptors (ARs) are being explored to generate non-opioid pain therapeutics. Vanilloid compounds, curcumin, capsaicin, and vanillin possess antinociceptive properties through their interactions with the transient receptor potential channel family. However, their binding with adenosine receptors has not been well studied. The hypothesis in this study was that a vanilloid compound, cis-trans curcumin (CTCUR), binds to each of the two Gi-linked AR subtypes (A1AR and A3AR). CTCUR was synthesized from curcumin (CUR) using the cavitand-mediated photoisomerization technique. The cell lines transfected with the specific receptor (A1AR or A3AR) were treated with CTCUR or CUR and the binding was analyzed using competitive assays, confocal microscopy, and docking. The binding assays and molecular docking indicated that CTCUR had Ki values of 306 nM (A1AR) and 400 nM (A3AR). These values suggest that CTCUR is selective for Gi-linked ARs (A1AR or A3AR) over Gs-linked ARs (A2AAR or A2BAR), based on our previous published research. In addition, the docking showed that CTCUR binds to the toggle switch domain of ARs. Curcumin (CUR) did not exhibit binding at any of these receptors. In summary, CTCUR and other modifications of CUR can be developed as novel therapeutic ligands for the Gi-linked ARs (A1AR and A3AR) involved with pain and cancer.

Role of adenosine A2a receptor in cancers and autoimmune diseases.

Adenosine receptors are P1 class of purinergic receptors that belong to G protein-coupled receptors. There are 4 subtypes of adenosine receptors, namely A1, A2A, A2B, and A3. A2AR has a high affinity for the ligand adenosine. Under pathological conditions or external stimuli, ATP is sequentially hydrolyzed to adenosine by CD39 and CD73. The combination of adenosine and A2AR can increase the concentration of cAMP and activate a series of downstream signaling pathways, and further playing the role of immunosuppression and promotion of tumor invasion. A2AR is expressed to some extent on various immune cells, where it is abnormally expressed on immune cells in cancers and autoimmune diseases. A2AR expression also correlates with disease progression. Inhibitors and agonists of A2AR may be potential new strategies for treatment of cancers and autoimmune diseases. We herein briefly reviewed the expression and distribution of A2AR, adenosine/A2AR signaling pathway, expression, and potential as a therapeutic target.

Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.